Corrinoid-dependent ethyl-transfer catalyzed by the methanol:coenzyme M methyl transferase from Methanosarcina acetivorans 

Corrinoid-dependent ethyl-transfer catalyzed by the methanol:coenzyme M methyl transferase from Methanosarcina acetivorans | 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 Corrinoid-dependent ethyl-transfer catalyzed by the methanol:coenzyme M methyl transferase from Methanosarcina acetivorans Tejas Somvanshi, Jichen Bao, Silvan Scheller This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4694130/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Corrinoid-dependent methyltransferases catalyze methyl-group transfer reactions in all domains of life. These enzymes are generally considered exclusive for C1-substrates (methyl-groups). However, in Methanosarcina trace ethane production from ethanol has been demonstrated in vivo , which led to the hypothesis that corrinoid-dependent methanol specific methyltransferases are promiscuous towards also accepting ethyl-groups. Here we show that the conversion of ethanol to trace amounts of ethane in Methanosarcina acetivorans proceeds via the known methanol-to-methane metabolism, involving the methanol:5-hydroxybenzimidazolylcobamide methyltransferase (MtaB) and a corrinoid-containing methyl-accepting protein (MtaC), but via transfer of ethyl groups instead of methyl groups. We demonstrate that all three isozymes of the methanol specific MtaB subunit and the corrinoid protein MtaC of M. acetivorans are promiscuous towards accepting ethanol, granting the microbe capacity of ethane production via promiscuity downstream in Co -methyl-5-hydroxybenzimidazolylcobamide:2-mercaptoethanesulfonate methyltransferase (MtaA) and methyl-coenzyme M reductase (Mcr). We assessed the ethyl-group transfer efficiency of each of the three isozymes and engineered chimeras that combine 2 different MtaA subunits with the 3 isoforms of MtaCB together to increase the ethane production capability of M. acetivorans . Demonstrating that corrinoid-dependent coenzyme M methyltransferases can catalyze transfer of higher alkyl groups extends the pool of reactions to be considered in metabolic networks. Ethane Promiscuity ethyl-transfer Methyltransferases Methanosarcina Corrinoids Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Described as the ‘nature’s most beautiful cofactor’, corrinoids are catalytic centers of non- S -adenosylmethionine dependent methyltransferases (Matthews 2001 ). Corrinoid-dependent methyltransferases are present in all 3 domains of life and are part of the energy metabolism of many anaerobic organisms (Matthews et al. 2008 ). The cobalt centre of the cobalamin acts as a shuttle for the methyl groups, cycling between its Co(I) and its Me-Co(III) state. Occasionally the Co(I) is oxidized to an inactive Co(II) state which requires reductive activation either dependent on adenosylmethionine or ATP to return to catalytically active Co(I) state (Matthews et al. 2008 ). In all methanogens and in anaerobic methanotrophic archaea, corrinoid dependent methyltransferases catalyze transfer of methyl group in at least one of the following catabolic reactions: (i) between tetrahydromethanopterin and coenzyme M in most methanogenesis pathways and in anaerobic methanotrophy (ii) from acetate to tetrahydromethanopterin in aceticlastic methanogenesis (iii) from methylated substrates to coenzyme M in methylotrophic methanogenesis and (iv) from a methylated corrinoid to homocysteine for methionine synthesis (Matthews et al. 2008 ). Although considered exclusive to methyl groups, methyltransferases have been hypothesized to also convert ethyl groups. Notably in bacteria where ethionine production and secretion has been studied (Loerch and Mallette 1963 ) and in biogenic ethane formation. While most ethane is formed by thermogenic or abiotic processes, evidence for biogenic ethane has been reported for anoxic slurries (Belay and Daniels 1987 ; Oremland et al. 1988 ; Hinrichs et al. 2006 ; Xie et al. 2013 ). Methanogens were identified as the source of biogenic ethane from ethanol when pure cultures of Methanosarcina barkeri strains showed trace ethane formation