Conversion and upgrading of S-lignin related syringate by Acinetobacter baylyi ADP1

Conversion and upgrading of S-lignin related syringate by Acinetobacter baylyi ADP1 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Conversion and upgrading of S-lignin related syringate by Acinetobacter baylyi ADP1 Heidi Tuomela, Johanna Koivisto, Elena Efimova, Suvi Santala This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6218493/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Sep, 2025 Read the published version in Microbial Cell Factories → Version 1 posted 9 You are reading this latest preprint version Abstract Background: Lignin holds great potential as an abundant and sustainable source of aromatic compounds, offering a viable alternative to fossil-based resources for producing chemicals and materials. Biological upgrading of lignin-derived aromatics can lead to more comprehensive lignocellulose utilization, thereby enhancing the overall feasibility of production. However, exploring a broader range of potential microbial hosts, pathways, and enzymes is crucial for developing efficient conversion processes. In particular, improving the conversion of S-lignin-related aromatics, such as syringate, remains a key area for future research. Results: In this study, we aimed to investigate the conversion of S-lignin-related syringate in Acinetobacter baylyi ADP1 by exploiting its native vanillate demethylase, VanAB. We discovered that the wild-type strain can efficiently O -demethylate syringate to 3-O-methylgallate (3MGA) and then to gallate, revealing a previously unknown activity of VanAB of A. baylyi ADP1. Conversion dynamics and in vitro characterization showed that VanAB prefers syringate as a substrate over 3MGA. Overexpression of vanAB resulted in simultaneous conversion of syringate and 3MGA, but negatively impacted growth, potentially due to toxic side product formaldehyde and redox imbalance caused by high NADH consumption of the O -demethylation reactions. Native vanAB expression resulted in 3MGA accumulation if syringate was available. We took advantage of this by constructing a strain with heterologous expression of galA , a gallate dioxygenase from Pseudomonas putida KT2440, and demonstrated the conversion of 3MGA into 2-pyrone-4,6,-dicarboxylate (PDC), a precursor for high-quality polyesters. Conclusions: In this study, we discovered a previously unknown activity of syringate conversion in A. baylyi ADP1. By adjusting the expression level of vanAB , syringate can be directed either into gallate or 3MGA, which could be further converted into PDC through the heterologous expression of galA. Our results further highlight the potential and versatility of A. baylyi ADP1 for lignin valorisation. Lignin syringate O-demethylation vanillate O-demethylase Acinetobacter baylyi ADP1 2-pyrone-4 6-dicarboxylate Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Background Lignin, comprising up to a third of lignocellulose, is a natural polymer found in all wood-like plants. It is well known for its tenacious structure that is formed by complex networks of three main subunits – p -coumaryl (H), coniferyl (G) and sinapyl (S) alcohol. While lignin provides protection and support for plants, it is considered a nuisance in industries that use lignocellulose as raw material. In these industries, burning lignin for power generation is the main means to derive value from it. Therefore, upgrading lignin to valuable products could improve the economic feasibility of biobased industries and contribute to more carbon-wise use of resources. Lignin also provides a renewable source of aromatics, an alternative to current fossil-based feedstocks for chemical production. Depolymerized lignin consists of a heterogeneous mixture of aromatic compounds, which varies across different plant types. Biological upgrading of lignin is a promising approach, as it allows these diverse aromatic compounds to be funnelled to specific, defined products. Recently, the native catabolic pathways of soil bacteria, such as Pseudomonas putida KT2440, Sphingobium sp. SYK-6, Novosphingobium aromaticivorans and Acinetobacter baylyi ADP1 have been utilized for upgrading lignin-related aromatic compounds into value-added products ( 1 , 2 ). For example, the biological production of plastic precursors such as cis,cis -muconate ( 3 , 4 ), adipate ( 5 , 6 ), gallate ( 7 , 8 ), and 2-pyrone-4,6-dicarboxylate (PDC) ( 9 , 10 ) has been extensively studied. Plastics produced from biobased precursors can have many advantages over their fossil-based counterparts, such as better biodegradability, and improved mechanical and adhesive properties ( 11 – 13 ). S-lignin constitutes a significant portion of the lignin polymer, particularly in grasses and hardwood ( 14 ). S-lignin derived aromatics, such as syringaldehyde and syringate, are prevalent in depolymerized lignin. However, microbial strains capable of catabolizing these compounds are scarce ( 15 , 16 ). The different lignin types are distinguished by the number of methoxy groups attached to the aromatic ring. S-lignin derived aromatics are the most challenging to catabolize because they contain two methoxy groups, compared to G-lignin with one methoxy group and H-lignin, which is not methoxylated. Consequently, O- demethylation of these methoxy groups is a crucial step in the upper pathways of aromatic catabolism. In aerobic microorganisms, O- demethylation is carried out by enzymes that can be divided into three groups: Rieske oxygenases (RO), cytochromes P450 (P450s), and tetrahydrofolate (THF)-dependent demethylases ( 17 ). THF-dependent demethylases transfer the methyl group non-oxidatively to THF ( 18 ), whereas ROs and P450s utilize NAD(P)H as a cofactor to oxidize the methyl group into formaldehyde ( 17 ). Formaldehyde, being highly toxic to cells, can be mitigated by more than one pathway ( 19 ). For example, in E. coli frmA encodes a glutathione dependent formaldehyde dehydrogenase that is coupled to restoring NAD + into NADH ( 20 ). The requirement for NADH increases for substrates with several methoxy groups, for which O- demethylating reactions have been shown to be energy-limited and cause cofactor imbalance in cells. Substrates (such as glucose) that drive primary metabolism are often required to support cell’s energy generation to obtain a balanced redox stoichiometry and sustain efficient utilization of the aromatic compounds ( 9 , 17 , 21 ). Rieske oxygenases consist of a reductase, an oxygenase, and in some cases an additional ferredoxin for electron transfer. The reductase transfers electrons from NAD(P)H to the oxygenase resulting in reduction of the mononuclear iron. Binding of the substrate leads to binding of O 2 at the iron-center, resulting in a high-valency ferryl species that can hydroxylate the substrate. The final product is obtained after rearrangement of the hydroxylated species ( 22 ). VanAB is a Rieske oxygenase that uses vanillate as its’ primary substrate (Fig. 1 A). Previously it has been shown that this enzyme pair can O- demethylate syringate into 3-O-methylgallate and subsequently into gallate in Pseudomonas sp . HR199, Pseudomonas putida KT2440 and Streptomyces sp. NL15-2K (Fig. 1 B) ( 21 , 23 – 25 ). Although the substrate range of VanAB in A. baylyi ADP1 (hereafter ADP1) has been investigated and includes several analogous substrates, no activity for syringate has been previously detected ( 26 ). This is unexpected given the high similarity between vanAB orthologues in the different strains. Many factors support the hypothesis of syringate O- demethylation by VanAB in ADP1. Amino acid sequence identity of the substrate binding VanA found in ADP1 to P. putida KT2440 and P. putida HR199 is very high, 75–78%. Known substrates of VanAB indicate that a methoxy group in meta position to a carboxyl group is necessary for the activity of the enzyme ( 26 ). This does not exclude syringate as a substrate. ADP1 has raised increasing interest as a potential microbial cell factory for its versatile metabolism and natural competence, which make metabolic engineering remarkably simple ( 27 – 29 ). ADP1 can utilize a variety of G- and H-type lignin monomers via the native β-ketoadipate pathway and has been shown to have ligninolytic effects on softwood ( 30 , 31 ). Previously, ADP1 has been engineered for the production of wax esters and alkanes ( 32 ), 1-alkenes ( 33 ), cis,cis -muconate ( 34 ), mevalonate ( 35 ), naringenin ( 36 ), and resveratrol and vanillin-glucoside ( 37 ) from lignin-related aromatic compounds, such as ferulate and p -coumarate. In addition, the strain has been previously evolved to tolerate very high concentrations of aromatic compounds ( 33 , 38 ) as well as exploited in the detoxification of lignocellulosic hydrolysates ( 39 – 42 ). In this study, our goal was to establish the conversion and upgrading of S-lignin-related syringate in ADP1. We first investigated the activity of VanAB both in vivo and in vitro under native and non-native expression systems. We showed that both syringate and 3MGA can be O- demethylated by ADP1. We also demonstrated the production of PDC, a precursor for high-quality polyesters, from syringate by the heterologous expression of gallate dioxygenase GalA from P. putida KT2440. Materials and methods Strains and media Escherichia coli XL1-Blue (Stratagene, USA) was used for plasmic construction and maintenance. Acinetobacter baylyi ADP1 (DSM 24193, Leibniz Institute DSMZ, Germany) was used to study the endogenous expression of VanAB and for strain construction. All the strains used and engineered in this study are listed in Table 1 . Table 1 Bacterial strains used in this study. Name Genotype Description Source ADP1 WT Wild-type A. baylyi ADP1 DSM 24193 DSMZ ASA1001 A. baylyi ADP1 ΔvanAB::tdk/kan r vanAB (ACIAD0979-0980) replaced with tdk/kan r cassette This study ASA1002 A. baylyi ADP1 ΔvanAB Strain with markerless vanAB deletion This study ASA1003 A. baylyi ADP1 ΔvanAB, pBAV1C-T5-vanAB/Cm Strain ASA1002 with vanAB overexpression from pBAV1C-T5-plasmid under constitutive T5 promoter. This study ASA1004 A. baylyi ADP1 ΔvanAB, pBAV1Cd-chn-vanAB/Cm Strain ASA1002 with vanAB overexpression from pBAV1Cd-plasmid under cyclohexanone inducible promoter. N-terminal His-tag in vanA for purification. This study ASA1005 A. baylyi ADP1 pKLxR5-luxR-galA/Gm Strain with plasmid galA overexpression. This study ASA1006 A. baylyi ADP1 ΔpoxB::luxR-kan r -galA Strain with genomic galA overexpression replacing ACIAD3381 ( poxB ). This study Pseudomonas putida KT2440 Wild-type Harbors galA gallate dioxygenase (PP_2518, glllA ) DSMZ Modified low salt lysogeny broth (LB) medium (10 g/L tryptone, 5 g/L yeast extract, 1 g/L NaCl) supplemented with 50 mM glucose was used to cultivate E. coli and ADP1 for strain construction. Mineral salts medium (MSM) supplemented with 50 mM (unless stated otherwise) glucose, 0.2% casein amino acids, and aromatics in specified concentrations was used in aromatics conversion experiments. MSM composition was 3.88 g/L K 2 HPO4, 1.63 g/L NaH 2 PO 4 , 2.00 g/L (NH 4 ) 2 SO 4 , 0.1 g/L MgCl 2 ·6 H2O, 10 mg/L ethylenediaminetetraacetic acid (EDTA), 2 mg/L ZnSO 4 ·7 H2O, 1 mg/L CaCl 2 ·2 H 2 O, 5 mg/L FeSO 4 ·7 H 2 O, 0.2 mg/L Na 2 MoO 4 ·2 H 2 O, 0.2 mg/L CuSO 4 ·5 H 2 O, 0.4 mg/L CoCl 2 ·6 H 2 O, and 1 mg/L MnCl 2 ·2 H 2 O. Antibiotics for E. coli and ADP1 were supplemented when needed in following concentrations: chloramphenicol, 25 µg/ml; gentamicin, 15 µg/ml; spectinomycin, 50 µg/ml; kanamycin 30 µg/ml. Zidovudine plates in concentration 400 µg/ml were prepared for counter selection of the gene deletions. N-(3-oxohexanoyl) homoserine lactone (AHL) and cyclohexanone (cyc) were used for induction in specified concentrations ranging from 1–10 µM. Aromatics stocks were prepared in following concentrations: vanillate 200 mM, syringate, 3MGA and gallate 100 mM. Correct amount of the aromatic compound was weighed and added to deionized water after which KOH was slowly added to equimolar or slightly excess amount to dissolve the compounds as potassium salts, with final pH reaching 8.2–8.5. Reagents were purchased from Sigma Aldrich (United States). Genetic engineering Genetic engineering was carried out using established methods for restriction-ligation, USER and NEBuilder® HiFi DNA Assembly cloning as well as overlap extension PCR. Reagents for molecular work were purchased from Thermo Scientific (USA) and New England Biolabs (USA) and used according to the manufacturer’s instruction. The primers used in this study are listed in Supplementary Table S1 (Additional File 1). Electroporation was used for the transformation of E. coli XL-1 Blue. ADP1 transformation and genomic editing by homologous recombination were carried out as described previously by ( 43 ). The transformants were screened on lysogeny agar (LA) plates with appropriate antibiotics. VanAB was deleted from ADP1 genome using a linear DNA cassette; regions of approximately 1 kb flanking vanAB to facilitate homologous recombination were amplified from ADP1 genome with primers VanB_P5-OE and VanB R2 (3’ segment), and VanA_P4-OE and VanA F2 (5’ segment). The fragment tdk/kan r carrying kanamycin resistance marker and tdk for counter-selection ( 44 ) was amplified with primers Tdk_kanF and Tdk_kanR from the genome of ADP1∆3383::tdk/kan r , (a kind gift from Dr. Veronique de Berardinis, Genoscope, France) ( 45 ). The cassette was assembled with overlap extension PCR and transformed into ADP1 WT resulting in strain ASA1001. Kanamycin was used for selection of successful clones. A rescue cassette to replace tdk/kan r was constructed in similar manner; flanking regions of vanAB were amplified with primers rescue cassette vanB forward and vanB R2 (3’ flanking) and vanA F2 and rescue cassette vanA reverse (5’ flanking). Restriction sites for MfeI, NotI and AvrII were designed into overlapping primers rescue cassette vanB forward and rescue cassette vanA reverse that join the flanking sequences together (Supplementary Table S1 , Additional File 1). Overlap extension PCR was used to combine the segments. The rescue cassette was transformed into ASA1001 resulting in ASA1002 with markerless vanAB deletion. Counterselection with zidovudine was used to select successful clones; After transformation with the rescue cassette, the cell culture was diluted 1:10, 1:100 and 100 µl volumes were spread on LA plates supplemented with 400 µg/ml zidovudine and 50 mM glucose. The plates were incubated at 30°C until colonies appeared. Colonies were then resuspended in LB media and divided into two cultures each, in LB media supplemented with 50 mM glucose and either 30 µg/ml kanamycin or no antibiotic. The cultures were incubated at 30°C, 300 rpm overnight, after which those that did not grow on kanamycin were selected. The genetic region was further confirmed with PCR using primers Ver_VanAB_F and Ver_VanAB_R binding outside the cassette region. Plasmid pBAV1Cd-chnR-vanAB for overexpression of vanAB was constructed by BioBrick cloning in the empty plasmid pBAV1Cd-chn ( 33 ). To clone vanAB from ADP1 genome it was amplified with primers VanAB_F_BB and VanAB_R_BB. A ribosome binding site, a new start codon, His( 6 ) -tag and Gly-Ser-Gly -linker sequence were included in the forward primer VanAB_F_BB annealing to vanA to enable purification with the histidine tag (Supplementary Table S1 , Additional File 1). The obtained plasmid pBAV1Cd-chn-vanAB was first transformed into E. coli XL1-Blue and then into ASA1002 resulting in ADP1Δ vanAB pBAV1Cd-chn-vanAB (ASA1004). Next, vanAB was cloned under a constitutive T5-promoter with USER cloning. VanAB from ADP1 genome was amplified with primers SS-21-03-IFU and SS-21-04-IRU. SS-21-01-VFU and SS-21-02-VRU were used to amplify the backbone of pBAV1C-T5-GFP ( 46 ). The obtained plasmid pBAV1C-T5-vanAB was first transformed into E. coli XL1-Blue and then into ASA1002 resulting in ADP1ΔvanAB pBAV1C-T5-vanAB (ASA1003). To overexpress galA , plasmid pKLxR5-galA was constructed with NEB HiFi assembly. GalA was amplified from P. putida KT2440 genome with primers GalA_F and GalA_rev . Plasmid pKLxR5-mRFP, a kind gift from Schuster and Reisch, Addgene #149465 ( 47 ) was utilized as backbone and amplified with primers pKLxR5_fwd and prR . The obtained plasmid pKLxR5-galA was transformed into E. coli XL1-Blue and then into ADP1 WT resulting in strain ASA1005. For the genomic expression of galA , a linear gene cassette was constructed based on a previously described cassette that creates a neutral gene knock-out of poxB (ACIAD3381) ( 48 ). LuxR-galA - region was amplified from pKLxR5-galA with primers HT-24-7-galA rev and HT-24-8-luxR fwd and the pIX-backbone with kan r and poxB -flankings was amplified with primers HT-24-5-pIX fwd and HT-24-6 pIX rev. The segments were then combined with USER cloning. The plasmid was first transformed into E. coli XL-1 Blue and then into ADP1 resulting in strain ASA1006. Growth experiments Characterization of syringate tolerance and growth on vanillate on microplates Effects of vanAB deletion and overexpression were characterized by cultivating the strains on vanillate as a sole carbon source. Overnight precultures were prepared in 5 ml volume of MSM, 0.2% casein amino acids, and 25 µg/ml chloramphenicol (dissolved in MQ) when appropriate. The following day, the precultures were used to inoculate culture medium containing MSM, 5 mM vanillate, 25 µg/ml chloramphenicol when appropriate, and 5 µM cyclohexanone for the induction of ASA1004. Each strain was cultivated in triplicates on 96-well plate (200 µl medium/well). The plates were incubated in Spark multimode microplate reader (Tecan, Switzerland) at 30°C. The cultures were mixed with double orbital shaking twice per hour with an amplitude of 6 mm and frequency of 54 rpm. Optical density at 600 nm was measured twice per hour. For studying the syringate tolerance, strains ADP1 WT, ASA1002, ASA1003 and ASA1004 were precultured overnight in 5 ml volume in MSM media with 0.2% casein amino acids, 20 mM glucose, and appropriate antibiotics. Each strain was cultivated in duplicates on a 96-well plate (200 µl medium/well) in the same media as the precultures supplemented with 0, 1, 2, 5, 10, and 20 mM syringate. The plates were incubated in Spark multimode microplate reader (Tecan, Switzerland) as described above. Batch cultivations for syringate conversion and production of PDC Overnight precultures were inoculated from single colonies on LA plates. Precultures were carried out in 5 ml MSM supplemented with 0.2% casein amino acids, 50 mM glucose, and appropriate antibiotics. The main cultivations were carried out in 15 ml MSM media in 50 ml Nunc™ bioreactor tubes (Thermo Scientific, United States). Culture media was supplemented with 0.2% casein amino acids, 50 mM glucose, appropriate antibiotics, and 5 mM syringate with a starting OD of 0.1–0.2. Cyclohexanone was added to the cultivations in concentrations 0–5 µM to induce vanAB expression in ASA1004. Induced cultivations were kept on separate incubator from uninduced ones to avoid any effect of the highly volatile cyclohexanone. The pH of the media was monitored in the beginning and at the end of the cultures with pH strips. For quantification of metabolites, 1 ml samples were collected and centrifuged at 14 000 g for 2 minutes. The supernatants were collected and stored at -20°C prior to analysis. The samples were diluted in deionized water to appropriate concentration (maximum 10-fold dilution) and filtered with 0.2 µm filters before analysis. The pellets were washed with and resuspended to MSM after which OD600 was measured because of the dark coloration formed during the cultivations. Cultivation for PDC production was carried out similarly. Precultures were continued overnight or for two days for ASA1004 pKLxR5 galA strain to reach sufficient cell density, after which appropriate volumes were used for inoculation to achieve initial OD of 0.1–0.2. 