Acyl CoA reductases useful for bioproduction of hydrocarbons

Acyl CoA reductases useful for bioproduction of hydrocarbons | 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 Acyl CoA reductases useful for bioproduction of hydrocarbons Masakazu Ito, Shigenobu Kishino, Masayoshi Muramatsu, Jun Ogawa This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8953641/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 11 You are reading this latest preprint version Abstract Background Hydrocarbon-based biofuels—so-called drop-in fuels—have gained attention as sustainable alternatives to petroleum-derived fuels, yet their biological production remains limited by the availability of efficient enzymatic pathways for generating hydrocarbon precursors. Medium-chain alkanes produced by microorganisms represent a promising target, but the aldehyde-producing capabilities of acyl-CoA reductases (ACRs) from bacteria, plants, and animals have not been systematically compared. Because ACRs generate fatty aldehydes—key intermediates in hydrocarbon biosynthesis—understanding their diversity is essential for expanding biological fuel production strategies. In this study, we performed a comprehensive screening of ACRs across diverse organisms to identify enzymes with promising aldehyde-producing activity and to advance the development of a new microbial hydrocarbon biosynthesis pathway. Results Sixteen acyl-CoA reductases (ACRs) from microorganisms, plants, and animals were cloned and expressed in Escherichia coli and evaluated by coexpressing each enzyme with a cyanobacterial aldehyde decarbonylase to enable hydrocarbon formation. Several Arabidopsis thaliana ACRs produced higher alkane levels than microbial and animal enzymes. To further examine plant-derived enzymes, ACR homologs with high amino acid similarity to A. thaliana ACR1 and ACR2 were cloned from multiple plant species and tested. Among these, ACR2 from Glycine max exhibited the highest alkane and alkene productivity, demonstrating that certain plant ACRs—known to generate long-chain alcohols—can also act on medium-chain fatty acids. Phylogenetic analysis of fourteen productive plant ACRs showed that ACRs similar to GmACR2 generated higher levels of C13 alkanes, although no clear trend was observed for C15 alkanes or C17 alkenes. Coexpression of GmACR2 with an aldehyde dehydrogenase from Schizosaccharomyces pombe enabled E. coli to produce C17 alkene from sugars. This demonstrates a previously unreported ACR–ALDH-based hydrocarbon biosynthesis pathway and expands the known enzymatic routes available for microbial hydrocarbon production. Conclusion This study identifies multiple microbial and plant-derived ACRs, particularly GmACR2, as effective catalysts for medium-chain hydrocarbon biosynthesis. Coexpression of GmACR2 with S. pombe aldehyde dehydrogenase shows that ACR–ALDH coexpression can enable microbial alkane and alkene production, which represents a previously unreported microbial hydrocarbon biosynthesis route. Because ACR and ALDH homologs are widely distributed across microorganisms, plants, and animals, these findings suggest that ACR–ALDH-based reductive processes may have contributed to the biogenic origin of petroleum, providing broader insight into both biofuel development and natural hydrocarbon formation. Drop-in fuels Hydrocarbon biosynthesis Acyl-CoA reductase Metabolic engineering Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Plant-derived biofuels are a promising alternative to fossil fuels ( 1 – 3 ). Since the carbon dioxide released during biofuel combustion is reabsorbed by plants through photosynthesis, biofuels result in significantly lower net carbon emissions compared to fossil fuels ( 4 ). This cycle helps reduce greenhouse gas emissions ( 5 – 7 ). However, current biofuels like ethanol and fatty acid methyl esters are unsuitable for internal combustion and jet engines due to issues such as metal corrosion ( 8 ), resin degradation ( 9 ), low energy density ( 10 , 11 ), and poor performance at low temperatures ( 12 – 14 ). Hydrocarbon-based biofuels, also known as “drop-in fuels”, are chemically similar to petroleum and have attracted significant attention ( 15 ). Common production methods include biohydrogen-refined diesel, produced by hydrogenating vegetable fats and oils ( 17 ), biomass-to-liquid processes that combine biomass gasification with Fischer-Tropsch synthesis ( 18 ), and hydrocarbon production via genetically modified organisms like algae ( 19 ), bacteria, yeast, and fungi ( 20 – 24 ). However, these technologies are still in their early stages and face challenges in terms of catalyst cost reduction, energy efficiency, and productivity. As a result, they are not yet economically viable. In our previous study, we screened 1,300 microorganisms, including bacteria, yeasts, molds, basidiomycetes, and actinomycetes, for the production of tetradecanal and/or tetradecanol from tetradecanoic acid ( 25 ). We isolated a novel aldehyde-producing acyl-CoA reductase gene ( acrI ) from Klebsiella pneumoniae subsp. pneumoniae NBRC3321, which exhibited significant tetradecanoic acid-transforming activity. Since acyl-CoA reductases (ACRs) producing aldehydes and/or alcohols are found in plants, animals, and bacteria, screening various ACRs is expected to identify enzymes with high aldehyde-producing capacity. In this study, we cloned several ACR genes from microorganisms, plants, and animals, and screened for enzymes with high aldehyde-producing activity. We also report the successful bioproduction of C17 alkene (heptadecene) by coexpressing an ACR from soybeans ( Glycine max ) and an aldehyde dehydrogenase (ALDH) with aldehyde decarbonylase activity from Schizosaccharomyces pombe 972h, as discovered in our previous study ( 26 ). This novel hydrocarbon biosynthesis pathway, using Escherichia coli transformants, represents a promising bioproduction method, as it utilizes ACR and ALDH, enzymes distributed across a wide range of species. Results Evaluation of the aldehyde-producing potential of ACR from microorganisms, plants and animals Sixteen ACRs from a wide range of species, including bacteria, plants, and animals, were selected from the U.S. National Center for Biotechnology Information (NCBI) database, and the corresponding genes were cloned into E. coli . We previously reported an acyl‑CoA reductase from Klebsiella pneumoniae NBRC3321 that produces aldehyde from acyl‑CoA ( 25 ). The bioproduction of alkanes requires aldehyde-producing ACRs that generate aldehydes, which are intermediates in alkane biosynthesis. Since intracellularly produced aldehydes are rapidly converted to