in an ethanol-supplemented medium. While the metabolic pathway responsible for this conversion has not been elucidated, it was hypothesized that methanol-specific methylotrophic methyltransferase system (Fig. 1 ) is involved, given that significant ethane production was only seen in methanogens capable of utilizing methanol and when grown using methanol (Belay and Daniels 1988 ). Here we focus on the methanol: coenzyme M methyltransferase (Mta) from Methanosarcina acetivorans , since this organism is a versatile, genetically tractable model methanogen capable of producing methane from methylated substrates such as methanol and methylamines (Sowers et al. 1984 ; Metcalf et al. 1997 ; Nayak and Metcalf 2017 ; Lyu and Liu 2019 ; Zhu et al. 2023 ; Bao et al. 2024 ). The methanol-specific methyltransferase is a 3-subunit protein complex (MtaABC) which catalyses methyl transfer from methanol to coenzyme M (CoM) forming methyl-CoM. M. acetivorans encodes 3 isoforms of MtaCB ( mtaCB1- ma_0455–0456, mtaCB2- ma_4391–4392, mtaCB3- ma_1616–1617 ), each with varying activity towards methanol and expression pattern (Pritchett and Metcalf 2005 ; Bose et al. 2006 ). Methyl coenzyme M reductase (Mcr) reduces the methyl-CoM to methane. Mcr has shown promiscuity towards ethyl-CoM (Goenrich et al. 2004 ; Scheller et al. 2013 ) and homologs of Mcr have been identified in other archaea catalyzing the reversible oxidation of non-methane alkanes (Laso-Pérez et al. 2016 , 2019 ; Chen et al. 2019 ; Hahn et al. 2020 ; Zehnle et al. 2023 ). In this study, we tested the microbial pathway of ethane production in M. acetivorans , showing differential promiscuity between isoforms towards ethanol conversion. We engineered chimeras between the individual Mta subunits to enhance the reactivity of the corrinoids towards ethane production from ethanol. Materials and methods Microbiological methods. Lysogenic broth containing 50 mg L − 1 ampicillin was used for plasmid construction in E. coli NEB5α. M. acetivorans was cultured in a high-salt medium tailored to the specific requirements of the experiment (Sowers et al. 1993 ). The optical density of the cultures was tracked using Eppendorf BioPhotometer plus spectrophotometer. Plasmid construction was carried out according to standard protocols. Liposome-mediated methods and polyethylene glycol-mediated methods were used to transform M. acetivorans (Metcalf et al. 1997 ). Resting cell suspension assays. 30 ml of mid-exponential phase cells grown in 60 mM methanol or 50 mM trimethylamine (TMA) with headspace 50% N 2 / 20% CO 2 / 30% of 1% H 2 S in N 2 at atmospheric pressure were centrifuged, washed twice and then resuspended in 6 ml of HS media with 33.6 µl of 99.99% ethanol (final concentration 96 mM) and 2 µl puromycin to block protein synthesis. The master mix was then divided to 3 Balch tubes (2 ml each), and the headspace was replaced by 50% N 2 / 20% CO 2 / 30% of 1% H 2 S in N 2 at atmospheric pressure. GC-FID quantification of methane and ethane. The T 0 sample was measured with GC-FID right after setting up the resting cell suspension and the tubes were incubated at 37˚C. Samples were taken periodically. The methane and ethane concentrations in the headspace gas samples were measured using a GC-FID (Agilent HP 6890 Gas Chromatograph, Hewlett-Packard), equipped with an HP-AL/KCL column (length, 50 m; diameter, 0.32 mm, thickness, 8 µm). The headspace gas samples were injected with a Gastight 1700 SampleLock Syringe (100 µL, PN81056) (Hamilton). A calibration curve was generated using methane standards. The ratio of the peak areas generated by methane and ethane for the same concentration each was used to quantify ethane. The detection limit for ethane was 3 nmol. in vivo methane and ethane production. The cultures were grown in Hungate tubes. 