De novo biosynthesis and whole-cell production of alkoxylated phenazine derivatives

De novo biosynthesis and whole-cell production of alkoxylated phenazine derivatives | 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 De novo biosynthesis and whole-cell production of alkoxylated phenazine derivatives chaozhi wang, Shuo Zhang, Sijia Xu, Chuanzeng Wang, Zhe Zhang, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9338442/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 7 You are reading this latest preprint version Abstract Background Phenazines are important nitrogen-containing heterocycles with diverse applications in the chemical and pharmaceutical industry. Alkoxylated phenazines, in particular, exhibit promising acaricidal and fungicidal properties. Currently, chemical synthesis is the main approach for alkoxylated phenazines and derivatives production. However, these processes are associated with harsh reaction conditions, accumulation of chemical waste (e.g., organic solvents, noble metal catalysts), and environmental concerns. Results In this study, we developed biocatalytic systems for the synthesis of alkoxylated phenazines by designing enzymatic cascades and de novo biosynthetic pathways. The O-methyltransferase LaphzM from Lysobacter antibioticus OH13 was identified to catalyze the alkoxylation of phenazines using SAM analogues generated in situ by halide methyltransferases (HMTs) from Burkholderia xenovorans and Arabidopsis thaliana . Using these enzymatic cascades, we successfully synthesized six alkoxylated phenazine derivatives, including three novel compounds. We further established de novo biosynthetic pathways for 1-ethoxyphenazine and 2-ethoxyphenazine in Pseudomonas chlororaphis via the direct sulfurylation pathway from S. cerevisiae . To improve production, we optimized whole-cell cascade systems, achieving 486.3 mg/L (54% yield) of 2-ethoxyphenazine, 218.1 mg/L (22.8% yield) of 2-propoxyphenazine and 378.2 mg/L (78.4% yield) of 1-ethoxyphenazine- N ′10-oxide within 7 h using microbially produced 2-hydroxyphenazine as the substrate along with EtI or PrI, respectively. Conclusions Overall, we successfully established green biocatalytic platforms for alkoxylated phenazines through enzymatic cascades and de novo biosynthetic pathways. Methyltransferases derived from diverse species, including LaphzM and HMTs, play key roles in the SAM-analogue-mediated alkylation of phenazines. This study provides a sustainable and efficient alternative to conventional chemical synthesis, with significant potential for green manufacturing of alkoxylated phenazine derivatives. alkoxylated phenazine derivatives enzyme cascade systems de novo biosynthesis whole-cell catalysis SAM analogues Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Phenazines are a class of nitrogen-containing heterocyclic aromatic compounds with broad applications in agriculture and pharmaceuticals due to their diverse biological activities [ 1 – 3 ]. Notably, phenazine-1-carboxylic acid (PCA) has been developed as an effective biopesticide named “Shenqinmycin” in China since 2011, and it has been widely applied in agricultural practices nationwide. To date, over 180 phenazine natural products have been discovered. Among them, methylation represents one of the most atom-efficient strategies for modifying the biological and physicochemical properties of these compounds. For instance, 1-methoxyphenazine exhibits significantly higher antifungal activity against Bipolaris maydis , Alternaria solani , and Aspergillus flavus than 1-hydroxyphenazine. Similarly, myxin and 1-methoxyphenazine N′ 10-oxide demonstrate stronger antimicrobial activities than their corresponding hydroxylated precursors [ 4 – 8 ]. Beyond methylation, alkoxylation with longer alkyl chains can further enhance biological activity. A previous study chemically synthesized a series of 1-n-alkoxyphenazines and 2-n-alkoxyphenazines and evaluated their acaricidal activity. Among them, 2-n-alkoxyphenazine derivatives with three- or four-carbon chains displayed the highest efficacy. Notably, 2-butoxyphenazine exhibited acaricidal activity four times higher than that of methyl parathion, a conventional organophosphate pesticide [ 9 ]. Despite their promising potential as biopesticides, the chemical synthesis of these compounds faces significant challenges, including harsh reaction conditions, the use of organic solvents and noble metal catalysts, and complex waste treatment and catalyst recovery processes [ 6 , 10 ]. In contrast, biocatalytic alkylation has gained increasing attention due to its high chemo-, regio-, and stereoselectivity under mild conditions [ 11 – 13 ]. In nature, S-adenosyl-L-methionine (SAM)-dependent methyltransferases are versatile biocatalysts capable of precisely transferring methyl groups to specific S, N, O, or C atoms with excellent chemo-selectivity, such as the alkylation of ambident nucleophiles, unsaturated heterocycles [ 14 – 16 ]. In the bioalkylation of phenazines, methylation is mainly catalyzed by O-methyltransferase LaphzM and N-methyltransferase PhzM, which utilize SAM as the methyl donor to synthesize compounds such as 1-methoxyphenazine, 1-methoxyphenazine N′ 10-oxide, myxin, pyocyanin [ 6 , 17 ]. Recent studies have further shown that many methyltransferases can catalyze not only methylation but also other alkylation reactions (such as ethylation, allylation, propylation) if the necessary SAM analogues are available [ 6 , 8 , 18 – 21 ]. For example, ethyl vanillin, 3′-O-ethylluteolin and 4-allyloxy-3-hydroxybenzaldehyde have been synthesized using halide methyltransferase and SAM-dependent methyltransferase (HMT-MT) cascade systems. In these systems, HMT is responsible for the synthesis of alkylated SAH from S-adenosyl-L-homocysteine (SAH) and alkyl iodides, while MT catalyzes mono-ethylation or mono-allylation [ 18 , 22 ]. Meanwhile, the similar strategy of “replacing the SAM with its fluorinated SAM analogues” has been developed for biocatalytic fluoroalkylation [ 23 , 24 ]. Novel and stable fluorinated SAM analogues, such as Te-adenosyl-L-(fluoromethyl) homotellurocysteine (FMeTeSAM), fluoro decarboxyl SAM (F-dcSAM) and fluoroethyl Se-adenosyl-L-selenomethionine (FEt-SeAM) have been synthesized and shown to be accepted by certain methyltransferases, enabling the transfer of fluoromethyl groups to oxygen, nitrogen, sulfur, and some carbon nucleophiles [ 25 – 27 ]. In recent years, fluorine has emerged as a privileged element in drug and agrochemical development. Compounds such as afloqualone, sevoflurane, fluticasone, fleroxacin, flutropium bromide, florbetapir-fluorine-18 exemplify how fluorine substitution can enhance metabolic stability, bioavailability, lipophilicity and cell permeability [ 28 – 34 ]. Most methods for introducing fluorine into organic molecules rely on chemical catalysis using Lewis acids, organic molecules, or transition metals [ 35 ], whereas enzymatic fluorination remains rare and challenging [ 36 , 37 ]. To date, no fluorinated phenazine natural products have been isolated, nor have biosynthetic routes for fluorinated phenazine derivatives been reported. More broadly, the biosynthesis of alkoxylated phenazine derivatives—particularly those with ethyl, propyl, and fluoromethyl groups—remains largely unexplored [ 35 – 37 ], which limits the development and application of these valuable compounds. In this article, enzymatic cascade systems and de novo pathway engineering were developed for synthesizing alkoxylated phenazines. We first constructed HMT-MT cascade systems using halide methyltransferases (HMTs) and O-methyltransferase LaphzM or monooxygenase NaphzNO1. These systems successfully catalyzed the alkoxylation of 1-hydroxyphenazine and 2-hydroxyphenazine using alkyl iodides and fluoro(iodo)methane as alkyl/fluoromethyl donors. To enable direct microbial production without exogenous HMTs-dependent alkylation, de novo biosynthetic pathways for 1-ethoxyphenazine and 2-ethoxyphenazine were engineered in P. chlororaphis by introducing the direct sulfurylation pathway, although the yields remained low. Building on these results, whole-cell cascade systems were developed for efficient production of alkylated phenazines with acaricidal and fungicidal potential. These results provide a foundation for the biocatalytic synthesis of alkylated and fluorinated phenazines with promising agricultural applications. Materials and methods Strains, plasmids and chemicals All host strains and plasmids used in this study were preserved in our laboratory. Escherichia coli DH5α was employed as the cloning host for gene manipulation, while E. coli BL21(DE3) served as the expression host for recombinant protein production. The vector pET-28a was utilized for gene cloning and expression. Detailed information regarding strains and plasmids is provided in Supplementary Table S1 . Restriction enzymes and DNA polymerase were sourced from Thermo Fisher Scientific (Waltham, MA, USA) and Takara Biomedical Technology (Dalian, China), respectively. A one-step cloning kit was obtained from Vazyme Biotech (Nanjing, China). All chemical reagents were purchased from Macklin Biochemical Co., Ltd. (Shanghai, China) and were of analytical grade unless otherwise specified. Overexpression and purification of enzymes LaphzM, HMT1, HMT2 and NaphzNO1 genes and PCR primers were chemically synthesized by Tsingke Biotechnology Co., Ltd. (Qingdao, China). LaphzM, HMT1, HMT2, NaphzNO1 were subcloned into the EcoRI/HindIII site of the pET28a (Novagen, Germany) expression vector, resulting in plasmids named pET28a-LaphzM, pET28a-HMT1, pET28a-HMT2, pET28a-NaphzNO1, respectively. And six his residues were fused at the N-terminus of every target protein. Then, the recombinant plasmids were introduced into E. coli BL21(DE3) for heterologous expression after verification of the sequences. The detailed information of plasmids, primers and genes were listed in Supplementary Table S2 and Table S3. The confirmed E. coli BL21(DE3) harboring LaphzM/HMT1/HMT2/NaphzNO1 was cultivated overnight at 37 ℃ in LB medium with 50 µg/mL kanamycin, respectively. Then 500 µL of the seed cultures transferred into 50 mL LB media containing appropriate antibiotics. When the OD600 of the culture broth reached 0.6–0.8, 0.1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) was added to the cells to induce the protein expression at 30 ℃ for 6 h. And then the cells were collected by centrifugation (8000 rpm, 10 min, 4 ℃), the resulting cell pellets were resuspended in 30 mL lysis buffer (25 mM HEPES, pH 7.5, 0.5 M NaCl), lysed by homogenization on ice. Cellular debris was removed by centrifugation (10000 rpm, 10 min, 4 ℃), the supernatant was incubated with 0.5 mL of Ni-NTA agarose resin at 4°C for 1 h, and loaded onto a gravity flow column. The proteins were washed with washing buffer (20 mM HEPES, pH 7.5, 300 mM NaCl and 20 mM imidazole) and elution buffer (20 mM HEPES, pH 7.5, 300 mM NaCl, and 150 mM imidazole). Protein expression and purification were confirmed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, 12%). And purified proteins were concentrated using Amicon Ultra filters (6000 rpm, 30 min, 4 ℃). The final proteins were frozen at -80 ℃ for further use. General procedure for the enzymatic reactions In vitro ethylation and propylation of phenazines catalyzed by HMT1 and LaphzM: the reaction mixtures contained 100 mM phosphate buffer (pH 8.0), 1 mM phenazine derivatives, 6 mM ethyl iodide (EtI) or propyl iodide (PrI), 50 µM SAH and purified enzymes (0.1 mg/mL HMT1 and LaphzM, respectively) in a final volume of 100 µL. Activity assays, initiated by the addition of enzymes, were performed at 30 ℃ and 220 rpm overnight. At least three independent replicates were performed for each assay. 100 µL methanol was added to stop the reaction. The supernatants were filtered, and substrate conversion was determined using high-performance liquid chromatography (HPLC) and high- resolution mass spectrometer (HR-MS). Ethylated phenazine N -oxides catalyzed by HMT1-LaphzM-NaphzNO1 cascade systems: the reaction mixture (150 µL) was composed of 100 mM phosphate buffer (pH 8.0), 1 mM phenazine derivatives, 6 mM ethyl iodide (EtI), 50 µM SAH, purified enzymes (0.1 mg/mL HMT1, LaphzM, and NaphzNO1, respectively), 100 µM flavin adenine dinucleotide (FAD), and 100 µM nicotinamide adenine dinucleotide (NADH). The first stage of the “two-step one-pot” was performed in a 100 µL reaction mixture containing 100 mM phosphate buffer (pH 8.0), 1 mM phenazine derivatives, purified enzymes (NaphzNO1), 100 µM FAD, and 100 µM NADH. After 1 h, purified enzymes (HMT1 and LaphzM), 6 mM EtI and 50 µM SAH were added for the subsequent alkylation of phenazine N-oxides at 30 ℃ and 220 rpm for overnight, and then the reactions were stopped by adding 150 µL of methanol. All assays were performed in triplicate. The reaction mixtures were centrifuged to collect the supernatant, and aliquots were analyzed by HPLC and HR-MS. Fluoromethylated phenazines catalyzed by HMT2 and LaphzM: reactions were performed according to the methods of ethylated and propylated phenazine derivatives. The reaction mixture (100 µL) contained HMT2 and LaphzM (or other modified enzymes), 1 mM phenazine derivatives, 6 mM fluoro(iodo)methane (FMeI), 50 µM SAH in 100 mM phosphate buffer, pH 8.0. At least three independent replicates were performed for each assay. The reactions were stopped by adding methanol (100 µL), and the precipitated protein was filtered before analysis via HPLC and HR-MS. Gene overexpression and fermentation Gene knockout and overexpression in P. chlororaphis H18 were carried out via homologous recombination with the plasmid pK18mobsacB, as described previously. For fermentation, P. chlororaphis H18 and its derivatives were first grown in 3 mL of KB medium at 30°C for 24 h with shaking at 200 rpm as seed cultures. Subsequently, 1 mL of the seed culture was inoculated into 50 mL of PPM medium (22 g/L tryptone, 20 g/L glucose, 5 g/L KNO₃, pH 7.0) in 250 mL flasks and incubated at 30°C for 48 h with shaking at 200 rpm. When required, kanamycin (100 µg/mL) or ampicillin (100 µg/mL) was added to the media. Whole-cell cascade reactions In one-pot enzymatic cascades, HMT1 catalyzes the conversion of SAH and alkyl halides into alkylated SAH, which subsequently serves as a substrate for methyltransferases to alkylated phenazine analogues. This process simultaneously regenerates SAH, enabling its continuous recycling within the reaction system. Therefore, whole cell reactions were prepared according to the established protocol, with the addition of a phosphate buffer washing step. The whole-cell reactions were performed at a final volume of 5 mL. The E. coli BL21(DE3) cells containing enzymes were resuspended in phosphate buffer (pH 8.0) containing phenazine derivatives, EtI, SAH. And then the reactions were incubated in a shaking incubator at 30 ℃, 200 rpm for overnight. The effects of HMT1/LaphzM ratio (1:2–4:1), and biocatalyst loading (10–40 g/L) were investigated. After biotransformation, the samples were thoroughly extracted by ethyl acetate. Subsequently, the organic phases were combined and concentrated under reduced pressure. The resultant residue was dissolved in methanol (1 mL) for HPLC analysis. Analytical Methods At the end of the reaction, methanol was added to the sample to stop the reaction. Subsequently, the mixtures were centrifuged at 12000g for 5 min. The supernatant was filtered through a nylon membrane filter (0.22 µm) and analyzed with HPLC and detected at 250 nm under the following conditions: an Agilent 5TC C18 (2) (250×4.6 mm) column was eluted with acetonitrile and water (0.1% (v/v) trifluoroacetic acid). HPLC procedure was performed at 1 mL/min with water and acetonitrile (v/v): 30–50% acetonitrile from 0 to 5 min, 50% acetonitrile from 5 to 10 min, 50–70% acetonitrile from 10 to 15 min, 70–80% acetonitrile from 15 to18 min, 80% acetonitrile from 18 to 21 min, 80 − 30% acetonitrile from 21 to 23 min, 30% acetonitrile from 23 to 29 min. Liquid chromatography high-resolution mass spectrometry (LC-HR-MS) analysis was performed with an Agilent Eclipse Plus C18 column (4.6×100 mm) on an Agilent Technologies 6520 Accurate-Mass Q-TOF LC-MS instrument. The reaction system described above was isolated and purified to obtain a sufficient amount of the phenazine derivatives for NMR spectroscopic analysis. The reaction system was extracted with an equal volume of ethyl acetate three times, and ethyl acetate was collected and evaporated under vacuum. The crude extract was dissolved in methanol and subsequently purified using semipreparative HPLC equipped with a reversed-phase C18 column (250 mm × 50 mm, 8 µm). The purification procedure was similar to that described with analytical methods of HPLC (solvent A: water; solvent B: acetonitrile; flow rate: 10 mL/min, gradient: 30–85% B in 25 min followed by 100% B for 5min). The purified compounds were further characterized by NMR spectroscopy (Bruker AVANCE III 400, equipped with a 5 mm PABBO probe). Spectroscopic data of prepared phenazine derivatives. 