Metabolic pathway analysis of an Acinetobacter strain capable of assimilating diverse hydrocarbons

Metabolic pathway analysis of an Acinetobacter strain capable of assimilating diverse hydrocarbons and aromatic compounds | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (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];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Metabolic pathway analysis of an Acinetobacter strain capable of assimilating diverse hydrocarbons and aromatic compounds View ORCID Profile Shori Inoue , View ORCID Profile Shogo Yoshimoto , Maiko Hattori , Shotaro Yamagishi , View ORCID Profile Katsutoshi Hori doi: https://doi.org/10.1101/2025.10.30.684093 Shori Inoue 1 Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya University , Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8603, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Shori Inoue Shogo Yoshimoto 1 Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya University , Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8603, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Shogo Yoshimoto Maiko Hattori 1 Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya University , Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8603, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Shotaro Yamagishi 1 Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya University , Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8603, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Katsutoshi Hori 1 Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya University , Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8603, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Katsutoshi Hori For correspondence: khori{at}chembio.nagoya-u.ac.jp Abstract Full Text Info/History Metrics Supplementary material Data/Code Preview PDF ABSTRACT Sustainable bioproduction requires developing robust microbial chassis with broad metabolic versatility and suitability for industrial applications. Acinetobacter sp. Tol 5 is a highly adhesive bacterium capable of utilizing various hydrocarbons, making it a promising chassis candidate for immobilized whole-cell catalysis. In this study, we characterized the carbon metabolism of Tol 5 by reconstructing metabolic pathway maps and analyzing the transcriptomes of cells grown on ethanol, hexadecane, toluene, and phenol. Genomic analysis revealed broad metabolic pathways for alcohols, alkanes, and aromatics, including five distinct aromatic degradation routes. Transcriptome data elucidated the specific genes governing the metabolic pathways for each carbon source. Notably, during growth on phenol, both the ortho - and meta - catechol cleavage pathways were induced. While the gene disruption of the meta - cleavage enzyme TodE had no effect, that of toluene dioxygenase resulted in an extended lag phase but a higher final cell yield on phenol, indicating that it facilitates rapid detoxification but diverts carbon into unassimilable by-products. Furthermore, genes involved in oxidative and osmotic stress resistance were coordinately upregulated on hydrocarbon sources. These results provide a comprehensive view of the carbon metabolism of Tol 5 and reveal new aspects of microbial metabolism of hydrocarbons and aromatic compounds. INTRODUCTION In recent years, there has been a global shift from petrochemical synthesis to more sustainable bioprocesses, driven by the need to reduce fossil resource consumption and lessen environmental impact. Microorganisms, especially bacteria, are considered useful hosts for bioproduction due to their rapid growth, ease of genetic manipulation, and capacity for engineering to improve the productivity of valuable compounds. Escherichia coli is one of the most widely used microbial chassis in bioproduction, owing to its well-characterized molecular biology and metabolic systems ( Pontrelli et al ., 2018 ). Concurrently, environmental microorganisms capable of assimilating non-sugar carbon substrates, such as C1 compounds, aromatic hydrocarbons, and oils, are increasingly valued for converting underutilized resources, including petrochemical by-products, agricultural waste, lignocellulosic biomass, and exhaust gases, into valuable products ( Liew et al ., 2022 , Liu et al ., 2024 , Martín-González et al ., 2025 ). For instance, Pseudomonas putida is recognized for its high organic solvent tolerance and versatile catabolic pathways and has been deployed in many applications, demonstrating the immense potential of environmental isolates ( Nakazawa, 2002 , Nelson et al ., 2002 , Ramos et al ., 2015 , de Lorenzo et al ., 2024 , Martínez-García & de Lorenzo, 2024 ). Therefore, it is essential to explore and develop new microbial chassis that can metabolize diverse substrates, tolerate harsh environmental conditions, and perform robustly in industrial bioprocesses. Members of the genus Acinetobacter are ubiquitous and found in diverse environments, including soil, oceans, freshwater, sediments, activated sludge, and clinical settings (5, 6). The genus possesses extensive genomic plasticity, which contributes to the pathogenic potential of some species. However, this trait, combined with a high tolerance to environmental stress, has also made it a focus of great interest in environmental microbiology, where several species have shown potential in bioproduction and bioremediation. For example, A. baylyi ADP1 has been studied as a host for the valorization of aromatic compounds via the β-ketoadipate pathway ( Biggs et al ., 2020 , Luo et al ., 2022 , Kurnia et al ., 2024 ). A. venetianus RAG-1, which has multiple alkane hydroxylase genes, exhibits a pronounced ability to degrade crude oil and has been employed in bioremediation applications ( Liu et al ., 2021 ). Furthermore, A. junii BP25 has been utilized for the production of polyhydroxyalkanoates (PHAs) from food waste-derived organic acids, while A. venetianus AMO1502 has been applied in the biosynthesis of bioemulsifiers ( Sabapathy et al ., 2019 , D’Almeida et al ., 2024 ). Acinetobacter sp. Tol 5, originally isolated as a toluene-degrading bacterium from an exhaust gas treatment reactor, can degrade various hydrocarbons, including benzene, toluene, and xylene (BTX) ( Hori et al ., 2001 ). In addition to metabolic versatility, Tol 5 exhibits rapid autoagglutination and high adhesiveness to various solid materials, such as plastics, glass, and metals ( Ishikawa et al ., 2012 ). This adhesive phenotype is mediated by its fibrous cell surface protein AtaA, a member of the trimeric autotransporter adhesin (TAA) family proteins ( Ishikawa et al ., 2012 ). Previous studies have revealed the functional domains and biophysical properties of AtaA ( Yoshimoto