4.5 ml cultures were set for the M. acetivorans WWM73 background test whereas 3 ml for the cultures comparing WWM73 to WWM13 and cultures expressing engineered Mta chimeras. The headspace was replaced by 70% N 2 / 20% CO 2 / 10% of 1% H 2 S in N 2 at atmospheric pressure. The substrates added for both in vivo tests were 120 mM methanol or 50 mM TMA with 580 mM ethanol supplemented where necessary. For the experiments with engineered chimeras of Mta subunits, the growth media consisted of 150 mM methanol and ethanol with puromycin. Construction of strains expressing engineered chimeras of MtaABC subunits. The plasmids pMSJ5 and pMSJ6 were derived from pM000 (Zhu et al. 2023 ). pMSJ5 contains the expression cassette P mtr_Mb fusaro - mtaA1 -T fpo_Mb 227 , while pMSJ6 hosts P mtr_Mb fusaro - mtaA2 -T fpo_Mb 227 . All chimeric plasmids containing mtaA1 were constructed based on pMSJ5. Briefly, the genes mtaC and mtaB were amplified using specific primers (Suppl Table. S2). mtaC was then fused to the P mcr−tetO1 promoter via overlap PCR, while mtaB was joined with T mcr_Mb fusaro . These two cassettes were assembled into SbfI-linearized pMSJ5 using the Gibson assembly method. Similarly, all chimeric plasmids containing mtaA2 were constructed based on pMSJ6 using the same methods described above. The resulting plasmids were transformed into M. acetivorans WWM73. Results and discussion Ethane production in resting cell suspensions and in vivo . Resting cell suspension of M. acetivorans WWM1 (containing all 3 methyltransferases) grown in methanol catalysed the conversion of ethanol to 28 ± 11 nmol of ethane (n = 3) in 118 hours of incubation. Because growth on TMA supresses expression of methanol-specific methyltransferases (Bose et al. 2006 ), M. acetivorans WWM1 cells grown in TMA showed no detectable ethane production from ethanol in 118 hours. M. acetivorans WWM13, a triple mtaCB mutant, also failed to show ethane production in presence of ethanol (Table 1 ). A similar experiment was repeated with M. acetivorans WWM73 and WWM13 grown in TMA to meaure in vivo ethane production (Fig. 2 ). M. acetivorans WWM13 did not show any ethane production after incubation for 31 days. Whereas M. acetivorans WWM73 showed in vivo ethane production, albeit reduced, that was absent in cell suspensions. This in vivo ethane production compared to its absence in resting cell suspension is likely due to the extended incubation period (744 hrs) and higher ethanol concentration (580 mM) along with absence of protein synthesis inhibitor in the in vivo studies. Necessity of mtaCB shows that the promiscuity of methanol-specific methyltransferases indeed leads to formation of ethane in M. acetivorans , and putatively also in all other methanol utilizing methanogens. Table 1 Resting cell suspension assay for ethane formation capability of M. acetivorans WWM1 and WWM13. Strain Genotype Growth media Accumulated ethane after 118 h incubation (in nmol) WWM1 ∆hpt Methanol Yes (28 ± 11 nmol) WWM1 ∆hpt TMA No (Not detectable) WWM13 ∆hpt ∆mtaCB1 ∆mtaCB2 ∆mtaCB3 TMA No (Not detectable) Testing the substrate promiscuity of Mta isoforms in resting cell suspensions. Because M. acetivorans encodes 3 different isozymes of MtaCB, we tested the promiscuity of each isozyme towards ethanol-to-ethane conversion. M. acetivorans mutant strains containing only one isozyme were used from a previous study (Pritchett and Metcalf 2005 ). All three isozymes showed ethane formation, but with different rate normalized to OD. The ethane formation rate was significantly higher in mutants expressing only MtaCB3 and MtaCB2 (WWM9 and WWM7 respectively) (Table 2 ). The mutant expressing only MtaCB3 isozyme also had the lowest methanol conversion rate whereas MtaCB1 had the highest, as shown in a previous study (Pritchett and Metcalf 2005 ). Table 2 Resting cell suspension assay to measure the rate of ethane production in strains expressing only one MtaCB isoform Strain Genotype Rate of ethane production (nmol h − 1 OD − 1 ) WWM5 ∆hpt ∆mtaCB2 ∆mtaCB3 0.20 ± 0.02 WWM7 ∆hpt ∆mtaCB1 ∆mtaCB3 0.53 ± 0.08 WWM9 ∆hpt ∆mtaCB1 ∆mtaCB2 0.51 ± 0.08 Testing strains that express engineered chimeras of MtaCB for their capabilities to produce ethane. The different ethane production rates of mutant strains containing only one isozyme of MtaCB motivated us to construct and test all possible combinations of individual subunits. The engineered chimeras of mtaCBs were expressed in M. acetivorans WWM73 from a plasmid. The in vivo ethane production