1-ethoxyphenazine ( 1a ). Green-yellow powder. HR-ESI-MS (m/z): 225.1024 [M + H] + , calculated for C 14 H 13 N 2 O, 225.1022. 1 H NMR (400 MHz, CDCl 3 , δ H ): δ 8.40 (dd, J = 8.4, 1.8 Hz, H-9), 8.23 (dd, J = 8.4, 1.8 Hz, H-6), 7.89–7.77 (m, H-4, H-7, H-8), 7.74 (dd, J = 8.9, 7.5 Hz, H-3), 7.07 (dd, J = 7.5, 1.1 Hz, H-2), 4.43 (q, J = 7.0 Hz, H-12), 1.69 (t, J = 7.0 Hz, H-11). 13 C NMR (150 MHz, CDCl 3, δ C ): 154.4 (C-1), 144.2 (C-4a), 143.3 (C-9a), 142.2 (C-5a), 137.1 (C-10a), 130.8 (C-7), 130.7 (C-3), 130.4 (C-9), 130.0 (C-8), 129.2 (C-6), 121.1 (C-4), 107.3 (C-2), 64.9 (C-11), 14.5 (C-12). 1-ethoxyphenazine N′10-oxide ( 1b ). Golden-yellow powder. HR-ESI-MS (m/z): 241.0966 [M + H] + , calculated for C 14 H 13 N 2 O 2 , 241.0972. ¹H NMR (400 MHz, CDCl₃, δ H ): δ 8.71–8.59 (m, H-9), 8.20 (d, J = 8.5 Hz, H-6), 7.89–7.76 (m, H-7, H-8), 7.75–7.64 (m, H-3, H-4), 7.00 (d, J = 7.9 Hz, H-2), 4.27 (q, J = 6.9 Hz, H-12), 1.66 (t, J = 6.9 Hz, H-11). 1-fluoromethoxyphenazine ( 1c ). Golden-yellow powder. HR-ESI-MS (m/z): 229.0763 [M + H] + , calculated for: C 13 H 10 OFN 2 , 229.0766. 1 H NMR (400 MHz, CDCl₃, δ H ): δ 8.44–8.34 (m, H-9), δ 8.31–8.22 (m, H-6), δ 7.93–7.84 (m, H-7, H-8), δ 8.03 (d, J = 8.8 Hz, H-4), δ 7.79 (dd, J = 8.8, 7.6 Hz, H-3), δ 7.49 (d, J = 7.5 Hz, H-2), δ 6.13 (d, J = 53.7 Hz, H-11). 13 C NMR (150 MHz, CDCl₃, δ C ): 151.9 (d, J = 3.2 Hz, C-1), 143.9 (C-4a), 143.5 (C-5a), 142.6 (C-9a), 136.5 (d, J = 1.5 Hz, C-10a), 131.2 (C-7), 130.8 (C-8), 130.1 (C-3, C-6), 129.4 (C-9), 124.6 (C-4), 112.3 (d, J = 2.2 Hz, C-2), 100.9 (d, J = 221.4 Hz, C-11). 2-fluoromethoxyphenazine ( 2b ). Yellow powder. HR-ESI-MS (m/z): 229.0766 [M + H] + , calculated for: C 13 H 10 OFN 2 , 229.0766. ¹H NMR (400 MHz, CDCl₃) δ 8.29–8.19 (m, H-1, H-4, H-6, H-9), 7.92–7.77 (m, H-7, H-8), 7.62 (dd, J = 9.5, 2.8 Hz, H-3), 5.96 (d, J = 53.4 Hz, H-11). Results Design of the enzymatic cascades for alkoxylation of phenazines. To date, the chemical synthesis of alkylated phenazines, particularly their mono-N-oxide or di-N-oxide derivatives, has faced considerable challenges. These include reliance on precious metal catalysts (Pd (II)-brettphos), harsh oxidative conditions using reagents like mCPBA, high temperatures and consumption of non-renewable or hazardous raw materials (Fig. 1 A). Furthermore, the generation of toxic intermediates and waste throughout the process raises environmental and safety concerns, limiting the sustainability and scalability of these routes. In contrast, enzyme-catalyzed reactions typically operate under mild aqueous conditions with high catalytic efficiency. Currently, the ability of HMT from Arabidopsis thaliana to form SAM analogues from SAH and alkyl iodide has been identified [ 18 ]. Its V140T variant (namely HMT1) could accept ethyl-, propyl-, and allyl iodide to produce the corresponding SAM analogs. And HMTs were utilized in one-pot cascade with the reported O-, N-, C-methyltransferases (such as, EgtD, SDMT, MrsA, TAMT, COMT, etc .) to synthesize alkylated chemicals [ 18 , 22 , 24 ]. Meanwhile the ability of HMT from Burkholderia xenovorans (protein family 05724, namely HMT2) to form fluorinated SAM from SAH and fluoromethyl iodide has also been identified [ 24 ]. Fluorinated SAM can serve as a substrate for SAM-dependent methyltransferases, enabling enzyme-catalyzed fluoromethylation. In addition, LaphzM gene from Lysobacter antibioticus OH13 encodes a SAM-dependent O-methyltransferase which is responsible for monomethoxy and dimethoxy formation in phenazines, such as myxin, 1-methoxyphenazine, 1-methoxyphenazine N ′ 10-oxide [ 13 , 17 ]. LaphzM demonstrates broad substrate flexibility, enabling the O-methylation of diverse phenazine scaffolds. According to the above information, HMT1 and HMT2 were utilized in one-pot cascades with LaphzM to synthesize alkoxylated phenazine derivatives in this study, respectively. Halide methytransferases (HMTs) and methyltransferases (MTs) cascade reactions for alkoxylated phenazine derivatives were designed as shown in Fig. 1 B. Synthesis of ethylated and propylated phenazines by HMT1-LaphzM cascade. First of all, we cloned the codon-optimized HMT1 and LaphzM genes into E. coli BL21(DE3) and obtained the soluble proteins. In a next step we explored whether LaphzM could catalyze ethylation of 1-hydroxyphenazine ( 1 ) using in situ-generated ethyl-SAH (SAE) as a co-substrate (Fig. 2 A). The cascade reaction containing 1 mM 1-hydroxyphenazine, 50 µM SAH, 6 mM EtI, 0.1 mg/mL HMT1 and 0.1 mg/mL LaphzM in 100 mM phosphate buffer (pH 8.0) was incubated at 30 ℃. After 7 h, 1-ethoxyphenazine ( 1a ) was synthesized in 96% yield, as confirmed by HPLC and HR-MS analysis. The MS spectrum of 1-ethoxyphenazine ( 1a ) showed significant peak of [M + H] + (m/z 225.1024, calculated for C 14 H 13 N 2 O, 225.1022), suggesting its molecular formula of C 14 H 12 N 2 O (Fig. 2 B and 2 C). The target compound was subsequently isolated from the cascade reaction, presenting as a green-yellow powder. Its structure was further characterized and confirmed through detailed NMR spectroscopy (Figure S1 ) [ 39 ]. The 1D and 2D NMR data (COSY, HSQC, HMBC) consistently support the structural assignment of the compound as 1-ethoxyphenazine (Figure S1 -S6 and Table S4). The HMT1-LaphzM cascade system was also employed to catalyze the conversion of 2-hydroxyphenazine into 2-ethoxyphenazine ( 2a1 , 21% yield). The target product was successfully identified and analyzed using HR-MS (Figure S7). These results indicate that the O-ethylation activity of LaphzM is relatively flexible. This approach, which utilizes propyl-SAH as a substitute for ethyl-SAH, was successfully extended to the synthesis of propylated derivatives. When PrI and 2-hydroxyphenazine were used as substrates, 2-propoxyphenazine ( 2a2 ) was detected by HPLC with a yield of 12% within 7 h. HR-MS analysis of 2-propoxyphenazine ( 2a2 ) revealed a prominent [M + H] ⁺ peak at m/z 239.1181 (calculated for C₁₅H₁₅N₂O, 239.1179), consistent with the molecular formula C₁₅H₁₄N₂O (Figure S8). The lower yield compared to ethylation may be attributed to increased steric hindrance from the larger alkyl group. And when allyl iodide, 2-iodopropane and 1-iodobutane were added to the reaction system to replace PrI, no corresponding products were detected by HPLC and HR-MS. Synthesis of ethylated phenazine N-oxides by HMT1-LaphzM-NaphzNO1 cascade. LaphzM can also accept mono-oxide or dioxide phenazines as substrates, suggesting that HMT1-LaphzM cascade systems could be used to synthesize ethylated N-oxide phenazines. However, these oxidized substrates are expensive and less accessible [ 40 ]. Previous studies showed that NaphzNO1 can convert 1-hydroxyphenazine to 1-hydroxyphenazine N ′10-oxide [ 13 , 14 ]. But, the N-oxidation activity of NaphzNO1 is relatively selective, requiring a free hydroxy group at carbon-1 position of the phenazine ring. Additionally, the engineered Pseudomonas strains still exhibit metabolic flux constraints in the biosynthesis of 1-hydroxyphenazine N′10-oxide and alkylated analogues [ 6 ]. To address these limitations, we proposed that ethylated mono-oxide could be produced by controlling the reaction sequence, and the “two-step one pot” synthetic strategy was designed (Fig. 2 D). First, NaphzNO1 was overexpressed in E. coli BL21(DE3), and the soluble proteins were obtained. NaphzNO1 was incubated with 1-hydroxyphenazine, FAD and NADH in phosphate buffer at 30 ℃ for 1 h to produce 1-hydroxyphenazine-N′10-oxide. Subsequently, HMT1-LaphzM cascade systems were added to the same reaction mixture with EtI and SAH. Finally, 1-ethoxyphenazine-N′10-oxide ( 1b ) was detected in 72% yield by HPLC and HR-MS/MS (Figure S9). The structure was further confirmed by NMR spectroscopy (Figure S10). Synthesis of fluoromethylated phenazines by HMT2-LaphzM cascade. The approach of substituting ethyl- and propyl-SAH with fluorinated SAM has been effectively employed to synthesize fluoromethylated phenazines (Fig. 3 ). HMT2 was initially overexpressed in E. coli BL21(DE3), yielding soluble protein for further use. The HMT2-LaphzM cascade reaction contained 1 mM 1-hydroxyphenazine, 50 µM SAH, 6 mM FMeI, 0.1 mg/mL HMT2 and 0.1 mg/mL LaphzM in 100 mM phosphate buffer (pH 8.0), incubated at 30 ℃ for 7 h. HPLC and LC-MS analysis indicated that a fluoromethylated 1-hydroxyphenazine compound ( 1c , 98% yield) might be produced in the reaction system. The HR-MS spectrum showed significant peak of [M + H] + (m/z 229.0763, calculated for C 13 H 10 FN 2 O, 229.0766), suggesting its molecular formula of C 13 H 9 FN 2 O (Fig. 3 B and 3 C). The target compound was successfully isolated from the cascade reaction. It was also obtained as yellow powder, and its structure was unambiguously identified as 1-fluoromethoxyphenazine by NMR spectroscopy (Figure S11-S16 and Table S5). Similarly, 2-hydroxyphenazine was converted to 2-fluoromethoxyphenazine ( 2b ) in 46% yield using the HMT2-LaphzM cascade system. HR-MS showed a peak of [M + H] ⁺ (m/z 229. 0766), consistent with the molecular formula C₁₃H₁₀FN₂O (calculated for 229.0766), confirming the presence of one fluorine atom. Comparison with the data for 1-fluoromethoxyphenazine reveals a similar set of signals but with distinct chemical shifts and coupling patterns, particularly in the aromatic region, suggesting an alternative substitution pattern. The number of protons and the magnitude of the H-F coupling (J = 53.4 Hz) are consistent with a fluoromethoxy substituent. The combined HR-MS and NMR data, especially the distinctive fluorine-proton coupling and aromatic substitution pattern, allow the compound to be identified as 2-fluoromethoxyphenazine (Figure S17 and S18). De novo biosynthesis for alkoxylated phenazines in Pseudomonas chlororaphis H18. Pseudomonas species have emerged as suitable chassis hosts for the synthesis of phenazine derivatives, owing to their rapid growth, facile genetic manipulation, and well-established fermentation systems. In particular, P. chlororaphis H18 has been demonstrated to biosynthesize various PCA-based derivatives, including 1-hydroxyphenazine, 2-hydroxyphenazine, 1-methoxyphenazine and 1-hydroxyphenazine-Nˊ10-oxide [ 6 , 44 ]. Notably, while 2-ethoxyphenazine ( 2a1 ) and 2-propoxyphenazine ( 2a2 ) exhibit promising acaricidal activity and are considered potential pesticide candidates, their microbial synthesis has not yet been achieved [ 9 ]. Here, we aimed to achieve in vivo synthesis of SAM analogues and subsequent alkyl chain transfer onto 1-hydroxyphenazine or 2-hydroxyphenazine in P. chlororaphis H18. To circumvent the toxicity of exogenous alkylating agents such as methyl iodide and ethyl iodide, we leveraged the endogenous SAM biosynthetic pathway of Pseudomonas to produce SAM analogues. Previous studies have demonstrated the substrate promiscuity of O-acetylhomoserine sulfhydrolase from S. cerevisiae (ScOAHS), which enables the in vivo production of methionine analogues from organic thiols [ 41 ]. To harness this property, we introduced the direct sulfurylation pathway, consisting ScOAHS and O-acetyltransferase (ScMET2) from S. cerevisiae , into P. chlororaphis . This allowed the production of ethionine from ethanethiol, which were then converted to SAE by the endogenous methionine adenosyltransferase (MAT). We therefore introduced ScOAHS and LaphzM into two P. chlororaphis strains: a high 1-hydroxyphenazine-producing strain ( P. chlororaphis H18-1-8) and a 2-hydroxyphenazine-producing strain ( P. chlororaphis H18), yielding P. chlororaphis H18-1-8n and H18n, respectively [ 6 , 44 ]. The cultures were then supplemented with ethanethiol (EtSH, 5 mM) in an attempt to produce 1-ethoxyphenazine ( 1a ) and 2-ethoxyphenazine ( 2a1 ), respectively (Fig. 4 ). However, this approach yielded only trace amounts of 1-ethoxyphenazine ( 1a ), with 1-methoxyphenazine remaining the predominant product, while 2-ethoxyphenazine ( 2a1 ) production also remained low by LC-MS. We attribute these outcomes to the robust endogenous SAM biosynthetic network in P. chlororaphis H18, which preferentially channels endogenous methionine toward SAM synthesis and subsequent methylation, outcompeting the pathway for methionine analogue incorporation. Consequently, enhancing the production of alkylated phenazine derivatives will require systematic engineering of the entire SAM metabolic network to redirect flux from endogenous methionine to the desired methionine analogues. Whole-cell cascade catalysis production of alkoxylated phenazines. Given that insufficient supply of SAM analogues in the recombinant strain may limit 2-ethoxyphenazine ( 2a1 ) and 2-propoxyphenazine ( 2a2 ) production, we explored an alternative whole-cell biocatalytic approach