1 µM AHL was added to strains with galA under LuxR-regulated promoter for induction. VanAB expression was not induced. Fed-batch cultivations for the production of PDC Bioreactor experiments for PDC production were carried out in small scale bioreactors (Applikon Biotechnology, Netherlands) with 250 ml working volume. The reactor was maintained at 30°C, 200 rpm and at a constant aeration. Dissolved oxygen and pH were monitored during the cultivations. When necessary, 10% antifoam A (Fluka Analytical) was added. Samples of 1–2 ml for analysis of OD600 and aromatics were collected and handled as described previously in batch cultivations. Starting volume of the cultivations was 50 ml of MSM media supplemented with 20 mM glucose, 0.2% casein amino acids, 2 mM syringate, 1 µM AHL, 30 µg/ml kanamycin and inoculant resulting in initial OD of approximately 3–4. Feed containing 15 mM syringate and 75 mM glucose was pumped to the reactor at 3.7 ml per hour. Fed media contained other components in same concentrations as in the initial media. Two independent biological replicates were produced by individual experiments. ASA1006 was inoculated in 5 ml of MSM supplemented with 50 mM glucose, 0.2% casein amino acids, and 30 µg/ml kanamycin at 30°C, 300 rpm. After overnight cultivation, the cells were inoculated in the second preculture media in 15 ml of MSM supplemented with 50 mM glucose, 0.2% casein amino acids, 30 µg/ml kanamycin, and 10 µM AHL to induce galA expression, as well as 1 mM vanillate to induce native vanAB expression, with initial OD 0.1–0.2. The cells were cultivated overnight in 50 ml bioreactor tubes in shaker 30°C, 300 rpm. Parallel cultivations were pelleted at 5000 g for 10 minutes and resuspended in 15 ml MSM to achieve initial OD of 3–4 in bioreactor after inoculation. Analytical methods Gallic acid (GA) was purchased from Merck (Switzerland), 3,4-dihydroxy-5-methoxybenzoic acid (3MGA) and syringic acid (SA) were purchased from Sigma (USA). Methanol (HPLC grade) was purchased from Honeywell (Germany). All standards of aromatic compounds were prepared as 20 mM stock solutions in water by adding of 5M KOH until full solubilization as described above. For calibration, working standard solutions were prepared by diluting of stock solutions with water to concentrations 0.5–5 mM and filtered using 0.2 µm filters. HPLC analysis of the aromatic compounds and PDC was performed on Shimadzu LC-40 (Japan), equipped with a photodiode array detector (PDA). The compounds were analysed on the column Rezex RFQ-Fast Acid H+ (8%), 100 x 7.8 mm, 55 o C (Phenomenex Inc., USA) in 5 mM H 2 SO 4 at the flow rate of 0.6 mL/min. The injection volume was 5 µL. Eluted GA, 3MGA and SA were monitored at wavelength 272 nm. Since a commercial PDC standard was not available, we used a sample collected from a cultivation of ADP1 containing PDC as a qualitative standard. The presence of PDC in the qualitative standard was proven by mass-spectrometry and nuclear magnetic resonance ( 1 H-NMR). Mass-spectrometric analysis was carried out using JEOL AccuTOF LCplus (JMS-T100LP) (Japan) in ESI- mode. The molecular ion of PDC was detected as [M-H] − 182.96624 and distinguished from the molecular ion of 3-O-methyl gallate [M-H] − 183.02990, the compound with almost equal molecular weight. As an additional proof, the 1 H-NMR spectra of the qualitative standard and 3-O-methyl gallate were recorded on a JEOL spectrometer ECZ500R 500MHz (Japan) in D 2 O. Characteristic signals of the two protons of ɑ-pyrone ring at 6.74 (s) and 7.23 (s) ppm were detected in the spectrum of the tested sample whereas characteristic signals of aromatic protons and methoxy protons of 3-O-methyl gallate (7.02, 7.07 and 3.78, respectively) were not found in the spectrum. Quantification of PDC was performed from its chromatographic peak area using extinction coefficient e = 6200 M − 1 .cm − 1 at 313 nm reported by Michinobu et al. (Bull. Chem. Soc. Jpn. Vol. 80, No. 12, 2436–2442 (2007)). VanAB expression and purification For VanAB expression, 10 ml overnight culture of ASA1004 was grown at 30°C in low salt LB medium supplemented with 50 mM glucose and 25 µg/ml chloramphenicol. The following day, cells were diluted in 100 ml of fresh medium (supplemented also with 20 µM FeSO 4 ) to initial OD 600 of 0.05, and grown at 30°C. When OD 600 reached 0.5–0.8, cyclohexanone was added as an inducer of vanAB expression to a final concentration of 5 µM. Cultivation was continued at 25°C for approximately 20 hours. Cells were harvested by centrifugation at 30 000 g for 30 min at 4°C and stored at -20°C. For the copurification of VanAB, the cell pellets were thawed and resuspended in BugBuster Protein Extraction Reagent (Novagen, USA) using 5 ml reagent per gram of wet cell paste, or more if necessary. Lysozyme was then added to a final concentration of 1–2 mg/ml and incubated while gently mixing for 30 minutes at room temperature. Disruption by sonication was performed in sequence of 15 s sonication followed by a 15 s pause on Fisherbrand™ Model 120 Sonic Dismembrator (Thermo Fisher Scientific, USA) while kept on ice. The cell lysate was cleared by centrifugation at 30 000 g for 20 min at 4°C. Imidazole was added to the cleared lysate to a final concentration of 20 mM. The cleared lysate was loaded on to a HisGraviTrap Ni-Sepharose column (GE, USA) and washed with binding/washing buffer (20 mM Tris, 500 mM NaCl, 20 mM imidazole, pH 8). VanAB was eluted with elution buffer (20 mM Tris, 500 mM NaCl, 500 mM imidazole, pH 8) and dithiothreitol (DTT) was added to a final concentration of 5 mM to increase the stability of the protein. Purity of the protein preparation was estimated by Sodium Dodecyl Sulfate Polyacrylamide Gel Electroforesis (SDS-PAGE) based on the method described by Laemmli ( 49 ) using a 12% precast polyacrylamide gel (Bio-Rad, USA). VanAB activity assay Activity assay for the purified VanAB was performed the following day due to loss of activity after longer storage. Prior to assay, PD MidiTrap G-25 (Cytiva, USA) column was used for buffer exchange into 20 mM Tris-HCl, 100 mM NaCl, 5mM DTT, pH 8. The activity assay was based on monitoring the consumption of NADH by measuring the absorbance at 340 nm. Reactions were performed at 25°C with a range of substrate concentrations in 20 mM Tris-HCl, 100 mM NaCl, 5 mM DTT, pH 8. When vanillate or syringate was used as a substrate, two-fold successive dilution series were prepared to give final concentrations in reactions ranging from 400 µM to 25 µM. For 3MGA, substrate concentrations in reactions starting from 3200 µM to 200 µM were used. Control reactions were performed with 400 µM PCA and without any substrate. All reactions contained initial 400 µM NADH. Reactions were initiated by the addition of 40 µl purified VanAB preparation into total reaction volume of 200 µl. The absorbance at 340 nm was measured with Spark multimode microplate reader (Tecan, Switzerland) every 3 minutes until 90 minutes in total. The oxidation of NADH was converted to the amount of consumed NADH. Initial velocities, v 1 , at different substrate concentrations, [S] were fitted to the Michaelis-Menten equation shown in Eq. 1 Where K M is the Michaelis-Menten constant and V max is the maximum velocity of the reaction. Results Characterization of the growth and conversion of vanillate and syringate by vanAB expressing strains We first investigated the O- demethylation activity of ADP1 VanAB by native and non-native expression systems. First, the native copy of vanAB was deleted from the genome resulting in strain ASA1002. Two plasmid-based overexpression systems were transformed into ASA1002, resulting in strains ASA1003 and ASA1004 with constitutive P T5 - vanAB and cyclohexanone-inducible P chn - vanAB , respectively. The strains were cultivated with vanillate, the known substrate for VanAB, as the sole carbon source (Fig. 2 ). Overexpression of vanAB resulted in nearly 10-hour lag phase, while for ADP1 WT noticeable lag phase was not observed. Interestingly, for the uninduced strain, the lag-phase was slightly shorter compared to overexpressing strains. A low expression level of vanAB can be expected due to leakiness of the chnR /P ChnB ( 50 ). As expected, ASA1002 with vanAB deletion did not grow on vanillate. Next, the syringate tolerance of ADP1 WT and the engineered strains was determined (Fig. 3 ). The media was supplemented with 50 mM glucose and syringate in concentrations ranging from 0 to 20 mM. At the highest concentration of syringate tested (20 mM), strains ADP1 WT, ASA1002, and ASA1003 had approximately a 6-hour lag phase, whereas the induced ASA1004 strain had even a longer lag phase, approximately 12 hours. In all strains, syringate negatively impacted growth in comparison to cultivations with only glucose, and the growth of induced ASA1004 was hindered even at low syringate concentrations. By contrast, for ASA1002, only the highest concentrations of syringate resulted in clearly reduced overall growth. Close to the end of the cultivation, a medium color change from clear to black was noticed in cultures of ADP1 WT and ASA1003 (Supplementary Figure S1 , Additional File 1). Syringate and 3MGA conversion by VanAB in wild-type and engineered ADP1 Based on the tolerance test, 5 mM concentration was selected for studying syringate conversion in ADP1 in vivo . We first tested the conversion in ADP1 WT and compared the performance to that of P. putida KT2440, which has been previously shown to O- demethylate syringate in the presence of an auxiliary carbon source, resulting in the accumulation of 3MGA ( 21 ). The strains were cultivated in MSM supplemented with 50 mM glucose, 0.2% casein amino acids, and 5 mM syringate (Fig. 4 ). Both syringate and 3MGA were completely O- demethylated by ADP1 WT within 24 hours. Only small amount of syringate was converted to 3MGA by P. putida , being in line with previous research ( 21 ). We then investigated the syringate conversion by ASA1003 and ASA1004. ASA1002 was used as the control. The strains were cultivated in MSM supplemented with 50 mM glucose, 0.2% casein amino acids, and 5 mM syringate and appropriate antibiotics (Fig. 5 ). Interestingly, the strains ASA1003 and ASA1004, which overexpress vanAB , exhibited different reaction dynamics compared to ADP1 WT. In these strains, syringate and 3MGA were O- demethylated simultaneously, unlike in ADP1 WT, where syringate was depleted first. Notably, the induction of ASA1004 caused an over 8-hour lag phase, resulting in much slower overall conversion of syringate. A very minor decrease in syringate concentration was observed in cultivations with ASA1002, likely due to abiotic degradation. These results indicate that VanAB is indeed responsible for syringate conversion. To screen for the optimal induction level in ASA1004, different cyclohexanone concentrations up to 5 µM were tested (Supplementary Figure S2, Additional File 1). We found that very low expression levels of vanAB , even without induction, are sufficient for the conversion, whereas too high expression almost completely inhibits growth (Supplementary Figure S3, Additional File 1). Again, dark coloration was observed after approximately 24 hours of cultivation most likely caused by the abiotic oxidation of gallate, which results in a dark gray or black coloration and precipitate ( 7 , 51 – 53 ). To confirm that the O- demethylation of 3MGA is also carried out by VanAB, we cultivated ADP1 WT and ASA1002 in media supplemented with 3MGA instead of syringate (Supplementary Figure S4, Additional File 1). As expected, ADP1 WT promptly O- demethylated 3MGA to gallate, while no conversion above the abiotic rate was detected for ASA1002. To prospect for the possibility of unknown enzymes capable of syringate conversion or further syringate metabolism that could enable growth, ADP1 WT, ASA1002, and ASA1004 were cultivated with syringate as the sole carbon source (Supplementary Figure S5, Additional File 1). Minor conversion of syringate into 3MGA was observed, but neither further conversion to gallate nor growth was detected. The findings indicate that syringate and 3MGA O- demethylation in ADP1 is exclusively carried out by VanAB. VanAB activity in vitro To investigate the substrate preference of VanAB, the enzymes were produced and co-purified for enzymatic activity assay. The purified protein preparation had a red-brown color, which is characteristic of Rieske-type oxygenases such as VanA ( 54 ). The activity of VanAB towards different substrates was assayed based on monitoring the consumption of NADH. The results indicated a clear coupling of NADH consumption by VanAB in the presence of vanillate or syringate in the reaction, as opposed to the minimal consumption of NADH in the presence of PCA or without substrate (Supplementary Figure S6, Additional File 1). The saturation curves for VanAB with vanillate and syringate as a substrate are shown in Fig. 6 . Using vanillate and syringate as substrates, the Michaelis-Menten constants ( K M ) were found to be 42 ± 15 µM and 38 ± 9 µM, respectively. These results align with previous studies on homologous enzyme from P. putida KT2440 ( 21 ). Interestingly, syringate appears to be as good a substrate as vanillate for the VanAB of ADP1. However, we were unable to detect in vitro activity when 3MGA was used as a substrate under the studied conditions, presumably because of the much lower catalytic ability of VanAB with 3MGA. Nevertheless, VanAB-mediated O- demethylation of 3MGA was observed in the in vivo cultivations (Fig. 4 and Supplementary Figure S4, Additional File 1). Production of PDC in ADP1 The robust conversion of syringate by VanAB encouraged us to further explore potential production pathways. PDC is an exciting product of aromatic catabolism, as several lignin-derived aromatics can be simultaneously funnelled towards its production. In the VanAB-mediated pathway, there are two known routes to PDC from syringate: through 3MGA and gallate ( 21 ). Protocatechuate dioxygenase PcaHG of P. putida KT2440 has been reported to convert gallate into PDC ( 21 ). To test the corresponding activity of the homologous PcaHG in ADP1, we first cultivated ADP1 WT and a control strain with pcaHG deletion in media supplemented with 5 mM syringate and 50 mM glucose. Small amounts of PDC were detected from ADP1 WT cultures (Fig. 7 ), indicating potential, albeit minor activity of PcaHG towards gallate. Given the high instability of gallate and the significant accumulation of 3MGA in the media during ADP1 WT cultivations before it is further O- demethylated to gallate, we opted to establish a PDC production pathway directly from 3MGA. Using 3MGA as a product precursor instead of gallate also eliminates the need for the second O -demethylation step. To that end, we expressed a gallate dioxygenase, galA , from P. putida KT2440 in ADP1. GalA is known to convert 3MGA into 4-carboxy-2-hydroxy-6-methyoxy-6-oxohexa-2,4-dienoate (CHMOD) which is non-enzymatically transformed into PDC ( 21 ). We cloned galA into pKLxR5 backbone under P LUXB promoter, and the expression plasmid was transformed into ADP1 WT, resulting in the strain ASA1005. In addition to the activity of GalA, small amounts of gallate may also be converted into PDC by the native PcaHG. Next, we compared ASA1005 expressing galA with and without induction to ADP1 WT in the same conditions. With induction, ASA1005 produced PDC from syringate with a molar yield of 38% (Fig. 8 ) Following the successful conversion of syringate into PDC via plasmid expression, we constructed a strain with genomic galA expression system to ensure stable and consistent expression, resulting in the strain designated as ASA1006. To characterize the LuxR mediated expression in ADP1 genome, a strain with mRFP expression was constructed. The expression with the tested inducer concentrations ranging from 1–100 µM AHL was even stronger compared to the plasmid-based expression (Supplementary Figure S7, Additional File 1). As high 3MGA concentrations are known to inhibit GalA ( 21 ), it may be beneficial to have a higher expression level of galA in relation to vanAB . Thus, 10 µM AHL concentration was chosen for the induction in the subsequent experiments. We hypothesized that in a bioreactor, syringate concentration could be maintained at a defined level for 3MGA to keep accumulating instead of being converted into gallate. In addition, glucose supplementation supporting the primary metabolism could enhance the demethylation reactions. The initial media in the bioreactor was supplemented with 2 mM syringate and 20 mM glucose and 10 µM AHL. The feed contained otherwise same components as the initial media with exception of 15 mM syringate and 75 mM glucose resulting in feeding rates of 0.058 mmol/h and 0.29 mmol/h, respectively. Despite continuous feeding, syringate was consumed rapidly, after which 3MGA was converted into gallate. The decrease in gallate concentration in the media seemed to proceed at higher rate than what could be expected from abiotic degradation or dilution caused by feeding. GalA can also oxidise gallate into 4-OMAmesaconate (OMA), which was not analysed from the samples, but this reaction is a plausible explanation for the decrease in gallate concentration. Accumulation of gallate did not completely inhibit the production of PDC, although the amount remained low at 9.2–10.8 mg/l (Fig. 9 ). Discussion ADP1 has previously shown great promise for the utilization of lignin-related aromatic compounds ( 30 , 32 – 37 ). However, O- demethylation of S-lignin derived aromatics by ADP1 has not been detected until now. In this study, we investigated the reactions carried out by VanAB, a promiscuous vanillate O- demethylase. Other VanAB homologs can O- demethylate many S-lignin derived aromatics, as has been demonstrated in Pseudomonas sp. HR199 (syringate) ( 23 ), Streptomyces sp . NL15-2K (syringate and 3MGA) ( 25 ) and most recently in P. putida KT2440 (syringate and 3MGA in the presence of an auxiliary carbon source) ( 21 , 24 ). In this context, more extensive study of VanAB of ADP1 seemed warranted. First, we examined how non-native expression of vanAB affects the growth of ADP1 on vanillate, a native carbon source of ADP1 (Fig. 2 ). As expected, the deletion of vanAB (strain ASA1002) prevented growth on vanillate as a sole carbon source. The strains overexpressing vanAB had significantly longer lag phases compared to ADP1 WT, and based on OD, the final biomasses were also slightly lower. Of the engineered strains, the uninduced ASA1004 strain performed the best, supporting the previous findings ( 21 ) that low expression level of vanAB is beneficial in terms of growth. The negative effects of vanAB overexpression on the growth can be explained by the increased NAD(P)H consumption and the rapid generation of formaldehyde formed in the O -demethylation reaction. In P. putida KT2440, providing additional carbon and energy source for the cells significantly improved growth on syringate with strains overexpressing vanAB , likely due to increased supply of cofactors ( 21 ). In addition, overexpression of vanAB per se can potentially have negative impact on growth, although such effect was not observed in ADP1 while producing VanAB for in vitro studies, when specific substrate for VanAB was not available. In the ADP1 WT, vanAB is repressed by a GntR-type transcriptional regulator VanR, which typically causes a 3–4-hour lag-phase preceding vanillate conversion ( 55 ). Overexpression of vanAB in strains ASA1003 and ASA1004 resulted in approximately 10-hour long lag-phase, despite that the expression is not under the regulation of VanR. Thus, the longer lag phase is potentially related to the redox-imbalance and toxicity of formaldehyde, as discussed above. Next, we explored syringate tolerance of the ADP1 strains. 20 mM of syringate caused an extended lag-phase in all strains. In concentrations 1–10 mM, the expression level of VanAB seemed to correlate with syringate tolerance and its effect on growth: For ASA1002 with vanAB deletion, the impact of syringate on the growth was more clearly dependent on the concentration, whereas with ADP1 WT and uninduced ASA1004, the inhibitory effect of syringate was similar in all concentrations. In contrast, when vanAB was overexpressed in ASA1004, syringate had a drastic effect on growth even at low concentrations. The growth of the induced ASA1004 was potentially hindered by the faster O- demethylation of syringate, similarly to what was observed in vanillate cultivations. Regardless, all the strains were able to grow in the concentrations tested. Tolerance to even higher concentrations could be further explored and improved in the future, for example by adaptive laboratory evolution, as we have previously demonstrated with ferulate ( 38 ). Growth in up to 120 mM syringate has been reported for P. putida KT2440, with overexpression of VanAB increasing tolerance ( 21 ). However, in ADP1, gallate is not further metabolized and its accumulation potentially adds to the toxicity of syringate. To explore the reactions carried out by natively expressed VanAB on non-native carbon sources, we set up cultivations of ADP1 WT and P. putida KT2440 with 5 mM syringate and 50 mM glucose supplementations. We found that syringate is quickly O- demethylated into 3MGA and then into gallate by ADP1, in contrast to previous research ( 26 ). Experimental methods could explain the contradiction to previous VanAB characterization in ADP1; In the paper of Morawski et al. ( 26 ), VanAB activity was measured from washed cell suspensions with the tested VanAB substrates as sole carbon sources. In this setup, for example the inability to refill NAD(P)H reservoirs could significantly reduce reaction capacity ( 26 ). In our experiments, we used glucose as an additional carbon source to provide carbon and energy for growth and for replenishing NAD(P)H. We detected high amounts of 3MGA in the ADP1 WT culture media prior to its further conversion to gallate. The demonstrated dynamic conversion, in which syringate is O- demethylated first is consistent with previous findings that suggest 3MGA is less preferred substrate for VanAB ( 21 , 23 ). In P. putida KT2440, syringate conversion was much slower than in ADP1 and 3MGA was not O- demethylated at all, being in line with the previous study ( 21 ). However, when vanillate was provided instead of glucose for P. putida KT2440, a higher syringate conversion rate was achieved, and with continuous vanillate feeding, both syringate and 3MGA were consumed completely ( 21 ). Thus, in P. putida KT2440, syringate conversion could be affected by glucose-induced carbon catabolite repression. For ADP1, neither glucose nor gluconate have been observed to repress the utilization of aromatic compounds ( 36 , 40 ). As previously mentioned, VanR is a negative transcriptional regulator that represses the expression of VanAB in the absence of an inducer, namely vanillate. ( 55 , 56 ) Interestingly, there are two variants of vanR found in ADP1, namely O24839 ( 57 ) and Q6FDI8 ( 58 ), that differ in length and in their orientation in respect to vanB . The differences may be result of genetic scrambling caused by a Tn5613 transposon located nearby ( 58 ). In O24839, vanR and vanB overlap, whereas in Q6FDI8 vanR and vanB are separated by 46 bp and vanR is 23 amino acids shorter. The strains used in this study contain the variant Q6FDI8, while vanAB repression has been previously studied with strain containing the variant O24839 ( 55 , 57 , 58 ). In our experiments, syringate was O- demethylated by VanAB robustly without induction by vanillate in ADP1 WT. Therefore, it appears that syringate can serve as an inducer for vanAB expression in ADP1. Amino acid sequence identity of VanR in ADP1 and P. putida KT2440 is 48% for variant Q6FDI8 and 49% for the variant O24839 used in the study ( 55 ) where VanR was previously characterized. Difference in the protein structures or potentially even the different gene organization of the van region described earlier could affect VanR binding to DNA or VanAB substrates and partially explain the differences in performance between P. putida and ADP1. Uptake and conversion of the compounds are also affected by specific transporters and porins ( 59 ). In ADP1, aromatic compounds are transported by four known acid:H + symporters, VanK, PcaK, BenK and MucK ( 60 ). VanK, a vanillate transporter and PcaK, a 4-hydroxybenzoate transporter are both promiscuous and overlap in activity, meaning they could potentially transport S-lignin derived aromatics as well ( 61 ). Porins might also facilitate uptake of syringate, 3MGA, or gallate: for example, VanP and HcaE are involved in vanillate transport ( 62 ). The robust conversion of syringate into 3MGA in ADP1 suggests sufficient transfer of the compounds into the cells. The efficient transport in ADP1 may also negatively affect the tolerance towards syringate, especially in elevated concentrations. Aromatic compounds can also cross the cell membrane