et al ., 2023 , Yoshimoto et al ., 2024 , Inoue et al ., 2025 ). Based on these findings, a reversible cell immobilization method was proposed to enhance the efficiency and productivity of bioproduction processes ( Yoshimoto et al ., 2017 ). To demonstrate its utility, Tol 5 expressing heterologous biosynthetic genes was immobilized on porous carriers and successfully utilized in an unconventional gas-phase bioreaction process to produce a high-value-added monoterpenoid, ( E )-geranic acid ( Usami et al ., 2020 ). Although these advances indicate that Tol 5 is a promising microbial chassis for innovative bioproduction, its central metabolic network remains largely uncharacterized. In this study, we characterized the carbon metabolism of Tol 5 by reconstructing metabolic pathways from the complete genome sequence and performing transcriptomic analyses of Tol 5 cells grown on ethanol, hexadecane, or aromatic compounds as the sole carbon source. MATERIALS AND METHODS Genome sequence data analysis The complete genome sequence of Tol 5, consisting of a chromosome and a plasmid, was obtained from the NCBI database (accession: AP024708 and AP024709 ). All amino acid sequences of the coding sequences (CDSs) were searched against the UniProtKB/Swiss-Prot database (as of August 2024) using BLASTP, and the best-scoring hit for each CDS was used for functional annotation. The sequences were further searched against the KEGG database using BlastKOALA and KEGG automatic annotation server (KAAS) (accession: T11084 ). For CDSs that could not be annotated with the KEGG database, additional searches were performed against the EggNOG database using eggNOG-mapper. Based on the annotations obtained, a central carbon metabolic pathway map was reconstructed using KEGG mapper. Peripheral metabolic pathways, including those involved in the degradation of alkanes and aromatic compounds, were inferred by comparing operon organization with those previously characterized in closely related bacterial species. Operon structures were predicted using two tools: Operon-mapper ( Taboada et al ., 2018 ), which infers operons based on intergenic distances and gene functions, and Rockhopper ( McClure et al ., 2013 ), which additionally incorporates transcriptomic data. Functional annotations, operon predictions, and metabolic pathways were manually curated based on information from relevant literature. Strains, plasmids, and culture conditions The primers, bacterial strains, and plasmids used in this study are shown in Tables S1 and S2. E. coli and its transformants were cultured in lysogeny broth (LB) medium (20066-95; Nacalai Tesque, Kyoto, Japan) at 37°C. Acinetobacter sp. Tol 5 and its mutants were cultured in LB medium or basal salt (BS) medium ( Hori et al ., 2001 ) at 28°C. Gene knockout The gene knockout mutants were generated using the cytidine base editing system for Acinetobacter according to the previous reports ( Wang et al ., 2019 , Inoue et al ., 2025 ). The DNA dimer fragment encoding the sequence of the single guide RNA was prepared by mixing oligo DNAs listed in Table S2 in 50 mM NaCl solution and gradually cooling from 95°C to 18°C at 0.1°C/s. This dimer was introduced into the BsaI site of pBECAb-apr. The constructed plasmid was electroporated into Tol 5 REK , a restriction-modification system and ataA -deficient strain ( Ishikawa & Hori, 2024 ). After overnight culture of Tol 5 REK harboring the plasmid in BS medium to promote mutation, the cells were spread on a BS agar plate containing 5 % (w/v) sucrose to remove the plasmid. Mutation of the target gene was confirmed by sequencing the DNA amplified from the genome by PCR using KOD FX Neo (Toyobo, Osaka, Japan). Bacterial cell growth For the wild type strain of Tol 5, the cells cultured in LB medium supplemented with each carbon source were collected by centrifugation, washed with BS medium, and resuspended in BS medium. A 0.20-mL aliquot of the suspension was inoculated into 20 mL of BS medium in 100-mL Erlenmeyer flasks. Four rectangular pieces of polyurethane foam support (10 × 10 × 10 mm, CFH-20, 20 pores/25 mm; Inoac Corporation, Nagoya, Japan) were added to each flask as carriers to immobilize the cells. Each carbon source was added at a carbon equivalent concentration of 3.3 × 10 -2 mol/L. The flasks were then capped with silicone stoppers for cultures containing sodium lactate, ethanol, and hexadecane, or with Viton rubber stoppers for those containing toluene and phenol. The cultures were incubated at 28°C with shaking at 115 rpm. The optical density at 660 nm (OD 660 ) was measured as previously reported ( Inoue et al ., 2025 ). At each time point, flasks were collected, and the cultures were mixed vigorously with 1 mL of 10% Casamino Acids technical grade (CA-T; Becton, Dickinson and Company, Franklin Lakes, NJ, USA). The CA-T solution was added to inhibit AtaA-mediated cell aggregation and to detach the bacterial cells from the support ( Ohara et al ., 2019 ). The OD 660 of the resulting cell suspension was measured using a UVLVis spectrophotometer (UV-1850; Shimadzu Corporation, Kyoto, Japan). For the Tol 5 REK mutants, the cells cultured in LB medium supplemented with each carbon source were collected by centrifugation, washed with BS medium, and resuspended in BS medium. A 0.25-mL aliquot of the suspension was inoculated into 5 mL of BS medium in a glass test tube. Each carbon source was added at a carbon equivalent concentration of 1.4 × 10 -2 mol/L, and the optical density (OD) was monitored every 60 min using the OD-monitorC&T system (Taitec, Saitama, Japan) during the culture. RNA sequencing Tol 5 cells were harvested from the polyurethane foam support during the late log phase (OD 660 = 0.2–0.5). The detached cells were collected by centrifugation (8,000 × g, 4°C, 3 min), and the cell pellets were stored at −80°C. For each growth condition, three biological replicates were prepared. Total RNA was extracted using the RNA Prep Kit (Cica Genus, Tokyo, Japan) according to the manufacturer’s protocol. Ribosomal RNA was removed using the NEBNext rRNA Depletion Kit (Bacteria) (New England Biolabs, Ipswich, MA, USA), and cDNA libraries were generated with the NEBNext Ultra II RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA). The cDNA libraries were sequenced on the Illumina NextSeq550. Raw FASTQ reads were quality-filtered using fastp (version 0.23.2) and mapped to the Tol 5 genome (accession: AP024708 and AP024709 ) using Bowtie2 (version 2.5.1) with default parameters. Mapped reads were counted using featureCounts (version 2.0.6). Differential gene-expression analysis Raw read counts of CDSs were analyzed in R using the edgeR package (version 3.42.4). Genes with extremely low expression were