by directly supplying 2-hydroxyphenazine as the precursor. Although the HMT1-LaphzM enzymatic cascade system was successfully developed to produce 2-ethoxyphenazine ( 2a1 , 45% yield) and 2-propoxyphenazine ( 2a2 , 12% yield), the product yields limit its broader applicability. Whole-cell cascade catalysis, which utilizes bacterial cells to catalyze chemical transformations, offers advantages such as enhanced robustness, high catalytic efficiency, and the elimination of labor-intensive protein purification steps [ 42 ]. To address the limitations of the enzymatic system, whole-cell reaction systems were designed to improve the production of alkylated phenazines and analogues. Along with the large quantity of PCA production through microbial fermentation and structure diversity of phenazine derivatives through combinatorial biology, these enable phenazine derivatives to be used as sustainable raw materials to synthesize value-added chemicals [ 43 ]. In our preliminary research, we constructed strains producing 1-hydroxyphenazine, 2-hydroxyphenazine and other phenazine derivatives [ 6 , 44 ]. And these compounds can be used as starting materials to directly synthesize alkylated phenazine derivatives via whole-cell cascade catalysis. HMT1 and LaphzM were overexpressed in E. coli BL21(DE3) to construct strain 1 and strain 2, respectively (Fig. 5 A). At 30 ℃, these strains (ratio 1:1) were then incubated with 1 mM 2-hydroxyphenazine, 100 µM SAH and 6 mM EtI in a 5 mL reaction volume with the cell loading of 10 g CDW (cell dry weight) per L. After 7 h, the substrates were successfully converted into 39.5 mg/L 2-ethoxyphenazine ( 2a1 , ~ 17.5% yield), but most of the substrates could not be converted. We proposed that the efficient production of 2-ethoxyphenazine ( 2a1 ) could be realized by controlling the ratio of two kinds of E. coli cells. And then, we conducted reactions using 10 g CDW per L strain 1 and strain 2 (at ratios 2:1 and 1:2, respectively), along with 1 mM 2-hydroxyphenazine, 100 µM SAH and 6 mM EtI in a 5 mL reaction volume. Results indicated that when the ratio of strain 1 to strain 2 cells was 2:1, the yield of 2-ethoxyphenazine ( 2a1 ) was increased, which may be attributed to the relatively low catalytic efficiency of HMT1 (Fig. 5 B). To further enhance the economic feasibility of the process, the one-pot catalytic reactions were systematically optimized under various conditions, as illustrated in Fig. 5 B. The yield of 2-ethoxyphenazine ( 2a1 ) was evaluated at different cell densities (10–40 g CDW per L), with a 3:1 ratio of strain 1 to strain 2 (30 g CDW per L) demonstrating the highest catalytic efficiency. Under these optimized conditions, 4 mM 2-hydroxyphenazine reacted to form 486.3 mg/L 2-ethoxyphenazine ( 2a1 , 2.16 mM, 54% yield) within 7 h, which was 12.3-fold higher than that of the original reaction. Furthermore, the whole-cell cascade system was also applied to synthesize 2-propoxyphenazine ( 2a2 , 218.1 mg/L, 22.8% yield) from 4 mM 2-hydroxyphenazine and PrI, and other phenazine derivatives (Fig. 6). NaphzNO1 was overexpressed in E. coli BL21(DE3) to construct strain 3 (Fig. 6C). To facilitate cofactor regeneration, a glucose dehydrogenase (GDH)-coupled NADH recycling system was integrated with NaphzNO1. The combined system achieved the production of 378.2 mg/L (78.4% yield) of 1-ethoxyphenazine-N′10-oxide ( 1b ). Notably, the reaction proceeded without the need for exogenous addition of FAD or NADH cofactors. However, the overall process yield was limited by the inherent instability of phenazine mono-N-oxides under whole-cell cascade conditions, which led to undesired reduction of the target products. Discussion As a large family of colored nitrogen-containing heterocyclic compounds, phenazines exhibit a wide range of valuable biological activities, underscoring their potential in medicine and agriculture. In particular, 2-ethoxyphenazine ( 2a1 ) and 2-propoxyphenazine ( 2a2 ) exhibit significant acaricidal activity, positioning them as promising candidate compounds for pesticide development [ 9 ]. Conventional chemical synthesis of 1-n-alkoxyphenazines and 2-n-alkoxyphenazines relies on fossil fuel-derived benzene derivatives, organic solvents, and noble metal catalysts, raising environmental and safety concerns [ 6 , 10 ]. In this study, we demonstrated that LaphzM can catalyze alkoxylation of 1-hydroxyphenazine, 2-hydroxyphenazine and other derivatives using SAM analogues. Enzymatic cascade systems were designed to produce six alkoxylated and fluoromethylated phenazines, including three novel compounds confirmed by HR-MS and NMR. Meanwhile, de novo biosynthetic pathways for 1-ethoxyphenazine ( 1a ) and 2-ethoxyphenazine ( 2a1 ) were constructed in P. chlororaphis H18, representing the first report of alkylated phenazine biosynthesis. To enhance production, we adopted a more cost-effective alkyl donor, EtI, in place of EtSH. The whole-cell one-pot catalysis has been developed to rapidly and efficiently convert 2-ethoxyphenazine ( 2a1 ) and 2-propoxyphenazine ( 2a2 ) using microorganism metabolite 2-hydroxyphenazine as the substrate. After the optimization of the reaction conditions, the 2-ethoxyphenazine titers could reach 486.3 mg/L ( 2a1 , 54% yield) within 7 h, a 12.3-fold improvement over the initial reaction. The whole-cell system was also successfully applied to synthesize 2-propoxyphenazine ( 2a2 , 218.1 mg/L, 22.8% yield) and 1-ethoxyphenazine-N′10-oxide ( 1b , 378.2 mg/L, 78.4% yield). The process is conducted under mild conditions, occurring in a neutral aqueous solution at room temperature, without the requirement for metal catalysts and high temperature. These features highlight its potential as a sustainable alternative for synthetic applications. However, the reliance on iodolkanes as alkyl donors in aqueous systems raises concerns regarding waste and safety. The high volatility and poor water solubility of alkyl iodides also contribute to the limited efficiency and yield of the biotransformation process investigated in this study. Therefore, developing more sustainable and environmentally benign alkyl donors is essential for advancing this green methodology. And future work should explore the family of halide methyltransferases to accept different alkylation reagents. Rational design and directed evolution strategies can be combined to accurately optimize key amino acid residues in halide methyltransferases and methyltransferases through structural and functional analysis. Computation simulations and high-throughput screening should be integrated to further accelerate the engineering of enzyme activity and substrate specificity [ 45 , 46 ]. Finally, enhancing the reusability of enzymatic components through advanced immobilization techniques and scaling up reaction processes will be crucial for significantly enhancing production yields. Conclusions This study reported the green biocatalytic systems of alkylated phenazines through two complementary strategies: enzymatic cascades (HMT-MTs systems and other modifying enzymes) and de novo biosynthetic pathway engineering. Using enzymatic cascades, we successfully synthesized six alkoxylated phenazine derivatives, three of which were identified as novel structures (1-ethoxyphenazine-N′10-oxide ( 1b ), 1-fluoromethoxyphenazine ( 1c ), and 2-fluoromethoxyphenazine ( 2b )) based on NMR and SciFinder analysis. And we constructed the de novo biosynthetic pathway of ethylated phenazines in P. chlororaphis for the direct production of ethylated phenazines. Moreover, whole-cell cascade biocatalytic synthesis ways to alkoxylated phenazines have been established and optimized. This study offers a sustainable and highly efficient alternative to conventional chemical manufacturing processes of alkoxylated phenazines, presenting a promising green solution. Abbreviations PCA phenazine-1-carboxylic acid SAH S-adenosyl-L-homocysteine SAM S-adenosyl-L-methionine MT methyltransferase HMT halide methyltransferase FMeTeSAM Te-adenosyl-L-(fluoromethyl) homotellurocysteine F-dcSAM fluoro decarboxyl SAM FEt-SeAM fluoroethyl Se-adenosyl-L-selenomethionine EtI ethyl iodide IPTG isopropyl-β-D-1-thiogalactopyranoside PrI propyl iodide HPLC high-performance liquid chromatography HRMS high- resolution mass spectrometer FAD flavin adenine dinucleotide NADH nicotinamide adenine dinucleotide FMeI fluoro(iodo)methane LC-HRMS liquid chromatography high-resolution mass spectrometry HRESIMS high resolution electrospray ionization mass spectrometry SAE ethyl-SAH mCPBA 3-chloroperbenzoic acid EtSH ethanethiol Declarations Ethics approval and consent to participate No applicable. Consent for publication No applicable. Competing interests No applicable. Funding This work was supported by grant from the Scientific Research Starting Foundation of Shandong University of Technology (4041/422046), the Natural Science Foundation of Shandong Province, China (ZR2024QC398). Author Contribution F.H. and W.H. conceived the project, F.H. and P.X. supervised the project, F.H., S.Z. and C.W. performed the experiments and analyzed the data; W.H. and F.H. wrote the manuscript; all authors checked and modified the manuscript. Acknowledgements No applicable. Data Availability The datasets analysed during the current study are available from the corresponding author upon reasonable request. References Krishnaiah M, de Almeida NR, Udumula V, et al. Synthesis, biological evaluation, and metabolic stability of phenazine derivatives as antibacterial agents. Eur J Med Chem. 2018;143:936–47. Serafim B, Bernardino AR, Freitas F, Torres CAV. Recent developments in the biological activities, bioproduction, and applications of Pseudomonas spp. phenazines. Molecules. 2023;28(3):1368. Thalhammer KO, Newman DK. A phenazine-inspired framework for identifying biological functions of microbial redox-active metabolites. Curr Opin Chem Biol. 2023;75:102320. Guttenberger N, Blankenfeldt W, Breinbauer R. Recent developments in the isolation, biological function, biosynthesis, and synthesis of phenazine natural products. Bioorg Med Chem. 2017;25:6149–66. Yan JJ, Liu WW, Cai JT, et al. Advances in phenazines over the past decade: review of their pharmacological activities, mechanisms of action, biosynthetic pathways and synthetic strategies. Mar Drugs. 2021;19(11):610. Wan YP, Liu HC, Xian M, Huang W. Biosynthetic pathway construction and production enhancement of 1-hydroxyphenazine derivatives in Pseudomonas chlororaphis H18. J Agric Food Chem. 2022;70(4):1223–31. Bennett MR, Shepherd SA, Cronin VA, Micklefield J. Recent advances in methyltransferase biocatalysis. Curr Opin Chem Biol. 2017;37:97–106. Schülke KH, Ospina F, Hörnschemeyer K, Gergel S, Hammer SC. Substrate profiling of anion methyltransferases for promiscuous synthesis of S-adenosylmethionine analogs from haloalkanes. ChemBioChem. 2022;23(4):e202100632. Cross B, Dunn CL, Payne DH, Tipton JD. Synthesis and pesticidal activity of phenazines. II. Alkyl, alkoxy, alkylthio and alkylsulphonyl phenazines. J Sci Food Agric. 1969;20(6):340–4. Tietze M, Iglesias A, Merisor E, Conrad J, Klaiber I, Beifuss U. Efficient methods for the synthesis of 2-hydroxyphenazine based on the Pd-catalyzed N-arylation of aryl bromides. Org Lett. 2005;7(8):1549–52. McKean IJW, Hoskisson PA, Burley GA. Biocatalytic alkylation cascades: Recent advances and future opportunities for late-stage functionalization. ChemBioChem. 2020;21:2890–7. Buller R, Lutz S, Kazlauskas RJ, Snajdrova R, Moore JC, Bornscheuer UT. From nature to industry: harnessing enzymes for biocatalysis. Science. 2023;382:eadh8615. Dong M, Horitani M, Dzikovski B, et al. Substrate-dependent cleavage site selection by unconventional radical S-adenosylmethionine enzymes in diphthamide biosynthesis. J Am Chem Soc. 2017;139(16):5680–3. Ospina F, Schülke KH, Hammer SC. Biocatalytic alkylation chemistry: building molecular complexity with high selectivity. ChemPlusChem. 2022;87(4):e202100454. Ospina F, Schülke KH, Schnutenhaus M, et al. Biocatalytic alkylation of ambident nucleophiles enables selective N-functionalization of heterocycles and late-stage modifications. Angew Chem Int Ed. 2025;64(10):e202510300. Ospina F, Schülke KH, Soler J, Klein A, et al. Selective biocatalytic N-methylation of unsaturated heterocycles. Angew Chem Int Ed. 2022;61(49):e202213056. Jiang J, Guiza Beltran D, Schacht A, Wright S, Zhang L, Du LM. Functional and structural analysis of phenazine O-methyltransferase LaphzM from Lysobacter antibioticus OH13 and one-pot enzymatic synthesis of the antibiotic myxin. ACS Chem Biol. 2018;13(4):1003–12. Tang QY, Grathwol CW, Aslan-Üzel AS, et al. Directed evolution of a halide methyltransferase enables biocatalytic synthesis of diverse SAM analogs. Angew Chem Int Ed. 2021;60(3):1524–7. Abdelraheem EMM, Thair B, Varela RF, Jockmann E, Popadić D, Hailes HC, et al. Methyltransferases: functions and applications. ChemBioChem. 2022;23(13):e202200212. Liao CS, Seebeck FP. S-adenosylhomocysteine as a methyl transfer catalyst in biocatalytic methylation reactions. Nat Catal. 2019;2(8):696–701. Gao CY, Yang GY, Ding XW, et al. Engineering of halide methyltransferase BxHMT through dynamic cross-correlation network analysis. Angew Chem Int Ed. 2024;63(19):e202401235. Yang GY, Zheng GW, Zeng BB, Xu JH, Chen Q. Engineering of halide methyltransferases for synthesis of SAE and its application in biosynthesis of ethyl vanillin. Mol Catal. 2023;550:113533. Ding WP, Zhou MQ, Li HY, et al. Biocatalytic fluoroalkylation using fluorinated S-adenosyl-L-methionine cofactors. Org Lett. 2023;25(30):5650–5. Peng JM, Liao CS, Bauer C, Seebeck FP. Fluorinated S-adenosylmethionine as a reagent for enzyme-catalyzed fluoromethylation. Angew Chem Int Ed. 2021;60(52):27178–83. Wang WR, Zhao HM, Yu NH, Chen F, Dong M. Stable S-adenosylmethionine analogue for enzymatic fluoromethylation. ACS Catal. 2023;13(20):13729–34. Neti SS, Wang B, Iwig DF, Onderko EL, Booker SJ. Enzymatic fluoromethylation enabled by the S-adenosylmethionine analog Te-adenosyl-L-(fluoromethyl)homotellurocysteine. ACS Cent Sci. 2023;9(5):905–14. Yu NH, Zhao HM, Wang WR, Dong M. Enzymatic fluoroethylation by a fluoroethyl selenium analogue of S-adenosylmethionine. ACS Catal. 2024;14(8):6211–6. Merkel L, Budisa N. Organic fluorine as a polypeptide building element: in vivo expression of fluorinated peptides, proteins and proteomes. Org Biomol Chem. 2012;10(36):7241–61. Barnes-Seeman D, Beck J, Springer C. Fluorinated compounds in medicinal chemistry: recent applications, synthetic advances and matched-pair analyses. Curr Top Med Chem. 2014;14(7):855–64. Chaudhari SB, Kumar A, Mankar VH, et al. Diverse role, structural trends, and applications of fluorinated sulphonamide compounds in agrochemical and pharmaceutical fields. Heliyon. 2024;10(12):e32434. Hoff BH, Sundby E. Preparation of pharmaceutical important fluorinated 1-arylethanols using isolated enzymes. Bioorg Chem. 2013;51:31–47. Inoue M, Sumii Y, Shibata N. Contribution of organofluorine compounds to pharmaceuticals. ACS Omega. 