passively. Next, we investigated syringate conversion by the strains overexpressing vanAB along with ASA1002 with vanAB deletion as a control. We found that overexpression enables simultaneous conversion of syringate and 3MGA, but for overall efficiency the lower expression level is better. As discussed above, the reason for this is likely that the O- demethylation reaction catalyzed by VanAB produces formaldehyde as a side product, which has been previously suggested to cause a redox imbalance and energy limitation in P. putida KT2440 ( 21 ). A conserved route for detoxification of formaldehyde utilizes a glutathione-dependent formaldehyde dehydrogenase (FrmA) and a S-formylglutathione hydrolase ( 20 ). In recombinant E. coli expressing vanAB from P. putida , deletion of FrmA led to accumulation of formaldehyde and halted vanillate O- demethylation ( 20 ). While the specific mechanism for eliminating formaldehyde in ADP1 has not been described, in A. baumannii , a close relative of ADP1, glutathione-dependent formaldehyde dehydrogenases adhC1 and adhC2 have been characterized ( 63 ). Interestingly, the expression of adhC1 in A. baumannii was found to be repressed in the presence of free inorganic iron ( 63 ). As VanAB requires iron in the iron-sulfur cluster [2Fe-2S] and in the active site, optimization might be required to balance O- demethylation and formaldehyde detoxification ( 17 ). Notonier et al. ( 21 ) supplemented P. putida KT2440 cultures with formate, which is oxidized in cells to generate NADH and thus improved the O- demethylation of syringate. For ADP1, we found no benefit from formate supplementation (data not shown), potentially explained by the possible VanAB preference for NADPH over NADH ( 20 ). In the future, identifying how formaldehyde is detoxified in ADP1 could help to overcome the inhibitory effects caused by VanAB overexpression. In addition to the in vivo experiments, activity of VanAB was evaluated in vitro. We were able to co-purify VanA and VanB with a N-terminal His-( 6 )-tag only in VanA, suggesting strong interaction between them. By the assay setup we showed that NADH consumption can be used to indirectly measure VanAB activity. We found that syringate is nearly as preferred substrate as vanillate. However, in contrast to the in vivo experiments, no activity for 3MGA was detected. In vitro activity towards 3MGA has been previously shown only for the VanAB homolog from P. putida KT2440 and with clear preference for vanillate and syringate as substrates over 3MGA ( 21 ). The purification and assay conditions here may have further negatively impacted the activity, as Rieske oxygenases are known to be sensitive to oxidation ( 23 , 64 ). Addition of sources of iron and sulfur, as well as dithiothreitol as an antioxidant have been shown to be beneficial, and systematic characterization of their effects in ADP1 could further improve the activity ( 23 ). The robust and complete O- demethylation of syringate and 3MGA in the in vivo experiments yielded approximately equimolar amounts of gallate in ADP1. Gallate itself is an interesting product, as it can be used for example in the production of antioxidants, pharmaceuticals and antimicrobials ( 65 , 66 ). However, gallate is known to oxidize readily, indicated by the change of color to dark brown or black (Supplementary Figure S1 , Additional File 1). Gallate can also react with media components and proteins and may form a precipitate that could be harmful for cell growth and prevent its further utilization ( 7 , 52 ). To that end, we wanted to explore further ways to utilize the robust and dynamic O- demethylation of syringate and 3MGA for production in ADP1. Production of PDC from lignin related aromatics has shown a lot of promise. PDC is an enticing target product for microbial valorisation of lignin; It has promising industrial applications such as high-quality biodegradable polyesters and polyurethanes ( 67 , 68 ). In addition, chemical synthesis of PDC is very difficult, making it a so called bioprivileged compound ( 67 ). PDC production has been demonstrated from single lignin monomers including vanillate, syringate, 4-hydroxybenzoate and p -coumarate and even lignin derived feedstocks. ( 10 , 69 – 75 ) The highest titer to date from lignin-related compounds as substrate, 99.9 g/L and productivity of 1.69 g/L/h, were achieved by engineered Sphingobium spp. SYK-6 with vanillate as a substrate ( 9 ). Directing 3MGA into PDC by expression of galA emerged as an attractive option as it would benefit from the accumulation of 3MGA that occurs when VanAB is natively expressed in ADP1. Conversion of 3MGA directly into PDC by GalA would also be beneficial because it circumvents the second O- demethylation step, reducing NAD(P)H consumption and production of toxic formaldehyde. Reported rate for this reaction has been low, in part due to substrate inhibition by 3MGA ( 21 ), but it had not been tested in ADP1. An alternative route would be via PcaHG found both in ADP1 and P. putida KT2440, in which it converts gallate into PDC. Despite we observed only modest conversion of gallate to PDC in ADP1 by PcaHG, it can potentially be exploited to support PDC production in combination with GalA: any 3MGA that is not directly converted into PDC by GalA could be salvaged by the conversion of gallate into PDC by PcaHG. By introducing galA from P. putida KT2440 into ADP1 we were able to demonstrate production of PDC from syringate in ADP1. The obtained yield was modest, approximately 38%. To improve PDC production, we integrated the galA expression system into ADP1 genome for stable and consistent expression. Additionally, we established a bioreactor cultivation system, aiming to reduce gallate formation and improve PDC production through controlled feeding of syringate and glucose as a supporting carbon source. Indeed, we found that maintaining a high concentration of syringate in the media hindered the conversion of 3MGA to gallate. However, PDC production remained unexpectedly low due to the limited conversion of 3MGA to PDC under the studied conditions. It is possible that the conditions favored the conversion of 3MGA to gallate by VanAB over its conversion to PDC by GalA. The formed gallate, serving as a native substrate for GalA, further blocked the conversion of 3MGA to PDC; we observed a decrease in gallate concentration in the media, possibly due to its conversion to 4-oxalomesaconate (OMA) by GalA, which also represents the first reaction in gallate catabolism by the gal operon ( 76 ). Indeed, in the future, the utilization of S-lignin derived aromatics for growth could be established in ADP1 by extending the pathway from gallate and OMA towards central metabolism. In future studies, several approaches could be applied to further improve PDC production in ADP1. For example, in this study, we used a galA gene directly from the genome of a GC-rich strain P. putida KT2440, while a synthetic gene codon optimized specifically for ADP1 could function more optimally. In addition, protein engineering of the substrate specificity of GalA towards 3MGA could be a potential strategy for improving the conversion. Furthermore, employing alternative enzymes such as LigAB and DesZ from Sphingobium spp. SYK-6 which can also carry out the ring-opening reaction ( 77 ), should be considered. Conclusions In this study, we established the conversion and upgrading of S-lignin derived syringate in Acinetobacter baylyi ADP1. We characterized the activity of a promiscuous vanillate O- demethylase VanAB in ADP1, demonstrating its efficient O- demethylation of both syringate and 3MGA. Overexpression of vanAB altered the conversion dynamics, resulting in the simultaneous conversion of 3MGA and syringate, unlike in the ADP1 WT where syringate is O- demethylated first. Excessive expression levels negatively impacted cell growth, likely due to the accumulation of toxic intermediates and the high energy demand of VanAB, as previously reported. Finally, we exploited the accumulation of 3MGA caused by the native VanAB expression by introducing gallate dioxygenase GalA from P. putida KT2440 to produce PDC, a bioprivileged plastic precursor. Our study expands the aromatic substrate range of ADP1 to include S-lignin derived aromatics for production and further promotes the use of this host for biological upgrading of lignin-derived compounds. Abbreviations ADP1 Acinetobacter baylyi ADP1 3MGA 3-O-methylgallate AHL N-(3-oxohexanoyl) homoserine lactone Cyc cyclohexanone DTT dithiothreitol GA gallate LA lysogeny agar LB lysogeny broth MSM mineral salts medium NAD(P)H nicotinamide adenine dinucleotide (phosphate) NMR 1 H nuclear magnetic resonance OD optical density P450 Cytochrome P450 PCR Polymerase chain reaction PDA photodiode array detector PDC 2-pyrone-4,6-dicarboxylic acid RO Rieske oxygenase SA syringate SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electroforesis THF tetrahydrofolate Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availibility of data and materials The data that support the findings of this study are included within the article or the additional files. The corresponding author is willing to provide the raw data related to this manuscript upon reasonable request. Competing interests The authors declare that they have no competing interests. Funding SS would like to thank the Novo Nordisk Foundation (grant NNF21OC0067758) and the Re-search Council of Finland (grant no. 347204 and 353587). Authors’ contributions HT, JK, and SS designed the study. HT and JK carried out the experimental research work. HT, JK, and SS analysed the data. EE and HT conducted the substrate and metabolite analyses. SS supervised the study and acquired funding. All authors participated in writing and reviewing the manuscript. All authors read and approved the final manuscript. Acknowledgements Not applicable. References Bugg TDH, Williamson JJ, Alberti F. Microbial hosts for metabolic engineering of lignin bioconversion to renewable chemicals. Renew Sustain Energy Rev. 2021;152:111674. Yaguchi AL, Lee SJ, Blenner MA. Synthetic Biology towards Engineering Microbial Lignin Biotransformation. Trends Biotechnol. 2021;39(10):1037–64. Kohlstedt M, Weimer A, Weiland F, Stolzenberger J, Selzer M, Sanz M, et al. Biobased PET from lignin using an engineered cis, cis-muconate-producing Pseudomonas putida strain with superior robustness, energy and redox properties. Metab Eng. 2022;72:337–52. Sonoki T, Takahashi K, Sugita H, Hatamura M, Azuma Y, Sato T, et al. Glucose-Free cis , cis -Muconic Acid Production via New Metabolic Designs Corresponding to the Heterogeneity of Lignin. ACS Sustain Chem Eng. 2018;6(1):1256–64. Niu W, Willett H, Mueller J, He X, Kramer L, Ma B, et al. Direct biosynthesis of adipic acid from lignin-derived aromatics using engineered Pseudomonas putida KT2440. Metab Eng. 2020;59:151–61. Zhao M, Huang D, Zhang X, Koffas MAG, Zhou J, Deng Y. Metabolic engineering of Escherichia coli for producing adipic acid through the reverse adipate-degradation pathway. Metab Eng. 2018;47:254–62. Cai C, Xu Z, Zhou H, Chen S, Jin M. Valorization of lignin components into gallate by integrated biological hydroxylation, O-demethylation, and aryl side-chain oxidation. Sci Adv. 2021;7(36):eabg4585. Dias FMS, Pantoja RK, Gomez JGC, Silva LF. From degrader to producer: reversing the gallic acid metabolism of Pseudomonas putida KT2440. Int Microbiol. 2022;26(2):243–55. Otsuka Y, Araki T, Suzuki Y, Nakamura M, Kamimura N, Masai E. High-level production of 2-pyrone-4,6-dicarboxylic acid from vanillic acid as a lignin-related aromatic compound by metabolically engineered fermentation to realize industrial valorization processes of lignin. Bioresour Technol. 2023;377:128956. Lee S, Jung YJ, Park SJ, Ryu MH, Kim JE, Song HM et al. Microbial production of 2-pyrone-4,6-dicarboxylic acid from lignin derivatives in an engineered Pseudomonas putida and its application for the synthesis of bio-based polyester. Bioresour Technol. 2022;352. Jin Y, Joshi M, Araki T, Kamimura N, Masai E, Nakamura M, et al. Click Synthesis of Triazole Polymers Based on Lignin-Derived Metabolic Intermediate and Their Strong Adhesive Properties to Cu Plate. Polymers. 2023;15(6):1349. Kang MJ, Kim HT, Lee MW, Kim KA, Khang TU, Song HM, et al. A chemo-microbial hybrid process for the production of 2-pyrone-4,6-dicarboxylic acid as a promising bioplastic monomer from PET waste. Green Chem. 2020;22(11):3461–9. Michinobu T, Hishida M, Sato M, Katayama Y, Masai E, Nakamura M, et al. Polyesters of 2-Pyrone-4,6-Dicarboxylic Acid (PDC) Obtained from a Metabolic Intermediate of Lignin. Polym J. 2008;40(1):68–75. Obrzut N, Hickmott R, Shure L, Gray KA. The effects of lignin source and extraction on the composition and properties of biorefined depolymerization products. RSC Sustain. 2023;1(9):2328–40. Constant S, Wienk HLJ, Frissen AE, Peinder PD, Boelens R, Van Es DS, et al. New insights into the structure and composition of technical lignins: a comparative characterisation study. Green Chem. 2016;18(9):2651–65. Li W, Wang Y, Tan X, Miao C, Ragauskas AJ, Zhuang X. Biocompatible organosolv fractionation via a novel alkaline lignin-first strategy towards lignocellulose valorization. Chem Eng J. 2024;481:148695. Erickson E, Bleem A, Kuatsjah E, Werner AZ, DuBois JL, McGeehan JE, et al. Critical enzyme reactions in aromatic catabolism for microbial lignin conversion. Nat Catal. 2022;5(2):86–98. Masai E, Sasaki M, Minakawa Y, Abe T, Sonoki T, Miyauchi K, et al. A Novel Tetrahydrofolate-Dependent O-Demethylase Gene Is Essential for Growth of Sphingomonas paucimobilis SYK-6 with Syringate. J Bacteriol. 2004;186(9):2757–65. Lee JA, Stolyar S, Marx CJ. Aerobic Methoxydotrophy: Growth on Methoxylated Aromatic Compounds by Methylobacteriaceae. Front Microbiol. 2022;13:849573. Hibi M, Sonoki T, Mori H. Functional coupling between vanillate- O -demethylase and formaldehyde detoxification pathway. FEMS Microbiol Lett. 2005;253(2):237–42. Notonier S, Werner AZ, Kuatsjah E, Dumalo L, Abraham PE, Hatmaker EA, et al. Metabolism of syringyl lignin-derived compounds in Pseudomonas putida enables convergent production of 2-pyrone-4,6-dicarboxylic acid. Metab Eng. 2021;65(C):111–22. Kovaleva EG, Lipscomb JD. Versatility of biological non-heme Fe(II) centers in oxygen activation reactions. Nat Chem Biol. 2008;4(3):186–93. Lanfranchi E, Trajković M, Barta K, de Vries JG, Janssen DB. Exploring the Selective Demethylation of Aryl Methyl Ethers with a Pseudomonas Rieske Monooxygenase. ChemBioChem. 2019;20(1):118–25. Mueller J, Willett H, Feist AM, Niu W. Engineering Pseudomonas putida for improved utilization of syringyl aromatics. Biotechnol Bioeng. 2022;119(9):2541–50. Nishimura M, Nishimura Y, Abe C, Kohhata M. Expression and Substrate Range of Streptomyces Vanillate Demethylase. Biol Pharm Bull. 2014;37(9):1564–8. Morawski B, Segura A, Ornston N. Substrate Range and Genetic Analysis of Acinetobacter Vanillate Demethylase. J Bacteriol. 2000;182(5):1383–9. Bedore SR, Neidle EL, Pardo I, Luo J, Baugh AC, Duscent-Maitland CV et al. Natural transformation as a tool in Acinetobacter baylyi: Streamlined engineering and mutational analysis. In: Methods in Microbiology [Internet]. Elsevier; 2023 [cited 2024 Sep 26]. pp. 207–34. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0580951723000028 Pardo I, Bedore SR, Tumen-Velasquez MP, Duscent-Maitland CV, Baugh AC, Santala S et al. Natural transformation as a tool in Acinetobacter baylyi: Evolution by amplification of gene copy number. In: Methods in Microbiology [Internet]. Elsevier; 2023 [cited 2024 Sep 26]. pp. 183–205. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0580951723000016 Santala S. Acinetobacter baylyi ADP1—naturally competent for synthetic biology. Essays Biochem. 2021;65(2):309–18. Salmela M, Lehtinen T, Efimova E, Santala S, Santala V. Towards bioproduction of poly-α-olefins from lignocellulose. Green Chem. 2020;22(15):5067–76. Salvachúa D, Karp EM, Nimlos CT, Vardon DR, Beckham GT. Towards lignin consolidated bioprocessing: simultaneous lignin depolymerization and product generation by bacteria. Green Chem. 2015;17(11):4951–67. Salmela M, Lehtinen T, Efimova E, Santala S, Santala V. Alkane and wax ester production from lignin-related aromatic compounds. Biotechnol Bioeng. 2019;116(8):1934–45. Luo J, Lehtinen T, Efimova E, Santala V, Santala S. Synthetic metabolic pathway for the production of 1-alkenes from lignin-derived molecules. Microb Cell Factories. 2019;18(1):48. Meriläinen E, Efimova E, Santala V, Santala S. Carbon-wise utilization of lignin-related compounds by synergistically employing anaerobic and aerobic bacteria. Biotechnol Biofuels Bioprod. 2024;17(1):78. Arvay E, Biggs BW, Guerrero L, Jiang V, Tyo K. Engineering Acinetobacter baylyi ADP1 for mevalonate production from lignin-derived aromatic compounds. Metab Eng Commun. 2021;13:e00173. Kurnia K, Efimova E, Santala V, Santala S. Metabolic engineering of Acinetobacter baylyi ADP1 for naringenin production. Metab Eng Commun. 2024;19:e00249. Biggs BW, Tyo KEJ. Aromatic natural products synthesis from aromatic lignin monomers using Acinetobacter baylyi ADP1 [Internet]. 2023 [cited 2024 Sep 26]. Available from: http://biorxiv.org/lookup/doi/ 10.1101/2023.08.24.554694 Luo J, McIntyre EA, Bedore SR, Santala V, Neidle EL, Santala S. Characterization of Highly Ferulate-Tolerant Acinetobacter baylyi ADP1 Isolates by a Rapid Reverse Engineering Method. Appl Environ Microbiol. 2022;88(2):e01780–21. Kannisto MS, Mangayil RK, Shrivastava-Bhattacharya A, Pletschke BI, Karp MT, Santala VP. Metabolic engineering of Acinetobacter baylyi ADP1 for removal of Clostridium butyricum growth inhibitors produced from lignocellulosic hydrolysates. Biotechnol Biofuels. 2015;8(1):198. Liu C, Choi B, Efimova E, Nygård Y, Santala S. Enhanced upgrading of lignocellulosic substrates by coculture of Saccharomyces cerevisiae and Acinetobacter baylyi ADP1. Biotechnol Biofuels Bioprod. 2024;17(1):61. Singh A, Bedore SR, Sharma NK, Lee SA, Eiteman MA, Neidle EL. Removal of aromatic inhibitors produced from lignocellulosic hydrolysates by Acinetobacter baylyi ADP1 with formation of ethanol by Kluyveromyces marxianus. Biotechnol Biofuels. 2019;12(1):91. Liu C, Efimova E, Santala V, Santala S. Analysis of detoxification kinetics and end products of furan aldehydes in Acinetobacter baylyi ADP1. Sci Rep. 2024;14(1):29678. Santala S, Efimova E, Kivinen V, Larjo A, Aho T, Karp M, et al. Improved Triacylglycerol Production in Acinetobacter baylyi ADP1 by Metabolic Engineering. Microb Cell Factories. 2011;10(1):36. Metzgar D. Acinetobacter sp. ADP1: an ideal model organism for genetic analysis and genome engineering. Nucleic Acids Res. 2004;32(19):5780–90. De Berardinis V, Vallenet D, Castelli V, Besnard M, Pinet A, Cruaud C, et al. A complete collection of single-gene deletion mutants of Acinetobacter baylyi ADP1. Mol Syst Biol. 2008;4(1):174. Santala S, Karp M, Santala V. Rationally Engineered Synthetic Coculture for Improved Biomass and Product Formation. PLoS ONE. 2014;9(12):e113786. Schuster LA, Reisch CR. A plasmid toolbox for controlled gene expression across the Proteobacteria. Nucleic Acids Res. 2021;49(12):7189–202. Santala S, Efimova E, Santala V. Dynamic decoupling of biomass and wax ester biosynthesis in Acinetobacter baylyi by an autonomously regulated switch. Metab Eng Commun. 2018;7:e00078. Laemmli UK. Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4. Nature. 1970;227(5259):680–5. Luo J, Efimova E, Volke DC, Santala V, Santala S. Engineering cell morphology by CRISPR interference in Acinetobacter baylyi ADP1. Microb Biotechnol. 2022;15(11):2800–18. Kutraite I, Malys N. Development and Application of Whole-Cell Biosensors for the Detection of Gallic Acid. ACS Synth Biol. 2023;12(2):533–43. Nogales J, Canales Á, Jiménez-Barbero J, Serra B, Pingarrón JM, García JL, et al. Unravelling the gallic acid degradation pathway in bacteria: the gal cluster from Pseudomonas putida . Mol Microbiol. 2011;79(2):359–74. Vona D, Buscemi G, Ragni R, Cantore M, Cicco SR, Farinola GM, et al. Synthesis of (poly)gallic acid in a bacterial growth medium. MRS Adv. 2020;5(18–19):957–63. D’Ordine RL, Rydel TJ, Storek MJ, Sturman EJ, Moshiri F, Bartlett RK, et al. Dicamba monooxygenase: structural insights into a dynamic Rieske oxygenase that catalyzes an exocyclic monooxygenation. J Mol Biol. 2009;392(2):481–97. Morawski B, Segura A, Ornston LN. Repression of Acinetobacter vanillate demethylase synthesis by VanR, a member of the GntR family of transcriptional regulators. FEMS Microbiol Lett. 2000;187(1):65–8. Tropel D, Van Der Meer JR. Bacterial Transcriptional Regulators for Degradation Pathways of Aromatic Compounds. Microbiol Mol Biol Rev. 2004;68(3):474–500. Segura A, Bünz PV, D’Argenio DA, Ornston LN. Genetic Analysis of a Chromosomal Region Containing vanA and vanB , Genes Required for Conversion of Either Ferulate or Vanillate to Protocatechuate in Acinetobacter . J Bacteriol. 1999;181(11):3494–504. Barbe V. Unique features revealed by the genome sequence of Acinetobacter sp. ADP1, a versatile and naturally transformation competent bacterium. Nucleic Acids Res. 2004;32(19):5766–79. Dal S, Steiner I, Gerischer U. Multiple operons connected with catabolism of aromatic compounds in Acinetobacter sp. strain ADP1 are under carbon catabolite repression. J Mol Microbiol Biotechnol. 2002;4(4):389–404. Pernstich C, Senior L, MacInnes KA, Forsaith M, Curnow P. Expression, purification and reconstitution of the 4-hydroxybenzoate transporter PcaK from Acinetobacter sp. ADP1. Protein Expr Purif. 2014;101:68–75. D’Argenio DA, Segura A, Coco WM, Bünz PV, Ornston LN. The Physiological Contribution of Acinetobacter PcaK, a Transport System That Acts upon Protocatechuate, Can Be Masked by the Overlapping Specificity of VanK. J Bacteriol. 1999;181(11):3505–15. Bleichrodt FS, Fischer R, Gerischer UC. The β-ketoadipate pathway of Acinetobacter baylyi undergoes carbon catabolite repression, cross-regulation and vertical regulation, and is affected by Crc. Microbiology. 2010;156(5):1313–22. Echenique JR, Dorsey CW, Patrito LC, Petroni A, Tolmasky ME, Actis LA. Acinetobacter baumannii has two genes encoding glutathione-dependent formaldehyde dehydrogenase: evidence for differential regulation in response to iron This paper is dedicated to the memory of Dr M. A. Vides, Facultad de Ciencias Quı́micas, Universidad Nacional de Córdoba, Argentina, who was a great mentor and colleague. The GenBank accession number for the sequence reported in this paper is AF130307. Microbiology. 