removed using the filterByExpr function. Normalization was performed using the trimmed mean of M-values (TMM) method implemented in the calcNormFactors function. Differential gene expression analysis was conducted using the quasi-likelihood pipeline. A design matrix was constructed incorporating the cells grown on six different carbon sources (n = 3), with the data from those grown on lactate set as the reference group. Dispersion estimates were obtained using the estimateDisp function, and model fitting was performed with the glmQLFit function. Differential expression between each condition and the control condition (lactate condition) was assessed using the glmQLFTest function. Genes with false discovery rate (FDR) 1, and log 2 counts per million (CPM) > 3 were extracted as significantly differentially expressed genes. RESULTS Central carbon metabolism The complete genome of Tol 5 consists of two circular DNA components, a chromosome of 4,681,789Lbp and a plasmid of 117,717Lbp, collectively encoding 4,404 predicted CDSs ( Ishikawa & Hori, 2021 ). We first evaluated the functional annotation of each CDS using multiple databases, including NCBI, UniProtKB/Swiss-Prot, KEGG, and EggNOG, as well as information from relevant literature (Table S3). Tol 5 encodes all the enzymes for gluconeogenesis, pentose phosphate pathway, tricarboxylic acid (TCA) cycle, glyoxylate shunt, purine and pyrimidine biosynthesis, β-oxidation, and fatty acid biosynthesis ( Fig. 1 , Table S4). In contrast, glycolysis via the Embden–Meyerhof–Parnas (EMP) pathway is incomplete due to the absence of hexokinase, phosphofructokinase, and pyruvate kinase, which is a trait commonly observed among Acinetobacter species ( Zhao et al ., 2023 ). While some Acinetobacter strains, such as A. baylyi ADP1 and A. baumannii , metabolize glucose via nonspecific pyrroloquinoline quinone-dependent aldose dehydrogenase and the Entner–Doudoroff (ED) pathway ( Barbe et al ., 2004 , Kannisto et al ., 2015 , Ren & Palmer Lauren, 2023 ), Tol 5 completely lacks these genes. Furthermore, A. baumannii ATCC 19606 metabolizes pentoses such as L-arabinose, D-xylose, and D-ribose via a pathway encoded by ara genes that converts them into α-ketoglutarate ( Alberti et al ., 2023 ), but Tol 5 also lacks the ara genes. These results suggest that Tol 5 has a very limited capacity for sugar utilization as carbon sources. Download figure Open in new tab Fig. 1. Prediction of the central metabolic pathway of Acinetobacter sp. Tol 5. Genes related to the central metabolic pathway were analyzed and listed in Table S4. Dashed arrows indicate metabolic reactions for which the corresponding genes are not found in Tol 5 genome. Abbreviations: ADH; alcohol dehydrogenase, AH; alkane hydroxylase, AKG; α-ketoglutarate, ALDH; aldehyde dehydrogenase, CIT; citrate, FUM; fumarate, ICIT; isocitrate, MAL; malate, OAA; oxaloacetate, PRPP; phosphoribosyl pyrophosphate, SUC; succinate, SUC-CoA; succinyl-CoA. Many Acinetobacter strains utilize organic acids and fatty acids as carbon sources ( Ren & Palmer Lauren, 2023 ). Tol 5 possesses three acetate assimilation routes, including the reversible acetate kinase-phosphotransacetylase (AckA-Pta) pathway, the irreversible acetyl-CoA synthetase (ACS) pathway, and the reversible pathway mediated by succinyl-CoA:coenzyme A transferase AarC ( Fig. 1 , Table S4). For fatty acid degradation, multiple candidate genes corresponding to each step of β-oxidation were identified, including two putative acyl-CoA ligases, twelve acyl-CoA dehydrogenases, four enoyl-CoA hydratases, and three thiolases. Propionyl-CoA generated during the β-oxidation of odd-chain fatty acids was predicted to be catabolized to succinate and pyruvate via the methylcitrate cycle, which consists of 2-methylisocitrate lyase (PrpB), 2-methylcitrate synthase (PrpC), 2-methylcitrate dehydratase (AcnD), 2-methylaconitate isomerase (PrpF), and bifunctional aconitate hydratase/isomerase (AcnA and/or AcnB). In addition, four extracellular lipase genes were identified in Tol 5 (Table S1), all of which are tandemly arranged ( TOL5_03740 – TOL5_03770 ) and share 44–52% amino acid sequence identities with the triacylglycerol lipase Lip reported in P. aeruginosa PAO1 ( Wohlfarth et al ., 1992 ). Tol 5 further encodes key enzymes that redirect fatty acid oxidation intermediates into carbon storage compounds. A bifunctional wax ester synthase/acyl-CoA:diacylglycerol acyltransferase (WS/DGAT, AtfA) mediates the production of wax esters and triacylglycerols, while polyhydroxyalkanoate (PHA) synthase (PhaC) is responsible for the production of PHAs. Alkane metabolism Under aerobic conditions, n -alkanes are first oxidized at their terminal methyl group to primary alcohols by alkane hydroxylases, and these alcohols are then further oxidized to aldehydes and finally to fatty acids ( Rojo, 2009 ). The Tol 5 chromosome encodes multiple alkane hydroxylases, including a membrane-bound non-heme iron alkane monooxygenase AlkB, and three flavin-containing monooxygenases encoded by almA , ladA_1 , ladA_2 (Table S4). Additionally, the plasmid of Tol 5 carries a gene encoding an alkane hydroxylase ( TOL5_43480 ), whose amino acid sequence shares 99% identity with that of a cytochrome P450 monooxygenase of the CYP153 family found in Acinetobacter sp. EB104 ( Maier et al ., 2001 ). It also includes genes encoding associated redox partners, ferredoxin ( TOL5_43490 ) and ferredoxin reductase ( TOL5_43470 ). AlkB and CYP153 typically oxidize short- and medium-chain n -alkanes (C16 or less), whereas AlmA and LadA act on long-chain n -alkanes (C20 or more) ( Nie et al ., 2014 , Kong et al ., 2021 , Chen et al ., 2024 ). The resulting primary alcohols are further oxidized by alcohol dehydrogenases (ADHs) and aldehyde dehydrogenases (ALDHs). In Tol 5 genome, at least six putative ADH genes and one ALDH gene were predicted to be involved in fatty alcohol and fatty aldehyde oxidation (Table S4). Although the functions of most ADHs in Tol 5 remain unclear, the iron-containing ADH encoded by yiaY_1 shares 91% amino acid sequence identity with ADH4 from A. baumannii ATCC 19606, which catalyzes the oxidation of ethanol, 1-propanol, and 1-butanol ( Lin et al ., 2021 ). The putative ALDH encoded by aldB (AldB) shares 87% amino acid identity with a long-chain ALDH, Ald1, reported in Acinetobacter sp. M-1 (46). Aromatic compounds metabolism Genes encoding multi-component oxygenases for aromatic compound oxidation, as well as enzymes for successive reaction steps leading to central metabolites, are generally organized into operons and regulated by specific transcriptional regulators ( Phale et al ., 2020 ). Based on the predicted operons involved in aromatic compound degradation (Table S4, Fig. 2 ), we reconstructed the aromatic degradation pathways in Tol 5, identifying five major aromatic degradation routes ( Fig. 3 ). Download figure Open in new tab Fig. 2. Genetic organization of genes involved in the degradation of aromatic compounds in Acinetobacter sp. Tol 5. Predicted operon structures are illustrated based on analyses using Operon-mapper and Rockhopper. Genes within the same operon are represented by arrows of the same color. Proposed gene names or predicted functions are labeled above each arrow, and genomic coordinates are indicated below. Download figure Open in new tab Fig. 3. Predicted degradation pathways of aromatic compounds in Acinetobacter sp. Tol 5, reconstructed based on the genes shown in Fig. 2 and Table S4. Question marks and dashed arrows indicate steps for which the genes encoding the corresponding enzymes have not been identified in Tol 5. The pca genes encode the enzymes responsible for the degradation of protocatechuate (PCA pathway) ( Fig. 3 ), forming a gene cluster together with the aromatic compounds transport protein PcaK and the transcriptional repressor PcaU ( Fig. 2 ). The cat gene cluster encodes the enzymes responsible for the degradation of catechol (CAT pathway) ( Fig. 3 ) and the transcriptional activator CatM ( Fig. 2 ). Protocatechuate and catechol are common intermediates derived from various aromatic compounds through various upstream pathways. The PCA and CAT pathways funnel these intermediates into acetyl-CoA and succinyl-CoA via oxidative ortho -cleavage of aromatic ring, serving as central routes for aromatic compound degradation that are widely conserved in Acinetobacter species ( Barbe et al ., 2004 , Fischer et al ., 2008 , Stuani et al ., 2014 , Breisch et al ., 2022 ). In Tol 5, the pca genes are located adjacent to the qui genes ( Fig. 2 ), which encode enzymes that convert quinate and shikimate to protocatechuate ( Fig. 3 ). The structure of this gene cluster is similar to that reported in A. baylyi ADP1 ( Dal et al ., 2005 ), and as described for ADP1, the pca and qui genes are likely organized as a large single operon under the regulator PcaU in Tol 5. Tol 5 also possesses the pob and van genes, which encode upstream pathways leading to protocatechuate, along with their respective transcriptional regulators (PobR and VanR) ( Fig. 2 and 3 ). The cat genes are located adjacent to the mph gene cluster, which encodes the components of the phenol monooxygenase complex MphKLMNOP, catechol 1,2-dioxygenase CatA (encoded by catA_2 ), and the regulatory proteins MphX (repressor) and MphR (activator) ( Fig. 2 ). The structure of the mph operon in Tol 5 resembles that of A. calcoaceticus PHEA-2 ( Yu et al ., 2011 ), but is distinguished by the presence of the additional catA gene ( catA_2 ) located immediately downstream in the same operon. In addition, Tol 5 possesses the ben and iac genes, which encode enzymes for the upstream pathways that lead to catechol, along with their respective transcriptional regulators (BenM and IacR) and transporters (BenE, BenK, and a putative transporter encoded by TOL5_19290 ) ( Fig. 2 and 3 ). Notably, the ben genes in Tol 5 are located over 900 kbp away from the cat operon, in contrast to A. baylyi ADP1, in which the ben genes are located adjacent to the cat genes to facilitate coordinated regulation of gene expression ( Bleichrodt et al ., 2010 ). Despite its distinct genomic location, the ben operon in Tol 5 includes the third catA gene (encoded by catA_4 ), indicating that coordinated regulation between the ben and cat genes is maintained. The tod operon encodes the enzymes responsible for the degradation of alkylbenzene (TOD pathway) ( Fig. 3 ) and is located between the todS - todT two-component regulatory system and the putative toluene transporter gene fadL2 , which is located in a separate operon ( Fig. 2 ). In the TOD pathway, alkylbenzene is oxidized into 3-alkylcatechol. The aromatic ring is then opened via oxidative meta -cleavage, and in the case of toluene, the intermediates are ultimately converted into acetate, pyruvate, and acetyl-CoA. Although rarely found in other Acinetobacter species, the tod genes of Tol 5 share high similarity with those of P. putida strains and play a central role in the degradation of toluene and benzene ( Yoshimoto et al ., 2025 ). Furthermore, Tol 5 possesses the paa genes, which encode the enzymes responsible for the degradation of phenylacetic acid (PAA pathway) ( Fig. 3 ), as well as the transcriptional repressor PaaX ( Fig. 2 ). The PAA pathway degrades phenylacetate via epoxidation of CoA thioesters. While this pathway in A. baumannii strains is thought to be primarily associated with phenylalanine degradation ( Teufel et al ., 2010 , Hooppaw Anna et al ., 2022 ), Tol 5 lacks the enzymes required to convert phenylalanine to phenylacetate. In addition, Tol 5 also possesses a gene cluster encoding a putative homogentisate degradation route (HMG pathway) ( Fig. 3 ), which includes 4-hydroxyphenylpyruvate dioxygenase (HPD), an IclR-type transcriptional repressor (IclR), maleylacetoacetate isomerase (MaiA), fumarylacetoacetase (FahA), 3-oxoacid CoA-transferase subunits A and B (ScoA and ScoB), and an aromatic amino acid transporter (AroP), as well as a putative homogentisate dioxygenase encoded by TOL5_11140 ( Fig. 2 ). Although the protein product of TOL5_11140 was annotated as a glyoxalase (Table S1), it shares 22.7% amino acid identity with homogentisate 1,2-dioxygenase HmgA (B7VA96) and 18.8% amino acid identity with metapyrocatechase XylE (Q04385) from P. putida . Based on the genomic context, the HMG pathway in Tol 5 is likely involved in tyrosine metabolism. Substrate-dependent transcriptomic responses to lactate, ethanol, and hexadecane While genomic analysis revealed that Tol 5 possesses multiple genes involved in the metabolism of alcohols and alkanes, the expression of key enzymes, such as alcohol dehydrogenases and alkane hydroxylases, is generally subject to strict regulation depending on the available carbon source. To identify the specific genes induced for their assimilation, we performed transcriptome analysis. Ethanol and hexadecane were selected as a representative short-chain alcohol and long-chain alkane, respectively, and Tol 5 cells were cultured in BS medium supplemented with each carbon source (Fig. S1A and B). Because Tol 5 cells exhibit autoagglutination and high adhesiveness in BS medium, polyurethane support was used to immobilize the cells as previously reported ( Hori et al ., 2011 ). Cells were harvested from the support during the late log phase, and their transcriptomes were compared with those of cells grown on lactate (Table S5 and S6). Differentially expressed