2020;5(19):10633–40. Johnson BM, Shu YZ, Zhuo XL, Meanwell NA. Metabolic and pharmaceutical aspects of fluorinated compounds. J Med Chem. 2020;63(12):6315–86. Hunter L. The C-F bond as a conformational tool in organic and biological chemistry. Beilstein J Org Chem. 2010;6:38. Chatterjee T, Iqbal N, You Y, Cho EJ. Controlled fluoroalkylation reactions by visible-light photoredox catalysis. Acc Chem Res. 2016;49(10):2284–94. Wu LR, Maglangit F, Deng H. Fluorine biocatalysis. Curr Opin Chem Biol. 2020;55:119–26. Nikel PI, Welner DH, Cros A, Volke DC. In vivo fluorine biocatalysis: Six enzymes in search of a cell factory. Chem Catal. 2022;2(10):2403–5. Gohain N, Thomashow LS, Mavrodi DV, Blankenfeldt W. The purification, crystallization and preliminary structural characterization of PhzM, a phenazine-modifying methyltransferase from Pseudomonas aeruginosa . Acta Crystallogr F. 2006;62(9):887–90. Liu HS, Zhu GL, Zhao SG, Fu P, Zhu WM. Bioactive natural products from the marine sponge-derived Nocardiopsis dassonvillei OUCMDZ-4534. Chin J Org Chem. 2019;39(2):507. Viktorsson EO, Melling Grøthe B, Aesoy R, et al. Total synthesis and antileukemic evaluations of the phenazine 5,10-dioxide natural products iodinin, myxin and their derivatives. Bioorg Med Chem. 2017;25(8):2285–93. Mohr MKF, Benčić P, Andexer JN. Doping In Vivo Alkylation in E. coli by Introducing the Direct Sulfurylation Pathway of S. cerevisiae. Angew Chem Int Ed Engl. 2025;64(2):e202414598. Hou FF, Xian M, Huang W. De novo biosynthesis and whole-cell catalytic production of paracetamol on a gram scale in Escherichia coli . Green Chem. 2021;23(20):8280–9. Huang W, Wan YP, Su H, et al. Recent advances in phenazine natural products: biosynthesis and metabolic engineering. J Agric Food Chem. 2024;72(39):21364–79. Wan YP, Liu HC, Xian M, Huang W. Biosynthesis and metabolic engineering of 1-hydroxyphenazine in Pseudomonas chlororaphis H18. Microb Cell Fact. 2021;20(1):235. Wen XJ, Leisinger F, Leopold V, Seebeck FP. Synthetic Reagents for Enzyme-Catalyzed Methylation. Angew Chem Int Ed. 2022;61(41):e202208746. Singh N, Lane S, Yu TH, et al. A generalized platform for artificial intelligence-powered autonomous enzyme engineering. Nat Commun. 2025;16(1):5648. Additional Declarations No competing interests reported. Supplementary Files SIalkyphz20260402.docx TOC.png Table of Contents Graphic: a dual-strategy platform for alkoxylated phenazines: an enzymatic cascade using methyltransferases and halide methyltransferases, and a de novo biosynthetic route in Pseudomonas chlororaphis H18 enabled by the direct sulfurylation pathway. Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 18 May, 2026 Reviews received at journal 18 May, 2026 Reviewers agreed at journal 27 Apr, 2026 Reviewers invited by journal 21 Apr, 2026 Editor assigned by journal 08 Apr, 2026 Submission checks completed at journal 08 Apr, 2026 First submitted to journal 06 Apr, 2026 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. 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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-9338442","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":630034742,"identity":"39cb3caa-0cc4-47d6-bbb6-2c03c45c1a2f","order_by":0,"name":"chaozhi wang","email":"","orcid":"","institution":"Shandong University of Technology","correspondingAuthor":false,"prefix":"","firstName":"chaozhi","middleName":"","lastName":"wang","suffix":""},{"id":630034743,"identity":"abb8ef80-ff76-4105-9be8-5178ae229763","order_by":1,"name":"Shuo Zhang","email":"","orcid":"","institution":"Shandong University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Shuo","middleName":"","lastName":"Zhang","suffix":""},{"id":630034745,"identity":"316fc3e6-ba3d-4b73-ad43-505eaed6e549","order_by":2,"name":"Sijia Xu","email":"","orcid":"","institution":"Shandong University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Sijia","middleName":"","lastName":"Xu","suffix":""},{"id":630034749,"identity":"8c0871b3-8247-468f-b19e-9dd2b7cbb9c7","order_by":3,"name":"Chuanzeng Wang","email":"","orcid":"","institution":"Shandong University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Chuanzeng","middleName":"","lastName":"Wang","suffix":""},{"id":630034750,"identity":"291796b3-42c6-4f9f-a35e-09970c69e59a","order_by":4,"name":"Zhe Zhang","email":"","orcid":"","institution":"CAS Key Lab of Biobased Materials, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Zhe","middleName":"","lastName":"Zhang","suffix":""},{"id":630034751,"identity":"6c4e2460-3546-4269-b68c-d89f7740e926","order_by":5,"name":"Mohd Sadeeq","email":"","orcid":"","institution":"Shandong University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Mohd","middleName":"","lastName":"Sadeeq","suffix":""},{"id":630034753,"identity":"0033fe46-c24e-4a45-83c0-0cbfe16be0b8","order_by":6,"name":"Yupeng Wan","email":"","orcid":"","institution":"CAS Key Lab of Biobased Materials, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Yupeng","middleName":"","lastName":"Wan","suffix":""},{"id":630034756,"identity":"5981bff9-b840-4bab-8814-192694be4297","order_by":7,"name":"Chen Gao","email":"","orcid":"","institution":"Jinan Central Hospital affiliated to Shandong university","correspondingAuthor":false,"prefix":"","firstName":"Chen","middleName":"","lastName":"Gao","suffix":""},{"id":630034758,"identity":"74f8026b-5d17-4aea-b64a-2a07e08a1429","order_by":8,"name":"Wei Huang","email":"","orcid":"","institution":"Shandong Freda Biotech Co.","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Huang","suffix":""},{"id":630034759,"identity":"27247c42-72ff-4028-92c6-e4f39957f8f4","order_by":9,"name":"Peng Xiong","email":"","orcid":"","institution":"Shandong University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Peng","middleName":"","lastName":"Xiong","suffix":""},{"id":630034761,"identity":"fd11bafe-c064-42ba-84b7-c90c985f8761","order_by":10,"name":"Feifei Hou","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuklEQVRIiWNgGAWjYBACAyD+UFHBwNgA4vEQqYVxxpkzQC1sJGk520aKFnP2HsOGg/PqZDfcb2B88LaNQd6ckBbLnjNALdsOG284xsBsOLeNwXBnAyGH3cgxf/xx24FEoBY2ad42hgSDA4S1AG2ZUwfSwv6bBC0NzGBbmInTcuZYYcOBY4eNZx5LbJacc07CcANBLcebNzYcqKmT7Tt8+OCHN2U28gRtQQLgBCBBvPpRMApGwSgYBbgBADpZSEiQHpiTAAAAAElFTkSuQmCC","orcid":"","institution":"Shandong University of Technology","correspondingAuthor":true,"prefix":"","firstName":"Feifei","middleName":"","lastName":"Hou","suffix":""}],"badges":[],"createdAt":"2026-04-07 02:38:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9338442/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9338442/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108114026,"identity":"05654135-55f3-4064-a17b-70a05b8e27ae","added_by":"auto","created_at":"2026-04-29 13:25:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1577522,"visible":true,"origin":"","legend":"\u003cp\u003eSynthesis routes used to prepare phenazine derivatives. (A): Chemical route for alkoxyphenazines production. (B): Halide methytransferases (HMT) and methyltransferases (MT) cascade reactions to produce alkylated phenazine derivatives. HMT-catalyzed fluoromethylation and alkylation of SAH provides SAM analogues. MT-catalyzed fluoromethylation and alkylation of phenazines. (R: alkyl and fluoromethyl groups.)\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9338442/v1/90d4b10fed8049fbc91a9406.png"},{"id":108114064,"identity":"02f84458-3898-4313-b444-c250ef591bd2","added_by":"auto","created_at":"2026-04-29 13:26:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1741904,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vitro \u003c/em\u003ecatalytic assay of HMT1 and LaphzM with the substrate 1-hydroxyphenazine.\u003cstrong\u003e \u003c/strong\u003e(A) Schematic representation of HMT1 and LaphzM-catalyzed reactions with 1-hydroxyphenazine as the substrate. (B) HPLC chromatograms of HMT1 and LaphzM-catalyzed reactions with 1-hydroxyphenazine as the substrate. (i) 1-ethoxyphenazine standard; (ii) HMT1 and LaphzM with 1-hydroxyphenazine; (iii) boiled HMT1 and LaphzM with 1-hydroxyphenazine. (C) HRMS analysis of 1-ethoxyphenazine. (D) Schematic representation of NaphzNO1-HMT1-LaphzM-catalyzed reactions using1-hydroxyphenazine as the substrate. Yields were shown.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9338442/v1/e3a43f27ff0e4f954d2339d4.png"},{"id":108113951,"identity":"3bfae192-c4e9-41f3-9750-8ba6fc2e81bb","added_by":"auto","created_at":"2026-04-29 13:25:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1367169,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vitro \u003c/em\u003ecatalytic assay of HMT2 and LaphzM using 1-hydroxyphenazine as the substrate.\u003cstrong\u003e \u003c/strong\u003e(A) Schematic diagram of the reactions catalyzed by HMT2 and LaphzM-catalyzed reactions with 1-hydroxyphenazine as the substrate. (B) HPLC chromatograms of the reactions catalyzed by HMT2 and LaphzM with 1-hydroxyphenazine as the substrate: (i) 1-fluoromethoxyphenazine standard; (ii) Reaction mixture containing HMT2, LaphzM and 1-hydroxyphenazine; (iii) Boiled HMT2 and LaphzM with 1-hydroxyphenazine. (C) HRMS analysis of the target product 1-fluoromethoxyphenazine. Yields were shown.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9338442/v1/488dec647fd4973d08badc9f.png"},{"id":108113953,"identity":"4bd75e23-8270-4578-80f6-caa9e2fe4384","added_by":"auto","created_at":"2026-04-29 13:25:45","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1159961,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eDe novo\u003c/em\u003e biosynthesis of alkoxylated phenazine derivatives in \u003cem\u003ePseudomonas chlororaphis\u003c/em\u003e H18\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9338442/v1/f26e3124ae82a3ff4f464197.png"},{"id":108114038,"identity":"479ae701-a2c8-4913-9eaf-91aff8e61e16","added_by":"auto","created_at":"2026-04-29 13:25:59","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1673423,"visible":true,"origin":"","legend":"\u003cp\u003eWhole-cell cascade catalysis for the production of 2-ethoxyphenazine. (A) Design of whole-cell cascade catalysis for 2-ethoxyphenazine production (\u003cem\u003eE. coli \u003c/em\u003estrain1 expressing LaphzM and srain2 expressing HMT1 for the biocatalysis of 2-ethoxyphenazine by one-step method). (B) Optimization of 2-ethoxyphenazine production conditions with different strain1: strain2 ratio and CDW.(C) Design of whole-cell cascade catalysis (NaphzNO1-HMT1-LaphzM-catalyzed reactions) using1-hydroxyphenazine as the substrate.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9338442/v1/0304fa58d05c050b7fcaab15.png"},{"id":108114417,"identity":"94216b45-dba9-4e5b-b9cf-5270ab8c3d5b","added_by":"auto","created_at":"2026-04-29 13:26:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7872146,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9338442/v1/716ce377-4a56-468f-ad61-6ef984c61413.pdf"},{"id":108113952,"identity":"3c1e03c2-0f96-490f-8f3e-6bbd6b6adf7d","added_by":"auto","created_at":"2026-04-29 13:25:44","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":12222124,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"SIalkyphz20260402.docx","url":"https://assets-eu.researchsquare.com/files/rs-9338442/v1/89f002c38cc294482f47f920.docx"},{"id":108113948,"identity":"54284336-1ab9-46b2-82d4-bde0dac0fd1f","added_by":"auto","created_at":"2026-04-29 13:25:43","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1677239,"visible":true,"origin":"","legend":"\u003cp\u003eTable of Contents Graphic: a dual-strategy platform for alkoxylated phenazines: an enzymatic cascade using methyltransferases and halide methyltransferases, and a \u003cem\u003ede novo\u003c/em\u003e biosynthetic route in \u003cem\u003ePseudomonas chlororaphis\u003c/em\u003e H18 enabled by the direct sulfurylation pathway.\u003c/p\u003e","description":"","filename":"TOC.png","url":"https://assets-eu.researchsquare.com/files/rs-9338442/v1/55cb9c1169816162ff91c218.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003e\u003cem\u003eDe novo \u003c/em\u003ebiosynthesis and whole-cell production of alkoxylated phenazine derivatives\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003ePhenazines are a class of nitrogen-containing heterocyclic aromatic compounds with broad applications in agriculture and pharmaceuticals due to their diverse biological activities [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Notably, phenazine-1-carboxylic acid (PCA) has been developed as an effective biopesticide named \u0026ldquo;Shenqinmycin\u0026rdquo; in China since 2011, and it has been widely applied in agricultural practices nationwide. To date, over 180 phenazine natural products have been discovered. Among them, methylation represents one of the most atom-efficient strategies for modifying the biological and physicochemical properties of these compounds. For instance, 1-methoxyphenazine exhibits significantly higher antifungal activity against \u003cem\u003eBipolaris maydis\u003c/em\u003e, \u003cem\u003eAlternaria solani\u003c/em\u003e, and \u003cem\u003eAspergillus flavus\u003c/em\u003e than 1-hydroxyphenazine. Similarly, myxin and 1-methoxyphenazine N\u0026prime; 10-oxide demonstrate stronger antimicrobial activities than their corresponding hydroxylated precursors [\u003cspan additionalcitationids=\"CR5 CR6 CR7\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Beyond methylation, alkoxylation with longer alkyl chains can further enhance biological activity. A previous study chemically synthesized a series of 1-n-alkoxyphenazines and 2-n-alkoxyphenazines and evaluated their acaricidal activity. Among them, 2-n-alkoxyphenazine derivatives with three- or four-carbon chains displayed the highest efficacy. Notably, 2-butoxyphenazine exhibited acaricidal activity four times higher than that of methyl parathion, a conventional organophosphate pesticide [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Despite their promising potential as biopesticides, the chemical synthesis of these compounds faces significant challenges, including harsh reaction conditions, the use of organic solvents and noble metal catalysts, and complex waste treatment and catalyst recovery processes [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn contrast, biocatalytic alkylation has gained increasing attention due to its high chemo-, regio-, and stereoselectivity under mild conditions [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In nature, S-adenosyl-L-methionine (SAM)-dependent methyltransferases are versatile biocatalysts capable of precisely transferring methyl groups to specific S, N, O, or C atoms with excellent chemo-selectivity, such as the alkylation of ambident nucleophiles, unsaturated heterocycles [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In the bioalkylation of phenazines, methylation is mainly catalyzed by