2001;147(10):2805–15. Dumitru R, Jiang WZ, Weeks DP, Wilson MA. Crystal structure of dicamba monooxygenase: a Rieske nonheme oxygenase that catalyzes oxidative demethylation. J Mol Biol. 2009;392(2):498–510. Badhani B, Sharma N, Kakkar R. Gallic acid: a versatile antioxidant with promising therapeutic and industrial applications. RSC Adv. 2015;5(35):27540–57. Fernandes FHA, Salgado HRN. Gallic Acid: Review of the Methods of Determination and Quantification. Crit Rev Anal Chem. 2016;46(3):257–65. Cheng Y, Kuboyama K, Akasaka S, Araki T, Masai E, Nakamura M, et al. Polyurethanes based on lignin-derived metabolic intermediate with strong adhesion to metals. Polym Chem. 2022;13(48):6589–98. Jin Y, Araki T, Kamimura N, Masai E, Nakamura M, Michinobu T. Biodegradable and wood adhesive polyesters based on lignin-derived 2-pyrone-4,6-dicarboxylic acid. RSC Sustain. 2024;2(7):1985–93. Hall BW, Kontur WS, Neri JC, Gille DM, Noguera DR, Donohue TJ. Production of carotenoids from aromatics and pretreated lignocellulosic biomass by Novosphingobium aromaticivorans . Appl Environ Microbiol. 2023;89(12):e01268–23. Johnson CW, Salvachúa D, Rorrer NA, Black BA, Vardon DR et al. St. John PC,. Innovative Chemicals and Materials from Bacterial Aromatic Catabolic Pathways. Joule. 2019;3(6):1523–37. Perez JM, Sener C, Misra S, Umana GE, Coplien J, Haak D, et al. Integrating lignin depolymerization with microbial funneling processes using agronomically relevant feedstocks. Green Chem. 2022;24(7):2795–811. Perez JM, Kontur WS, Gehl C, Gille DM, Ma Y, Niles AV, et al. Redundancy in Aromatic O -Demethylation and Ring-Opening Reactions in Novosphingobium aromaticivorans and Their Impact in the Metabolism of Plant-Derived Phenolics. Appl Environ Microbiol. 2021;87(8):e02794–20. Perez JM, Kontur WS, Alherech M, Coplien J, Karlen SD, Stahl SS, et al. Funneling aromatic products of chemically depolymerized lignin into 2-pyrone-4-6-dicarboxylic acid with Novosphingobium aromaticivorans . Green Chem. 2019;21(6):1340–50. Qian Y, Otsuka Y, Sonoki T, Mukhopadhyay B, Nakamura M, Jellison J, et al. Engineered Microbial Production of 2-Pyrone-4,6-Dicarboxylic Acid from Lignin Residues for Use as an Industrial Platform Chemical. BioResources. 2016;11(3):6097–109. Suzuki Y, Okamura-Abe Y, Otsuka Y, Araki T, Nojiri M, Kamimura N, et al. Integrated process development for grass biomass utilization through enzymatic saccharification and upgrading hydroxycinnamic acids via microbial funneling. Bioresour Technol. 2022;363:127836. Nogales J, Canales Á, Jiménez-Barbero J, García JL, Díaz E. Molecular Characterization of the Gallate Dioxygenase from Pseudomonas putida KT2440. J Biol Chem. 2005;280(42):35382–90. Kasai D, Masai E, Miyauchi K, Katayama Y, Fukuda M. Characterization of the 3-O-methylgallate dioxygenase gene and evidence of multiple 3-O-methylgallate catabolic pathways in Sphingomonas paucimobilis SYK-6. J Bacteriol. 2004;186(15):4951–9. Additional Declarations No competing interests reported. Supplementary Files Additionalfile1.pdf Additional file 1.pdf Title of data: Supplemental information Description of data: Primers, supplementary results and figures. Cite Share Download PDF Status: Published Journal Publication published 29 Sep, 2025 Read the published version in Microbial Cell Factories → Version 1 posted Editorial decision: Revision requested 07 Apr, 2025 Reviews received at journal 07 Apr, 2025 Reviews received at journal 05 Apr, 2025 Reviewers agreed at journal 17 Mar, 2025 Reviewers agreed at journal 16 Mar, 2025 Reviewers invited by journal 16 Mar, 2025 Editor assigned by journal 15 Mar, 2025 Submission checks completed at journal 15 Mar, 2025 First submitted to journal 13 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6218493","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":430175795,"identity":"c5b55a81-01a4-425d-84f5-044d0310fa43","order_by":0,"name":"Heidi Tuomela","email":"","orcid":"","institution":"Tampere University","correspondingAuthor":false,"prefix":"","firstName":"Heidi","middleName":"","lastName":"Tuomela","suffix":""},{"id":430175797,"identity":"ddf02ef5-7940-4ed0-a6ca-e5400b223aa0","order_by":1,"name":"Johanna Koivisto","email":"","orcid":"","institution":"Tampere University","correspondingAuthor":false,"prefix":"","firstName":"Johanna","middleName":"","lastName":"Koivisto","suffix":""},{"id":430175799,"identity":"dad65865-fc6e-4ab5-bd73-8a835a8c70a2","order_by":2,"name":"Elena Efimova","email":"","orcid":"","institution":"Tampere University","correspondingAuthor":false,"prefix":"","firstName":"Elena","middleName":"","lastName":"Efimova","suffix":""},{"id":430175803,"identity":"3b68237f-e23f-4bce-a43a-516eb10365c8","order_by":3,"name":"Suvi Santala","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4UlEQVRIie2OvwrCMBCHLwR0KXYVOvQV4qb471VyFHQpLo4OVgrpEnDt5qtEOrj4AB0tvkDBRbCDqeLgkjg65FuOg/vu9wNwOP4U9R40gVqPDpCL+knxgCQk561CmVWBj0I9/opjxsswjwcKmtlq3j3ubtN7seoBJcYUVsZMERGtPQ/TIObFWhcDs9JfcKUroQRMWgUF+MqohLlWoNmi9Kv0MXwplhQoI/2zU6DsowjgF4Wdr0qhOKEsKzGSiyUKalHCDNO6bjaY7aOivE/GeMhSWhuLtfCvjVrvHQ6Hw2HjCaEwTKhLqiY9AAAAAElFTkSuQmCC","orcid":"","institution":"Tampere University","correspondingAuthor":true,"prefix":"","firstName":"Suvi","middleName":"","lastName":"Santala","suffix":""}],"badges":[],"createdAt":"2025-03-13 09:23:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6218493/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6218493/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12934-025-02839-1","type":"published","date":"2025-09-29T15:57:08+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":78755503,"identity":"d8d7ee92-fe13-4ad1-86cf-c4d2dfb6b6b7","added_by":"auto","created_at":"2025-03-18 12:51:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":44615,"visible":true,"origin":"","legend":"\u003cp\u003eThe enzymatic reaction performed by VanAB. A) VanAB O-demethylates its substrate vanillate by using NADH and oxygen. Protocatechuate (PCA), NAD\u003csup\u003e+\u003c/sup\u003e, and formaldehyde are formed as products. B) The chemical structures of syringate, 3-O-methylgallate (3MGA) and gallate. O-demethylation of syringate produces 3MGA, and further O-demethylation of 3MGA produces gallate.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-6218493/v1/615b435039357b02b1a153d5.png"},{"id":78755215,"identity":"9dbafd58-40dc-4006-a869-fc1f6db7eca8","added_by":"auto","created_at":"2025-03-18 12:43:13","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":86729,"visible":true,"origin":"","legend":"\u003cp\u003eThe growth of ADP1 WT, ASA1002, ASA1003, induced ASA1004 IND (5 µM cyclohexanone), and uninduced ASA1004 on vanillate as a sole carbon source. The strains were cultivated in MSM supplemented with 5 mM vanillate, and 25 µg/ml chloramphenicol for ASA1004. The strains were cultivated in 200 µl on a 96 well plate at 30 °C with shaking for 24 hours. The mean values and error bars representing the standard deviations from three parallel cultures are shown.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-6218493/v1/f89b6c3ed951edaffa8700c2.png"},{"id":78755218,"identity":"97d597e7-d4fc-4022-92c9-a1ad01bfd15a","added_by":"auto","created_at":"2025-03-18 12:43:13","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":91192,"visible":true,"origin":"","legend":"\u003cp\u003eSyringate tolerance of A: ADP1 WT, B: ASA1002, C: ASA1003 and D: ASA1004 (induced with 5 µM cyclohexanone). The strains were cultivated in mineral salts medium supplemented with 0 -20 mM syringate, 50 mM glucose, 0.2 % casein amino acids and 25 µg/ml chloramphenicol where required. The strains were cultivated in 200 µl media on a 96 well plate for 24 hours. The mean values and error bars representing the standard deviations from two parallel cultures are shown.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-6218493/v1/6950c9bc7031e1480000207e.png"},{"id":78755219,"identity":"15fca48d-c4ef-437c-af2b-78daf042d389","added_by":"auto","created_at":"2025-03-18 12:43:13","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":121688,"visible":true,"origin":"","legend":"\u003cp\u003eO-demethylation of syringate by A: ADP1 WT, B: P. putida KT2440. The strains were cultivated in 15 ml mineral salts medium supplemented with 5 mM syringate, 50 mM glucose and 0.2 % casein amino acids. Samples were collected at the indicated timepoints to monitor growth (OD600) and metabolite concentrations in the media (analysed with HPLC). The mean values and error bars representing the standard deviations from two parallel cultures are shown. 3MGA: 3-O-methylgallate\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6218493/v1/ceb614137894cf93615c82d8.png"},{"id":78755505,"identity":"919a110b-a85a-4649-acbd-fb298288f937","added_by":"auto","created_at":"2025-03-18 12:51:13","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":198535,"visible":true,"origin":"","legend":"\u003cp\u003eO-demethylation of syringate by A: ASA1002 B: ASA1003, C: ASA1004, D: ASA1004 induced with 5 µM cyclohexanone. The strains were cultivated in 15 ml mineral salts medium supplemented with 5 mM syringate, 50 mM glucose, 0.2 % casein amino acids and 25 µg/ml chloramphenicol when required. Samples were collected at the indicated timepoints to monitor growth (OD600) and metabolite concentrations in the media (analysed with HPLC). The mean values and error bars representing the standard deviations from two parallel cultures are shown. Abbreviations: 3MGA: 3-O-methylgallate.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-6218493/v1/b61ae0b6113222e8f2b00d99.png"},{"id":78756369,"identity":"9e1a07d1-01d4-44b5-b3f3-190cee17f10b","added_by":"auto","created_at":"2025-03-18 12:59:13","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":124909,"visible":true,"origin":"","legend":"\u003cp\u003eThe saturation curves for VanAB with A: vanillate and B: syringate as a substrate. The curves are generated from fitting Eq. 1 of v\u003csub\u003e1\u003c/sub\u003e of consumed NADH versus [S]. Experiments were performed as described in Materials and methods. Duplicate reactions at each substrate concentration were performed.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-6218493/v1/4c9cdcf7d419913c4424d615.png"},{"id":78755507,"identity":"1ad92819-4ab9-498a-8bfe-1ad7cedfb0c5","added_by":"auto","created_at":"2025-03-18 12:51:14","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":110657,"visible":true,"origin":"","legend":"\u003cp\u003eSyringate conversion into PDC by A: ADP1 WT B: ADP1 ΔpcaHG. The strains were cultivated in 50 ml bioreactor tubes with 15 ml mineral salts medium supplemented with 5 mM syringate, 50 mM glucose and 0.2 % casein amino acids. Samples were collected at the indicated timepoints to monitor growth (OD600) and metabolite concentrations in the media (analysed with HPLC). The mean values and error bars representing the standard deviations from three parallel cultures are shown. 3MGA: 3-O-methylgallate; PDC: 2-pyrone-4,6-dicarboxylate.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-6218493/v1/5cc0a8a789a8e7738bac5fd3.png"},{"id":78755230,"identity":"8f5279da-85df-4f08-aa18-3ab199646341","added_by":"auto","created_at":"2025-03-18 12:43:14","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":154776,"visible":true,"origin":"","legend":"\u003cp\u003eSyringate conversion into PDC by A: ADP1 WT, B: ASA1005 C: ASA1005 induced with 1 µM AHL. The strains were cultivated in 50 ml bioreactor tubes in 15 ml mineral salts media supplemented with 5 mM syringate, 50 mM glucose and 0.2 % casein amino acids. In addition, for ASA1005 15 µg/ml gentamicin and 1 µM AHL for the induction of galA were supplemented. Samples were collected at the indicated timepoints to monitor growth (OD600) and metabolite concentrations in the media (analysed with HPLC). The mean values and error bars representing the standard deviations from three parallel cultures are shown. 3MGA: 3-O-methylgallate; PDC: 2-pyrone-4,6-dicarboxylate.\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-6218493/v1/850183c3575ee959bdf5257b.png"},{"id":78755231,"identity":"bac5cb2d-6275-42b8-851b-eb45313f6a80","added_by":"auto","created_at":"2025-03-18 12:43:14","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":122429,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003ePDC production in a bioreactor. The graphs show two individual biological replicates. The reactor was maintained at 30 °C, 200 rpm and at constant aeration. Starting volume of the cultivations was 50 ml of MSM media supplemented with 20 mM glucose, 0.2 % casein amino acids, 2 mM syringate, 10 µM AHL, 30 µg/ml kanamycin, and inoculant resulting in initial OD of approximately 3-4. Syringate and glucose were fed to the reactor at rates of 0.058 mmol/h and 0.29 mmol/h, respectively. Feeding stop time is indicated by a gray vertical line. Abbreviations: 3MGA (3-O-methylgallate), PDC (2-pyrone-4,6-dicarboxylate).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image10.png","url":"https://assets-eu.researchsquare.com/files/rs-6218493/v1/690108465ce0e455a10e6b2b.png"},{"id":92883692,"identity":"2dffc0d0-9a1a-4b74-bac5-71698db7f4b9","added_by":"auto","created_at":"2025-10-06 16:07:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2378791,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6218493/v1/554486b2-554e-4d18-843b-92877c95b977.pdf"},{"id":78755221,"identity":"d8801a62-d081-4cca-9180-978cb2ca951e","added_by":"auto","created_at":"2025-03-18 12:43:13","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":790129,"visible":true,"origin":"","legend":"\u003cp\u003eAdditional file 1.pdf\u003c/p\u003e\n\u003cp\u003eTitle of data: Supplemental information\u003c/p\u003e\n\u003cp\u003eDescription of data: Primers, supplementary results and figures.\u003c/p\u003e","description":"","filename":"Additionalfile1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6218493/v1/985393eabdb14bdb4a16d942.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Conversion and upgrading of S-lignin related syringate by Acinetobacter baylyi ADP1","fulltext":[{"header":"Background","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eLignin, comprising up to a third of lignocellulose, is a natural polymer found in all wood-like plants. It is well known for its tenacious structure that is formed by complex networks of three main subunits \u0026ndash; \u003cem\u003ep\u003c/em\u003e-coumaryl (H), coniferyl (G) and sinapyl (S) alcohol. While lignin provides protection and support for plants, it is considered a nuisance in industries that use lignocellulose as raw material. In these industries, burning lignin for power generation is the main means to derive value from it. Therefore, upgrading lignin to valuable products could improve the economic feasibility of biobased industries and contribute to more carbon-wise use of resources. Lignin also provides a renewable source of aromatics, an alternative to current fossil-based feedstocks for chemical production.\u003c/p\u003e \u003cp\u003eDepolymerized lignin consists of a heterogeneous mixture of aromatic compounds, which varies across different plant types. Biological upgrading of lignin is a promising approach, as it allows these diverse aromatic compounds to be funnelled to specific, defined products. Recently, the native catabolic pathways of soil bacteria, such as \u003cem\u003ePseudomonas putida\u003c/em\u003e KT2440, \u003cem\u003eSphingobium sp.\u003c/em\u003e SYK-6, \u003cem\u003eNovosphingobium aromaticivorans\u003c/em\u003e and \u003cem\u003eAcinetobacter baylyi\u003c/em\u003e ADP1 have been utilized for upgrading lignin-related aromatic compounds into value-added products (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). For example, the biological production of plastic precursors such as \u003cem\u003ecis,cis\u003c/em\u003e-muconate (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e), adipate (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e), gallate (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e), and 2-pyrone-4,6-dicarboxylate (PDC) (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e) has been extensively studied. Plastics produced from biobased precursors can have many advantages over their fossil-based counterparts, such as better biodegradability, and improved mechanical and adhesive properties (\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eS-lignin constitutes a significant portion of the lignin polymer, particularly in grasses and hardwood (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). S-lignin derived aromatics, such as syringaldehyde and syringate, are prevalent in depolymerized lignin. However, microbial strains capable of catabolizing these compounds are scarce (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). The different lignin types are distinguished by the number of methoxy groups attached to the aromatic ring. S-lignin derived aromatics are the most challenging to catabolize because they contain two methoxy groups, compared to G-lignin with one methoxy group and H-lignin, which is not methoxylated. Consequently, \u003cem\u003eO-\u003c/em\u003edemethylation of these methoxy groups is a crucial step in the upper pathways of aromatic catabolism.\u003c/p\u003e \u003cp\u003eIn aerobic microorganisms, \u003cem\u003eO-\u003c/em\u003edemethylation is carried out by enzymes that can be divided into three groups: Rieske oxygenases (RO), cytochromes P450 (P450s), and tetrahydrofolate (THF)-dependent demethylases (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). THF-dependent demethylases transfer the methyl group non-oxidatively to THF (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e), whereas ROs and P450s utilize NAD(P)H as a cofactor to oxidize the methyl group into formaldehyde (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Formaldehyde, being highly toxic to cells, can be mitigated by more than one pathway (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). For example, in \u003cem\u003eE. coli frmA\u003c/em\u003e encodes a glutathione dependent formaldehyde dehydrogenase that is coupled to restoring NAD\u0026thinsp;+\u0026thinsp;into NADH (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). The requirement for NADH increases for substrates with several methoxy groups, for which \u003cem\u003eO-\u003c/em\u003edemethylating reactions have been shown to be energy-limited and cause cofactor imbalance in cells. Substrates (such as glucose) that drive primary metabolism are often required to support cell\u0026rsquo;s energy generation to obtain a balanced redox stoichiometry and sustain efficient utilization of the aromatic compounds (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Rieske oxygenases consist of a reductase, an oxygenase, and in some cases an additional ferredoxin for electron transfer. The reductase transfers electrons from NAD(P)H to the oxygenase resulting in reduction of the mononuclear iron. Binding of the substrate leads to binding of O\u003csub\u003e2\u003c/sub\u003e at the iron-center, resulting in a high-valency ferryl species that can hydroxylate the substrate. The final product is obtained after rearrangement of the hydroxylated species (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). VanAB is a Rieske oxygenase that uses vanillate as its\u0026rsquo; primary substrate (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Previously it has been shown that this enzyme pair can \u003cem\u003eO-\u003c/em\u003edemethylate syringate into 3-O-methylgallate and subsequently into gallate in \u003cem\u003ePseudomonas sp\u003c/em\u003e. HR199, \u003cem\u003ePseudomonas putida\u003c/em\u003e KT2440 and \u003cem\u003eStreptomyces\u003c/em\u003e sp. NL15-2K (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAlthough the substrate range of VanAB in \u003cem\u003eA. baylyi\u003c/em\u003e ADP1 (hereafter ADP1) has been investigated and includes several analogous substrates, no activity for syringate has been previously detected (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). This is unexpected given the high similarity between \u003cem\u003evanAB\u003c/em\u003e orthologues in the different strains. Many factors support the hypothesis of syringate \u003cem\u003eO-\u003c/em\u003edemethylation by VanAB in ADP1. Amino acid sequence identity of the substrate binding VanA found in ADP1 to \u003cem\u003eP. putida\u003c/em\u003e KT2440 and \u003cem\u003eP. putida\u003c/em\u003e HR199 is very high, 75\u0026ndash;78%. Known substrates of VanAB indicate that a methoxy group in meta position to a carboxyl group is necessary for the activity of the enzyme (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). This does not exclude syringate as a substrate.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003eADP1 has raised increasing interest as a potential microbial cell factory for its versatile metabolism and natural competence, which make metabolic engineering remarkably simple (\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). ADP1 can utilize a variety of G- and H-type lignin monomers via the native β-ketoadipate pathway and has been shown to have ligninolytic effects on softwood (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Previously, ADP1 has been engineered for the production of wax esters and alkanes (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e), 1-alkenes (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e), \u003cem\u003ecis,cis\u003c/em\u003e-muconate (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e), mevalonate (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e), naringenin (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e), and resveratrol and vanillin-glucoside (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e) from lignin-related aromatic compounds, such as ferulate and \u003cem\u003ep\u003c/em\u003e-coumarate. In addition, the strain has been previously evolved to tolerate very high concentrations of aromatic compounds (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e) as well as exploited in the detoxification of lignocellulosic hydrolysates (\u003cspan additionalcitationids=\"CR40 CR41\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, our goal was to establish the conversion and upgrading of S-lignin-related syringate in ADP1. We first investigated the activity of VanAB both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e under native and non-native expression systems. We showed that both syringate and 3MGA can be \u003cem\u003eO-\u003c/em\u003edemethylated by ADP1. We also demonstrated the production of PDC, a precursor for high-quality polyesters, from syringate by the heterologous expression of gallate dioxygenase GalA from \u003cem\u003eP. putida\u003c/em\u003e KT2440.