genes were defined by the thresholds of |log 2 FC| > 1, log 2 CPM > 3, and FDR < 0.01 ( Fig. 4A , Table S7). Download figure Open in new tab Fig. 4. Transcriptome analysis of Acinetobacter sp. Tol 5 grown on lactate, ethanol, and hexadecane. ( A ) Volcano plots comparing gene expression between lactate and ethanol (left) or hexadecane (right) as sole carbon sources (n = 3 biologically independent samples). Red and blue dots represent significantly upregulated and downregulated genes, respectively (FDR 1). ( B , C ) Proposed ethanol ( B ) and hexadecane ( C ) degradation pathways in Tol 5 based on transcriptome analysis. Genes encoding enzymes significantly upregulated under each condition are shown in red. In ethanol culture, 14 genes were significantly upregulated, including yiaY_1 and aldB , which encode an iron-containing ADH and a putative ALDH, respectively (Table S7). Since yiaY_1 was the only ADH gene induced by ethanol, it is likely the primary ADH responsible for ethanol oxidation in Tol 5 ( Fig. 4B ). Although AldB shares 87% amino acid identity with Ald1 from Acinetobacter sp. M-1, which has been characterized as a long-chain ALDH that is inactive toward short-chain substrates such as acetaldehyde ( Ishige et al ., 2000 ), its induction during growth on ethanol suggests that AldB functions differently from Ald1 and catalyzes acetaldehyde oxidation in Tol 5. Regarding the conversion of acetate into acetyl-CoA, although Tol 5 possesses both the AckA-Pta pathway and the ACS pathway ( Fig. 1 ), genes in neither pathway were significantly upregulated ( Fig. 4B ). Thus, the primary acetate conversion pathway into acetyl-CoA in Tol 5 during growth on ethanol could not be determined in this study. Additionally, aceA , which encodes isocitrate lyase of the glyoxylate shunt that bypasses the decarboxylation steps of the TCA cycle and conserves carbon flux for gluconeogenesis ( Fig. 1 ), was upregulated. A similar regulatory pattern was observed in Acinetobacter oleivorans DR1, where the aceA was upregulated during growth on acetate ( Park et al ., 2019 ), suggesting that Tol 5 employs a similar strategy for assimilating acetate generated from ethanol. In hexadecane culture, 266 genes were significantly upregulated (Table S7). Among the multiple alkane hydroxylase genes in Tol 5, alkB and the CYP153 gene were specifically induced, indicating their involvement in the initial hydroxylation of hexadecane ( Fig. 4C ). The gene yiaY_3 , encoding an iron-containing ADH, and calB , encoding a putative coniferyl aldehyde dehydrogenase, were also upregulated, suggesting their roles in the oxidation of hexadecanol to hexadecanal and subsequently to hexadecanoic acid. The genes involved in every step of the β-oxidation pathway were upregulated. Among the multiple predicted enzymes for each step, only fagA , fadB , fadD4 (one of the two predicted acyl-CoA ligase genes), fadE , and ydiO_5 (two of the twelve predicted acyl-CoA dehydrogenase genes) were specifically induced ( Fig. 4C ). Twenty-one genes were downregulated in both ethanol and hexadecane cultures compared to lactate culture, implying that these genes are specifically upregulated during growth on lactate (Table S7 and S8). Among them, the lldP , lldR , lldD_1 , and dld genes ( Fig. 1 and Table S8), which form an operon similar to the lldPRD operon involved in lactate metabolism reported in A. baumannii, were identified ( Morris et al ., 2024 ). The aceE and aceF genes, which encode components of pyruvate dehydrogenase, were also upregulated during growth on lactate. The pca and qui genes, responsible for quinate and shikimate utilization, were downregulated in ethanol and hexadecane cultures, indicating catabolite repression similar to that reported for A. baylyi ADP1, where these genes are repressed in the presence of preferred carbon sources ( Fischer et al ., 2008 , Bleichrodt et al ., 2010 ). Substrate-specific regulation of aromatics degradation pathways Subsequently, we focused on the transcriptional regulation of the aromatic degradation pathways in Tol 5, particularly for the degradation of toluene and phenol, which are valuable substrates for the bioproduction of aromatic-derived fine chemicals. Tol 5 cells were cultured in BS medium supplemented with toluene or phenol (Fig. S1C) and subjected to transcriptome analysis using the same procedure described above. The resulting data were also compared with those of cells grown on lactate ( Fig. 5A , Table S6). Download figure Open in new tab Fig. 5. Differential gene expression of Acinetobacter sp. Tol 5 grown on various aromatics, ethanol, and hexadecane, using lactate as the reference condition. ( A ) Volcano plots comparing gene expression between lactate and toluene (left) or phenol (right) as sole carbon sources (n = 3 biologically independent samples). Red and blue dots represent significantly upregulated and downregulated genes, respectively (FDR 1). ( B ) Heat maps showing the average log□ FC of the genes involved in the degradation of toluene and phenol ( tod , cat , and mph genes). The color gradient indicates the magnitude of the log□ FC values, with the deepest red representing ≥ 10 and the deepest blue representing ≤ −10. Asterisks (*) denote differentially expressed genes defined by |log 2 FC| > 1, log 2 CPM > 3, and FDR < 0.01. ( C, D ) Proposed degradation pathways of toluene ( C ) and phenol ( D ) in Tol 5 based on transcriptome data. Genes encoding enzymes significantly upregulated under each condition are shown in red. In toluene and phenol cultures, 349 and 643 genes were upregulated, while 292 and 578 genes were downregulated, respectively (Table S7). Regarding the aromatic degradation pathways, toluene culture strongly induced only the tod operon, including all of the TOD pathway genes ( Fig. 5B ). Expression levels of genes in the other aromatic degradation pathways did not differ significantly ( Fig. 5B and S2). Although genes in the mph operon (encoding phenol monooxygenase) and the paa operon (encoding the PAA pathway) showed slight upregulation, their induction levels were negligible compared to those of the tod operon. The fadL2 , which is located adjacent to the tod operon and encodes a putative toluene transporter ( Fig. 2 ), was also upregulated ( Fig. 5B ). Although it was reported that its deletion did not affect growth on toluene ( Yoshimoto et al ., 2025 ), this strong induction suggests its involvement in toluene assimilation. A previous study has shown that disruption of todC1 completely abolishes growth on toluene ( Yoshimoto et al ., 2025 ). Consistent with this phenotypic evidence and our genomic predictions, these transcriptional responses indicate that toluene degradation proceeds exclusively through