O-methyltransferase LaphzM and N-methyltransferase PhzM, which utilize SAM as the methyl donor to synthesize compounds such as 1-methoxyphenazine, 1-methoxyphenazine N\u0026prime; 10-oxide, myxin, pyocyanin [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Recent studies have further shown that many methyltransferases can catalyze not only methylation but also other alkylation reactions (such as ethylation, allylation, propylation) if the necessary SAM analogues are available [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan additionalcitationids=\"CR19 CR20\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. For example, ethyl vanillin, 3\u0026prime;-O-ethylluteolin and 4-allyloxy-3-hydroxybenzaldehyde have been synthesized using halide methyltransferase and SAM-dependent methyltransferase (HMT-MT) cascade systems. In these systems, HMT is responsible for the synthesis of alkylated SAH from S-adenosyl-L-homocysteine (SAH) and alkyl iodides, while MT catalyzes mono-ethylation or mono-allylation [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Meanwhile, the similar strategy of \u0026ldquo;replacing the SAM with its fluorinated SAM analogues\u0026rdquo; has been developed for biocatalytic fluoroalkylation [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Novel and stable fluorinated SAM analogues, such as Te-adenosyl-L-(fluoromethyl) homotellurocysteine (FMeTeSAM), fluoro decarboxyl SAM (F-dcSAM) and fluoroethyl Se-adenosyl-L-selenomethionine (FEt-SeAM) have been synthesized and shown to be accepted by certain methyltransferases, enabling the transfer of fluoromethyl groups to oxygen, nitrogen, sulfur, and some carbon nucleophiles [\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In recent years, fluorine has emerged as a privileged element in drug and agrochemical development. Compounds such as afloqualone, sevoflurane, fluticasone, fleroxacin, flutropium bromide, florbetapir-fluorine-18 exemplify how fluorine substitution can enhance metabolic stability, bioavailability, lipophilicity and cell permeability [\u003cspan additionalcitationids=\"CR29 CR30 CR31 CR32 CR33\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Most methods for introducing fluorine into organic molecules rely on chemical catalysis using Lewis acids, organic molecules, or transition metals [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], whereas enzymatic fluorination remains rare and challenging [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. To date, no fluorinated phenazine natural products have been isolated, nor have biosynthetic routes for fluorinated phenazine derivatives been reported. More broadly, the biosynthesis of alkoxylated phenazine derivatives\u0026mdash;particularly those with ethyl, propyl, and fluoromethyl groups\u0026mdash;remains largely unexplored [\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], which limits the development and application of these valuable compounds.\u003c/p\u003e \u003cp\u003eIn this article, enzymatic cascade systems and \u003cem\u003ede novo\u003c/em\u003e pathway engineering were developed for synthesizing alkoxylated phenazines. We first constructed HMT-MT cascade systems using halide methyltransferases (HMTs) and O-methyltransferase LaphzM or monooxygenase NaphzNO1. These systems successfully catalyzed the alkoxylation of 1-hydroxyphenazine and 2-hydroxyphenazine using alkyl iodides and fluoro(iodo)methane as alkyl/fluoromethyl donors. To enable direct microbial production without exogenous HMTs-dependent alkylation, \u003cem\u003ede novo\u003c/em\u003e biosynthetic pathways for 1-ethoxyphenazine and 2-ethoxyphenazine were engineered in \u003cem\u003eP. chlororaphis\u003c/em\u003e by introducing the direct sulfurylation pathway, although the yields remained low. Building on these results, whole-cell cascade systems were developed for efficient production of alkylated phenazines with acaricidal and fungicidal potential. These results provide a foundation for the biocatalytic synthesis of alkylated and fluorinated phenazines with promising agricultural applications.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStrains, plasmids and chemicals\u003c/h2\u003e \u003cp\u003eAll host strains and plasmids used in this study were preserved in our laboratory. \u003cem\u003eEscherichia coli\u003c/em\u003e DH5α was employed as the cloning host for gene manipulation, while \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3) served as the expression host for recombinant protein production. The vector pET-28a was utilized for gene cloning and expression. Detailed information regarding strains and plasmids is provided in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Restriction enzymes and DNA polymerase were sourced from Thermo Fisher Scientific (Waltham, MA, USA) and Takara Biomedical Technology (Dalian, China), respectively. A one-step cloning kit was obtained from Vazyme Biotech (Nanjing, China). All chemical reagents were purchased from Macklin Biochemical Co., Ltd. (Shanghai, China) and were of analytical grade unless otherwise specified.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eOverexpression and purification of enzymes\u003c/h3\u003e\n\u003cp\u003eLaphzM, HMT1, HMT2 and NaphzNO1 genes and PCR primers were chemically synthesized by Tsingke Biotechnology Co., Ltd. (Qingdao, China). LaphzM, HMT1, HMT2, NaphzNO1 were subcloned into the EcoRI/HindIII site of the pET28a (Novagen, Germany) expression vector, resulting in plasmids named pET28a-LaphzM, pET28a-HMT1, pET28a-HMT2, pET28a-NaphzNO1, respectively. And six his residues were fused at the N-terminus of every target protein. Then, the recombinant plasmids were introduced into \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3) for heterologous expression after verification of the sequences. The detailed information of plasmids, primers and genes were listed in Supplementary Table S2 and Table S3.\u003c/p\u003e \u003cp\u003eThe confirmed \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3) harboring LaphzM/HMT1/HMT2/NaphzNO1 was cultivated overnight at 37 ℃ in LB medium with 50 \u0026micro;g/mL kanamycin, respectively. Then 500 \u0026micro;L of the seed cultures transferred into 50 mL LB media containing appropriate antibiotics. When the OD600 of the culture broth reached 0.6\u0026ndash;0.8, 0.1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) was added to the cells to induce the protein expression at 30 ℃ for 6 h. And then the cells were collected by centrifugation (8000 rpm, 10 min, 4 ℃), the resulting cell pellets were resuspended in 30 mL lysis buffer (25 mM HEPES, pH 7.5, 0.5 M NaCl), lysed by homogenization on ice. Cellular debris was removed by centrifugation (10000 rpm, 10 min, 4 ℃), the supernatant was incubated with 0.5 mL of Ni-NTA agarose resin at 4\u0026deg;C for 1 h, and loaded onto a gravity flow column. The proteins were washed with washing buffer (20 mM HEPES, pH 7.5, 300 mM NaCl and 20 mM imidazole) and elution buffer (20 mM HEPES, pH 7.5, 300 mM NaCl, and 150 mM imidazole). Protein expression and purification were confirmed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, 12%). And purified proteins were concentrated using Amicon Ultra filters (6000 rpm, 30 min, 4 ℃). The final proteins were frozen at -80 ℃ for further use.\u003c/p\u003e\n\u003ch3\u003eGeneral procedure for the enzymatic reactions\u003c/h3\u003e\n\u003cp\u003e \u003cem\u003eIn vitro\u003c/em\u003e ethylation and propylation of phenazines catalyzed by HMT1 and LaphzM: the reaction mixtures contained 100 mM phosphate buffer (pH 8.0), 1 mM phenazine derivatives, 6 mM ethyl iodide (EtI) or propyl iodide (PrI), 50 \u0026micro;M SAH and purified enzymes (0.1 mg/mL HMT1 and LaphzM, respectively) in a final volume of 100 \u0026micro;L. Activity assays, initiated by the addition of enzymes, were performed at 30 ℃ and 220 rpm overnight. At least three independent replicates were performed for each assay. 100 \u0026micro;L methanol was added to stop the reaction. The supernatants were filtered, and substrate conversion was determined using high-performance liquid chromatography (HPLC) and high- resolution mass spectrometer (HR-MS).\u003c/p\u003e \u003cp\u003eEthylated phenazine \u003cem\u003eN\u003c/em\u003e-oxides catalyzed by HMT1-LaphzM-NaphzNO1 cascade systems: the reaction mixture (150 \u0026micro;L) was composed of 100 mM phosphate buffer (pH 8.0), 1 mM phenazine derivatives, 6 mM ethyl iodide (EtI), 50 \u0026micro;M SAH, purified enzymes (0.1 mg/mL HMT1, LaphzM, and NaphzNO1, respectively), 100 \u0026micro;M flavin adenine dinucleotide (FAD), and 100 \u0026micro;M nicotinamide adenine dinucleotide (NADH). The first stage of the \u0026ldquo;two-step one-pot\u0026rdquo; was performed in a 100 \u0026micro;L reaction mixture containing 100 mM phosphate buffer (pH 8.0), 1 mM phenazine derivatives, purified enzymes (NaphzNO1), 100 \u0026micro;M FAD, and 100 \u0026micro;M NADH. After 1 h, purified enzymes (HMT1 and LaphzM), 6 mM EtI and 50 \u0026micro;M SAH were added for the subsequent alkylation of phenazine N-oxides at 30 ℃ and 220 rpm for overnight, and then the reactions were stopped by adding 150 \u0026micro;L of methanol. All assays were performed in triplicate. The reaction mixtures were centrifuged to collect the supernatant, and aliquots were analyzed by HPLC and HR-MS.\u003c/p\u003e \u003cp\u003eFluoromethylated phenazines catalyzed by HMT2 and LaphzM: reactions were performed according to the methods of ethylated and propylated phenazine derivatives. The reaction mixture (100 \u0026micro;L) contained HMT2 and LaphzM (or other modified enzymes), 1 mM phenazine derivatives, 6 mM fluoro(iodo)methane (FMeI), 50 \u0026micro;M SAH in 100 mM phosphate buffer, pH 8.0. At least three independent replicates were performed for each assay. The reactions were stopped by adding methanol (100 \u0026micro;L), and the precipitated protein was filtered before analysis via HPLC and HR-MS.\u003c/p\u003e\n\u003ch3\u003eGene overexpression and fermentation\u003c/h3\u003e\n\u003cp\u003eGene knockout and overexpression in \u003cem\u003eP. chlororaphis\u003c/em\u003e H18 were carried out via homologous recombination with the plasmid pK18mobsacB, as described previously. For fermentation, \u003cem\u003eP. chlororaphis\u003c/em\u003e H18 and its derivatives were first grown in 3 mL of KB medium at 30\u0026deg;C for 24 h with shaking at 200 rpm as seed cultures. Subsequently, 1 mL of the seed culture was inoculated into 50 mL of PPM medium (22 g/L tryptone, 20 g/L glucose, 5 g/L KNO₃, pH 7.0) in 250 mL flasks and incubated at 30\u0026deg;C for 48 h with shaking at 200 rpm. When required, kanamycin (100 \u0026micro;g/mL) or ampicillin (100 \u0026micro;g/mL) was added to the media.\u003c/p\u003e\n\u003ch3\u003eWhole-cell cascade reactions\u003c/h3\u003e\n\u003cp\u003eIn one-pot enzymatic cascades, HMT1 catalyzes the conversion of SAH and alkyl halides into alkylated SAH, which subsequently serves as a substrate for methyltransferases to alkylated phenazine analogues. This process simultaneously regenerates SAH, enabling its continuous recycling within the reaction system. Therefore, whole cell reactions were prepared according to the established protocol, with the addition of a phosphate buffer washing step. The whole-cell reactions were performed at a final volume of 5 mL. The \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3) cells containing enzymes were resuspended in phosphate buffer (pH 8.0) containing phenazine derivatives, EtI, SAH. And then the reactions were incubated in a shaking incubator at 30 ℃, 200 rpm for overnight. The effects of HMT1/LaphzM ratio (1:2\u0026ndash;4:1), and biocatalyst loading (10\u0026ndash;40 g/L) were investigated. After biotransformation, the samples were thoroughly extracted by ethyl acetate. Subsequently, the organic phases were combined and concentrated under reduced pressure. The resultant residue was dissolved in methanol (1 mL) for HPLC analysis.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAnalytical Methods\u003c/h2\u003e \u003cp\u003eAt the end of the reaction, methanol was added to the sample to stop the reaction. Subsequently, the mixtures were centrifuged at 12000g for 5 min. The supernatant was filtered through a nylon membrane filter (0.22 \u0026micro;m) and analyzed with HPLC and detected at 250 nm under the following conditions: an Agilent 5TC C18 (2) (250\u0026times;4.6 mm) column was eluted with acetonitrile and water (0.1% (v/v) trifluoroacetic acid). HPLC procedure was performed at 1 mL/min with water and acetonitrile (v/v): 30\u0026ndash;50% acetonitrile from 0 to 5 min, 50% acetonitrile from 5 to 10 min, 50\u0026ndash;70% acetonitrile from 10 to 15 min, 70\u0026ndash;80% acetonitrile from 15 to18 min, 80% acetonitrile from 18 to 21 min, 80\u0026thinsp;\u0026minus;\u0026thinsp;30% acetonitrile from 21 to 23 min, 30% acetonitrile from 23 to 29 min. Liquid chromatography high-resolution mass spectrometry (LC-HR-MS) analysis was performed with an Agilent Eclipse Plus C18 column (4.6\u0026times;100 mm) on an Agilent Technologies 6520 Accurate-Mass Q-TOF LC-MS instrument.\u003c/p\u003e \u003cp\u003eThe reaction system described above was isolated and purified to obtain a sufficient amount of the phenazine derivatives for NMR spectroscopic analysis. The reaction system was extracted with an equal volume of ethyl acetate three times, and ethyl acetate was collected and evaporated under vacuum. The crude extract was dissolved in methanol and subsequently purified using semipreparative HPLC equipped with a reversed-phase C18 column (250 mm \u0026times; 50 mm, 8 \u0026micro;m). The purification procedure was similar to that described with analytical methods of HPLC (solvent A: water; solvent B: acetonitrile; flow rate: 10 mL/min, gradient: 30\u0026ndash;85% B in 25 min followed by 100% B for 5min). The purified compounds were further characterized by NMR spectroscopy (Bruker AVANCE III 400, equipped with a 5 mm PABBO probe).\u003c/p\u003e \u003cp\u003eSpectroscopic data of prepared phenazine derivatives. \u003cem\u003e1-ethoxyphenazine\u003c/em\u003e (\u003cb\u003e1a\u003c/b\u003e). Green-yellow powder. HR-ESI-MS (m/z): 225.1024 [M\u0026thinsp;+\u0026thinsp;H] \u003csup\u003e+\u003c/sup\u003e, calculated for C\u003csub\u003e14\u003c/sub\u003eH\u003csub\u003e13\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO, 225.1022. \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e, δ\u003csub\u003eH\u003c/sub\u003e): δ 8.40 (dd, J\u0026thinsp;=\u0026thinsp;8.4, 1.8 Hz, H-9), 8.23 (dd, J\u0026thinsp;=\u0026thinsp;8.4, 1.8 Hz, H-6), 7.89\u0026ndash;7.77 (m, H-4, H-7, H-8), 7.74 (dd, J\u0026thinsp;=\u0026thinsp;8.9, 7.5 Hz, H-3), 7.07 (dd, J\u0026thinsp;=\u0026thinsp;7.5, 1.1 Hz, H-2), 4.43 (q, J\u0026thinsp;=\u0026thinsp;7.0 Hz, H-12), 1.69 (t, J\u0026thinsp;=\u0026thinsp;7.0 Hz, H-11). \u003csup\u003e13\u003c/sup\u003eC NMR (150 MHz, CDCl\u003csub\u003e3,\u003c/sub\u003e δ\u003csub\u003eC\u003c/sub\u003e): 154.4 (C-1), 144.2 (C-4a), 143.3 (C-9a), 142.2 (C-5a), 137.1 (C-10a), 130.8 (C-7), 130.7 (C-3), 130.4 (C-9), 130.0 (C-8), 129.2 (C-6), 121.1 (C-4), 107.3 (C-2), 64.9 (C-11), 14.5 (C-12).