alcohols by alcohol dehydrogenase activity, ACRs from various species were coexpressed with a cyanobacterial aldehyde decarbonylase that converts intracellular aldehydes into alkanes and alkenes. We then evaluated the aldehyde-producing capacity of the ACRs based on hydrocarbon production (Fig. 1 ). The results showed that some ACRs from Arabidopsis thaliana exhibited higher alkane productivity compared to ACRs from microorganisms and animals (Fig. 2 ). E. coli strains were coexpressed with plasmids carrying acyl-CoA reductase (ACR) genes from Klebsiella pneumoniae MGH 78578 (KpACR), Acinetobacter sp. ADP1 (AsACR), Shewanella woodyi ATCC 51908 (SwLUX), Photorhabdus luminescens subsp. laumondii TTO1 (PlLUX), Vibrio fischeri ES114 (VfLUX), Vibrio campbellii ATCC BAA-1116 (VhLUX), Arabidopsis thaliana (AtACR1, AtACR2, AtCER4, AtACR4, AtACR5, AtACR6, AtACR7, AtACR8), and Mus musculus (MmACR1, MmACR2), along with an alkane synthase gene from Nostoc punctiforme PCC 73102 (NpAD) for hydrocarbon production. Error bars represent the standard deviation of three biological replicates. Evaluation of the aldehyde-producing potential of ACRs from various plants Using the KEGG genome information for Arabidopsis thaliana ( https://www.kegg.jp/kegg-bin/show_organism?org=T00041 ), we identified eight different ACRs, but only ACR1 and ACR2 were capable of producing alkanes or alkenes. Therefore, we cloned enzymes with high amino acid sequence similarity (70–95%) to ACR1 and ACR2 of A. thaliana from other plant species and compared their hydrocarbon production by coexpressing them with cyanobacterial aldehyde decarbonylase in E. coli . The results showed that ACR2 from Glycine max (soybean) exhibited the highest productivity of both alkane and alkene compared to the other ACRs (Fig. 3 ). Although plants are generally known to produce long-chain alcohols (C20 or higher), it is notable that they also exhibit substrate specificity for medium-chain fatty acid (C14-18). E. coli strains were coexpressed with plasmids carrying acyl-CoA reductase (ACR) genes from Arabidopsis thaliana (AtACR1, AtACR2), Brachypodium distachyon (BdACR), Cicer arietinum (CaACR), Cucumis sativus (CsACR), Fragaria vesca (FvACR1, FvACR2, FvACR3), Glycine max (GmACR1, GmACR2), Oryza sativa japonica (OjACR), Populus trichocarpa (PtACR1, PtACR2), Ricinus communis (RcACR1, RcACR2, RcACR3, RcACR4), Setaria italica (SiACR), Solanum lycopersicum (SlACR1, SlACR2, SlACR3), Sorghum bicolor (SbACR), and Vitis vinifera (VvACR1, VvACR2, VvACR3, VvACR4, VvACR5), along with an alkane synthase gene from Nostoc punctiforme PCC 73102 (NpAD) for hydrocarbon production. Error bars represent the standard deviation of three biological replicates. Phylogenetic tree analysis of plant-derived ACRs Phylogenetic tree analysis of the amino acid sequences was performed on 14 plant-derived ACRs that produced hydrocarbons, and their hydrocarbon productivity was compared. The results showed that ACRs with similarity to GmACR2 were relatively more active, producing C13 alkanes at concentrations ranging from 0.011 to 0.064 µg/ml. In contrast, no significant trend was observed for the production of C15 alkanes and C17 alkenes (Fig. 4 .), suggesting that these ACRs produce aldehydes with longer carbon chains without a clear sequence bias. Discussion In this study, we established a screening method for aldehyde-producing acyl-CoA reductases (ACRs) by coexpressing aldehyde decarbonylase from Nostoc punctiforme PCC 73102 in Escherichia coli . This approach enabled the direct evaluation of hydrocarbon-producing potential among diverse ACR candidates by allowing their aldehyde products to accumulate as hydrocarbons, and allowed us to identify two microbial ACRs and fourteen plant-derived ACRs capable of generating aldehydes that are applicable for hydrocarbon biosynthesis. Generally, plants are known to produce long-chain alcohols ranging from C16 to C32, and their fatty acyl-CoA/ACP reductases are assumed to have specificity toward long-chain substrates ( 27 ). However, in this study, we also identified enzymes that also exhibit substrate specificity toward medium-chain fatty acids, such as C14, converting them into aldehydes. This is a novel finding that broadens our understanding of the diversity and substrate range of plant-derived ACRs. Notably, our phylogenetic analysis revealed that high C13 and C15 alkane productivity tended to be higher within the GmACR-like group, whereas no clear phylogenetic bias was observed for total hydrocarbon production (Fig. 4 ). This suggests that, among the ACRs evaluated in this study, overall sequence similarity does not necessarily correlate with hydrocarbon synthesis activity. Instead, subtle amino acid substitutions or local structural features around the active site may play a more significant role in determining enzyme activity. To elucidate these mechanisms in detail, further studies performing structural modeling, molecular docking analyses, and site-directed mutagenesis will be required. On the other hand, the absence of a strong sequence-based bias can be considered advantageous for future enzyme screening, as it suggests that valuable ACRs may be found across a wide range of phylogenetic lineages. This broadens the possibilities for identifying novel enzymes from unexplored biological resources and metagenomic datasets, and will be highly beneficial for optimizing industrial hydrocarbon production through expanded screening efforts. In a previous study, we cloned several aldehyde dehydrogenases (ALDHs) from bacteria and yeast that possess aldehyde decarbonylase activity, enabling the conversion of aldehydes to alkanes ( 25 ). Based on these findings, we constructed a recombinant E. coli strain coexpressing the ALDH from S. pombe (SpALDH)—which had shown the highest aldehyde decarbonylase activity in our previous work—and the ACR from G. max (GmACR2), which exhibited the highest activity among the plant-derived ACRs identified in this study. This coexpression allowed us to investigate whether hydrocarbons could be produced from sugars. The coexpressing strain successfully produced C17 alkene (heptadecene), indicating that the enzymes coupled effectively for the production of alkenes (Fig. 5 ) and suggesting that GmACR2 may also act on C18 fatty acids. This is the first example of hydrocarbon production by fermentation using ALDH and ACR. Currently, the production of hydrocarbons using cyanobacterial enzymes is superior ( 28 – 29 ); however, we believe that pursuing this pathway is valuable because ALDHs and ACRs are widely distributed across species, providing a vast enzyme screening pool. In our newly established