the TOD pathway ( Fig. 5C ). In contrast, in phenol culture, both the mph-cat gene cluster, which comprises the predicted phenol degradation pathway, and the tod operon were significantly upregulated ( Fig. 5B ). Notably, the induction levels of the tod genes reached those observed in toluene culture. Most genes of the PCA, PAA, and HMG pathways were not significantly changed in phenol culture (Fig. S2). Although phenol is predicted to be metabolized via hydroxylation by phenol monooxygenase and the CAT pathway, encoded by the mph-cat gene cluster ( Fig. 3 ), this transcriptional cross-regulation suggests that phenol might also be degraded via tod enzymes, including toluene dioxygenase and the catechol meta -cleavage pathway ( Fig. 5D ). Aromatic substrates also influenced downstream central metabolic pathways (Fig. S3, Table S7). Growth on these compounds triggered a differential response in aconitate hydratase isozymes; acnA_2 was upregulated, whereas the expression of acnB was not significantly changed. Given that this pattern was also observed during growth on hexadecane, the upregulation of acnA_2 appears to be a common response to hydrocarbon utilization. Concurrently, pckG, encoding the key gluconeogenic enzyme phosphoenolpyruvate carboxykinase ( Sauer & Eikmanns, 2005 ) ( Fig. 1 ), was downregulated in both toluene and phenol cultures, indicating reduced carbon flux from the TCA cycle into gluconeogenesis compared to cells grown on lactate. In phenol culture, mqo , encoding malate:quinone oxidoreductase, was specifically upregulated, while mdh , encoding NAD-dependent malate dehydrogenase, was downregulated (Fig. S3). Both enzymes catalyze the conversion of malate to oxaloacetate; however, Mqo transfers electrons directly to the quinone pool, while Mdh reduces NAD + to generate NADH. This transcriptional switching suggests that during phenol assimilation, Tol 5 modulates malate conversion routes to manage the cellular redox balance. Expression of intracellular stress resistance genes under different carbon sources In addition to carbon metabolic pathways, genes involved in environmental stress resistance showed significant transcriptional changes depending on the carbon source ( Fig. 6 , Table S7). Notably, several genes related to oxidative stress resistance were upregulated in Tol 5 cells grown on hexadecane, toluene, and phenol. Genes encoding monofunctional heme catalases ( katA and/or katE ) were highly upregulated under all three conditions. In hexadecane culture, the Mn/Fe superoxide dismutase genes ( sodA_1 and/or sodA_2 ) were repressed. In contrast, during growth on phenol, the sodA_2 was significantly upregulated along with the periplasmic Cu/Zn superoxide dismutase gene ( sodC ). These superoxide dismutase genes were also weakly upregulated in toluene culture. The alkyl hydroperoxide reductase gene ( ahpC_1 ), which plays a role in peroxide detoxification, was highly upregulated in phenol culture. Concurrently, the trehalose synthesis genes ( otsA and otsB ), associated with osmotic stress resistance ( Zeidler et al ., 2017 , Liu et al ., 2025 ), were also upregulated during growth on all three alkane and aromatic carbon sources. The otsB gene, which encodes trehalose-6-phosphate phosphatase, exhibited low expression levels (logL CPM) but showed high upregulation (log 2 FC). The upregulation of oxidative stress resistance and trehalose synthesis genes has been reported in A. oleivorans DR1 during triacontane metabolism ( Park et al ., 2019 ). Our findings demonstrate that the upregulation of stress resistance genes represents a more general adaptation strategy induced not only by alkanes but also by aromatic compounds such as toluene and phenol, suggesting that these stress responses constitute a fundamental mechanism required for cells to metabolize such substrates. Download figure Open in new tab Fig. 6. Heat map showing the average log 2 FC of genes involved in oxidative stress and osmotic stress resistance. The color gradient indicates the magnitude of the log□ FC values, with the deepest red representing ≥ 5 and the deepest blue representing ≤ –5. Asterisks (*) denote differentially expressed genes defined by |log 2 FC| > 1, log 2 CPM > 3, and FDR < 0.01. Growth abilities of tod and mph gene disruption mutants on toluene and phenol Transcriptome analysis of Tol 5 cells grown on aromatic substrates revealed that while toluene culture induces only the tod genes with high specificity, phenol culture induces both the tod operon and the mph-cat gene cluster. This transcriptional cross-regulation suggested multiple potential phenol assimilation routes; specifically, phenol might also be degraded via tod enzymes, including toluene dioxygenase and the meta -cleavage pathway ( Fig. 5D ). Thus, to evaluate the contribution of these pathways to growth on phenol, we used gene disruption mutants of the Tol 5 REK strain targeting the tod genes (Δ todC1 and Δ todE ) and the mph gene (Δ mphN ). Tol 5 REK carries mutations in the restriction-modification system and the ataA gene, enabling easier genetic manipulation and reducing cell adhesion, which is necessary for the accuracy of growth evaluation. The todC1 and todE encode the large subunit of the toluene dioxygenase complex and a catechol 2,3-dioxygenase, respectively, while mphN encodes the large subunit of the phenol monooxygenase complex. In phenol culture, the Δ todC1 mutant exhibited retarded growth but a higher cell yield ( Fig. 7 ). This indicates that while toluene dioxygenase facilitates phenol utilization, likely through phenol hydroxylation, its activity simultaneously reduces the efficiency of assimilating the carbon substrate into cellular components. However, the disruption of mphN (Δ mphN ) completely abolished growth. This indicates that the phenol monooxygenase is the primary phenol hydroxylation route and that toluene dioxygenase cannot fully compensate for its absence. Furthermore, the Δ todE mutant showed no significant change in growth rate in phenol culture. This suggests that while the upstream toluene dioxygenase complex functions to hydroxylate phenol, the subsequent meta -cleavage reaction is not essential for phenol assimilation in Tol 5. Download figure Open in new tab Fig. 7. Growth on different carbon sources. Tol 5 REK (circles), the Δ todC1 mutant (squares), the Δ todE mutant (triangles), and the Δ mphN mutant (diamonds) were inoculated to BS medium containing phenol, toluene, or sodium lactate at a carbon equivalent concentration of 1.4 × 10 -2 mol/L. Data are presented as the means□±□SEMs (biological replicates n = 3). On the other hand, consistent with the specific induction of the tod operon observed in our transcriptome analysis, the disruption