\u003c/p\u003e \u003cp\u003e \u003cem\u003e1-ethoxyphenazine N\u0026prime;10-oxide\u003c/em\u003e (\u003cb\u003e1b\u003c/b\u003e). Golden-yellow powder. HR-ESI-MS (m/z): 241.0966 [M\u0026thinsp;+\u0026thinsp;H] \u003csup\u003e+\u003c/sup\u003e, calculated for C\u003csub\u003e14\u003c/sub\u003eH\u003csub\u003e13\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, 241.0972. \u0026sup1;H NMR (400 MHz, CDCl₃, δ\u003csub\u003eH\u003c/sub\u003e): δ 8.71\u0026ndash;8.59 (m, H-9), 8.20 (d, J\u0026thinsp;=\u0026thinsp;8.5 Hz, H-6), 7.89\u0026ndash;7.76 (m, H-7, H-8), 7.75\u0026ndash;7.64 (m, H-3, H-4), 7.00 (d, J\u0026thinsp;=\u0026thinsp;7.9 Hz, H-2), 4.27 (q, J\u0026thinsp;=\u0026thinsp;6.9 Hz, H-12), 1.66 (t, J\u0026thinsp;=\u0026thinsp;6.9 Hz, H-11).\u003c/p\u003e \u003cp\u003e \u003cem\u003e1-fluoromethoxyphenazine\u003c/em\u003e (\u003cb\u003e1c\u003c/b\u003e). Golden-yellow powder. HR-ESI-MS (m/z): 229.0763 [M\u0026thinsp;+\u0026thinsp;H] \u003csup\u003e+\u003c/sup\u003e, calculated for: C\u003csub\u003e13\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eOFN\u003csub\u003e2\u003c/sub\u003e, 229.0766. \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, CDCl₃, δ\u003csub\u003eH\u003c/sub\u003e): δ 8.44\u0026ndash;8.34 (m, H-9), δ 8.31\u0026ndash;8.22 (m, H-6), δ 7.93\u0026ndash;7.84 (m, H-7, H-8), δ 8.03 (d, J\u0026thinsp;=\u0026thinsp;8.8 Hz, H-4), δ 7.79 (dd, J\u0026thinsp;=\u0026thinsp;8.8, 7.6 Hz, H-3), δ 7.49 (d, J\u0026thinsp;=\u0026thinsp;7.5 Hz, H-2), δ 6.13 (d, J\u0026thinsp;=\u0026thinsp;53.7 Hz, H-11). \u003csup\u003e13\u003c/sup\u003eC NMR (150 MHz, CDCl₃, δ\u003csub\u003eC\u003c/sub\u003e): 151.9 (d, J\u0026thinsp;=\u0026thinsp;3.2 Hz, C-1), 143.9 (C-4a), 143.5 (C-5a), 142.6 (C-9a), 136.5 (d, J\u0026thinsp;=\u0026thinsp;1.5 Hz, C-10a), 131.2 (C-7), 130.8 (C-8), 130.1 (C-3, C-6), 129.4 (C-9), 124.6 (C-4), 112.3 (d, J\u0026thinsp;=\u0026thinsp;2.2 Hz, C-2), 100.9 (d, J\u0026thinsp;=\u0026thinsp;221.4 Hz, C-11).\u003c/p\u003e \u003cp\u003e \u003cem\u003e2-fluoromethoxyphenazine\u003c/em\u003e (\u003cb\u003e2b\u003c/b\u003e). Yellow powder. HR-ESI-MS (m/z): 229.0766 [M\u0026thinsp;+\u0026thinsp;H] \u003csup\u003e+\u003c/sup\u003e, calculated for: C\u003csub\u003e13\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eOFN\u003csub\u003e2\u003c/sub\u003e, 229.0766. \u0026sup1;H NMR (400 MHz, CDCl₃) δ 8.29\u0026ndash;8.19 (m, H-1, H-4, H-6, H-9), 7.92\u0026ndash;7.77 (m, H-7, H-8), 7.62 (dd, J\u0026thinsp;=\u0026thinsp;9.5, 2.8 Hz, H-3), 5.96 (d, J\u0026thinsp;=\u0026thinsp;53.4 Hz, H-11).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eDesign of the enzymatic cascades for alkoxylation of phenazines.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo date, the chemical synthesis of alkylated phenazines, particularly their mono-N-oxide or di-N-oxide derivatives, has faced considerable challenges. These include reliance on precious metal catalysts (Pd (II)-brettphos), harsh oxidative conditions using reagents like mCPBA, high temperatures and consumption of non-renewable or hazardous raw materials (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Furthermore, the generation of toxic intermediates and waste throughout the process raises environmental and safety concerns, limiting the sustainability and scalability of these routes. In contrast, enzyme-catalyzed reactions typically operate under mild aqueous conditions with high catalytic efficiency. Currently, the ability of HMT from \u003cem\u003eArabidopsis thaliana\u003c/em\u003e to form SAM analogues from SAH and alkyl iodide has been identified [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Its V140T variant (namely HMT1) could accept ethyl-, propyl-, and allyl iodide to produce the corresponding SAM analogs. And HMTs were utilized in one-pot cascade with the reported O-, N-, C-methyltransferases (such as, EgtD, SDMT, MrsA, TAMT, COMT, \u003cem\u003eetc\u003c/em\u003e.) to synthesize alkylated chemicals [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Meanwhile the ability of HMT from \u003cem\u003eBurkholderia xenovorans\u003c/em\u003e (protein family 05724, namely HMT2) to form fluorinated SAM from SAH and fluoromethyl iodide has also been identified [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Fluorinated SAM can serve as a substrate for SAM-dependent methyltransferases, enabling enzyme-catalyzed fluoromethylation. In addition, LaphzM gene from \u003cem\u003eLysobacter antibioticus\u003c/em\u003e OH13 encodes a SAM-dependent O-methyltransferase which is responsible for monomethoxy and dimethoxy formation in phenazines, such as myxin, 1-methoxyphenazine, 1-methoxyphenazine \u003cem\u003eN\u003c/em\u003e\u0026prime; 10-oxide [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. LaphzM demonstrates broad substrate flexibility, enabling the O-methylation of diverse phenazine scaffolds. According to the above information, HMT1 and HMT2 were utilized in one-pot cascades with LaphzM to synthesize alkoxylated phenazine derivatives in this study, respectively. Halide methytransferases (HMTs) and methyltransferases (MTs) cascade reactions for alkoxylated phenazine derivatives were designed as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eB.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSynthesis of ethylated and propylated phenazines by HMT1-LaphzM cascade.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFirst of all, we cloned the codon-optimized HMT1 and LaphzM genes into \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3) and obtained the soluble proteins. In a next step we explored whether LaphzM could catalyze ethylation of 1-hydroxyphenazine (\u003cb\u003e1\u003c/b\u003e) using in situ-generated ethyl-SAH (SAE) as a co-substrate (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The cascade reaction containing 1 mM 1-hydroxyphenazine, 50 \u0026micro;M SAH, 6 mM EtI, 0.1 mg/mL HMT1 and 0.1 mg/mL LaphzM in 100 mM phosphate buffer (pH 8.0) was incubated at 30 ℃. After 7 h, 1-ethoxyphenazine (\u003cb\u003e1a\u003c/b\u003e) was synthesized in 96% yield, as confirmed by HPLC and HR-MS analysis. The MS spectrum of 1-ethoxyphenazine (\u003cb\u003e1a\u003c/b\u003e) showed significant peak of [M\u0026thinsp;+\u0026thinsp;H] \u003csup\u003e+\u003c/sup\u003e (m/z 225.1024, calculated for C\u003csub\u003e14\u003c/sub\u003eH\u003csub\u003e13\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO, 225.1022), suggesting its molecular formula of C\u003csub\u003e14\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The target compound was subsequently isolated from the cascade reaction, presenting as a green-yellow powder. Its structure was further characterized and confirmed through detailed NMR spectroscopy (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The 1D and 2D NMR data (COSY, HSQC, HMBC) consistently support the structural assignment of the compound as 1-ethoxyphenazine (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e-S6 and Table S4). The HMT1-LaphzM cascade system was also employed to catalyze the conversion of 2-hydroxyphenazine into 2-ethoxyphenazine (\u003cb\u003e2a1\u003c/b\u003e, 21% yield). The target product was successfully identified and analyzed using HR-MS (Figure S7). These results indicate that the O-ethylation activity of LaphzM is relatively flexible.\u003c/p\u003e \u003cp\u003eThis approach, which utilizes propyl-SAH as a substitute for ethyl-SAH, was successfully extended to the synthesis of propylated derivatives. When PrI and 2-hydroxyphenazine were used as substrates, 2-propoxyphenazine (\u003cb\u003e2a2\u003c/b\u003e) was detected by HPLC with a yield of 12% within 7 h. HR-MS analysis of 2-propoxyphenazine (\u003cb\u003e2a2\u003c/b\u003e) revealed a prominent [M\u0026thinsp;+\u0026thinsp;H] ⁺ peak at m/z 239.1181 (calculated for C₁₅H₁₅N₂O, 239.1179), consistent with the molecular formula C₁₅H₁₄N₂O (Figure S8). The lower yield compared to ethylation may be attributed to increased steric hindrance from the larger alkyl group. And when allyl iodide, 2-iodopropane and 1-iodobutane were added to the reaction system to replace PrI, no corresponding products were detected by HPLC and HR-MS.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSynthesis of ethylated phenazine N-oxides by HMT1-LaphzM-NaphzNO1 cascade.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eLaphzM can also accept mono-oxide or dioxide phenazines as substrates, suggesting that HMT1-LaphzM cascade systems could be used to synthesize ethylated N-oxide phenazines. However, these oxidized substrates are expensive and less accessible [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Previous studies showed that NaphzNO1 can convert 1-hydroxyphenazine to 1-hydroxyphenazine \u003cem\u003eN\u003c/em\u003e\u0026prime;10-oxide [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. But, the N-oxidation activity of NaphzNO1 is relatively selective, requiring a free hydroxy group at carbon-1 position of the phenazine ring. Additionally, the engineered \u003cem\u003ePseudomonas\u003c/em\u003e strains still exhibit metabolic flux constraints in the biosynthesis of 1-hydroxyphenazine N\u0026prime;10-oxide and alkylated analogues [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. To address these limitations, we proposed that ethylated mono-oxide could be produced by controlling the reaction sequence, and the \u0026ldquo;two-step one pot\u0026rdquo; synthetic strategy was designed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). First, NaphzNO1 was overexpressed in \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3), and the soluble proteins were obtained. NaphzNO1 was incubated with 1-hydroxyphenazine, FAD and NADH in phosphate buffer at 30 ℃ for 1 h to produce 1-hydroxyphenazine-N\u0026prime;10-oxide. Subsequently, HMT1-LaphzM cascade systems were added to the same reaction mixture with EtI and SAH. Finally, 1-ethoxyphenazine-N\u0026prime;10-oxide (\u003cb\u003e1b\u003c/b\u003e) was detected in 72% yield by HPLC and HR-MS/MS (Figure S9). The structure was further confirmed by NMR spectroscopy (Figure S10).\u003c/p\u003e \u003cp\u003e \u003cb\u003eSynthesis of fluoromethylated phenazines by HMT2-LaphzM cascade.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe approach of substituting ethyl- and propyl-SAH with fluorinated SAM has been effectively employed to synthesize fluoromethylated phenazines (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003e). HMT2 was initially overexpressed in \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3), yielding soluble protein for further use. The HMT2-LaphzM cascade reaction contained 1 mM 1-hydroxyphenazine, 50 \u0026micro;M SAH, 6 mM FMeI, 0.1 mg/mL HMT2 and 0.1 mg/mL LaphzM in 100 mM phosphate buffer (pH 8.0), incubated at 30 ℃ for 7 h. HPLC and LC-MS analysis indicated that a fluoromethylated 1-hydroxyphenazine compound (\u003cb\u003e1c\u003c/b\u003e, 98% yield) might be produced in the reaction system. The HR-MS spectrum showed significant peak of [M\u0026thinsp;+\u0026thinsp;H] \u003csup\u003e+\u003c/sup\u003e (m/z 229.0763, calculated for C\u003csub\u003e13\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eFN\u003csub\u003e2\u003c/sub\u003eO, 229.0766), suggesting its molecular formula of C\u003csub\u003e13\u003c/sub\u003eH\u003csub\u003e9\u003c/sub\u003eFN\u003csub\u003e2\u003c/sub\u003eO (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). The target compound was successfully isolated from the cascade reaction. It was also obtained as yellow powder, and its structure was unambiguously identified as 1-fluoromethoxyphenazine by NMR spectroscopy (Figure S11-S16 and Table S5). Similarly, 2-hydroxyphenazine was converted to 2-fluoromethoxyphenazine (\u003cb\u003e2b\u003c/b\u003e) in 46% yield using the HMT2-LaphzM cascade system. HR-MS showed a peak of [M\u0026thinsp;+\u0026thinsp;H] ⁺ (m/z 229. 0766), consistent with the molecular formula C₁₃H₁₀FN₂O (calculated for 229.0766), confirming the presence of one fluorine atom. Comparison with the data for 1-fluoromethoxyphenazine reveals a similar set of signals but with distinct chemical shifts and coupling patterns, particularly in the aromatic region, suggesting an alternative substitution pattern. The number of protons and the magnitude of the H-F coupling (J\u0026thinsp;=\u0026thinsp;53.4 Hz) are consistent with a fluoromethoxy substituent. The combined HR-MS and NMR data, especially the distinctive fluorine-proton coupling and aromatic substitution pattern, allow the compound to be identified as 2-fluoromethoxyphenazine (Figure S17 and S18).\u003c/p\u003e \u003cp\u003e \u003cb\u003eDe novo\u003c/b\u003e \u003cb\u003ebiosynthesis for alkoxylated phenazines in\u003c/b\u003e \u003cb\u003ePseudomonas chlororaphis\u003c/b\u003e \u003cb\u003eH18.\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003ePseudomonas\u003c/em\u003e species have emerged as suitable chassis hosts for the synthesis of phenazine derivatives, owing to their rapid growth, facile genetic manipulation, and well-established fermentation systems. In particular, \u003cem\u003eP. chlororaphis\u003c/em\u003e H18 has been demonstrated to biosynthesize various PCA-based derivatives, including 1-hydroxyphenazine, 2-hydroxyphenazine, 1-methoxyphenazine and 1-hydroxyphenazine-Nˊ10-oxide [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Notably, while 2-ethoxyphenazine (\u003cb\u003e2a1\u003c/b\u003e) and 2-propoxyphenazine (\u003cb\u003e2a2\u003c/b\u003e) exhibit promising acaricidal activity and are considered potential pesticide candidates, their microbial synthesis has not yet been achieved [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Here, we aimed to achieve \u003cem\u003ein vivo\u003c/em\u003e synthesis of SAM analogues and subsequent alkyl chain transfer onto 1-hydroxyphenazine or 2-hydroxyphenazine in \u003cem\u003eP. chlororaphis\u003c/em\u003e H18.\u003c/p\u003e \u003cp\u003eTo circumvent the toxicity of exogenous alkylating agents such as methyl iodide and ethyl iodide, we leveraged the endogenous SAM biosynthetic pathway of \u003cem\u003ePseudomonas\u003c/em\u003e to produce SAM analogues. Previous studies have demonstrated the substrate promiscuity of O-acetylhomoserine sulfhydrolase from \u003cem\u003eS. cerevisiae\u003c/em\u003e (ScOAHS), which enables the \u003cem\u003ein vivo\u003c/em\u003e production of methionine analogues from organic thiols [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. To harness this property, we introduced the direct sulfurylation pathway, consisting ScOAHS and O-acetyltransferase (ScMET2) from \u003cem\u003eS. cerevisiae\u003c/em\u003e, into \u003cem\u003eP. chlororaphis\u003c/em\u003e. This allowed the production of ethionine from ethanethiol, which were then converted to SAE by the endogenous methionine adenosyltransferase (MAT).