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStrains and media\u003c/h2\u003e \u003cp\u003e \u003cem\u003eEscherichia coli\u003c/em\u003e XL1-Blue (Stratagene, USA) was used for plasmic construction and maintenance. \u003cem\u003eAcinetobacter baylyi\u003c/em\u003e ADP1 (DSM 24193, Leibniz Institute DSMZ, Germany) was used to study the endogenous expression of VanAB and for strain construction. All the strains used and engineered in this study are listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBacterial strains used in this study.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eName\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGenotype\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDescription\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSource\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eADP1 WT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWild-type \u003cem\u003eA. baylyi\u003c/em\u003e ADP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDSM 24193\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDSMZ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eASA1001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eA. baylyi\u003c/em\u003e ADP1 ΔvanAB::tdk/kan\u003csup\u003er\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003evanAB\u003c/em\u003e (ACIAD0979-0980) replaced with tdk/kan\u003csup\u003er\u003c/sup\u003e cassette\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eASA1002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eA. baylyi\u003c/em\u003e ADP1 ΔvanAB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eStrain with markerless \u003cem\u003evanAB\u003c/em\u003e deletion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eASA1003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eA. baylyi\u003c/em\u003e ADP1 ΔvanAB, pBAV1C-T5-vanAB/Cm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eStrain ASA1002 with \u003cem\u003evanAB\u003c/em\u003e overexpression from pBAV1C-T5-plasmid under constitutive T5 promoter.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eASA1004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eA. baylyi\u003c/em\u003e ADP1 ΔvanAB, pBAV1Cd-chn-vanAB/Cm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eStrain ASA1002 with \u003cem\u003evanAB\u003c/em\u003e overexpression from pBAV1Cd-plasmid under cyclohexanone inducible promoter. N-terminal His-tag in \u003cem\u003evanA\u003c/em\u003e for purification.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eASA1005\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eA. baylyi\u003c/em\u003e ADP1 pKLxR5-luxR-galA/Gm\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eStrain with plasmid \u003cem\u003egalA\u003c/em\u003e overexpression.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eASA1006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eA. baylyi\u003c/em\u003e ADP1 ΔpoxB::luxR-kan\u003csup\u003er\u003c/sup\u003e-galA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eStrain with genomic \u003cem\u003egalA\u003c/em\u003e overexpression replacing ACIAD3381 (\u003cem\u003epoxB\u003c/em\u003e).\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePseudomonas putida\u003c/em\u003e KT2440\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWild-type\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHarbors \u003cem\u003egalA\u003c/em\u003e gallate dioxygenase (PP_2518, \u003cem\u003eglllA\u003c/em\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDSMZ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eModified low salt lysogeny broth (LB) medium (10 g/L tryptone, 5 g/L yeast extract, 1 g/L NaCl) supplemented with 50 mM glucose was used to cultivate \u003cem\u003eE. coli\u003c/em\u003e and ADP1 for strain construction. Mineral salts medium (MSM) supplemented with 50 mM (unless stated otherwise) glucose, 0.2% casein amino acids, and aromatics in specified concentrations was used in aromatics conversion experiments. MSM composition was 3.88 g/L K\u003csub\u003e2\u003c/sub\u003eHPO4, 1.63 g/L NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 2.00 g/L (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, 0.1 g/L MgCl\u003csub\u003e2\u003c/sub\u003e\u0026middot;6 H2O, 10 mg/L ethylenediaminetetraacetic acid (EDTA), 2 mg/L ZnSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7 H2O, 1 mg/L CaCl\u003csub\u003e2\u003c/sub\u003e\u0026middot;2 H\u003csub\u003e2\u003c/sub\u003eO, 5 mg/L FeSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7 H\u003csub\u003e2\u003c/sub\u003eO, 0.2 mg/L Na\u003csub\u003e2\u003c/sub\u003eMoO\u003csub\u003e4\u003c/sub\u003e\u0026middot;2 H\u003csub\u003e2\u003c/sub\u003eO, 0.2 mg/L CuSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;5 H\u003csub\u003e2\u003c/sub\u003eO, 0.4 mg/L CoCl\u003csub\u003e2\u003c/sub\u003e\u0026middot;6 H\u003csub\u003e2\u003c/sub\u003eO, and 1 mg/L MnCl\u003csub\u003e2\u003c/sub\u003e\u0026middot;2 H\u003csub\u003e2\u003c/sub\u003eO. Antibiotics for \u003cem\u003eE. coli\u003c/em\u003e and ADP1 were supplemented when needed in following concentrations: chloramphenicol, 25 \u0026micro;g/ml; gentamicin, 15 \u0026micro;g/ml; spectinomycin, 50 \u0026micro;g/ml; kanamycin 30 \u0026micro;g/ml. Zidovudine plates in concentration 400 \u0026micro;g/ml were prepared for counter selection of the gene deletions. N-(3-oxohexanoyl) homoserine lactone (AHL) and cyclohexanone (cyc) were used for induction in specified concentrations ranging from 1\u0026ndash;10 \u0026micro;M.\u003c/p\u003e \u003cp\u003eAromatics stocks were prepared in following concentrations: vanillate 200 mM, syringate, 3MGA and gallate 100 mM. Correct amount of the aromatic compound was weighed and added to deionized water after which KOH was slowly added to equimolar or slightly excess amount to dissolve the compounds as potassium salts, with final pH reaching 8.2\u0026ndash;8.5. Reagents were purchased from Sigma Aldrich (United States).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGenetic engineering\u003c/h3\u003e\n\u003cp\u003eGenetic engineering was carried out using established methods for restriction-ligation, USER and NEBuilder\u0026reg; HiFi DNA Assembly cloning as well as overlap extension PCR. Reagents for molecular work were purchased from Thermo Scientific (USA) and New England Biolabs (USA) and used according to the manufacturer\u0026rsquo;s instruction. The primers used in this study are listed in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e (Additional File 1). Electroporation was used for the transformation of \u003cem\u003eE. coli\u003c/em\u003e XL-1 Blue. ADP1 transformation and genomic editing by homologous recombination were carried out as described previously by (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). The transformants were screened on lysogeny agar (LA) plates with appropriate antibiotics.\u003c/p\u003e \u003cp\u003e \u003cem\u003eVanAB\u003c/em\u003e was deleted from ADP1 genome using a linear DNA cassette; regions of approximately 1 kb flanking \u003cem\u003evanAB\u003c/em\u003e to facilitate homologous recombination were amplified from ADP1 genome with primers \u003cem\u003eVanB_P5-OE\u003c/em\u003e and \u003cem\u003eVanB R2\u003c/em\u003e (3\u0026rsquo; segment), and \u003cem\u003eVanA_P4-OE\u003c/em\u003e and \u003cem\u003eVanA F2\u003c/em\u003e (5\u0026rsquo; segment). The fragment tdk/kan\u003csup\u003er\u003c/sup\u003e carrying kanamycin resistance marker and \u003cem\u003etdk\u003c/em\u003e for counter-selection (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e) was amplified with primers Tdk_kanF and Tdk_kanR from the genome of ADP1∆3383::tdk/kan\u003csup\u003er\u003c/sup\u003e, (a kind gift from Dr. Veronique de Berardinis, Genoscope, France) (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). The cassette was assembled with overlap extension PCR and transformed into ADP1 WT resulting in strain ASA1001. Kanamycin was used for selection of successful clones. A rescue cassette to replace tdk/kan\u003csup\u003er\u003c/sup\u003e was constructed in similar manner; flanking regions of \u003cem\u003evanAB\u003c/em\u003e were amplified with primers \u003cem\u003erescue cassette vanB forward\u003c/em\u003e and \u003cem\u003evanB R2\u003c/em\u003e (3\u0026rsquo; flanking) and \u003cem\u003evanA F2\u003c/em\u003e and \u003cem\u003erescue cassette vanA reverse\u003c/em\u003e (5\u0026rsquo; flanking). Restriction sites for MfeI, NotI and AvrII were designed into overlapping primers \u003cem\u003erescue cassette vanB forward\u003c/em\u003e and \u003cem\u003erescue cassette vanA reverse\u003c/em\u003e that join the flanking sequences together (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, Additional File 1). Overlap extension PCR was used to combine the segments.\u003c/p\u003e \u003cp\u003eThe rescue cassette was transformed into ASA1001 resulting in ASA1002 with markerless \u003cem\u003evanAB\u003c/em\u003e deletion. Counterselection with zidovudine was used to select successful clones; After transformation with the rescue cassette, the cell culture was diluted 1:10, 1:100 and 100 \u0026micro;l volumes were spread on LA plates supplemented with 400 \u0026micro;g/ml zidovudine and 50 mM glucose. The plates were incubated at 30\u0026deg;C until colonies appeared. Colonies were then resuspended in LB media and divided into two cultures each, in LB media supplemented with 50 mM glucose and either 30 \u0026micro;g/ml kanamycin or no antibiotic. The cultures were incubated at 30\u0026deg;C, 300 rpm overnight, after which those that did not grow on kanamycin were selected. The genetic region was further confirmed with PCR using primers \u003cem\u003eVer_VanAB_F\u003c/em\u003e and \u003cem\u003eVer_VanAB_R\u003c/em\u003e binding outside the cassette region.\u003c/p\u003e \u003cp\u003ePlasmid pBAV1Cd-chnR-vanAB for overexpression of \u003cem\u003evanAB\u003c/em\u003e was constructed by BioBrick cloning in the empty plasmid pBAV1Cd-chn (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). To clone \u003cem\u003evanAB\u003c/em\u003e from ADP1 genome it was amplified with primers \u003cem\u003eVanAB_F_BB\u003c/em\u003e and \u003cem\u003eVanAB_R_BB.\u003c/em\u003e A ribosome binding site, a new start codon, His(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e) -tag and Gly-Ser-Gly -linker sequence were included in the forward primer \u003cem\u003eVanAB_F_BB\u003c/em\u003e annealing to \u003cem\u003evanA\u003c/em\u003e to enable purification with the histidine tag (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, Additional File 1). The obtained plasmid pBAV1Cd-chn-vanAB was first transformed into \u003cem\u003eE. coli\u003c/em\u003e XL1-Blue and then into ASA1002 resulting in ADP1Δ\u003cem\u003evanAB\u003c/em\u003e pBAV1Cd-chn-vanAB (ASA1004).\u003c/p\u003e \u003cp\u003eNext, \u003cem\u003evanAB\u003c/em\u003e was cloned under a constitutive T5-promoter with USER cloning. \u003cem\u003eVanAB\u003c/em\u003e from ADP1 genome was amplified with primers SS-21-03-IFU and SS-21-04-IRU. SS-21-01-VFU and SS-21-02-VRU were used to amplify the backbone of pBAV1C-T5-GFP (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). The obtained plasmid pBAV1C-T5-vanAB was first transformed into \u003cem\u003eE. coli\u003c/em\u003e XL1-Blue and then into ASA1002 resulting in ADP1ΔvanAB pBAV1C-T5-vanAB (ASA1003).\u003c/p\u003e \u003cp\u003eTo overexpress \u003cem\u003egalA\u003c/em\u003e, plasmid pKLxR5-galA was constructed with NEB HiFi assembly. \u003cem\u003eGalA\u003c/em\u003e was amplified from \u003cem\u003eP. putida\u003c/em\u003e KT2440 genome with primers \u003cem\u003eGalA_F\u003c/em\u003e and \u003cem\u003eGalA_rev\u003c/em\u003e. Plasmid pKLxR5-mRFP, a kind gift from Schuster and Reisch, Addgene #149465 (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e) was utilized as backbone and amplified with primers \u003cem\u003epKLxR5_fwd\u003c/em\u003e and \u003cem\u003eprR\u003c/em\u003e. The obtained plasmid pKLxR5-galA was transformed into \u003cem\u003eE. coli\u003c/em\u003e XL1-Blue and then into ADP1 WT resulting in strain ASA1005.\u003c/p\u003e \u003cp\u003eFor the genomic expression of \u003cem\u003egalA\u003c/em\u003e, a linear gene cassette was constructed based on a previously described cassette that creates a neutral gene knock-out of \u003cem\u003epoxB\u003c/em\u003e (ACIAD3381) (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). \u003cem\u003eLuxR-galA -\u003c/em\u003eregion was amplified from pKLxR5-galA with primers HT-24-7-galA rev and HT-24-8-luxR fwd and the pIX-backbone with kan\u003csup\u003er\u003c/sup\u003e and \u003cem\u003epoxB\u003c/em\u003e-flankings was amplified with primers HT-24-5-pIX fwd and HT-24-6 pIX rev. The segments were then combined with USER cloning. The plasmid was first transformed into \u003cem\u003eE. coli\u003c/em\u003e XL-1 Blue and then into ADP1 resulting in strain ASA1006.\u003c/p\u003e\n\u003ch3\u003eGrowth experiments\u003c/h3\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization of syringate tolerance and growth on vanillate on microplates\u003c/h2\u003e \u003cp\u003eEffects of \u003cem\u003evanAB\u003c/em\u003e deletion and overexpression were characterized by cultivating the strains on\u003c/p\u003e \u003cp\u003evanillate as a sole carbon source. Overnight precultures were prepared in 5 ml volume of MSM, 0.2% casein amino acids, and 25 \u0026micro;g/ml chloramphenicol (dissolved in MQ) when appropriate. The following day, the precultures were used to inoculate culture medium containing MSM, 5 mM vanillate, 25 \u0026micro;g/ml chloramphenicol when appropriate, and 5 \u0026micro;M cyclohexanone for the induction of ASA1004. Each strain was cultivated in triplicates on 96-well plate (200 \u0026micro;l medium/well). The plates were incubated in Spark multimode microplate reader (Tecan, Switzerland) at 30\u0026deg;C. The cultures were mixed with double orbital shaking twice per hour with an amplitude of 6 mm and frequency of 54 rpm. Optical density at 600 nm was measured twice per hour.\u003c/p\u003e \u003cp\u003eFor studying the syringate tolerance, strains ADP1 WT, ASA1002, ASA1003 and ASA1004 were precultured overnight in 5 ml volume in MSM media with 0.2% casein amino acids, 20 mM glucose, and appropriate antibiotics. Each strain was cultivated in duplicates on a 96-well plate (200 \u0026micro;l medium/well) in the same media as the precultures supplemented with 0, 1, 2, 5, 10, and 20 mM syringate. The plates were incubated in Spark multimode microplate reader (Tecan, Switzerland) as described above.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eBatch cultivations for syringate conversion and production of PDC\u003c/h3\u003e\n\u003cp\u003eOvernight precultures were inoculated from single colonies on LA plates. Precultures were carried out in 5 ml MSM supplemented with 0.2% casein amino acids, 50 mM glucose, and appropriate antibiotics. The main cultivations were carried out in 15 ml MSM media in 50 ml Nunc\u0026trade; bioreactor tubes (Thermo Scientific, United States).\u003c/p\u003e \u003cp\u003eCulture media was supplemented with 0.2% casein amino acids, 50 mM glucose, appropriate antibiotics, and 5 mM syringate with a starting OD of 0.1\u0026ndash;0.2. Cyclohexanone was added to the cultivations in concentrations 0\u0026ndash;5 \u0026micro;M to induce \u003cem\u003evanAB\u003c/em\u003e expression in ASA1004. Induced cultivations were kept on separate incubator from uninduced ones to avoid any effect of the highly volatile cyclohexanone. The pH of the media was monitored in the beginning and at the end of the cultures with pH strips.\u003c/p\u003e \u003cp\u003eFor quantification of metabolites, 1 ml samples were collected and centrifuged at 14 000 g for 2 minutes. The supernatants were collected and stored at -20\u0026deg;C prior to analysis. The samples were diluted in deionized water to appropriate concentration (maximum 10-fold dilution) and filtered with 0.2 \u0026micro;m filters before analysis. The pellets were washed with and resuspended to MSM after which OD600 was measured because of the dark coloration formed during the cultivations.\u003c/p\u003e \u003cp\u003eCultivation for PDC production was carried out similarly. Precultures were continued overnight or for two days for ASA1004 pKLxR5 galA strain to reach sufficient cell density, after which appropriate volumes were used for inoculation to achieve initial OD of 0.1\u0026ndash;0.2. 1 \u0026micro;M AHL was added to strains with \u003cem\u003egalA\u003c/em\u003e under LuxR-regulated promoter for induction. \u003cem\u003eVanAB\u003c/em\u003e expression was not induced.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eFed-batch cultivations for the production of PDC\u003c/h2\u003e \u003cp\u003eBioreactor experiments for PDC production were carried out in small scale bioreactors (Applikon Biotechnology, Netherlands) with 250 ml working volume. The reactor was maintained at 30\u0026deg;C, 200 rpm and at a constant aeration. Dissolved oxygen and pH were monitored during the cultivations. When necessary, 10% antifoam A (Fluka Analytical) was added. Samples of 1\u0026ndash;2 ml for analysis of OD600 and aromatics were collected and handled as described previously in batch cultivations. Starting volume of the cultivations was 50 ml of MSM media supplemented with 20 mM glucose, 0.2% casein amino acids, 2 mM syringate, 1 \u0026micro;M AHL, 30 \u0026micro;g/ml kanamycin and inoculant resulting in initial OD of approximately 3\u0026ndash;4. Feed containing 15 mM syringate and 75 mM glucose was pumped to the reactor at 3.7 ml per hour. Fed media contained other components in same concentrations as in the initial media. Two independent biological replicates were produced by individual experiments.\u003c/p\u003e \u003cp\u003eASA1006 was inoculated in 5 ml of MSM supplemented with 50 mM glucose, 0.2% casein amino acids, and 30 \u0026micro;g/ml kanamycin at 30\u0026deg;C, 300 rpm. After overnight cultivation, the cells were inoculated in the second preculture media in 15 ml of MSM supplemented with 50 mM glucose, 0.2% casein amino acids, 30 \u0026micro;g/ml kanamycin, and 10 \u0026micro;M AHL to induce \u003cem\u003egalA\u003c/em\u003e expression, as well as 1 mM vanillate to induce native \u003cem\u003evanAB\u003c/em\u003e expression, with initial OD 0.1\u0026ndash;0.2. The cells were cultivated overnight in 50 ml bioreactor tubes in shaker 30\u0026deg;C, 300 rpm. Parallel cultivations were pelleted at 5000 g for 10 minutes and resuspended in 15 ml MSM to achieve initial OD of 3\u0026ndash;4 in bioreactor after inoculation.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAnalytical methods\u003c/h3\u003e\n\u003cp\u003eGallic acid (GA) was purchased from Merck (Switzerland), 3,4-dihydroxy-5-methoxybenzoic acid (3MGA) and syringic acid (SA) were purchased from Sigma (USA). Methanol (HPLC grade) was purchased from Honeywell (Germany). All standards of aromatic compounds were prepared as 20 mM stock solutions in water by adding of 5M KOH until full solubilization as described above. For calibration, working standard solutions were prepared by diluting of stock solutions with water to concentrations 0.5\u0026ndash;5 mM and filtered using 0.2 \u0026micro;m filters. HPLC analysis of the aromatic compounds and PDC was performed on Shimadzu LC-40 (Japan), equipped with a photodiode array detector (PDA). The compounds were analysed on the column Rezex RFQ-Fast Acid H+ (8%), 100 x 7.8 mm, 55\u003csup\u003eo\u003c/sup\u003eC (Phenomenex Inc., USA) in 5 mM H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e at the flow rate of 0.6 mL/min. The injection volume was 5 \u0026micro;L. Eluted GA, 3MGA and SA were monitored at wavelength 272 nm.\u003c/p\u003e \u003cp\u003eSince a commercial PDC standard was not available, we used a sample collected from a cultivation of ADP1 containing PDC as a qualitative standard. The presence of PDC in the qualitative standard was proven by mass-spectrometry and nuclear magnetic resonance (\u003csup\u003e1\u003c/sup\u003eH-NMR). Mass-spectrometric analysis was carried out using JEOL AccuTOF LCplus (JMS-T100LP) (Japan) in ESI- mode. The molecular ion of PDC was detected as [M-H]\u003csup\u003e\u0026minus;\u003c/sup\u003e 182.96624 and distinguished from the molecular ion of 3-O-methyl gallate [M-H]\u003csup\u003e\u0026minus;\u003c/sup\u003e 183.02990, the compound with almost equal molecular weight. As an additional proof, the \u003csup\u003e1\u003c/sup\u003eH-NMR spectra of the qualitative standard and 3-O-methyl gallate were recorded on a JEOL spectrometer ECZ500R 500MHz (Japan) in D\u003csub\u003e2\u003c/sub\u003eO. Characteristic signals of the two protons of ɑ-pyrone ring at 6.74 (s) and 7.23 (s) ppm were detected in the spectrum of the tested sample whereas characteristic signals of aromatic protons and methoxy protons of 3-O-methyl gallate (7.02, 7.07 and 3.78, respectively) were not found in the spectrum. Quantification of PDC was performed from its chromatographic peak area using extinction coefficient e\u0026thinsp;=\u0026thinsp;6200 M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e .cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at 313 nm reported by Michinobu et al. (Bull. Chem. Soc. Jpn. Vol. 80, No. 12, 2436\u0026ndash;2442 (2007)).\u003c/p\u003e\n\u003ch3\u003eVanAB expression and purification\u003c/h3\u003e\n\u003cp\u003eFor VanAB expression, 10 ml overnight culture of ASA1004 was grown at 30\u0026deg;C in low salt LB medium supplemented with 50 mM glucose and 25 \u0026micro;g/ml chloramphenicol. The following day, cells were diluted in 100 ml of fresh medium (supplemented also with 20 \u0026micro;M FeSO\u003csub\u003e4\u003c/sub\u003e) to initial OD\u003csub\u003e600\u003c/sub\u003e of 0.05, and grown at 30\u0026deg;C. When OD\u003csub\u003e600\u003c/sub\u003e reached 0.5\u0026ndash;0.8, cyclohexanone was added as an inducer of \u003cem\u003evanAB\u003c/em\u003e expression to a final concentration of 5 \u0026micro;M. Cultivation was continued at 25\u0026deg;C for approximately 20 hours. Cells were harvested by centrifugation at 30 000 \u003cem\u003eg\u003c/em\u003e for 30 min at 4\u0026deg;C and stored at -20\u0026deg;C.