of todC1 abolished growth in toluene culture, while the mphN disruption had no effect on growth in toluene culture ( Fig. 7 ). None of these mutations affected growth in lactate culture. These results demonstrated that the observed growth defects are not due to a general loss of cellular viability. DISCUSSION Members of the genus Acinetobacter inhabit diverse environments, including soil, oceans, freshwater, sediments, activated sludge, and polluted sites ( Mateo-Estrada et al ., 2019 , Zhao et al ., 2023 ). Several Acinetobacter strains, such as the A. calcoaceticus–A. baumannii complex, are also opportunistic nosocomial pathogens that exhibit multidrug resistance ( Vijayakumar et al ., 2019 ). Consequently, most previous research on Acinetobacter species has focused on antibiotic resistance, genomic plasticity, horizontal gene transfer, and pathogenicity, whereas the metabolic capabilities of this genus remain comparatively underexplored ( Mateo-Estrada et al ., 2019 , Zhao et al ., 2023 ). Although A. baylyi ADP1 is a widely used model strain and its 3.5 Mb genome has been well characterized ( Barbe et al ., 2004 , Durot et al ., 2008 ), this genome is smaller than those of many other Acinetobacter species, and thus represents only a subset of the metabolic potential present across the genus ( Barbe et al ., 2004 , Zhao et al ., 2023 ). In this study, we characterized the carbon metabolism of the highly adhesive hydrocarbon-degrading Acinetobacter strain Tol 5, which has a 4.8 Mb genome ( Ishikawa & Hori, 2021 ). Genome analyses revealed that Tol 5 has limited capability to metabolize sugars but possesses an extensive repertoire of genes for alkane and aromatic metabolism. Tol 5 has multiple types of hydrocarbon oxygenases, including alkane monooxygenase, aromatic oxygenase, and cytochrome P450, which allow this strain to assimilate a wide range of hydrocarbons and their oxidized derivatives. Notably, we also identified the five distinct aromatic degradation routes, combining pathways broadly conserved in the genus Acinetobacter with additional pathways (such as TOD and HMG) that are rare in this genus. This indicates metabolic versatility comparable to that of P. putida strains, widely used chassis for aromatic compound bioconversion ( Nelson et al ., 2002 , Nogales et al ., 2017 , Martínez-García & de Lorenzo, 2024 ), highlighting its utility as a microbial chassis for bioproduction using aromatic compounds. Acinetobacter species widely exhibit the capacity for alkane degradation, as more than 95% of strains encode the alkane monooxygenases AlkB and AlmA ( Zhao et al ., 2023 ). Oil-degrading strains such as A. venetianus RAG-1 and A. oleivorans DR1 possess multiple AlkB homologs, enabling efficient utilization of n -alkanes ( Park et al ., 2017 , Liu et al ., 2021 ). Although Tol 5 possesses only one alkB gene on the genome, it also carries a plasmid-encoded cytochrome P450 monooxygenase of the CYP153 family. CYP153, which is a distinct alkane hydroxylase targeting short- and medium-chain n -alkanes, is typically found in alkane-degrading bacteria lacking AlkB but is relatively rare among Acinetobacter genomes ( van Beilen Jan et al ., 2006 , Nie et al ., 2014 ). Both alkB and the CYP153 gene were induced in Tol 5 grown on hexadecane, indicating that Tol 5 employs both types of alkane hydroxylases to oxidize hexadecane efficiently ( Fig. 4C and Table S7). Regarding aromatic metabolism in Acinetobacter species, two main ortho -cleavage pathways of catechols, encoded by pca and cat genes, have been mainly studied ( Barbe et al ., 2004 , Fischer et al ., 2008 , Stuani et al ., 2014 , Breisch et al ., 2022 ). Typically, the cat operon is located adjacent to the ben operon, and their expression is coordinately regulated to prevent the accumulation of catechol, which is toxic at high concentration ( Jiménez et al ., 2014 ). Specifically, the catechol 1,2-dioxygenase, encoded by catA within the ben operon, converts the catechol generated by benzoate oxidation into cis , cis -muconate, which subsequently activates the downstream cat operon ( Cosper Nathaniel et al ., 2000 , Bundy et al ., 2002 , Silva-Rocha & de Lorenzo, 2012 ). In Tol 5, however, the cat operon is located adjacent to the mph operon instead of the ben operon ( Fig. 2 ). Furthermore, this adjacent mph operon contains an additional catA gene ( catA_2 ), representing a genetic organization distinct from the mph operon previously reported in A. calcoaceticus PHEA-2 ( Yu et al ., 2011 ). Analogous to the coordinated regulation seen in typical ben-cat clusters, the presence of this catA gene within the mph operon is likely essential for proper coordination with the downstream cat operon, ensuring the smooth conversion of the catechol derived from phenol without toxic accumulation. In addition, Tol 5 possesses a separate ben operon that also contains a catA gene ( catA_4 ) located at a different chromosomal locus ( Fig. 2 ). Therefore, the unique organization of the mph operon as well as the ben operon in Tol 5 likely enables fine-tuned transcription of the genes involved in the degradation of phenol and probably benzoate, representing an optimized gene organization for the degradation of aromatic compounds. Aromatic compounds with alkyl substitutes are typically degraded via the meta -cleavage pathway, which has mainly been studied in P. putida ( Zylstra et al ., 1988 , Assinder & Williams, 1990 ). Tol 5 likely acquired these tod genes through horizontal gene transfer, making this strain unique within the genus Acinetobacter ( Yoshimoto et al ., 2025 ). In the TOD pathway, the genes encoding all reaction steps for toluene degradation, from the initial oxidation catalyzed by toluene dioxygenase to the generation of pyruvate and acetyl-CoA, are clustered in a single tod operon ( Fig. 2 ). Transcriptome analysis of Tol 5 revealed that growth on toluene strongly induced the tod operon, whereas the mph operon was slightly induced ( Fig. 5B ). This regulatory response is likely essential for survival because if alkyl-substituted catechols derived from toluene were processed via the ortho -cleavage pathway, they would be converted into dead-end metabolites such as methylmuconolactone ( Taeger et al ., 1988 ). Thus, the specific induction of the tod operon prevents the metabolic flux from entering the unproductive ortho -pathway. In contrast to the response to toluene, Tol 5 exhibited a distinct transcriptional pattern during growth on phenol. Phenol induced not only the cat and mph genes but also the tod genes ( Fig. 5B ). Interestingly, the disruption of todC1 , encoding the large subunit of toluene dioxygenase, resulted in retarded growth and a higher final cell yield on phenol ( Fig. 7 ). Previous