\u003c/p\u003e \u003cp\u003eWe therefore introduced ScOAHS and LaphzM into two \u003cem\u003eP. chlororaphis\u003c/em\u003e strains: a high 1-hydroxyphenazine-producing strain (\u003cem\u003eP. chlororaphis\u003c/em\u003e H18-1-8) and a 2-hydroxyphenazine-producing strain (\u003cem\u003eP. chlororaphis\u003c/em\u003e H18), yielding \u003cem\u003eP. chlororaphis\u003c/em\u003e H18-1-8n and H18n, respectively [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The cultures were then supplemented with ethanethiol (EtSH, 5 mM) in an attempt to produce 1-ethoxyphenazine (\u003cb\u003e1a\u003c/b\u003e) and 2-ethoxyphenazine (\u003cb\u003e2a1\u003c/b\u003e), respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e). However, this approach yielded only trace amounts of 1-ethoxyphenazine (\u003cb\u003e1a\u003c/b\u003e), with 1-methoxyphenazine remaining the predominant product, while 2-ethoxyphenazine (\u003cb\u003e2a1\u003c/b\u003e) production also remained low by LC-MS. We attribute these outcomes to the robust endogenous SAM biosynthetic network in \u003cem\u003eP. chlororaphis\u003c/em\u003e H18, which preferentially channels endogenous methionine toward SAM synthesis and subsequent methylation, outcompeting the pathway for methionine analogue incorporation. Consequently, enhancing the production of alkylated phenazine derivatives will require systematic engineering of the entire SAM metabolic network to redirect flux from endogenous methionine to the desired methionine analogues.\u003c/p\u003e \u003cp\u003e \u003cb\u003eWhole-cell cascade catalysis production of alkoxylated phenazines.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eGiven that insufficient supply of SAM analogues in the recombinant strain may limit 2-ethoxyphenazine (\u003cb\u003e2a1\u003c/b\u003e) and 2-propoxyphenazine (\u003cb\u003e2a2\u003c/b\u003e) production, we explored an alternative whole-cell biocatalytic approach by directly supplying 2-hydroxyphenazine as the precursor. Although the HMT1-LaphzM enzymatic cascade system was successfully developed to produce 2-ethoxyphenazine (\u003cb\u003e2a1\u003c/b\u003e, 45% yield) and 2-propoxyphenazine (\u003cb\u003e2a2\u003c/b\u003e, 12% yield), the product yields limit its broader applicability. Whole-cell cascade catalysis, which utilizes bacterial cells to catalyze chemical transformations, offers advantages such as enhanced robustness, high catalytic efficiency, and the elimination of labor-intensive protein purification steps [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. To address the limitations of the enzymatic system, whole-cell reaction systems were designed to improve the production of alkylated phenazines and analogues. Along with the large quantity of PCA production through microbial fermentation and structure diversity of phenazine derivatives through combinatorial biology, these enable phenazine derivatives to be used as sustainable raw materials to synthesize value-added chemicals [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. In our preliminary research, we constructed strains producing 1-hydroxyphenazine, 2-hydroxyphenazine and other phenazine derivatives [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. And these compounds can be used as starting materials to directly synthesize alkylated phenazine derivatives via whole-cell cascade catalysis. HMT1 and LaphzM were overexpressed in \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3) to construct strain 1 and strain 2, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). At 30 ℃, these strains (ratio 1:1) were then incubated with 1 mM 2-hydroxyphenazine, 100 \u0026micro;M SAH and 6 mM EtI in a 5 mL reaction volume with the cell loading of 10 g CDW (cell dry weight) per L. After 7 h, the substrates were successfully converted into 39.5 mg/L 2-ethoxyphenazine (\u003cb\u003e2a1\u003c/b\u003e, ~\u0026thinsp;17.5% yield), but most of the substrates could not be converted. We proposed that the efficient production of 2-ethoxyphenazine (\u003cb\u003e2a1\u003c/b\u003e) could be realized by controlling the ratio of two kinds of \u003cem\u003eE. coli\u003c/em\u003e cells. And then, we conducted reactions using 10 g CDW per L strain 1 and strain 2 (at ratios 2:1 and 1:2, respectively), along with 1 mM 2-hydroxyphenazine, 100 \u0026micro;M SAH and 6 mM EtI in a 5 mL reaction volume. Results indicated that when the ratio of strain 1 to strain 2 cells was 2:1, the yield of 2-ethoxyphenazine (\u003cb\u003e2a1\u003c/b\u003e) was increased, which may be attributed to the relatively low catalytic efficiency of HMT1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). To further enhance the economic feasibility of the process, the one-pot catalytic reactions were systematically optimized under various conditions, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e5\u003c/span\u003eB. The yield of 2-ethoxyphenazine (\u003cb\u003e2a1\u003c/b\u003e) was evaluated at different cell densities (10\u0026ndash;40 g CDW per L), with a 3:1 ratio of strain 1 to strain 2 (30 g CDW per L) demonstrating the highest catalytic efficiency. Under these optimized conditions, 4 mM 2-hydroxyphenazine reacted to form 486.3 mg/L 2-ethoxyphenazine (\u003cb\u003e2a1\u003c/b\u003e, 2.16 mM, 54% yield) within 7 h, which was 12.3-fold higher than that of the original reaction. Furthermore, the whole-cell cascade system was also applied to synthesize 2-propoxyphenazine (\u003cb\u003e2a2\u003c/b\u003e, 218.1 mg/L, 22.8% yield) from 4 mM 2-hydroxyphenazine and PrI, and other phenazine derivatives (Fig.\u0026nbsp;6). NaphzNO1 was overexpressed in \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3) to construct strain 3 (Fig.\u0026nbsp;6C). To facilitate cofactor regeneration, a glucose dehydrogenase (GDH)-coupled NADH recycling system was integrated with NaphzNO1. The combined system achieved the production of 378.2 mg/L (78.4% yield) of 1-ethoxyphenazine-N\u0026prime;10-oxide (\u003cb\u003e1b\u003c/b\u003e). Notably, the reaction proceeded without the need for exogenous addition of FAD or NADH cofactors. However, the overall process yield was limited by the inherent instability of phenazine mono-N-oxides under whole-cell cascade conditions, which led to undesired reduction of the target products.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAs a large family of colored nitrogen-containing heterocyclic compounds, phenazines exhibit a wide range of valuable biological activities, underscoring their potential in medicine and agriculture. In particular, 2-ethoxyphenazine (\u003cb\u003e2a1\u003c/b\u003e) and 2-propoxyphenazine (\u003cb\u003e2a2\u003c/b\u003e) exhibit significant acaricidal activity, positioning them as promising candidate compounds for pesticide development [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Conventional chemical synthesis of 1-n-alkoxyphenazines and 2-n-alkoxyphenazines relies on fossil fuel-derived benzene derivatives, organic solvents, and noble metal catalysts, raising environmental and safety concerns [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In this study, we demonstrated that LaphzM can catalyze alkoxylation of 1-hydroxyphenazine, 2-hydroxyphenazine and other derivatives using SAM analogues. Enzymatic cascade systems were designed to produce six alkoxylated and fluoromethylated phenazines, including three novel compounds confirmed by HR-MS and NMR. Meanwhile, \u003cem\u003ede novo\u003c/em\u003e biosynthetic pathways for 1-ethoxyphenazine (\u003cb\u003e1a\u003c/b\u003e) and 2-ethoxyphenazine (\u003cb\u003e2a1\u003c/b\u003e) were constructed in \u003cem\u003eP. chlororaphis\u003c/em\u003e H18, representing the first report of alkylated phenazine biosynthesis. To enhance production, we adopted a more cost-effective alkyl donor, EtI, in place of EtSH. The whole-cell one-pot catalysis has been developed to rapidly and efficiently convert 2-ethoxyphenazine (\u003cb\u003e2a1\u003c/b\u003e) and 2-propoxyphenazine (\u003cb\u003e2a2\u003c/b\u003e) using microorganism metabolite 2-hydroxyphenazine as the substrate. After the optimization of the reaction conditions, the 2-ethoxyphenazine titers could reach 486.3 mg/L (\u003cb\u003e2a1\u003c/b\u003e, 54% yield) within 7 h, a 12.3-fold improvement over the initial reaction. The whole-cell system was also successfully applied to synthesize 2-propoxyphenazine (\u003cb\u003e2a2\u003c/b\u003e, 218.1 mg/L, 22.8% yield) and 1-ethoxyphenazine-N\u0026prime;10-oxide (\u003cb\u003e1b\u003c/b\u003e, 378.2 mg/L, 78.4% yield). The process is conducted under mild conditions, occurring in a neutral aqueous solution at room temperature, without the requirement for metal catalysts and high temperature. These features highlight its potential as a sustainable alternative for synthetic applications.\u003c/p\u003e \u003cp\u003eHowever, the reliance on iodolkanes as alkyl donors in aqueous systems raises concerns regarding waste and safety. The high volatility and poor water solubility of alkyl iodides also contribute to the limited efficiency and yield of the biotransformation process investigated in this study. Therefore, developing more sustainable and environmentally benign alkyl donors is essential for advancing this green methodology. And future work should explore the family of halide methyltransferases to accept different alkylation reagents. Rational design and directed evolution strategies can be combined to accurately optimize key amino acid residues in halide methyltransferases and methyltransferases through structural and functional analysis. Computation simulations and high-throughput screening should be integrated to further accelerate the engineering of enzyme activity and substrate specificity [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Finally, enhancing the reusability of enzymatic components through advanced immobilization techniques and scaling up reaction processes will be crucial for significantly enhancing production yields.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study reported the green biocatalytic systems of alkylated phenazines through two complementary strategies: enzymatic cascades (HMT-MTs systems and other modifying enzymes) and \u003cem\u003ede novo\u003c/em\u003e biosynthetic pathway engineering. Using enzymatic cascades, we successfully synthesized six alkoxylated phenazine derivatives, three of which were identified as novel structures (1-ethoxyphenazine-N\u0026prime;10-oxide (\u003cb\u003e1b\u003c/b\u003e), 1-fluoromethoxyphenazine (\u003cb\u003e1c\u003c/b\u003e), and 2-fluoromethoxyphenazine (\u003cb\u003e2b\u003c/b\u003e)) based on NMR and SciFinder analysis. And we constructed the \u003cem\u003ede novo\u003c/em\u003e biosynthetic pathway of ethylated phenazines in \u003cem\u003eP. chlororaphis\u003c/em\u003e for the direct production of ethylated phenazines. Moreover, whole-cell cascade biocatalytic synthesis ways to alkoxylated phenazines have been established and optimized. This study offers a sustainable and highly efficient alternative to conventional chemical manufacturing processes of alkoxylated phenazines, presenting a promising green solution.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePCA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ephenazine-1-carboxylic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSAH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eS-adenosyl-L-homocysteine\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSAM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eS-adenosyl-L-methionine\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emethyltransferase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHMT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehalide methyltransferase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFMeTeSAM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTe-adenosyl-L-(fluoromethyl) homotellurocysteine\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eF-dcSAM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003efluoro decarboxyl SAM\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFEt-SeAM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003efluoroethyl Se-adenosyl-L-selenomethionine\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEtI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eethyl iodide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIPTG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eisopropyl-β-D-1-thiogalactopyranoside\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePrI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epropyl iodide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHPLC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehigh-performance liquid chromatography\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHRMS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehigh- resolution mass spectrometer\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFAD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eflavin adenine dinucleotide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNADH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003enicotinamide adenine dinucleotide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFMeI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003efluoro(iodo)methane\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLC-HRMS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eliquid chromatography high-resolution mass spectrometry\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHRESIMS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehigh resolution electrospray ionization mass spectrometry\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSAE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eethyl-SAH\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003emCPBA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e3-chloroperbenzoic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEtSH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eethanethiol\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003eNo applicable.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNo applicable.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eNo applicable.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by grant from the Scientific Research Starting Foundation of Shandong University of Technology (4041/422046), the Natural Science Foundation of Shandong Province, China (ZR2024QC398).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eF.H. and W.H. conceived the project, F.H. and P.X. supervised the project, F.H., S.Z. and C.W. performed the experiments and analyzed the data; W.H. and F.H. wrote the manuscript; all authors checked and modified the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eNo applicable.