\u003c/p\u003e \u003cp\u003eFor the copurification of VanAB, the cell pellets were thawed and resuspended in BugBuster Protein Extraction Reagent (Novagen, USA) using 5 ml reagent per gram of wet cell paste, or more if necessary. Lysozyme was then added to a final concentration of 1\u0026ndash;2 mg/ml and incubated while gently mixing for 30 minutes at room temperature. Disruption by sonication was performed in sequence of 15 s sonication followed by a 15 s pause on Fisherbrand\u0026trade; Model 120 Sonic Dismembrator (Thermo Fisher Scientific, USA) while kept on ice. The cell lysate was cleared by centrifugation at 30 000 \u003cem\u003eg\u003c/em\u003e for 20 min at 4\u0026deg;C. Imidazole was added to the cleared lysate to a final concentration of 20 mM. The cleared lysate was loaded on to a HisGraviTrap Ni-Sepharose column (GE, USA) and washed with binding/washing buffer (20 mM Tris, 500 mM NaCl, 20 mM imidazole, pH 8). VanAB was eluted with elution buffer (20 mM Tris, 500 mM NaCl, 500 mM imidazole, pH 8) and dithiothreitol (DTT) was added to a final concentration of 5 mM to increase the stability of the protein. Purity of the protein preparation was estimated by Sodium Dodecyl Sulfate Polyacrylamide Gel Electroforesis (SDS-PAGE) based on the method described by Laemmli (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e) using a 12% precast polyacrylamide gel (Bio-Rad, USA).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eVanAB activity assay\u003c/h2\u003e \u003cp\u003eActivity assay for the purified VanAB was performed the following day due to loss of activity after longer storage. Prior to assay, PD MidiTrap G-25 (Cytiva, USA) column was used for buffer exchange into 20 mM Tris-HCl, 100 mM NaCl, 5mM DTT, pH 8. The activity assay was based on monitoring the consumption of NADH by measuring the absorbance at 340 nm. Reactions were performed at 25\u0026deg;C with a range of substrate concentrations in 20 mM Tris-HCl, 100 mM NaCl, 5 mM DTT, pH 8. When vanillate or syringate was used as a substrate, two-fold successive dilution series were prepared to give final concentrations in reactions ranging from 400 \u0026micro;M to 25 \u0026micro;M. For 3MGA, substrate concentrations in reactions starting from 3200 \u0026micro;M to 200 \u0026micro;M were used. Control reactions were performed with 400 \u0026micro;M PCA and without any substrate. All reactions contained initial 400 \u0026micro;M NADH. Reactions were initiated by the addition of 40 \u0026micro;l purified VanAB preparation into total reaction volume of 200 \u0026micro;l. The absorbance at 340 nm was measured with Spark multimode microplate reader (Tecan, Switzerland) every 3 minutes until 90 minutes in total. The oxidation of NADH was converted to the amount of consumed NADH. Initial velocities, \u003cem\u003ev\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e, at different substrate concentrations, [S] were fitted to the Michaelis-Menten equation shown in Eq.\u0026nbsp;1\u003c/p\u003e\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\"\u003e\u003c/p\u003e \u003cp\u003eWhere \u003cem\u003eK\u003c/em\u003e\u003csub\u003eM\u003c/sub\u003e is the Michaelis-Menten constant and \u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e is the maximum velocity of the reaction.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eCharacterization of the growth and conversion of vanillate and syringate by\u003c/b\u003e \u003cb\u003evanAB\u003c/b\u003e \u003cb\u003eexpressing strains\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe first investigated the \u003cem\u003eO-\u003c/em\u003edemethylation activity of ADP1 VanAB by native and non-native expression systems. First, the native copy of \u003cem\u003evanAB\u003c/em\u003e was deleted from the genome resulting in strain ASA1002. Two plasmid-based overexpression systems were transformed into ASA1002, resulting in strains ASA1003 and ASA1004 with constitutive P\u003csub\u003eT5\u003c/sub\u003e-\u003cem\u003evanAB\u003c/em\u003e and cyclohexanone-inducible P\u003csub\u003echn\u003c/sub\u003e-\u003cem\u003evanAB\u003c/em\u003e, respectively. The strains were cultivated with vanillate, the known substrate for VanAB, as the sole carbon source (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Overexpression of \u003cem\u003evanAB\u003c/em\u003e resulted in nearly 10-hour lag phase, while for ADP1 WT noticeable lag phase was not observed. Interestingly, for the uninduced strain, the lag-phase was slightly shorter compared to overexpressing strains. A low expression level of \u003cem\u003evanAB\u003c/em\u003e can be expected due to leakiness of the \u003cem\u003echnR\u003c/em\u003e/P\u003csub\u003eChnB\u003c/sub\u003e (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). As expected, ASA1002 with \u003cem\u003evanAB\u003c/em\u003e deletion did not grow on vanillate.\u003c/p\u003e\u003cp\u003eNext, the syringate tolerance of ADP1 WT and the engineered strains was determined (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The media was supplemented with 50 mM glucose and syringate in concentrations ranging from 0 to 20 mM. At the highest concentration of syringate tested (20 mM), strains ADP1 WT, ASA1002, and ASA1003 had approximately a 6-hour lag phase, whereas the induced ASA1004 strain had even a longer lag phase, approximately 12 hours. In all strains, syringate negatively impacted growth in comparison to cultivations with only glucose, and the growth of induced ASA1004 was hindered even at low syringate concentrations. By contrast, for ASA1002, only the highest concentrations of syringate resulted in clearly reduced overall growth. Close to the end of the cultivation, a medium color change from clear to black was noticed in cultures of ADP1 WT and ASA1003 (Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, Additional File 1).\u003c/p\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eSyringate and 3MGA conversion by VanAB in wild-type and engineered ADP1\u003c/h2\u003e \u003cp\u003eBased on the tolerance test, 5 mM concentration was selected for studying syringate conversion in ADP1 \u003cem\u003ein vivo\u003c/em\u003e. We first tested the conversion in ADP1 WT and compared the performance to that of \u003cem\u003eP. putida\u003c/em\u003e KT2440, which has been previously shown to \u003cem\u003eO-\u003c/em\u003edemethylate syringate in the presence of an auxiliary carbon source, resulting in the accumulation of 3MGA (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). The strains were cultivated in MSM supplemented with 50 mM glucose, 0.2% casein amino acids, and 5 mM syringate (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Both syringate and 3MGA were completely \u003cem\u003eO-\u003c/em\u003edemethylated by ADP1 WT within 24 hours. Only small amount of syringate was converted to 3MGA by \u003cem\u003eP. putida\u003c/em\u003e, being in line with previous research (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWe then investigated the syringate conversion by ASA1003 and ASA1004. ASA1002 was used as the control. The strains were cultivated in MSM supplemented with 50 mM glucose, 0.2% casein amino acids, and 5 mM syringate and appropriate antibiotics (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Interestingly, the strains ASA1003 and ASA1004, which overexpress \u003cem\u003evanAB\u003c/em\u003e, exhibited different reaction dynamics compared to ADP1 WT. In these strains, syringate and 3MGA were \u003cem\u003eO-\u003c/em\u003edemethylated simultaneously, unlike in ADP1 WT, where syringate was depleted first. Notably, the induction of ASA1004 caused an over 8-hour lag phase, resulting in much slower overall conversion of syringate. A very minor decrease in syringate concentration was observed in cultivations with ASA1002, likely due to abiotic degradation. These results indicate that VanAB is indeed responsible for syringate conversion.\u003c/p\u003e \u003cp\u003eTo screen for the optimal induction level in ASA1004, different cyclohexanone concentrations up to 5 \u0026micro;M were tested (Supplementary Figure S2, Additional File 1). We found that very low expression levels of \u003cem\u003evanAB\u003c/em\u003e, even without induction, are sufficient for the conversion, whereas too high expression almost completely inhibits growth (Supplementary Figure S3, Additional File 1). Again, dark coloration was observed after approximately 24 hours of cultivation most likely caused by the abiotic oxidation of gallate, which results in a dark gray or black coloration and precipitate (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan additionalcitationids=\"CR52\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo confirm that the \u003cem\u003eO-\u003c/em\u003edemethylation of 3MGA is also carried out by VanAB, we cultivated ADP1 WT and ASA1002 in media supplemented with 3MGA instead of syringate (Supplementary Figure S4, Additional File 1). As expected, ADP1 WT promptly \u003cem\u003eO-\u003c/em\u003edemethylated 3MGA to gallate, while no conversion above the abiotic rate was detected for ASA1002. To prospect for the possibility of unknown enzymes capable of syringate conversion or further syringate metabolism that could enable growth, ADP1 WT, ASA1002, and ASA1004 were cultivated with syringate as the sole carbon source (Supplementary Figure S5, Additional File 1). Minor conversion of syringate into 3MGA was observed, but neither further conversion to gallate nor growth was detected. The findings indicate that syringate and 3MGA \u003cem\u003eO-\u003c/em\u003edemethylation in ADP1 is exclusively carried out by VanAB.\u003c/p\u003e \u003cp\u003e \u003cb\u003eVanAB activity\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the substrate preference of VanAB, the enzymes were produced and co-purified for enzymatic activity assay. The purified protein preparation had a red-brown color, which is characteristic of Rieske-type oxygenases such as VanA (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e). The activity of VanAB towards different substrates was assayed based on monitoring the consumption of NADH. The results indicated a clear coupling of NADH consumption by VanAB in the presence of vanillate or syringate in the reaction, as opposed to the minimal consumption of NADH in the presence of PCA or without substrate (Supplementary Figure S6, Additional File 1). The saturation curves for VanAB with vanillate and syringate as a substrate are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. Using vanillate and syringate as substrates, the Michaelis-Menten constants (\u003cem\u003eK\u003c/em\u003e\u003csub\u003eM\u003c/sub\u003e) were found to be 42\u0026thinsp;\u0026plusmn;\u0026thinsp;15 \u0026micro;M and 38\u0026thinsp;\u0026plusmn;\u0026thinsp;9 \u0026micro;M, respectively. These results align with previous studies on homologous enzyme from \u003cem\u003eP. putida\u003c/em\u003e KT2440 (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Interestingly, syringate appears to be as good a substrate as vanillate for the VanAB of ADP1. However, we were unable to detect \u003cem\u003ein vitro\u003c/em\u003e activity when 3MGA was used as a substrate under the studied conditions, presumably because of the much lower catalytic ability of VanAB with 3MGA. Nevertheless, VanAB-mediated \u003cem\u003eO-\u003c/em\u003edemethylation of 3MGA was observed in the \u003cem\u003ein vivo\u003c/em\u003e cultivations (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Supplementary Figure S4, Additional File 1).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eProduction of PDC in ADP1\u003c/h2\u003e \u003cp\u003eThe robust conversion of syringate by VanAB encouraged us to further explore potential production pathways. PDC is an exciting product of aromatic catabolism, as several lignin-derived aromatics can be simultaneously funnelled towards its production. In the VanAB-mediated pathway, there are two known routes to PDC from syringate: through 3MGA and gallate (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Protocatechuate dioxygenase PcaHG of \u003cem\u003eP. putida\u003c/em\u003e KT2440 has been reported to convert gallate into PDC (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). To test the corresponding activity of the homologous PcaHG in ADP1, we first cultivated ADP1 WT and a control strain with \u003cem\u003epcaHG\u003c/em\u003e deletion in media supplemented with 5 mM syringate and 50 mM glucose. Small amounts of PDC were detected from ADP1 WT cultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), indicating potential, albeit minor activity of PcaHG towards gallate.\u003c/p\u003e \u003cp\u003eGiven the high instability of gallate and the significant accumulation of 3MGA in the media during ADP1 WT cultivations before it is further \u003cem\u003eO-\u003c/em\u003edemethylated to gallate, we opted to establish a PDC production pathway directly from 3MGA. Using 3MGA as a product precursor instead of gallate also eliminates the need for the second \u003cem\u003eO\u003c/em\u003e-demethylation step. To that end, we expressed a gallate dioxygenase, \u003cem\u003egalA\u003c/em\u003e, from \u003cem\u003eP. putida\u003c/em\u003e KT2440 in ADP1. GalA is known to convert 3MGA into 4-carboxy-2-hydroxy-6-methyoxy-6-oxohexa-2,4-dienoate (CHMOD) which is non-enzymatically transformed into PDC (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). We cloned \u003cem\u003egalA\u003c/em\u003e into pKLxR5 backbone under P\u003csub\u003eLUXB\u003c/sub\u003e promoter, and the expression plasmid was transformed into ADP1 WT, resulting in the strain ASA1005. In addition to the activity of GalA, small amounts of gallate may also be converted into PDC by the native PcaHG. Next, we compared ASA1005 expressing \u003cem\u003egalA\u003c/em\u003e with and without induction to ADP1 WT in the same conditions. With induction, ASA1005 produced PDC from syringate with a molar yield of 38% (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eFollowing the successful conversion of syringate into PDC via plasmid expression, we constructed a strain with genomic \u003cem\u003egalA\u003c/em\u003e expression system to ensure stable and consistent expression, resulting in the strain designated as ASA1006.\u003c/p\u003e \u003cp\u003eTo characterize the LuxR mediated expression in ADP1 genome, a strain with \u003cem\u003emRFP\u003c/em\u003e expression was constructed. The expression with the tested inducer concentrations ranging from 1\u0026ndash;100 \u0026micro;M AHL was even stronger compared to the plasmid-based expression (Supplementary Figure S7, Additional File 1). As high 3MGA concentrations are known to inhibit GalA (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e), it may be beneficial to have a higher expression level of \u003cem\u003egalA\u003c/em\u003e in relation to \u003cem\u003evanAB\u003c/em\u003e. Thus, 10 \u0026micro;M AHL concentration was chosen for the induction in the subsequent experiments.\u003c/p\u003e \u003cp\u003eWe hypothesized that in a bioreactor, syringate concentration could be maintained at a defined level for 3MGA to keep accumulating instead of being converted into gallate. In addition, glucose supplementation supporting the primary metabolism could enhance the demethylation reactions. The initial media in the bioreactor was supplemented with 2 mM syringate and 20 mM glucose and 10 \u0026micro;M AHL. The feed contained otherwise same components as the initial media with exception of 15 mM syringate and 75 mM glucose resulting in feeding rates of 0.058 mmol/h and 0.29 mmol/h, respectively. Despite continuous feeding, syringate was consumed rapidly, after which 3MGA was converted into gallate. The decrease in gallate concentration in the media seemed to proceed at higher rate than what could be expected from abiotic degradation or dilution caused by feeding. GalA can also oxidise gallate into 4-OMAmesaconate (OMA), which was not analysed from the samples, but this reaction is a plausible explanation for the decrease in gallate concentration. Accumulation of gallate did not completely inhibit the production of PDC, although the amount remained low at 9.2\u0026ndash;10.8 mg/l (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eADP1 has previously shown great promise for the utilization of lignin-related aromatic compounds (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan additionalcitationids=\"CR33 CR34 CR35 CR36\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). However, \u003cem\u003eO-\u003c/em\u003edemethylation of S-lignin derived aromatics by ADP1 has not been detected until now. In this study, we investigated the reactions carried out by VanAB, a promiscuous vanillate \u003cem\u003eO-\u003c/em\u003edemethylase. Other VanAB homologs can \u003cem\u003eO-\u003c/em\u003edemethylate many S-lignin derived aromatics, as has been demonstrated in \u003cem\u003ePseudomonas sp.\u003c/em\u003e HR199 (syringate) (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e), \u003cem\u003eStreptomyces sp\u003c/em\u003e. NL15-2K (syringate and 3MGA) (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e) and most recently in \u003cem\u003eP. putida\u003c/em\u003e KT2440 (syringate and 3MGA in the presence of an auxiliary carbon source) (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). In this context, more extensive study of VanAB of ADP1 seemed warranted.\u003c/p\u003e \u003cp\u003eFirst, we examined how non-native expression of \u003cem\u003evanAB\u003c/em\u003e affects the growth of ADP1 on vanillate, a native carbon source of ADP1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). As expected, the deletion of \u003cem\u003evanAB\u003c/em\u003e (strain ASA1002) prevented growth on vanillate as a sole carbon source. The strains overexpressing \u003cem\u003evanAB\u003c/em\u003e had significantly longer lag phases compared to ADP1 WT, and based on OD, the final biomasses were also slightly lower. Of the engineered strains, the uninduced ASA1004 strain performed the best, supporting the previous findings (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e) that low expression level of \u003cem\u003evanAB\u003c/em\u003e is beneficial in terms of growth. The negative effects of \u003cem\u003evanAB\u003c/em\u003e overexpression on the growth can be explained by the increased NAD(P)H consumption and the rapid generation of formaldehyde formed in the \u003cem\u003eO\u003c/em\u003e-demethylation reaction. In \u003cem\u003eP. putida\u003c/em\u003e KT2440, providing additional carbon and energy source for the cells significantly improved growth on syringate with strains overexpressing \u003cem\u003evanAB\u003c/em\u003e, likely due to increased supply of cofactors (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). In addition, overexpression of \u003cem\u003evanAB per se\u003c/em\u003e can potentially have negative impact on growth, although such effect was not observed in ADP1 while producing VanAB for \u003cem\u003ein vitro\u003c/em\u003e studies, when specific substrate for VanAB was not available.\u003c/p\u003e \u003cp\u003eIn the ADP1 WT, \u003cem\u003evanAB\u003c/em\u003e is repressed by a GntR-type transcriptional regulator VanR, which typically causes a 3\u0026ndash;4-hour lag-phase preceding vanillate conversion (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e). Overexpression of \u003cem\u003evanAB\u003c/em\u003e in strains ASA1003 and ASA1004 resulted in approximately 10-hour long lag-phase, despite that the expression is not under the regulation of VanR. Thus, the longer lag phase is potentially related to the redox-imbalance and toxicity of formaldehyde, as discussed above.\u003c/p\u003e \u003cp\u003eNext, we explored syringate tolerance of the ADP1 strains. 20 mM of syringate caused an extended lag-phase in all strains. In concentrations 1\u0026ndash;10 mM, the expression level of VanAB seemed to correlate with syringate tolerance and its effect on growth: For ASA1002 with \u003cem\u003evanAB\u003c/em\u003e deletion, the impact of syringate on the growth was more clearly dependent on the concentration, whereas with ADP1 WT and uninduced ASA1004, the inhibitory effect of syringate was similar in all concentrations. In contrast, when \u003cem\u003evanAB\u003c/em\u003e was overexpressed in ASA1004, syringate had a drastic effect on growth even at low concentrations. The growth of the induced ASA1004 was potentially hindered by the faster \u003cem\u003eO-\u003c/em\u003edemethylation of syringate, similarly to what was observed in vanillate cultivations.\u003c/p\u003e \u003cp\u003eRegardless, all the strains were able to grow in the concentrations tested. Tolerance to even higher concentrations could be further explored and improved in the future, for example by adaptive laboratory evolution, as we have previously demonstrated with ferulate (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Growth in up to 120 mM syringate has been reported for \u003cem\u003eP. putida\u003c/em\u003e KT2440, with overexpression of VanAB increasing tolerance (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). However, in ADP1, gallate is not further metabolized and its accumulation potentially adds to the toxicity of syringate.\u003c/p\u003e \u003cp\u003eTo explore the reactions carried out by natively expressed VanAB on non-native carbon sources, we set up cultivations of ADP1 WT and \u003cem\u003eP. putida\u003c/em\u003e KT2440 with 5 mM syringate and 50 mM glucose supplementations. We found that syringate is quickly \u003cem\u003eO-\u003c/em\u003edemethylated into 3MGA and then into gallate by ADP1, in contrast to previous research (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Experimental methods could explain the contradiction to previous VanAB characterization in ADP1; In the paper of Morawski et al. (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e), VanAB activity was measured from washed cell suspensions with the tested VanAB substrates as sole carbon sources. In this setup, for example the inability to refill NAD(P)H reservoirs could significantly reduce reaction capacity (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). In our experiments, we used glucose as an additional carbon source to provide carbon and energy for growth and for replenishing NAD(P)H.