process, alcohol accumulates at levels two orders of magnitude higher than hydrocarbons. This may be due to the weaker activity of aldehyde decarbonylating ALDH, which converts aldehydes to alkanes, compared to alcohol dehydrogenase, which produces alcohols from aldehydes. To address this, we aim to convert alcohol back to aldehyde by introducing an alcohol dehydrogenase, enabling subsequent conversion of the aldehyde into hydrocarbons by aldehyde-decarbonylating enzymes ( 30 – 31 ). We intend to improve hydrocarbon production to an industrial scale by employing metabolic engineering to optimize aldehyde intermediate accumulation, alongside screening for aldehyde decarbonylases with higher activity for hydrocarbon production. Interestingly, hydrocarbons could be produced not by the aldehyde decarbonylase found only in restricted groups such as cyanobacteria, but by ACR and ALDH, enzymes widely distributed among diverse microorganisms. These observations imply that microorganisms possessing ACR–ALDH–based reductive hydrocarbon-producing capability may have contributed to biogenic petroleum formation ( 32 ). Conclusions This study demonstrates the feasibility of using acyl-CoA reductases (ACRs) from diverse biological sources—including plants, microorganisms, and animals—for medium-chain hydrocarbon biosynthesis. Through systematic screening and coexpression with cyanobacterial aldehyde decarbonylase, we identified ACR2 from Glycine max as a highly active enzyme for aldehyde production. Furthermore, the successful fermentation-based synthesis of C17 alkene (heptadecene) via coexpression of G. max ACR2 and Schizosaccharomyces pombe aldehyde dehydrogenase establishes a novel enzymatic route for hydrocarbon production. Given the broad phylogenetic distribution of ACRs and ALDHs, this approach offers a promising platform for future biofuel development and metabolic engineering, particularly for drop-in fuel applications. Material and Methods Strains, plasmids and chemical reagents The bacterial strains used in this study are listed in Table S1. ACR genes were optimized for E. coli codon usage, artificially synthesized, and cloned into the NdeI site of the pCDFDuet-1 plasmid using the In-Fusion HD Cloning Kit (Clontech Laboratories, Inc., California, United States), with each gene expressed under the control of the T7 promoter. This construct, along with the alkane synthase gene from Nostoc punctiforme PCC 73102, was inserted into the Nde I site and transformed into E. coli BL21 (DE3). For coexpression of ACR and ALDH genes in pRSFduet-1, the ACR genes were inserted into the Nde I site of multiple cloning site 2, and the ALDH homologue genes were inserted into the Nco I site of multiple cloning site 1. These plasmids were used to transform E. coli BL21(DE3) (Novagen, Wisconsin, United States) for further hydrocarbon production tests. Plasmids, primers, and synthetic DNA used in this study are listed in Tables S2 and S3. Standard samples for gas chromatography-mass spectrometry (GC-MS) were purchased from Tokyo Chemical Industry Co., Japan. All other reagents were purchased from Nacalai Tesque Inc., Japan. Alkane production test E. coli BL21(DE3) strains coexpressing pCDF-ACR and pRSF-NpAD were cultured overnight in 0.5 ml LB medium (streptomycin 30 µg/ml, kanamycin 50 µg/ml ) at 37°C, 130 rpm with shaking. The overnight culture was inoculated into 2 ml of M9 medium (2% glucose, 0.1% yeast extract, 30 µg/ml streptomycin, 50 µg/ml kanamycin) at 1% volume and incubated at 37°C, 130 rpm for approximately 4 hours, until the final OD 600 reached 0.4–0.6. IPTG (Isopropyl β-D-1-thiogalactopyranoside) was then added to a final concentration of 1 mM, and the culture was incubated for an additional 48 hours at 37°C, 130 rpm. E. coli BL21 (DE3) with ACR and ALDH genes cloned in the pRSFduet vector was cultured overnight in 0.5 ml LB medium (kanamycin 50 µg/ml ) at 37°C, 130 rpm. The overnight culture was inoculated into 2 ml M9 medium (2% glucose, 0.1% yeast extract, 50 µg/ml kanamycin) at 1% volume and incubated at 37°C, 130 rpm for approximately 4 hours, until the final OD 600 reached 0.4–0.6. IPTG was added to a final concentration of 1 mM, and the culture was incubated for 48 hours at 37°C, 130 rpm. GC-MS analysis Alkane production was measured using GC-MS with an HP6890/5973 system (Agilent) equipped with a headspace sampler (HP7694, Agilent) and an HP-INNOWAX analytical column (0.32 mm i.d. x 30 m x 0.5 µm thickness, Agilent). The supernatant was harvested by centrifuging 1 ml post-culture at 8,000 × g for 5 minutes and transferring into a 20 ml headspace vial, which was tightly sealed with a 20 mm aluminum crimp cap-PTFE/silicone septum. The sealed vial was heated at 80°C for 15 minutes and pressurized to 15 psi with a helium purge. The sample was then analyzed using the following GC-MS conditions: injection time of 1 min, fill flow at 50 ml/min, loop temperature at 150°C, transfer line temperature at 200°C, and carrier gas (helium) at 1.0 ml/min. The oven temperature was set to 60°C for 10 minutes, then increased at a rate of 25°C/min to 260°C, holding at 260°C for 1 minute. Declarations Ethical Approval and consent to participate Not applicable Consent for publication Not applicable Availability of data and materials All data presented in this study are included in the manuscript or the supplementary information files. The data supporting the findings are available from the corresponding author upon reasonable request. Competing interests The authors declare that they have no competing interests. Funding A part of this research was supported by the Japan Society for the Promotion of Science 500 KAKENHI (24H00501 to J.O.). Authors’ contributions: Masakazu Ito and Jun Ogawa designed the research. Masakazu Ito and Masayoshi Muramatsu performed the research. Masakazu Ito and Masayoshi Muramatsu analyzed the data. Masakazu Ito, Shigenobu Kishino, and Jun Ogawa wrote the paper. All authors read and approved the final manuscript. Acknowledgments We express our sincere gratitude to Ohto Chikara for his invaluable advice during our research discussions. We also thank Ai Sawagashira for her exceptional support with the experiments and data analyses. References Callegari A, Bolognesi S, Cecconet D, Capodaglio A. Production technologies, current role, and future prospects of biofuels feedstocks: A state-of-the-art review. Crit Rev Environ Sci Technol. 2020;50:384–436. Okolie J, Mukherjee A, Nanda S, Dalai A, Kozinski J. 