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets analysed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKrishnaiah M, de Almeida NR, Udumula V, et al. Synthesis, biological evaluation, and metabolic stability of phenazine derivatives as antibacterial agents. Eur J Med Chem. 2018;143:936\u0026ndash;47.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSerafim B, Bernardino AR, Freitas F, Torres CAV. Recent developments in the biological activities, bioproduction, and applications of \u003cem\u003ePseudomonas\u003c/em\u003e spp. phenazines. Molecules. 2023;28(3):1368.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThalhammer KO, Newman DK. A phenazine-inspired framework for identifying biological functions of microbial redox-active metabolites. Curr Opin Chem Biol. 2023;75:102320.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuttenberger N, Blankenfeldt W, Breinbauer R. Recent developments in the isolation, biological function, biosynthesis, and synthesis of phenazine natural products. Bioorg Med Chem. 2017;25:6149\u0026ndash;66.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYan JJ, Liu WW, Cai JT, et al. Advances in phenazines over the past decade: review of their pharmacological activities, mechanisms of action, biosynthetic pathways and synthetic strategies. Mar Drugs. 2021;19(11):610.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWan YP, Liu HC, Xian M, Huang W. Biosynthetic pathway construction and production enhancement of 1-hydroxyphenazine derivatives in \u003cem\u003ePseudomonas chlororaphis\u003c/em\u003e H18. J Agric Food Chem. 2022;70(4):1223\u0026ndash;31.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBennett MR, Shepherd SA, Cronin VA, Micklefield J. Recent advances in methyltransferase biocatalysis. Curr Opin Chem Biol. 2017;37:97\u0026ndash;106.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSch\u0026uuml;lke KH, Ospina F, H\u0026ouml;rnschemeyer K, Gergel S, Hammer SC. Substrate profiling of anion methyltransferases for promiscuous synthesis of S-adenosylmethionine analogs from haloalkanes. ChemBioChem. 2022;23(4):e202100632.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCross B, Dunn CL, Payne DH, Tipton JD. Synthesis and pesticidal activity of phenazines. II. Alkyl, alkoxy, alkylthio and alkylsulphonyl phenazines. J Sci Food Agric. 1969;20(6):340\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTietze M, Iglesias A, Merisor E, Conrad J, Klaiber I, Beifuss U. Efficient methods for the synthesis of 2-hydroxyphenazine based on the Pd-catalyzed N-arylation of aryl bromides. Org Lett. 2005;7(8):1549\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcKean IJW, Hoskisson PA, Burley GA. Biocatalytic alkylation cascades: Recent advances and future opportunities for late-stage functionalization. ChemBioChem. 2020;21:2890\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBuller R, Lutz S, Kazlauskas RJ, Snajdrova R, Moore JC, Bornscheuer UT. From nature to industry: harnessing enzymes for biocatalysis. Science. 2023;382:eadh8615.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDong M, Horitani M, Dzikovski B, et al. Substrate-dependent cleavage site selection by unconventional radical S-adenosylmethionine enzymes in diphthamide biosynthesis. J Am Chem Soc. 2017;139(16):5680\u0026ndash;3.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOspina F, Sch\u0026uuml;lke KH, Hammer SC. Biocatalytic alkylation chemistry: building molecular complexity with high selectivity. ChemPlusChem. 2022;87(4):e202100454.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOspina F, Sch\u0026uuml;lke KH, Schnutenhaus M, et al. Biocatalytic alkylation of ambident nucleophiles enables selective N-functionalization of heterocycles and late-stage modifications. Angew Chem Int Ed. 2025;64(10):e202510300.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOspina F, Sch\u0026uuml;lke KH, Soler J, Klein A, et al. Selective biocatalytic N-methylation of unsaturated heterocycles. Angew Chem Int Ed. 2022;61(49):e202213056.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang J, Guiza Beltran D, Schacht A, Wright S, Zhang L, Du LM. Functional and structural analysis of phenazine O-methyltransferase LaphzM from \u003cem\u003eLysobacter antibioticus\u003c/em\u003e OH13 and one-pot enzymatic synthesis of the antibiotic myxin. ACS Chem Biol. 2018;13(4):1003\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTang QY, Grathwol CW, Aslan-\u0026Uuml;zel AS, et al. Directed evolution of a halide methyltransferase enables biocatalytic synthesis of diverse SAM analogs. Angew Chem Int Ed. 2021;60(3):1524\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbdelraheem EMM, Thair B, Varela RF, Jockmann E, Popadić D, Hailes HC, et al. Methyltransferases: functions and applications. ChemBioChem. 2022;23(13):e202200212.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiao CS, Seebeck FP. S-adenosylhomocysteine as a methyl transfer catalyst in biocatalytic methylation reactions. Nat Catal. 2019;2(8):696\u0026ndash;701.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGao CY, Yang GY, Ding XW, et al. Engineering of halide methyltransferase BxHMT through dynamic cross-correlation network analysis. Angew Chem Int Ed. 2024;63(19):e202401235.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang GY, Zheng GW, Zeng BB, Xu JH, Chen Q. Engineering of halide methyltransferases for synthesis of SAE and its application in biosynthesis of ethyl vanillin. Mol Catal. 2023;550:113533.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDing WP, Zhou MQ, Li HY, et al. Biocatalytic fluoroalkylation using fluorinated S-adenosyl-L-methionine cofactors. Org Lett. 2023;25(30):5650\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePeng JM, Liao CS, Bauer C, Seebeck FP. Fluorinated S-adenosylmethionine as a reagent for enzyme-catalyzed fluoromethylation. Angew Chem Int Ed. 2021;60(52):27178\u0026ndash;83.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang WR, Zhao HM, Yu NH, Chen F, Dong M. Stable S-adenosylmethionine analogue for enzymatic fluoromethylation. ACS Catal. 2023;13(20):13729\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNeti SS, Wang B, Iwig DF, Onderko EL, Booker SJ. Enzymatic fluoromethylation enabled by the S-adenosylmethionine analog Te-adenosyl-L-(fluoromethyl)homotellurocysteine. ACS Cent Sci. 2023;9(5):905\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu NH, Zhao HM, Wang WR, Dong M. Enzymatic fluoroethylation by a fluoroethyl selenium analogue of S-adenosylmethionine. ACS Catal. 2024;14(8):6211\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMerkel L, Budisa N. Organic fluorine as a polypeptide building element: in vivo expression of fluorinated peptides, proteins and proteomes. Org Biomol Chem. 2012;10(36):7241\u0026ndash;61.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarnes-Seeman D, Beck J, Springer C. Fluorinated compounds in medicinal chemistry: recent applications, synthetic advances and matched-pair analyses. Curr Top Med Chem. 2014;14(7):855\u0026ndash;64.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChaudhari SB, Kumar A, Mankar VH, et al. Diverse role, structural trends, and applications of fluorinated sulphonamide compounds in agrochemical and pharmaceutical fields. Heliyon. 2024;10(12):e32434.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHoff BH, Sundby E. Preparation of pharmaceutical important fluorinated 1-arylethanols using isolated enzymes. Bioorg Chem. 2013;51:31\u0026ndash;47.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eInoue M, Sumii Y, Shibata N. Contribution of organofluorine compounds to pharmaceuticals. ACS Omega. 2020;5(19):10633\u0026ndash;40.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJohnson BM, Shu YZ, Zhuo XL, Meanwell NA. Metabolic and pharmaceutical aspects of fluorinated compounds. J Med Chem. 2020;63(12):6315\u0026ndash;86.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHunter L. The C-F bond as a conformational tool in organic and biological chemistry. Beilstein J Org Chem. 2010;6:38.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChatterjee T, Iqbal N, You Y, Cho EJ. Controlled fluoroalkylation reactions by visible-light photoredox catalysis. Acc Chem Res. 2016;49(10):2284\u0026ndash;94.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu LR, Maglangit F, Deng H. Fluorine biocatalysis. Curr Opin Chem Biol. 2020;55:119\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNikel PI, Welner DH, Cros A, Volke DC. In vivo fluorine biocatalysis: Six enzymes in search of a cell factory. Chem Catal. 2022;2(10):2403\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGohain N, Thomashow LS, Mavrodi DV, Blankenfeldt W. The purification, crystallization and preliminary structural characterization of PhzM, a phenazine-modifying methyltransferase from \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e. Acta Crystallogr F. 2006;62(9):887\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu HS, Zhu GL, Zhao SG, Fu P, Zhu WM. Bioactive natural products from the marine sponge-derived \u003cem\u003eNocardiopsis dassonvillei\u003c/em\u003e OUCMDZ-4534. Chin J Org Chem. 2019;39(2):507.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eViktorsson EO, Melling Gr\u0026oslash;the B, Aesoy R, et al. Total synthesis and antileukemic evaluations of the phenazine 5,10-dioxide natural products iodinin, myxin and their derivatives. Bioorg Med Chem. 2017;25(8):2285\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMohr MKF, Benčić P, Andexer JN. Doping In Vivo Alkylation in E. coli by Introducing the Direct Sulfurylation Pathway of S. cerevisiae. Angew Chem Int Ed Engl. 2025;64(2):e202414598.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHou FF, Xian M, Huang W. De novo biosynthesis and whole-cell catalytic production of paracetamol on a gram scale in \u003cem\u003eEscherichia coli\u003c/em\u003e. Green Chem. 2021;23(20):8280\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang W, Wan YP, Su H, et al. Recent advances in phenazine natural products: biosynthesis and metabolic engineering. J Agric Food Chem. 2024;72(39):21364\u0026ndash;79.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWan YP, Liu HC, Xian M, Huang W. Biosynthesis and metabolic engineering of 1-hydroxyphenazine in \u003cem\u003ePseudomonas chlororaphis\u003c/em\u003e H18. Microb Cell Fact. 2021;20(1):235.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWen XJ, Leisinger F, Leopold V, Seebeck FP. Synthetic Reagents for Enzyme-Catalyzed Methylation. Angew Chem Int Ed. 2022;61(41):e202208746.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSingh N, Lane S, Yu TH, et al. A generalized platform for artificial intelligence-powered autonomous enzyme engineering. Nat Commun. 2025;16(1):5648.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"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":"alkoxylated phenazine derivatives, enzyme cascade systems, de novo biosynthesis, whole-cell catalysis, SAM analogues","lastPublishedDoi":"10.21203/rs.3.rs-9338442/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9338442/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003ePhenazines are important nitrogen-containing heterocycles with diverse applications in the chemical and pharmaceutical industry. Alkoxylated phenazines, in particular, exhibit promising acaricidal and fungicidal properties. Currently, chemical synthesis is the main approach for alkoxylated phenazines and derivatives production. However, these processes are associated with harsh reaction conditions, accumulation of chemical waste (e.g., organic solvents, noble metal catalysts), and environmental concerns.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn this study, we developed biocatalytic systems for the synthesis of alkoxylated phenazines by designing enzymatic cascades and \u003cem\u003ede novo\u003c/em\u003e biosynthetic pathways. The O-methyltransferase LaphzM from \u003cem\u003eLysobacter antibioticus\u003c/em\u003e OH13 was identified to catalyze the alkoxylation of phenazines using SAM analogues generated \u003cem\u003ein situ\u003c/em\u003e by halide methyltransferases (HMTs) from \u003cem\u003eBurkholderia xenovorans\u003c/em\u003e and \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. Using these enzymatic cascades, we successfully synthesized six alkoxylated phenazine derivatives, including three novel compounds. We further established \u003cem\u003ede novo\u003c/em\u003e biosynthetic pathways for 1-ethoxyphenazine and 2-ethoxyphenazine in \u003cem\u003ePseudomonas chlororaphis\u003c/em\u003e via the direct sulfurylation pathway from \u003cem\u003eS. cerevisiae\u003c/em\u003e. To improve production, we optimized whole-cell cascade systems, achieving 486.3 mg/L (54% yield) of 2-ethoxyphenazine, 218.1 mg/L (22.8% yield) of 2-propoxyphenazine and 378.2 mg/L (78.4% yield) of 1-ethoxyphenazine-\u003cem\u003eN\u003c/em\u003e\u0026prime;10-oxide within 7 h using microbially produced 2-hydroxyphenazine as the substrate along with EtI or PrI, respectively.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eOverall, we successfully established green biocatalytic platforms for alkoxylated phenazines through enzymatic cascades and \u003cem\u003ede novo\u003c/em\u003e biosynthetic pathways. Methyltransferases derived from diverse species, including LaphzM and HMTs, play key roles in the SAM-analogue-mediated alkylation of phenazines. This study provides a sustainable and efficient alternative to conventional chemical synthesis, with significant potential for green manufacturing of alkoxylated phenazine derivatives.\u003c/p\u003e","manuscriptTitle":"De novo biosynthesis and whole-cell production of alkoxylated phenazine derivatives","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-29 13:24:36","doi":"10.21203/rs.3.rs-9338442/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-18T11:09:43+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-18T09:49:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"118272080466325409760185642053488526941","date":"2026-04-27T06:05:09+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-21T10:23:18+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-08T14:16:40+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-08T14:16:19+00:00","index":"","fulltext":""},{"type":"submitted","content":"Microbial Cell Factories","date":"2026-04-07T02:25:42+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":"278ee8e1-7ccc-453b-86b4-1e13d1c0ca57","owner":[],"postedDate":"April 29th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-18T11:09:43+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-18T09:49:41+00:00","index":33,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-05-18T11:23:40+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-29 13:24:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9338442","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9338442","identity":"rs-9338442","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}