\u003c/p\u003e \u003cp\u003eWe detected high amounts of 3MGA in the ADP1 WT culture media prior to its further conversion to gallate. The demonstrated dynamic conversion, in which syringate is \u003cem\u003eO-\u003c/em\u003edemethylated first is consistent with previous findings that suggest 3MGA is less preferred substrate for VanAB (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). In \u003cem\u003eP. putida\u003c/em\u003e KT2440, syringate conversion was much slower than in ADP1 and 3MGA was not \u003cem\u003eO-\u003c/em\u003edemethylated at all, being in line with the previous study (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). However, when vanillate was provided instead of glucose for \u003cem\u003eP. putida\u003c/em\u003e KT2440, a higher syringate conversion rate was achieved, and with continuous vanillate feeding, both syringate and 3MGA were consumed completely (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Thus, in \u003cem\u003eP. putida\u003c/em\u003e KT2440, syringate conversion could be affected by glucose-induced carbon catabolite repression. For ADP1, neither glucose nor gluconate have been observed to repress the utilization of aromatic compounds (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAs previously mentioned, VanR is a negative transcriptional regulator that represses the expression of \u003cem\u003eVanAB\u003c/em\u003e in the absence of an inducer, namely vanillate. (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e) Interestingly, there are two variants of \u003cem\u003evanR\u003c/em\u003e found in ADP1, namely O24839 (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e) and Q6FDI8 (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e), that differ in length and in their orientation in respect to \u003cem\u003evanB\u003c/em\u003e. The differences may be result of genetic scrambling caused by a Tn5613 transposon located nearby (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e). In O24839, \u003cem\u003evanR\u003c/em\u003e and \u003cem\u003evanB\u003c/em\u003e overlap, whereas in Q6FDI8 \u003cem\u003evanR\u003c/em\u003e and \u003cem\u003evanB\u003c/em\u003e are separated by 46 bp and \u003cem\u003evanR\u003c/em\u003e is 23 amino acids shorter. The strains used in this study contain the variant Q6FDI8, while \u003cem\u003evanAB\u003c/em\u003e repression has been previously studied with strain containing the variant O24839 (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e). In our experiments, syringate was \u003cem\u003eO-\u003c/em\u003edemethylated by VanAB robustly without induction by vanillate in ADP1 WT. Therefore, it appears that syringate can serve as an inducer for \u003cem\u003evanAB\u003c/em\u003e expression in ADP1. Amino acid sequence identity of VanR in ADP1 and \u003cem\u003eP. putida\u003c/em\u003e KT2440 is 48% for variant Q6FDI8 and 49% for the variant O24839 used in the study (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e) where VanR was previously characterized. Difference in the protein structures or potentially even the different gene organization of the \u003cem\u003evan\u003c/em\u003e region described earlier could affect VanR binding to DNA or VanAB substrates and partially explain the differences in performance between \u003cem\u003eP. putida\u003c/em\u003e and ADP1.\u003c/p\u003e \u003cp\u003eUptake and conversion of the compounds are also affected by specific transporters and porins (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e). In ADP1, aromatic compounds are transported by four known acid:H\u0026thinsp;+\u0026thinsp;symporters, VanK, PcaK, BenK and MucK (\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e). VanK, a vanillate transporter and PcaK, a 4-hydroxybenzoate transporter are both promiscuous and overlap in activity, meaning they could potentially transport S-lignin derived aromatics as well (\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e). Porins might also facilitate uptake of syringate, 3MGA, or gallate: for example, VanP and HcaE are involved in vanillate transport (\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e). The robust conversion of syringate into 3MGA in ADP1 suggests sufficient transfer of the compounds into the cells. The efficient transport in ADP1 may also negatively affect the tolerance towards syringate, especially in elevated concentrations. Aromatic compounds can also cross the cell membrane passively.\u003c/p\u003e \u003cp\u003eNext, we investigated syringate conversion by the strains overexpressing \u003cem\u003evanAB\u003c/em\u003e along with ASA1002 with \u003cem\u003evanAB\u003c/em\u003e deletion as a control. We found that overexpression enables simultaneous conversion of syringate and 3MGA, but for overall efficiency the lower expression level is better. As discussed above, the reason for this is likely that the \u003cem\u003eO-\u003c/em\u003edemethylation reaction catalyzed by VanAB produces formaldehyde as a side product, which has been previously suggested to cause a redox imbalance and energy limitation in \u003cem\u003eP. putida\u003c/em\u003e KT2440 (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). A conserved route for detoxification of formaldehyde utilizes a glutathione-dependent formaldehyde dehydrogenase (FrmA) and a S-formylglutathione hydrolase (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). In recombinant \u003cem\u003eE. coli\u003c/em\u003e expressing \u003cem\u003evanAB\u003c/em\u003e from \u003cem\u003eP. putida\u003c/em\u003e, deletion of FrmA led to accumulation of formaldehyde and halted vanillate \u003cem\u003eO-\u003c/em\u003edemethylation (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). While the specific mechanism for eliminating formaldehyde in ADP1 has not been described, in \u003cem\u003eA. baumannii\u003c/em\u003e, a close relative of ADP1, glutathione-dependent formaldehyde dehydrogenases \u003cem\u003eadhC1\u003c/em\u003e and \u003cem\u003eadhC2\u003c/em\u003e have been characterized (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e). Interestingly, the expression of \u003cem\u003eadhC1\u003c/em\u003e in \u003cem\u003eA. baumannii\u003c/em\u003e was found to be repressed in the presence of free inorganic iron (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e). As VanAB requires iron in the iron-sulfur cluster [2Fe-2S] and in the active site, optimization might be required to balance \u003cem\u003eO-\u003c/em\u003edemethylation and formaldehyde detoxification (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Notonier et al. (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e) supplemented \u003cem\u003eP. putida\u003c/em\u003e KT2440 cultures with formate, which is oxidized in cells to generate NADH and thus improved the \u003cem\u003eO-\u003c/em\u003edemethylation of syringate. For ADP1, we found no benefit from formate supplementation (data not shown), potentially explained by the possible VanAB preference for NADPH over NADH (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). In the future, identifying how formaldehyde is detoxified in ADP1 could help to overcome the inhibitory effects caused by VanAB overexpression.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eIn addition to the \u003cem\u003ein vivo\u003c/em\u003e experiments, activity of VanAB was evaluated \u003cem\u003ein vitro.\u003c/em\u003e We were able to co-purify VanA and VanB with a N-terminal His-(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e)-tag only in VanA, suggesting strong interaction between them. By the assay setup we showed that NADH consumption can be used to indirectly measure VanAB activity. We found that syringate is nearly as preferred substrate as vanillate. However, in contrast to the \u003cem\u003ein vivo\u003c/em\u003e experiments, no activity for 3MGA was detected. \u003cem\u003eIn vitro\u003c/em\u003e activity towards 3MGA has been previously shown only for the VanAB homolog from \u003cem\u003eP. putida\u003c/em\u003e KT2440 and with clear preference for vanillate and syringate as substrates over 3MGA (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). The purification and assay conditions here may have further negatively impacted the activity, as Rieske oxygenases are known to be sensitive to oxidation (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e). Addition of sources of iron and sulfur, as well as dithiothreitol as an antioxidant have been shown to be beneficial, and systematic characterization of their effects in ADP1 could further improve the activity (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe robust and complete \u003cem\u003eO-\u003c/em\u003edemethylation of syringate and 3MGA in the \u003cem\u003ein vivo\u003c/em\u003e experiments yielded approximately equimolar amounts of gallate in ADP1. Gallate itself is an interesting product, as it can be used for example in the production of antioxidants, pharmaceuticals and antimicrobials (\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e). However, gallate is known to oxidize readily, indicated by the change of color to dark brown or black (Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, Additional File 1). Gallate can also react with media components and proteins and may form a precipitate that could be harmful for cell growth and prevent its further utilization (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e). To that end, we wanted to explore further ways to utilize the robust and dynamic \u003cem\u003eO-\u003c/em\u003edemethylation of syringate and 3MGA for production in ADP1.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eProduction of PDC from lignin related aromatics has shown a lot of promise. PDC is an enticing target product for microbial valorisation of lignin; It has promising industrial applications such as high-quality biodegradable polyesters and polyurethanes (\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e). In addition, chemical synthesis of PDC is very difficult, making it a so called bioprivileged compound (\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e). PDC production has been demonstrated from single lignin monomers including vanillate, syringate, 4-hydroxybenzoate and \u003cem\u003ep\u003c/em\u003e-coumarate and even lignin derived feedstocks. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan additionalcitationids=\"CR70 CR71 CR72 CR73 CR74\" citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e) The highest titer to date from lignin-related compounds as substrate, 99.9 g/L and productivity of 1.69 g/L/h, were achieved by engineered \u003cem\u003eSphingobium spp.\u003c/em\u003e SYK-6 with vanillate as a substrate (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDirecting 3MGA into PDC by expression of \u003cem\u003egalA\u003c/em\u003e emerged as an attractive option as it would benefit from the accumulation of 3MGA that occurs when VanAB is natively expressed in ADP1. Conversion of 3MGA directly into PDC by GalA would also be beneficial because it circumvents the second \u003cem\u003eO-\u003c/em\u003edemethylation step, reducing NAD(P)H consumption and production of toxic formaldehyde. Reported rate for this reaction has been low, in part due to substrate inhibition by 3MGA (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e), but it had not been tested in ADP1. An alternative route would be via PcaHG found both in ADP1 and \u003cem\u003eP. putida\u003c/em\u003e KT2440, in which it converts gallate into PDC. Despite we observed only modest conversion of gallate to PDC in ADP1 by PcaHG, it can potentially be exploited to support PDC production in combination with GalA: any 3MGA that is not directly converted into PDC by GalA could be salvaged by the conversion of gallate into PDC by PcaHG.\u003c/p\u003e \u003cp\u003eBy introducing \u003cem\u003egalA\u003c/em\u003e from \u003cem\u003eP. putida\u003c/em\u003e KT2440 into ADP1 we were able to demonstrate production of PDC from syringate in ADP1. The obtained yield was modest, approximately 38%. To improve PDC production, we integrated the \u003cem\u003egalA\u003c/em\u003e expression system into ADP1 genome for stable and consistent expression. Additionally, we established a bioreactor cultivation system, aiming to reduce gallate formation and improve PDC production through controlled feeding of syringate and glucose as a supporting carbon source. Indeed, we found that maintaining a high concentration of syringate in the media hindered the conversion of 3MGA to gallate. However, PDC production remained unexpectedly low due to the limited conversion of 3MGA to PDC under the studied conditions. It is possible that the conditions favored the conversion of 3MGA to gallate by VanAB over its conversion to PDC by GalA. The formed gallate, serving as a native substrate for GalA, further blocked the conversion of 3MGA to PDC; we observed a decrease in gallate concentration in the media, possibly due to its conversion to 4-oxalomesaconate (OMA) by GalA, which also represents the first reaction in gallate catabolism by the \u003cem\u003egal\u003c/em\u003e operon (\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e). Indeed, in the future, the utilization of S-lignin derived aromatics for growth could be established in ADP1 by extending the pathway from gallate and OMA towards central metabolism.\u003c/p\u003e \u003cp\u003eIn future studies, several approaches could be applied to further improve PDC production in ADP1. For example, in this study, we used a \u003cem\u003egalA\u003c/em\u003e gene directly from the genome of a GC-rich strain \u003cem\u003eP. putida\u003c/em\u003e KT2440, while a synthetic gene codon optimized specifically for ADP1 could function more optimally. In addition, protein engineering of the substrate specificity of GalA towards 3MGA could be a potential strategy for improving the conversion. Furthermore, employing alternative enzymes such as LigAB and DesZ from \u003cem\u003eSphingobium spp.\u003c/em\u003e SYK-6 which can also carry out the ring-opening reaction (\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e), should be considered.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn this study, we established the conversion and upgrading of S-lignin derived syringate in \u003cem\u003eAcinetobacter baylyi\u003c/em\u003e ADP1. We characterized the activity of a promiscuous vanillate \u003cem\u003eO-\u003c/em\u003edemethylase VanAB in ADP1, demonstrating its efficient \u003cem\u003eO-\u003c/em\u003edemethylation of both syringate and 3MGA. Overexpression of \u003cem\u003evanAB\u003c/em\u003e altered the conversion dynamics, resulting in the simultaneous conversion of 3MGA and syringate, unlike in the ADP1 WT where syringate is \u003cem\u003eO-\u003c/em\u003edemethylated first. Excessive expression levels negatively impacted cell growth, likely due to the accumulation of toxic intermediates and the high energy demand of VanAB, as previously reported. Finally, we exploited the accumulation of 3MGA caused by the native VanAB expression by introducing gallate dioxygenase GalA from \u003cem\u003eP. putida\u003c/em\u003e KT2440 to produce PDC, a bioprivileged plastic precursor. Our study expands the aromatic substrate range of ADP1 to include S-lignin derived aromatics for production and further promotes the use of this host for biological upgrading of lignin-derived compounds.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eADP1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003eAcinetobacter baylyi\u003c/em\u003e ADP1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e3MGA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e3-O-methylgallate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAHL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eN-(3-oxohexanoyl) homoserine lactone\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCyc\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecyclohexanone\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDTT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edithiothreitol\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003egallate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003elysogeny agar\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003elysogeny broth\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMSM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emineral salts medium\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNAD(P)H\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003enicotinamide adenine dinucleotide (phosphate)\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNMR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003csup\u003e1\u003c/sup\u003eH nuclear magnetic resonance\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eOD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eoptical density\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eP450\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCytochrome P450\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePCR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePolymerase chain reaction\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePDA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ephotodiode array detector\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePDC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e2-pyrone-4,6-dicarboxylic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRieske oxygenase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esyringate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSDS-PAGE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSodium Dodecyl Sulfate Polyacrylamide Gel Electroforesis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTHF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etetrahydrofolate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailibility of data and materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are included within the article\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eor the additional files. The corresponding author is willing to provide the raw\u0026nbsp;\u003c/p\u003e\n\u003cp\u003edata related to this manuscript upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSS would like to thank the Novo Nordisk Foundation (grant NNF21OC0067758) and the Re-search Council of Finland (grant no. 347204 and 353587).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHT, JK, and SS designed the study. HT and JK carried out the experimental research work. HT, JK, and SS analysed the data. EE and HT conducted the substrate and metabolite analyses. SS supervised the study and acquired funding. All authors participated in writing and reviewing the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBugg TDH, Williamson JJ, Alberti F. Microbial hosts for metabolic engineering of lignin bioconversion to renewable chemicals. Renew Sustain Energy Rev. 2021;152:111674.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYaguchi AL, Lee SJ, Blenner MA. Synthetic Biology towards Engineering Microbial Lignin Biotransformation. Trends Biotechnol. 2021;39(10):1037\u0026ndash;64.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKohlstedt M, Weimer A, Weiland F, Stolzenberger J, Selzer M, Sanz M, et al. Biobased PET from lignin using an engineered cis, cis-muconate-producing Pseudomonas putida strain with superior robustness, energy and redox properties. Metab Eng. 2022;72:337\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSonoki T, Takahashi K, Sugita H, Hatamura M, Azuma Y, Sato T, et al. Glucose-Free \u003cem\u003ecis\u003c/em\u003e, \u003cem\u003ecis\u003c/em\u003e -Muconic Acid Production via New Metabolic Designs Corresponding to the Heterogeneity of Lignin. ACS Sustain Chem Eng. 2018;6(1):1256\u0026ndash;64.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNiu W, Willett H, Mueller J, He X, Kramer L, Ma B, et al. Direct biosynthesis of adipic acid from lignin-derived aromatics using engineered Pseudomonas putida KT2440. Metab Eng. 2020;59:151\u0026ndash;61.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao M, Huang D, Zhang X, Koffas MAG, Zhou J, Deng Y. Metabolic engineering of Escherichia coli for producing adipic acid through the reverse adipate-degradation pathway. Metab Eng. 2018;47:254\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCai C, Xu Z, Zhou H, Chen S, Jin M. Valorization of lignin components into gallate by integrated biological hydroxylation, O-demethylation, and aryl side-chain oxidation. Sci Adv. 2021;7(36):eabg4585.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDias FMS, Pantoja RK, Gomez JGC, Silva LF. From degrader to producer: reversing the gallic acid metabolism of Pseudomonas putida KT2440. Int Microbiol. 2022;26(2):243\u0026ndash;55.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOtsuka Y, Araki T, Suzuki Y, Nakamura M, Kamimura N, Masai E. High-level production of 2-pyrone-4,6-dicarboxylic acid from vanillic acid as a lignin-related aromatic compound by metabolically engineered fermentation to realize industrial valorization processes of lignin. Bioresour Technol. 2023;377:128956.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee S, Jung YJ, Park SJ, Ryu MH, Kim JE, Song HM et al. Microbial production of 2-pyrone-4,6-dicarboxylic acid from lignin derivatives in an engineered Pseudomonas putida and its application for the synthesis of bio-based polyester. Bioresour Technol. 2022;352.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJin Y, Joshi M, Araki T, Kamimura N, Masai E, Nakamura M, et al. Click Synthesis of Triazole Polymers Based on Lignin-Derived Metabolic Intermediate and Their Strong Adhesive Properties to Cu Plate. Polymers. 2023;15(6):1349.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKang MJ, Kim HT, Lee MW, Kim KA, Khang TU, Song HM, et al. A chemo-microbial hybrid process for the production of 2-pyrone-4,6-dicarboxylic acid as a promising bioplastic monomer from PET waste. Green Chem. 2020;22(11):3461\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMichinobu T, Hishida M, Sato M, Katayama Y, Masai E, Nakamura M, et al. Polyesters of 2-Pyrone-4,6-Dicarboxylic Acid (PDC) Obtained from a Metabolic Intermediate of Lignin. Polym J. 2008;40(1):68\u0026ndash;75.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eObrzut N, Hickmott R, Shure L, Gray KA. The effects of lignin source and extraction on the composition and properties of biorefined depolymerization products. RSC Sustain. 2023;1(9):2328\u0026ndash;40.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eConstant S, Wienk HLJ, Frissen AE, Peinder PD, Boelens R, Van Es DS, et al. New insights into the structure and composition of technical lignins: a comparative characterisation study. Green Chem. 2016;18(9):2651\u0026ndash;65.