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Supplementary Files SupplementaryInformation.docx Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 30 Mar, 2026 Reviews received at journal 30 Mar, 2026 Reviews received at journal 25 Mar, 2026 Reviews received at journal 22 Mar, 2026 Reviewers agreed at journal 04 Mar, 2026 Reviewers agreed at journal 02 Mar, 2026 Reviewers agreed at journal 01 Mar, 2026 Reviewers invited by journal 27 Feb, 2026 Editor assigned by journal 25 Feb, 2026 Submission checks completed at journal 25 Feb, 2026 First submitted to journal 24 Feb, 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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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-8953641","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":599648629,"identity":"60a64100-00dc-48c6-90b3-62d2e94812be","order_by":0,"name":"Masakazu Ito","email":"","orcid":"","institution":"R-Frontier division, Toyota Motor Corporation","correspondingAuthor":false,"prefix":"","firstName":"Masakazu","middleName":"","lastName":"Ito","suffix":""},{"id":599648630,"identity":"bd825463-88ca-48dc-a3ce-a08a7c0fae8c","order_by":1,"name":"Shigenobu Kishino","email":"","orcid":"","institution":"Kyoto University","correspondingAuthor":false,"prefix":"","firstName":"Shigenobu","middleName":"","lastName":"Kishino","suffix":""},{"id":599648631,"identity":"23135698-e9f3-439e-9c30-85d348fd59df","order_by":2,"name":"Masayoshi Muramatsu","email":"","orcid":"","institution":"R-Frontier division, Toyota Motor Corporation","correspondingAuthor":false,"prefix":"","firstName":"Masayoshi","middleName":"","lastName":"Muramatsu","suffix":""},{"id":599648632,"identity":"1d91cc75-0152-469c-8972-ef1b2cee79fa","order_by":3,"name":"Jun Ogawa","email":"data:image/png;base64,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","orcid":"","institution":"Kyoto University","correspondingAuthor":true,"prefix":"","firstName":"Jun","middleName":"","lastName":"Ogawa","suffix":""}],"badges":[],"createdAt":"2026-02-24 06:54:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8953641/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8953641/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103895078,"identity":"6ce725ef-02d3-413c-aef1-6866abb9f0b1","added_by":"auto","created_at":"2026-03-04 08:49:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":223631,"visible":true,"origin":"","legend":"\u003cp\u003eScheme of aldehyde metabolism and alkane biosynthesis.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8953641/v1/c3986a2cf343caeccf01c79e.png"},{"id":103895081,"identity":"ac6fed46-c868-43f1-9301-5ecb8574c67b","added_by":"auto","created_at":"2026-03-04 08:49:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":171787,"visible":true,"origin":"","legend":"\u003cp\u003eHydrocarbon productivity of \u003cem\u003eE. coli\u003c/em\u003e coexpressing plasmids carrying ACR genes from various microorganisms and plants with alkane synthase (NpAD) gene.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8953641/v1/ecf39e6aeb73d2fe7253fc39.png"},{"id":103895079,"identity":"0a5aac17-c0b0-420e-ad91-f786a7779b52","added_by":"auto","created_at":"2026-03-04 08:49:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":236559,"visible":true,"origin":"","legend":"\u003cp\u003eHydrocarbon productivity of \u003cem\u003eE. coli \u003c/em\u003ecoexpressing ACR genes from various plants and alkane synthase (NpAD) gene.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8953641/v1/952ed99923dd6874d2c51673.png"},{"id":104401366,"identity":"42b72879-5f16-47bc-962f-e327ee0734ee","added_by":"auto","created_at":"2026-03-11 12:12:30","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":334139,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree analysis of the ACRs. Analysis was performed on 14 plant-derived ACRs that produced hydrocarbons.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8953641/v1/12d908e671acfc38d93146bc.png"},{"id":103895083,"identity":"467dfd58-7aff-4b72-b047-b8880cebaf63","added_by":"auto","created_at":"2026-03-04 08:49:21","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":595168,"visible":true,"origin":"","legend":"\u003cp\u003eHydrocarbon production by \u003cem\u003eE. coli\u003c/em\u003e expressing plasmids with ACR gene from \u003cem\u003eG. max\u003c/em\u003e(soybean) and ALDH gene from \u003cem\u003eS. pombe\u003c/em\u003e. (a) hydrocarbon standard, (b) ALDH gene from \u003cem\u003eS. pombe\u003c/em\u003e (SpADLH) and ACR gene from \u003cem\u003eG. max\u003c/em\u003e (GmACR2).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8953641/v1/1fde5fa78e1c4df048250da4.png"},{"id":104408104,"identity":"3469cf31-e9cc-4a63-bfb1-7dfd2724b47e","added_by":"auto","created_at":"2026-03-11 12:41:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2132626,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8953641/v1/0b3ce4fc-39f1-4ae9-9265-758dbb5bd7cc.pdf"},{"id":103895082,"identity":"b653b3ac-b46c-4228-9b41-ab777e0f16a1","added_by":"auto","created_at":"2026-03-04 08:49:21","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":161047,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-8953641/v1/34457d562ba6b4bed0941186.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Acyl CoA reductases useful for bioproduction of hydrocarbons","fulltext":[{"header":"Background","content":"\u003cp\u003ePlant-derived biofuels are a promising alternative to fossil fuels (\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Since the carbon dioxide released during biofuel combustion is reabsorbed by plants through photosynthesis, biofuels result in significantly lower net carbon emissions compared to fossil fuels (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). This cycle helps reduce greenhouse gas emissions (\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). However, current biofuels like ethanol and fatty acid methyl esters are unsuitable for internal combustion and jet engines due to issues such as metal corrosion (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e), resin degradation (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e), low energy density (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e), and poor performance at low temperatures (\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHydrocarbon-based biofuels, also known as \u0026ldquo;drop-in fuels\u0026rdquo;, are chemically similar to petroleum and have attracted significant attention (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Common production methods include biohydrogen-refined diesel, produced by hydrogenating vegetable fats and oils (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e), biomass-to-liquid processes that combine biomass gasification with Fischer-Tropsch synthesis (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e), and hydrocarbon production via genetically modified organisms like algae (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e), bacteria, yeast, and fungi (\u003cspan additionalcitationids=\"CR21 CR22 CR23\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). However, these technologies are still in their early stages and face challenges in terms of catalyst cost reduction, energy efficiency, and productivity. As a result, they are not yet economically viable.