studies have demonstrated that the toluene dioxygenase complex TodABC1C2 oxidizes phenol to both catechol and hydroquinone ( Höring et al ., 2016 ). Based on this, we hypothesize that, in Tol 5, the upregulation of toluene dioxygenase facilitates rapid cell growth by promoting the hydroxylation of toxic phenol. However, this rapid hydroxylation comes at the cost of substrate conversion efficiency, as a portion of the phenol is converted into a likely unavailable by-product, such as hydroquinone, diverting the carbon source away from the productive ortho -cleavage pathway. Furthermore, a mutant lacking phenol monooxygenase (Δ mphN ) completely failed to grow on phenol ( Fig. 7 ). This indicates that the conversion of phenol by toluene dioxygenase alone is insufficient for its utilization as a carbon source, possibly because a high proportion of phenol is converted into hydroquinone, for which no assimilation routes were identified in Tol 5. Taken together, these results indicate that the phenol monooxygenase is the primary route for phenol metabolism in Tol 5, while toluene dioxygenase functions primarily as an auxiliary enzyme that supports rapid degradation. This stands in contrast to P. putida F1, where the toluene dioxygenase has been recognized as the primary enzyme for phenol degradation ( Spain et al ., 1989 , Höring et al ., 2016 ). Therefore, we propose a fundamentally different physiological role for the tod genes in Tol 5, with the toluene dioxygenase functioning primarily as a rapid detoxification mechanism rather than an assimilatory pathway. Alkane and aromatic oxygenases catalyze the initial oxidation of their substrates by forming transient radicals at the active site, which can lead to the leakage of reactive oxygen species (ROS) ( Sazykin et al ., 2016 ). Consequently, ROS are considered to be by-products of alkane and aromatic hydroxylation, and cells must deploy protective mechanisms to scavenge them. For instance, A. calcoaceticus grown on hydrocarbons exhibits increased superoxide dismutase activity, while both A. oleivorans DR1 cultured on triacontane and Paraburkholderia xenovorans p2-fldX1 grown on 4-hydroxyphenylacetate upregulate multiple ROS scavengers ( Sazykin et al ., 2016 , Park et al ., 2017 , Rodríguez-Castro et al ., 2024 ). In Tol 5 grown on hexadecane or aromatic compounds, oxidative stress resistance genes, including sodA_2 , sodC , katA , katE, and ahpC_1 , were induced ( Fig. 6 ). Furthermore, the expression of TCA cycle enzymes is also likely to be adjusted according to the intracellular oxidative stress. In E. coli , ROS-sensitive aconitate hydratase AcnB functions as the primary aconitate hydratase during the growth phase, while ROS-tolerant isozyme AcnA is induced under oxidative stress conditions to maintain the TCA cycle ( Cunningham et al ., 1997 , Jordan et al ., 1999 ). Thus, the expression shift from acnB to acnA_2 in the TCA cycle of Tol 5 grown on aromatic compounds suggests that Tol 5 also switches metabolic enzymes to maintain central metabolism (Fig. S3). In summary, Tol 5 responds to oxidative stress according to the available carbon source, not only by activating stress resistance genes but also by shifting reliance to ROS-tolerant isozymes to sustain central metabolism. In conclusion, Tol 5 possesses a diverse set of metabolic pathways for alkanes and aromatic compounds, regulates these pathways according to available carbon sources, and activates stress resistance genes. Owing to its strong adhesiveness, ability to metabolize alkanes and aromatic compounds, and high tolerance to toxic intermediates, Tol 5 represents a promising microbial chassis for bioprocesses utilizing non-sugar substrates. AUTHOR CONTRIBUTIONS Shori Inoue: Conceptualization, Formal analysis, Investigation, Data curation, Writing - Original Draft. Shogo Yoshimoto: Conceptualization, Writing - Review & Editing. Maiko Hattori: Investigation. Shotaro Yamagishi: Investigation. Katsutoshi Hori: Conceptualization, Supervision, Project administration, Writing - Review & Editing. DATA AVAILABILITY RNA-seq data reported are available in the DDBJ Sequenced Read Archive under the accession numbers PRJDB35996. ACKNOWLEDGMENTS The authors wish to acknowledge the Center for Gene Research, Nagoya University, for technical support with the RNA sequencing. The authors also thank Yuki Ohara for kindly discussing genome sequence data analysis and reviewing the manuscript. This research was supported by the Graduate Program of Transformative Chem-Bio Research at Nagoya University supported by MEXT (WISE Program) to SI, the Japan Science and Technology Agency (JST) SPRING (Grant Number JPMJSP2125) to SI, the Japan Society for the Promotion of Science (JSPS) KAKENHI (Grant Number JP24H00043 and JP25K24646) to KH and SY, and the GteX Program Japan Grant number JPMJGX23B4 to KH. Funder Information Declared Japan Society for the Promotion of Science , JP24H00043 , JP25K24646 Footnotes Minor revisions were made throughout the manuscript, including the title, abstract, and main text. In addition, two authors were added to the author list. REFERENCES ↵ Alberti L , König P , Zeidler S , Poehlein A , Daniel R , Averhoff B & Müller V ( 2023 ) Identification and characterization of a novel pathway for aldopentose degradation in Acinetobacter baumannii . Environmental Microbiology 25 : 2416 – 2430 . 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Share Metabolic pathway analysis of an Acinetobacter strain capable of assimilating diverse hydrocarbons and aromatic compounds Shori Inoue , Shogo Yoshimoto , Maiko Hattori , Shotaro Yamagishi , Katsutoshi Hori bioRxiv 2025.10.30.684093; doi: https://doi.org/10.1101/2025.10.30.684093 Share This Article: Copy Citation Tools Metabolic pathway analysis of an Acinetobacter strain capable of assimilating diverse hydrocarbons and aromatic compounds Shori Inoue , Shogo Yoshimoto , Maiko Hattori , Shotaro Yamagishi , Katsutoshi Hori bioRxiv 2025.10.30.684093; doi: https://doi.org/10.1101/2025.10.30.684093 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Microbiology Subject Areas All Articles Animal Behavior and Cognition (7642) Biochemistry (17715) Bioengineering (13907) Bioinformatics (42003) Biophysics (21470) Cancer Biology (18624) Cell Biology (25533) Clinical Trials (138) Developmental Biology (13390) Ecology (19935) Epidemiology (2067) Evolutionary Biology (24356) Genetics (15617) Genomics (22529) Immunology (17753) Microbiology (40432) Molecular Biology (17200) Neuroscience (88681) Paleontology (667) Pathology (2840) Pharmacology and Toxicology (4828) Physiology (7653) Plant Biology (15171) Scientific Communication and Education (2046) Synthetic Biology (4304) Systems Biology (9826) Zoology (2271)