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi W, Wang Y, Tan X, Miao C, Ragauskas AJ, Zhuang X. Biocompatible organosolv fractionation via a novel alkaline lignin-first strategy towards lignocellulose valorization. Chem Eng J. 2024;481:148695.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eErickson E, Bleem A, Kuatsjah E, Werner AZ, DuBois JL, McGeehan JE, et al. Critical enzyme reactions in aromatic catabolism for microbial lignin conversion. Nat Catal. 2022;5(2):86\u0026ndash;98.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMasai E, Sasaki M, Minakawa Y, Abe T, Sonoki T, Miyauchi K, et al. A Novel Tetrahydrofolate-Dependent O-Demethylase Gene Is Essential for Growth of Sphingomonas paucimobilis SYK-6 with Syringate. J Bacteriol. 2004;186(9):2757\u0026ndash;65.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee JA, Stolyar S, Marx CJ. Aerobic Methoxydotrophy: Growth on Methoxylated Aromatic Compounds by Methylobacteriaceae. Front Microbiol. 2022;13:849573.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHibi M, Sonoki T, Mori H. Functional coupling between vanillate- \u003cem\u003eO\u003c/em\u003e -demethylase and formaldehyde detoxification pathway. FEMS Microbiol Lett. 2005;253(2):237\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNotonier S, Werner AZ, Kuatsjah E, Dumalo L, Abraham PE, Hatmaker EA, et al. Metabolism of syringyl lignin-derived compounds in Pseudomonas putida enables convergent production of 2-pyrone-4,6-dicarboxylic acid. Metab Eng. 2021;65(C):111\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKovaleva EG, Lipscomb JD. Versatility of biological non-heme Fe(II) centers in oxygen activation reactions. Nat Chem Biol. 2008;4(3):186\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLanfranchi E, Trajković M, Barta K, de Vries JG, Janssen DB. Exploring the Selective Demethylation of Aryl Methyl Ethers with a \u003cem\u003ePseudomonas\u003c/em\u003e Rieske Monooxygenase. ChemBioChem. 2019;20(1):118\u0026ndash;25.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMueller J, Willett H, Feist AM, Niu W. Engineering Pseudomonas putida for improved utilization of syringyl aromatics. Biotechnol Bioeng. 2022;119(9):2541\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNishimura M, Nishimura Y, Abe C, Kohhata M. Expression and Substrate Range of \u003cem\u003eStreptomyces\u003c/em\u003e Vanillate Demethylase. Biol Pharm Bull. 2014;37(9):1564\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorawski B, Segura A, Ornston N. Substrate Range and Genetic Analysis of Acinetobacter Vanillate Demethylase. J Bacteriol. 2000;182(5):1383\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBedore SR, Neidle EL, Pardo I, Luo J, Baugh AC, Duscent-Maitland CV et al. Natural transformation as a tool in Acinetobacter baylyi: Streamlined engineering and mutational analysis. In: Methods in Microbiology [Internet]. Elsevier; 2023 [cited 2024 Sep 26]. pp. 207\u0026ndash;34. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://linkinghub.elsevier.com/retrieve/pii/S0580951723000028\u003c/span\u003e\u003cspan address=\"https://linkinghub.elsevier.com/retrieve/pii/S0580951723000028\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePardo I, Bedore SR, Tumen-Velasquez MP, Duscent-Maitland CV, Baugh AC, Santala S et al. Natural transformation as a tool in Acinetobacter baylyi: Evolution by amplification of gene copy number. In: Methods in Microbiology [Internet]. Elsevier; 2023 [cited 2024 Sep 26]. pp. 183\u0026ndash;205. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://linkinghub.elsevier.com/retrieve/pii/S0580951723000016\u003c/span\u003e\u003cspan address=\"https://linkinghub.elsevier.com/retrieve/pii/S0580951723000016\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSantala S. \u003cem\u003eAcinetobacter baylyi\u003c/em\u003e ADP1\u0026mdash;naturally competent for synthetic biology. Essays Biochem. 2021;65(2):309\u0026ndash;18.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSalmela M, Lehtinen T, Efimova E, Santala S, Santala V. Towards bioproduction of poly-α-olefins from lignocellulose. Green Chem. 2020;22(15):5067\u0026ndash;76.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSalvach\u0026uacute;a D, Karp EM, Nimlos CT, Vardon DR, Beckham GT. Towards lignin consolidated bioprocessing: simultaneous lignin depolymerization and product generation by bacteria. Green Chem. 2015;17(11):4951\u0026ndash;67.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSalmela M, Lehtinen T, Efimova E, Santala S, Santala V. Alkane and wax ester production from lignin-related aromatic compounds. Biotechnol Bioeng. 2019;116(8):1934\u0026ndash;45.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLuo J, Lehtinen T, Efimova E, Santala V, Santala S. Synthetic metabolic pathway for the production of 1-alkenes from lignin-derived molecules. Microb Cell Factories. 2019;18(1):48.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeril\u0026auml;inen E, Efimova E, Santala V, Santala S. Carbon-wise utilization of lignin-related compounds by synergistically employing anaerobic and aerobic bacteria. Biotechnol Biofuels Bioprod. 2024;17(1):78.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArvay E, Biggs BW, Guerrero L, Jiang V, Tyo K. Engineering Acinetobacter baylyi ADP1 for mevalonate production from lignin-derived aromatic compounds. Metab Eng Commun. 2021;13:e00173.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKurnia K, Efimova E, Santala V, Santala S. Metabolic engineering of Acinetobacter baylyi ADP1 for naringenin production. Metab Eng Commun. 2024;19:e00249.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBiggs BW, Tyo KEJ. Aromatic natural products synthesis from aromatic lignin monomers using \u003cem\u003eAcinetobacter baylyi\u003c/em\u003e ADP1 [Internet]. 2023 [cited 2024 Sep 26]. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://biorxiv.org/lookup/doi/\u003c/span\u003e\u003cspan address=\"http://biorxiv.org/lookup/doi/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1101/2023.08.24.554694\u003c/span\u003e\u003cspan address=\"10.1101/2023.08.24.554694\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLuo J, McIntyre EA, Bedore SR, Santala V, Neidle EL, Santala S. Characterization of Highly Ferulate-Tolerant Acinetobacter baylyi ADP1 Isolates by a Rapid Reverse Engineering Method. Appl Environ Microbiol. 2022;88(2):e01780\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKannisto MS, Mangayil RK, Shrivastava-Bhattacharya A, Pletschke BI, Karp MT, Santala VP. Metabolic engineering of Acinetobacter baylyi ADP1 for removal of Clostridium butyricum growth inhibitors produced from lignocellulosic hydrolysates. Biotechnol Biofuels. 2015;8(1):198.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu C, Choi B, Efimova E, Nyg\u0026aring;rd Y, Santala S. Enhanced upgrading of lignocellulosic substrates by coculture of Saccharomyces cerevisiae and Acinetobacter baylyi ADP1. Biotechnol Biofuels Bioprod. 2024;17(1):61.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSingh A, Bedore SR, Sharma NK, Lee SA, Eiteman MA, Neidle EL. Removal of aromatic inhibitors produced from lignocellulosic hydrolysates by Acinetobacter baylyi ADP1 with formation of ethanol by Kluyveromyces marxianus. Biotechnol Biofuels. 2019;12(1):91.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu C, Efimova E, Santala V, Santala S. Analysis of detoxification kinetics and end products of furan aldehydes in Acinetobacter baylyi ADP1. Sci Rep. 2024;14(1):29678.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSantala S, Efimova E, Kivinen V, Larjo A, Aho T, Karp M, et al. Improved Triacylglycerol Production in Acinetobacter baylyi ADP1 by Metabolic Engineering. Microb Cell Factories. 2011;10(1):36.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMetzgar D. Acinetobacter sp. ADP1: an ideal model organism for genetic analysis and genome engineering. Nucleic Acids Res. 2004;32(19):5780\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDe Berardinis V, Vallenet D, Castelli V, Besnard M, Pinet A, Cruaud C, et al. A complete collection of single-gene deletion mutants of \u003cem\u003eAcinetobacter baylyi\u003c/em\u003e ADP1. Mol Syst Biol. 2008;4(1):174.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSantala S, Karp M, Santala V. Rationally Engineered Synthetic Coculture for Improved Biomass and Product Formation. PLoS ONE. 2014;9(12):e113786.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchuster LA, Reisch CR. A plasmid toolbox for controlled gene expression across the Proteobacteria. Nucleic Acids Res. 2021;49(12):7189\u0026ndash;202.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSantala S, Efimova E, Santala V. Dynamic decoupling of biomass and wax ester biosynthesis in Acinetobacter baylyi by an autonomously regulated switch. Metab Eng Commun. 2018;7:e00078.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLaemmli UK. Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4. Nature. 1970;227(5259):680\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLuo J, Efimova E, Volke DC, Santala V, Santala S. Engineering cell morphology by CRISPR interference in \u003cem\u003eAcinetobacter baylyi\u003c/em\u003e ADP1. Microb Biotechnol. 2022;15(11):2800\u0026ndash;18.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKutraite I, Malys N. Development and Application of Whole-Cell Biosensors for the Detection of Gallic Acid. ACS Synth Biol. 2023;12(2):533\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNogales J, Canales \u0026Aacute;, Jim\u0026eacute;nez-Barbero J, Serra B, Pingarr\u0026oacute;n JM, Garc\u0026iacute;a JL, et al. Unravelling the gallic acid degradation pathway in bacteria: the \u003cem\u003egal\u003c/em\u003e cluster from \u003cem\u003ePseudomonas putida\u003c/em\u003e. Mol Microbiol. 2011;79(2):359\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVona D, Buscemi G, Ragni R, Cantore M, Cicco SR, Farinola GM, et al. Synthesis of (poly)gallic acid in a bacterial growth medium. MRS Adv. 2020;5(18\u0026ndash;19):957\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eD\u0026rsquo;Ordine RL, Rydel TJ, Storek MJ, Sturman EJ, Moshiri F, Bartlett RK, et al. Dicamba monooxygenase: structural insights into a dynamic Rieske oxygenase that catalyzes an exocyclic monooxygenation. J Mol Biol. 2009;392(2):481\u0026ndash;97.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorawski B, Segura A, Ornston LN. Repression of \u003cem\u003eAcinetobacter\u003c/em\u003e vanillate demethylase synthesis by VanR, a member of the GntR family of transcriptional regulators. FEMS Microbiol Lett. 2000;187(1):65\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTropel D, Van Der Meer JR. Bacterial Transcriptional Regulators for Degradation Pathways of Aromatic Compounds. Microbiol Mol Biol Rev. 2004;68(3):474\u0026ndash;500.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSegura A, B\u0026uuml;nz PV, D\u0026rsquo;Argenio DA, Ornston LN. Genetic Analysis of a Chromosomal Region Containing \u003cem\u003evanA\u003c/em\u003e and \u003cem\u003evanB\u003c/em\u003e, Genes Required for Conversion of Either Ferulate or Vanillate to Protocatechuate in \u003cem\u003eAcinetobacter\u003c/em\u003e. J Bacteriol. 1999;181(11):3494\u0026ndash;504.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarbe V. Unique features revealed by the genome sequence of Acinetobacter sp. ADP1, a versatile and naturally transformation competent bacterium. Nucleic Acids Res. 2004;32(19):5766\u0026ndash;79.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDal S, Steiner I, Gerischer U. Multiple operons connected with catabolism of aromatic compounds in Acinetobacter sp. strain ADP1 are under carbon catabolite repression. J Mol Microbiol Biotechnol. 2002;4(4):389\u0026ndash;404.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePernstich C, Senior L, MacInnes KA, Forsaith M, Curnow P. Expression, purification and reconstitution of the 4-hydroxybenzoate transporter PcaK from Acinetobacter sp. ADP1. Protein Expr Purif. 2014;101:68\u0026ndash;75.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eD\u0026rsquo;Argenio DA, Segura A, Coco WM, B\u0026uuml;nz PV, Ornston LN. The Physiological Contribution of \u003cem\u003eAcinetobacter\u003c/em\u003e PcaK, a Transport System That Acts upon Protocatechuate, Can Be Masked by the Overlapping Specificity of VanK. J Bacteriol. 1999;181(11):3505\u0026ndash;15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBleichrodt FS, Fischer R, Gerischer UC. The β-ketoadipate pathway of Acinetobacter baylyi undergoes carbon catabolite repression, cross-regulation and vertical regulation, and is affected by Crc. Microbiology. 2010;156(5):1313\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEchenique JR, Dorsey CW, Patrito LC, Petroni A, Tolmasky ME, Actis LA. Acinetobacter baumannii has two genes encoding glutathione-dependent formaldehyde dehydrogenase: evidence for differential regulation in response to iron This paper is dedicated to the memory of Dr M. A. Vides, Facultad de Ciencias Quı́micas, Universidad Nacional de C\u0026oacute;rdoba, Argentina, who was a great mentor and colleague. The GenBank accession number for the sequence reported in this paper is AF130307. Microbiology. 2001;147(10):2805\u0026ndash;15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDumitru R, Jiang WZ, Weeks DP, Wilson MA. Crystal structure of dicamba monooxygenase: a Rieske nonheme oxygenase that catalyzes oxidative demethylation. J Mol Biol. 2009;392(2):498\u0026ndash;510.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBadhani B, Sharma N, Kakkar R. Gallic acid: a versatile antioxidant with promising therapeutic and industrial applications. RSC Adv. 2015;5(35):27540\u0026ndash;57.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFernandes FHA, Salgado HRN. Gallic Acid: Review of the Methods of Determination and Quantification. Crit Rev Anal Chem. 2016;46(3):257\u0026ndash;65.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheng Y, Kuboyama K, Akasaka S, Araki T, Masai E, Nakamura M, et al. Polyurethanes based on lignin-derived metabolic intermediate with strong adhesion to metals. Polym Chem. 2022;13(48):6589\u0026ndash;98.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJin Y, Araki T, Kamimura N, Masai E, Nakamura M, Michinobu T. Biodegradable and wood adhesive polyesters based on lignin-derived 2-pyrone-4,6-dicarboxylic acid. RSC Sustain. 2024;2(7):1985\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHall BW, Kontur WS, Neri JC, Gille DM, Noguera DR, Donohue TJ. Production of carotenoids from aromatics and pretreated lignocellulosic biomass by \u003cem\u003eNovosphingobium aromaticivorans\u003c/em\u003e. Appl Environ Microbiol. 2023;89(12):e01268\u0026ndash;23.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJohnson CW, Salvach\u0026uacute;a D, Rorrer NA, Black BA, Vardon DR et al. St. John PC,. Innovative Chemicals and Materials from Bacterial Aromatic Catabolic Pathways. Joule. 2019;3(6):1523\u0026ndash;37.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePerez JM, Sener C, Misra S, Umana GE, Coplien J, Haak D, et al. Integrating lignin depolymerization with microbial funneling processes using agronomically relevant feedstocks. Green Chem. 2022;24(7):2795\u0026ndash;811.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePerez JM, Kontur WS, Gehl C, Gille DM, Ma Y, Niles AV, et al. Redundancy in Aromatic \u003cem\u003eO\u003c/em\u003e -Demethylation and Ring-Opening Reactions in \u003cem\u003eNovosphingobium aromaticivorans\u003c/em\u003e and Their Impact in the Metabolism of Plant-Derived Phenolics. Appl Environ Microbiol. 2021;87(8):e02794\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePerez JM, Kontur WS, Alherech M, Coplien J, Karlen SD, Stahl SS, et al. Funneling aromatic products of chemically depolymerized lignin into 2-pyrone-4-6-dicarboxylic acid with \u003cem\u003eNovosphingobium aromaticivorans\u003c/em\u003e. Green Chem. 2019;21(6):1340\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQian Y, Otsuka Y, Sonoki T, Mukhopadhyay B, Nakamura M, Jellison J, et al. Engineered Microbial Production of 2-Pyrone-4,6-Dicarboxylic Acid from Lignin Residues for Use as an Industrial Platform Chemical. BioResources. 2016;11(3):6097\u0026ndash;109.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSuzuki Y, Okamura-Abe Y, Otsuka Y, Araki T, Nojiri M, Kamimura N, et al. Integrated process development for grass biomass utilization through enzymatic saccharification and upgrading hydroxycinnamic acids via microbial funneling. Bioresour Technol. 2022;363:127836.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNogales J, Canales \u0026Aacute;, Jim\u0026eacute;nez-Barbero J, Garc\u0026iacute;a JL, D\u0026iacute;az E. Molecular Characterization of the Gallate Dioxygenase from Pseudomonas putida KT2440. J Biol Chem. 2005;280(42):35382\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKasai D, Masai E, Miyauchi K, Katayama Y, Fukuda M. Characterization of the 3-O-methylgallate dioxygenase gene and evidence of multiple 3-O-methylgallate catabolic pathways in Sphingomonas paucimobilis SYK-6. J Bacteriol. 2004;186(15):4951\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"microbial-cell-factories","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"micf","sideBox":"Learn more about [Microbial Cell Factories](http://microbialcellfactories.biomedcentral.com/)","snPcode":"12934","submissionUrl":"https://submission.nature.com/new-submission/12934/3","title":"Microbial Cell Factories","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Lignin, syringate, O-demethylation, vanillate O-demethylase, Acinetobacter baylyi ADP1, 2-pyrone-4,6-dicarboxylate","lastPublishedDoi":"10.21203/rs.3.rs-6218493/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6218493/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground:\u003c/h2\u003e \u003cp\u003eLignin holds great potential as an abundant and sustainable source of aromatic compounds, offering a viable alternative to fossil-based resources for producing chemicals and materials. Biological upgrading of lignin-derived aromatics can lead to more comprehensive lignocellulose utilization, thereby enhancing the overall feasibility of production. However, exploring a broader range of potential microbial hosts, pathways, and enzymes is crucial for developing efficient conversion processes. In particular, improving the conversion of S-lignin-related aromatics, such as syringate, remains a key area for future research.\u003c/p\u003e\u003ch2\u003eResults:\u003c/h2\u003e \u003cp\u003eIn this study, we aimed to investigate the conversion of S-lignin-related syringate in \u003cem\u003eAcinetobacter baylyi\u003c/em\u003e ADP1 by exploiting its native vanillate demethylase, VanAB. We discovered that the wild-type strain can efficiently \u003cem\u003eO\u003c/em\u003e-demethylate syringate to 3-O-methylgallate (3MGA) and then to gallate, revealing a previously unknown activity of VanAB of \u003cem\u003eA. baylyi\u003c/em\u003e ADP1. Conversion dynamics and \u003cem\u003ein vitro\u003c/em\u003e characterization showed that VanAB prefers syringate as a substrate over 3MGA. Overexpression of \u003cem\u003evanAB\u003c/em\u003e resulted in simultaneous conversion of syringate and 3MGA, but negatively impacted growth, potentially due to toxic side product formaldehyde and redox imbalance caused by high NADH consumption of the \u003cem\u003eO\u003c/em\u003e-demethylation reactions. Native \u003cem\u003evanAB\u003c/em\u003e expression resulted in 3MGA accumulation if syringate was available. We took advantage of this by constructing a strain with heterologous expression of \u003cem\u003egalA\u003c/em\u003e, a gallate dioxygenase from \u003cem\u003ePseudomonas putida\u003c/em\u003e KT2440, and demonstrated the conversion of 3MGA into 2-pyrone-4,6,-dicarboxylate (PDC), a precursor for high-quality polyesters.\u003c/p\u003e\u003ch2\u003eConclusions:\u003c/h2\u003e \u003cp\u003eIn this study, we discovered a previously unknown activity of syringate conversion in \u003cem\u003eA. baylyi\u003c/em\u003e ADP1. By adjusting the expression level of \u003cem\u003evanAB\u003c/em\u003e, syringate can be directed either into gallate or 3MGA, which could be further converted into PDC through the heterologous expression of \u003cem\u003egalA.\u003c/em\u003e Our results further highlight the potential and versatility of \u003cem\u003eA. baylyi\u003c/em\u003e ADP1 for lignin valorisation.\u003c/p\u003e","manuscriptTitle":"Conversion and upgrading of S-lignin related syringate by Acinetobacter baylyi ADP1","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-18 12:43:09","doi":"10.21203/rs.3.rs-6218493/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-07T14:57:30+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-07T13:46:53+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-05T08:20:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"182541363130738099486401210162307345851","date":"2025-03-18T00:35:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"163110528926740957780406336104598219155","date":"2025-03-17T01:52:54+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-16T14:34:58+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-15T18:29:56+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-15T18:26:13+00:00","index":"","fulltext":""},{"type":"submitted","content":"Microbial Cell Factories","date":"2025-03-13T09:22:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"microbial-cell-factories","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"micf","sideBox":"Learn more about [Microbial Cell Factories](http://microbialcellfactories.biomedcentral.com/)","snPcode":"12934","submissionUrl":"https://submission.nature.com/new-submission/12934/3","title":"Microbial Cell Factories","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a1ddbad1-c5c8-45cf-b877-57176f83adc0","owner":[],"postedDate":"March 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-10-06T15:59:59+00:00","versionOfRecord":{"articleIdentity":"rs-6218493","link":"https://doi.org/10.1186/s12934-025-02839-1","journal":{"identity":"microbial-cell-factories","isVorOnly":false,"title":"Microbial Cell Factories"},"publishedOn":"2025-09-29 15:57:08","publishedOnDateReadable":"September 29th, 2025"},"versionCreatedAt":"2025-03-18 12:43:09","video":"","vorDoi":"10.1186/s12934-025-02839-1","vorDoiUrl":"https://doi.org/10.1186/s12934-025-02839-1","workflowStages":[]},"version":"v1","identity":"rs-6218493","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6218493","identity":"rs-6218493","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}