\u003c/p\u003e \u003cp\u003eIn our previous study, we screened 1,300 microorganisms, including bacteria, yeasts, molds, basidiomycetes, and actinomycetes, for the production of tetradecanal and/or tetradecanol from tetradecanoic acid (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). We isolated a novel aldehyde-producing acyl-CoA reductase gene (\u003cem\u003eacrI\u003c/em\u003e) from \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e subsp. \u003cem\u003epneumoniae\u003c/em\u003e NBRC3321, which exhibited significant tetradecanoic acid-transforming activity.\u003c/p\u003e \u003cp\u003eSince acyl-CoA reductases (ACRs) producing aldehydes and/or alcohols are found in plants, animals, and bacteria, screening various ACRs is expected to identify enzymes with high aldehyde-producing capacity. In this study, we cloned several ACR genes from microorganisms, plants, and animals, and screened for enzymes with high aldehyde-producing activity. We also report the successful bioproduction of C17 alkene (heptadecene) by coexpressing an ACR from soybeans (\u003cem\u003eGlycine max\u003c/em\u003e) and an aldehyde dehydrogenase (ALDH) with aldehyde decarbonylase activity from \u003cem\u003eSchizosaccharomyces pombe\u003c/em\u003e 972h, as discovered in our previous study (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). This novel hydrocarbon biosynthesis pathway, using \u003cem\u003eEscherichia coli\u003c/em\u003e transformants, represents a promising bioproduction method, as it utilizes ACR and ALDH, enzymes distributed across a wide range of species.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation of the aldehyde-producing potential of ACR from microorganisms, plants and animals\u003c/h2\u003e \u003cp\u003eSixteen ACRs from a wide range of species, including bacteria, plants, and animals, were selected from the U.S. National Center for Biotechnology Information (NCBI) database, and the corresponding genes were cloned into \u003cem\u003eE. coli\u003c/em\u003e. We previously reported an acyl‑CoA reductase from \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e NBRC3321 that produces aldehyde from acyl‑CoA (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). The bioproduction of alkanes requires aldehyde-producing ACRs that generate aldehydes, which are intermediates in alkane biosynthesis. Since intracellularly produced aldehydes are rapidly converted to alcohols by alcohol dehydrogenase activity, ACRs from various species were coexpressed with a cyanobacterial aldehyde decarbonylase that converts intracellular aldehydes into alkanes and alkenes. We then evaluated the aldehyde-producing capacity of the ACRs based on hydrocarbon production (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The results showed that some ACRs from \u003cem\u003eArabidopsis thaliana\u003c/em\u003e exhibited higher alkane productivity compared to ACRs from microorganisms and animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eE. coli\u003c/em\u003e strains were coexpressed with plasmids carrying acyl-CoA reductase (ACR) genes from \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e MGH 78578 (KpACR), \u003cem\u003eAcinetobacter\u003c/em\u003e sp. ADP1 (AsACR), \u003cem\u003eShewanella woodyi\u003c/em\u003e ATCC 51908 (SwLUX), \u003cem\u003ePhotorhabdus luminescens\u003c/em\u003e subsp. \u003cem\u003elaumondii\u003c/em\u003e TTO1 (PlLUX), \u003cem\u003eVibrio fischeri\u003c/em\u003e ES114 (VfLUX), \u003cem\u003eVibrio campbellii\u003c/em\u003e ATCC BAA-1116 (VhLUX), \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (AtACR1, AtACR2, AtCER4, AtACR4, AtACR5, AtACR6, AtACR7, AtACR8), and \u003cem\u003eMus musculus\u003c/em\u003e (MmACR1, MmACR2), along with an alkane synthase gene from \u003cem\u003eNostoc punctiforme\u003c/em\u003e PCC 73102 (NpAD) for hydrocarbon production. Error bars represent the standard deviation of three biological replicates.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEvaluation of the aldehyde-producing potential of ACRs from various plants\u003c/h3\u003e\n\u003cp\u003eUsing the KEGG genome information for \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.kegg.jp/kegg-bin/show_organism?org=T00041\u003c/span\u003e\u003cspan address=\"https://www.kegg.jp/kegg-bin/show_organism?org=T00041\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), we identified eight different ACRs, but only ACR1 and ACR2 were capable of producing alkanes or alkenes. Therefore, we cloned enzymes with high amino acid sequence similarity (70\u0026ndash;95%) to ACR1 and ACR2 of \u003cem\u003eA. thaliana\u003c/em\u003e from other plant species and compared their hydrocarbon production by coexpressing them with cyanobacterial aldehyde decarbonylase in \u003cem\u003eE. coli\u003c/em\u003e. The results showed that ACR2 from \u003cem\u003eGlycine max\u003c/em\u003e (soybean) exhibited the highest productivity of both alkane and alkene compared to the other ACRs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Although plants are generally known to produce long-chain alcohols (C20 or higher), it is notable that they also exhibit substrate specificity for medium-chain fatty acid (C14-18).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eE. coli\u003c/em\u003e strains were coexpressed with plasmids carrying acyl-CoA reductase (ACR) genes from \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (AtACR1, AtACR2), \u003cem\u003eBrachypodium distachyon\u003c/em\u003e (BdACR), \u003cem\u003eCicer arietinum\u003c/em\u003e (CaACR), \u003cem\u003eCucumis sativus\u003c/em\u003e (CsACR), \u003cem\u003eFragaria vesca\u003c/em\u003e (FvACR1, FvACR2, FvACR3), \u003cem\u003eGlycine max\u003c/em\u003e (GmACR1, GmACR2), \u003cem\u003eOryza sativa japonica\u003c/em\u003e (OjACR), \u003cem\u003ePopulus trichocarpa\u003c/em\u003e (PtACR1, PtACR2), \u003cem\u003eRicinus communis\u003c/em\u003e (RcACR1, RcACR2, RcACR3, RcACR4), \u003cem\u003eSetaria italica\u003c/em\u003e (SiACR), \u003cem\u003eSolanum lycopersicum\u003c/em\u003e (SlACR1, SlACR2, SlACR3), \u003cem\u003eSorghum bicolor\u003c/em\u003e (SbACR), and \u003cem\u003eVitis vinifera\u003c/em\u003e (VvACR1, VvACR2, VvACR3, VvACR4, VvACR5), along with an alkane synthase gene from \u003cem\u003eNostoc punctiforme\u003c/em\u003e PCC 73102 (NpAD) for hydrocarbon production. Error bars represent the standard deviation of three biological replicates.\u003c/p\u003e\n\u003ch3\u003ePhylogenetic tree analysis of plant-derived ACRs\u003c/h3\u003e\n\u003cp\u003ePhylogenetic tree analysis of the amino acid sequences was performed on 14 plant-derived ACRs that produced hydrocarbons, and their hydrocarbon productivity was compared. The results showed that ACRs with similarity to GmACR2 were relatively more active, producing C13 alkanes at concentrations ranging from 0.011 to 0.064 \u0026micro;g/ml. In contrast, no significant trend was observed for the production of C15 alkanes and C17 alkenes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.), suggesting that these ACRs produce aldehydes with longer carbon chains without a clear sequence bias.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we established a screening method for aldehyde-producing acyl-CoA reductases (ACRs) by coexpressing aldehyde decarbonylase from \u003cem\u003eNostoc punctiforme\u003c/em\u003e PCC 73102 in \u003cem\u003eEscherichia coli\u003c/em\u003e. This approach enabled the direct evaluation of hydrocarbon-producing potential among diverse ACR candidates by allowing their aldehyde products to accumulate as hydrocarbons, and allowed us to identify two microbial ACRs and fourteen plant-derived ACRs capable of generating aldehydes that are applicable for hydrocarbon biosynthesis. Generally, plants are known to produce long-chain alcohols ranging from C16 to C32, and their fatty acyl-CoA/ACP reductases are assumed to have specificity toward long-chain substrates (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). However, in this study, we also identified enzymes that also exhibit substrate specificity toward medium-chain fatty acids, such as C14, converting them into aldehydes. This is a novel finding that broadens our understanding of the diversity and substrate range of plant-derived ACRs.\u003c/p\u003e \u003cp\u003eNotably, our phylogenetic analysis revealed that high C13 and C15 alkane productivity tended to be higher within the GmACR-like group, whereas no clear phylogenetic bias was observed for total hydrocarbon production (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). This suggests that, among the ACRs evaluated in this study, overall sequence similarity does not necessarily correlate with hydrocarbon synthesis activity. Instead, subtle amino acid substitutions or local structural features around the active site may play a more significant role in determining enzyme activity. To elucidate these mechanisms in detail, further studies performing structural modeling, molecular docking analyses, and site-directed mutagenesis will be required. On the other hand, the absence of a strong sequence-based bias can be considered advantageous for future enzyme screening, as it suggests that valuable ACRs may be found across a wide range of phylogenetic lineages. This broadens the possibilities for identifying novel enzymes from unexplored biological resources and metagenomic datasets, and will be highly beneficial for optimizing industrial hydrocarbon production through expanded screening efforts.\u003c/p\u003e \u003cp\u003eIn a previous study, we cloned several aldehyde dehydrogenases (ALDHs) from bacteria and yeast that possess aldehyde decarbonylase activity, enabling the conversion of aldehydes to alkanes (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Based on these findings, we constructed a recombinant \u003cem\u003eE. coli\u003c/em\u003e strain coexpressing the ALDH from \u003cem\u003eS. pombe\u003c/em\u003e (SpALDH)\u0026mdash;which had shown the highest aldehyde decarbonylase activity in our previous work\u0026mdash;and the ACR from \u003cem\u003eG. max\u003c/em\u003e (GmACR2), which exhibited the highest activity among the plant-derived ACRs identified in this study. This coexpression allowed us to investigate whether hydrocarbons could be produced from sugars. The coexpressing strain successfully produced C17 alkene (heptadecene), indicating that the enzymes coupled effectively for the production of alkenes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) and suggesting that GmACR2 may also act on C18 fatty acids. This is the first example of hydrocarbon production by fermentation using ALDH and ACR. Currently, the production of hydrocarbons using cyanobacterial enzymes is superior (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e); however, we believe that pursuing this pathway is valuable because ALDHs and ACRs are widely distributed across species, providing a vast enzyme screening pool.\u003c/p\u003e \u003cp\u003eIn our newly established process, alcohol accumulates at levels two orders of magnitude higher than hydrocarbons. This may be due to the weaker activity of aldehyde decarbonylating ALDH, which converts aldehydes to alkanes, compared to alcohol dehydrogenase, which produces alcohols from aldehydes. To address this, we aim to convert alcohol back to aldehyde by introducing an alcohol dehydrogenase, enabling subsequent conversion of the aldehyde into hydrocarbons by aldehyde-decarbonylating enzymes (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). We intend to improve hydrocarbon production to an industrial scale by employing metabolic engineering to optimize aldehyde intermediate accumulation, alongside screening for aldehyde decarbonylases with higher activity for hydrocarbon production.\u003c/p\u003e \u003cp\u003eInterestingly, hydrocarbons could be produced not by the aldehyde decarbonylase found only in restricted groups such as cyanobacteria, but by ACR and ALDH, enzymes widely distributed among diverse microorganisms. These observations imply that microorganisms possessing ACR\u0026ndash;ALDH\u0026ndash;based reductive hydrocarbon-producing capability may have contributed to biogenic petroleum formation (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study demonstrates the feasibility of using acyl-CoA reductases (ACRs) from diverse biological sources\u0026mdash;including plants, microorganisms, and animals\u0026mdash;for medium-chain hydrocarbon biosynthesis. Through systematic screening and coexpression with cyanobacterial aldehyde decarbonylase, we identified ACR2 from \u003cem\u003eGlycine max\u003c/em\u003e as a highly active enzyme for aldehyde production. Furthermore, the successful fermentation-based synthesis of C17 alkene (heptadecene) via coexpression of \u003cem\u003eG. max\u003c/em\u003e ACR2 and \u003cem\u003eSchizosaccharomyces pombe\u003c/em\u003e aldehyde dehydrogenase establishes a novel enzymatic route for hydrocarbon production. Given the broad phylogenetic distribution of ACRs and ALDHs, this approach offers a promising platform for future biofuel development and metabolic engineering, particularly for drop-in fuel applications.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStrains, plasmids and chemical reagents\u003c/h2\u003e \u003cp\u003eThe bacterial strains used in this study are listed in Table S1. ACR genes were optimized for \u003cem\u003eE. coli\u003c/em\u003e codon usage, artificially synthesized, and cloned into the NdeI site of the pCDFDuet-1 plasmid using the In-Fusion HD Cloning Kit (Clontech Laboratories, Inc., California, United States), with each gene expressed under the control of the T7 promoter. This construct, along with the alkane synthase gene from \u003cem\u003eNostoc punctiforme\u003c/em\u003e PCC 73102, was inserted into the \u003cem\u003eNde\u003c/em\u003e I site and transformed into \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3).\u003c/p\u003e \u003cp\u003eFor coexpression of ACR and ALDH genes in pRSFduet-1, the ACR genes were inserted into the \u003cem\u003eNde\u003c/em\u003eI site of multiple cloning site 2, and the ALDH homologue genes were inserted into the \u003cem\u003eNco\u003c/em\u003eI site of multiple cloning site 1. These plasmids were used to transform \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3) (Novagen, Wisconsin, United States) for further hydrocarbon production tests.\u003c/p\u003e \u003cp\u003ePlasmids, primers, and synthetic DNA used in this study are listed in Tables S2 and S3.\u003c/p\u003e \u003cp\u003eStandard samples for gas chromatography-mass spectrometry (GC-MS) were purchased from Tokyo Chemical Industry Co., Japan. All other reagents were purchased from Nacalai Tesque Inc., Japan.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAlkane production test\u003c/h3\u003e\n\u003cp\u003e \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3) strains coexpressing pCDF-ACR and pRSF-NpAD were cultured overnight in 0.5 ml LB medium (streptomycin 30 \u0026micro;g/ml, kanamycin 50 \u0026micro;g/ml ) at 37\u0026deg;C, 130 rpm with shaking. The overnight culture was inoculated into 2 ml of M9 medium (2% glucose, 0.1% yeast extract, 30 \u0026micro;g/ml streptomycin, 50 \u0026micro;g/ml kanamycin) at 1% volume and incubated at 37\u0026deg;C, 130 rpm for approximately 4 hours, until the final OD\u003csub\u003e600\u003c/sub\u003e reached 0.4\u0026ndash;0.6. IPTG (Isopropyl β-D-1-thiogalactopyranoside) was then added to a final concentration of 1 mM, and the culture was incubated for an additional 48 hours at 37\u0026deg;C, 130 rpm.\u003c/p\u003e \u003cp\u003e \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3) with ACR and ALDH genes cloned in the pRSFduet vector was cultured overnight in 0.5 ml LB medium (kanamycin 50 \u0026micro;g/ml ) at 37\u0026deg;C, 130 rpm. The overnight culture was inoculated into 2 ml M9 medium (2% glucose, 0.1% yeast extract, 50 \u0026micro;g/ml kanamycin) at 1% volume and incubated at 37\u0026deg;C, 130 rpm for approximately 4 hours, until the final OD\u003csub\u003e600\u003c/sub\u003e reached 0.4\u0026ndash;0.6. IPTG was added to a final concentration of 1 mM, and the culture was incubated for 48 hours at 37\u0026deg;C, 130 rpm.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eGC-MS analysis\u003c/h2\u003e \u003cp\u003eAlkane production was measured using GC-MS with an HP6890/5973 system (Agilent) equipped with a headspace sampler (HP7694, Agilent) and an HP-INNOWAX analytical column (0.32 mm i.d. x 30 m x 0.5 \u0026micro;m thickness, Agilent). The supernatant was harvested by centrifuging 1 ml post-culture at 8,000 \u0026times; g for 5 minutes and transferring into a 20 ml headspace vial, which was tightly sealed with a 20 mm aluminum crimp cap-PTFE/silicone septum. The sealed vial was heated at 80\u0026deg;C for 15 minutes and pressurized to 15 psi with a helium purge. The sample was then analyzed using the following GC-MS conditions: injection time of 1 min, fill flow at 50 ml/min, loop temperature at 150\u0026deg;C, transfer line temperature at 200\u0026deg;C, and carrier gas (helium) at 1.0 ml/min. The oven temperature was set to 60\u0026deg;C for 10 minutes, then increased at a rate of 25\u0026deg;C/min to 260\u0026deg;C, holding at 260\u0026deg;C for 1 minute.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical Approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data presented in this study are included in the manuscript or the supplementary information files. The data supporting the findings are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA part of this research was supported by the Japan Society for the Promotion of Science 500 KAKENHI (24H00501 to J.O.). \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions: \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMasakazu Ito and Jun Ogawa designed the research. Masakazu Ito and Masayoshi Muramatsu performed the research. Masakazu Ito and Masayoshi Muramatsu analyzed the data. Masakazu Ito, Shigenobu Kishino, and Jun Ogawa wrote the paper. All authors read and approved the final manuscript.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe express our sincere gratitude to Ohto Chikara for his invaluable advice during our research discussions. We also thank Ai Sawagashira for her exceptional support with the experiments and data analyses.\u003c/p\u003e\n\n"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCallegari A, Bolognesi S, Cecconet D, Capodaglio A. Production technologies, current role, and future prospects of biofuels feedstocks: A state-of-the-art review. Crit Rev Environ Sci Technol. 2020;50:384\u0026ndash;436.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOkolie J, Mukherjee A, Nanda S, Dalai A, Kozinski J. Next-generation biofuels and platform biochemicals from lignocellulosic biomass. 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Fuel. 2014;115:88\u0026ndash;96.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBurimsitthigul T, Yoosuk B, Ngamcharussrivichai C, Prasassarakich P. Hydrocarbon biofuel from hydrotreating of palm oil over unsupported Ni\u0026ndash;Mo sulfide catalysts. Renewable Energy. 2021;163:1648\u0026ndash;59.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVan VOP, Faaij AP. Turkenburg WC. Fischer\u0026ndash;Tropsch diesel production in a well-to-wheel perspective: A carbon, energy flow and cost analysis. Energ Convers Manag. 2009;50:855\u0026ndash;76.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSorigu\u0026eacute; D, L\u0026eacute;geret B, Cuin\u0026eacute; S, Morales P, Mirabella B, Gu\u0026eacute;deney G. Microalgae synthesize hydrocarbons from long-chain fatty acids via a light-dependent pathway. 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The Plant Fatty Acyl Reductases. Int J Mol Sci. 2022;23:16156.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchirmer A. (2010). Microbial biosynthesis of alkanes. Science. 329,559\u0026thinsp;\u0026ndash;\u0026thinsp;62.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePark AK. Crystal structures of aldehyde deformylating oxygenase from \u003cem\u003eLimnothrix\u003c/em\u003e sp. KNUA012 and \u003cem\u003eOscillatoria\u003c/em\u003e sp. KNUA011. Biochem Biophys Res Commun. 2016;477:395\u0026ndash;400.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSui YA, Kishino S, Maruyama S, Ito M, Muramatsu M, Obata S, Ogawa J. Utilizing Alcohol for Alkane Biosynthesis by Introducing a Fatty Alcohol Dehydrogenase. Appl Environ Microbiol. 2022;88:e0126422.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSui YA, Maruyama S, Okada N, Ito M, Muramatsu M, Obata S, Ogawa J. Alkane production from fatty alcohols by the combined reactions catalyzed by an alcohol dehydrogenase and an aldehyde-deformylating oxygenase. Biosci Biotechnol Biochem. 2023;87:925\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Z, et al. Biological origin and depositional environment of crude oils in the Qiongdongnan Basin: Insights from molecular biomarkers and whole oil carbon isotope. PETROL SCI. 2024;21:3029\u0026ndash;46.\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":"Drop-in fuels, Hydrocarbon biosynthesis, Acyl-CoA reductase, Metabolic engineering","lastPublishedDoi":"10.21203/rs.3.rs-8953641/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8953641/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eHydrocarbon-based biofuels\u0026mdash;so-called drop-in fuels\u0026mdash;have gained attention as sustainable alternatives to petroleum-derived fuels, yet their biological production remains limited by the availability of efficient enzymatic pathways for generating hydrocarbon precursors. Medium-chain alkanes produced by microorganisms represent a promising target, but the aldehyde-producing capabilities of acyl-CoA reductases (ACRs) from bacteria, plants, and animals have not been systematically compared. Because ACRs generate fatty aldehydes\u0026mdash;key intermediates in hydrocarbon biosynthesis\u0026mdash;understanding their diversity is essential for expanding biological fuel production strategies. In this study, we performed a comprehensive screening of ACRs across diverse organisms to identify enzymes with promising aldehyde-producing activity and to advance the development of a new microbial hydrocarbon biosynthesis pathway.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eSixteen acyl-CoA reductases (ACRs) from microorganisms, plants, and animals were cloned and expressed in \u003cem\u003eEscherichia coli\u003c/em\u003e and evaluated by coexpressing each enzyme with a cyanobacterial aldehyde decarbonylase to enable hydrocarbon formation. Several \u003cem\u003eArabidopsis thaliana\u003c/em\u003e ACRs produced higher alkane levels than microbial and animal enzymes. To further examine plant-derived enzymes, ACR homologs with high amino acid similarity to \u003cem\u003eA. thaliana\u003c/em\u003e ACR1 and ACR2 were cloned from multiple plant species and tested. Among these, ACR2 from \u003cem\u003eGlycine max\u003c/em\u003e exhibited the highest alkane and alkene productivity, demonstrating that certain plant ACRs\u0026mdash;known to generate long-chain alcohols\u0026mdash;can also act on medium-chain fatty acids. Phylogenetic analysis of fourteen productive plant ACRs showed that ACRs similar to GmACR2 generated higher levels of C13 alkanes, although no clear trend was observed for C15 alkanes or C17 alkenes. Coexpression of GmACR2 with an aldehyde dehydrogenase from \u003cem\u003eSchizosaccharomyces pombe\u003c/em\u003e enabled \u003cem\u003eE. coli\u003c/em\u003e to produce C17 alkene from sugars. This demonstrates a previously unreported ACR\u0026ndash;ALDH-based hydrocarbon biosynthesis pathway and expands the known enzymatic routes available for microbial hydrocarbon production.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThis study identifies multiple microbial and plant-derived ACRs, particularly GmACR2, as effective catalysts for medium-chain hydrocarbon biosynthesis. Coexpression of GmACR2 with \u003cem\u003eS. pombe\u003c/em\u003e aldehyde dehydrogenase shows that ACR\u0026ndash;ALDH coexpression can enable microbial alkane and alkene production, which represents a previously unreported microbial hydrocarbon biosynthesis route. Because ACR and ALDH homologs are widely distributed across microorganisms, plants, and animals, these findings suggest that ACR\u0026ndash;ALDH-based reductive processes may have contributed to the biogenic origin of petroleum, providing broader insight into both biofuel development and natural hydrocarbon formation.\u003c/p\u003e","manuscriptTitle":"Acyl CoA reductases useful for bioproduction of hydrocarbons","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-04 08:49:16","doi":"10.21203/rs.3.rs-8953641/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-30T11:42:19+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-30T10:56:55+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-25T04:09:35+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-22T08:37:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"135168591072928887834404093469057782947","date":"2026-03-04T15:52:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"36380958436364092745346027274790739128","date":"2026-03-03T02:06:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"158324904016565055367992083090521237221","date":"2026-03-01T21:25:34+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-27T15:29:49+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-25T10:30:38+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-25T10:24:42+00:00","index":"","fulltext":""},{"type":"submitted","content":"Microbial Cell Factories","date":"2026-02-24T06:42:23+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":"9ffd54b8-9b42-491b-be87-9d3bc92e1d80","owner":[],"postedDate":"March 4th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-05-19T05:40:07+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-04 08:49:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8953641","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8953641","identity":"rs-8953641","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}