capability of the plasmid-free M. acetivorans WWM73 grown in methanol and TMA was tested as a background (Fig. 3 ). The engineered chimeras included MtaA subunits (mtaA subunits ma_0855 – mtaA1 or ma_4379 – mtaA2 ) to ensure that the ethane formation rate would not be limited due to the lack availability of MtaA. A total of 18 chimeras of different combinations of MtaABC isoforms were compared for the total amount of methane and ethane formation (Fig. 4 ). Compared to the control, the MtaA1B2C3 construct showed the highest ethane production, almost two times relative to the control (Fig. 4 ). Even if the construction of the chimera of MtaA1B2C3 enhanced ethane production, there is only trace levels of ethane produced. The final ethane concentrations are not only dependent on the ethyltransferase activity and ethanol concentrations (Belay 1995 ), but also on the incubation period. Our construction of the chimeras shows that the MtaB and MtaC subunits can work with each other even if originated from different operons and by mixing and matching, the overall substrate promiscuity of the enzyme can be increased. MtaA1 is a pyrrolysine-containing methyltransferase that was shown to play an important role in methylotrophic methanogenesis in pylT deletion strain of M. acetivorans (O’Donoghue et al. 2014 ). In the top six of the 18 combinations regarding ethyl-group transfer, MtaA1 showed up five times, which indicates that MtaA1 is more promiscuous to transfer ethyl group from MtaC to CoM. The higher substrate promiscuity of mixed Mta combinations could be due to the formation of more flexible Mta complexes, which could form larger catalytic pockets with enhanced accessibility for ethyl groups. We modelled the structures in AplhaFold (SI Text). Our estimations of the catalytic pockets’ volumes in the isozymes and the chimeras relying on AlphaFold-predicted structures (Varadi et al. 2022 )​​ and the MtaBC crystal structure from M. barkeri (Hagemeier et al. 2006 )​ show differences between 40 to 200 Å 3 between each other (Suppl Fig. S1 ; Suppl Table. S1). However, the volume near the cobalt core of the cobalamin cofactor where the catalytic zinc would be hosted seems consistent among the tested predicted structures. Given that methyltransferases rely on a wide molecular motion for catalysis (Hagemeier et al. 2006 ), structural studies showing different movement states instead of static predicted structures would be warranted to understand the difference in the catalytic activity of the isozymes and the chimeras. Conclusion Lack of ethane production from ethanol in M. acetivorans WWM13 shows that the ethanol-to-ethane metabolism in M. acetivorans follows the terminal portion of methylotrophic methanogenesis via substrate promiscuity for transferring an ethyl group. The three isozymes of the multisubunit methanol-specific methyltransferase showed distinct levels of substrate promiscuity where strains containing only MtaCB2 and MtaCB3 had a higher rate of ethane production. Synthetic chimera combining individual subunits from different operons, MtaA1B2C3, showed increased ethane production from ethanol. Our work highlights that ethyl transfer reactions via corrinoid-dependent enzymes need to be considered as additional reactions in metabolic networks. In the metabolism of non-methane alkane-degrading archaea, where the alkane is oxidized to alkyl-CoM as the first step, this alkyl group needs to be further transferred via steps that are currently considered unknown (Wegener et al. 2022 ). This transfer in some archaea may rely on a corrinoid protein that has evolved from transferring methyl-groups towards catalyzing a longer alkyl group. Declarations Author Contribution J.B., T.S., and S.S. conceptualized the study. J.B. and T.S. performed the experiments and analyzed the data. S.S supervised the experiments. T.S., J.B., and S.S, wrote the manuscript. All authors read and approved the manuscript. Acknowledgement This research has received financial support from the NovoNordisk foundation (Grant no NNF19OC0054329 to S.S.) and the Research Council of Finland (Grant no 329510 to S.S.). The authors thank Maxime Laird (Aalto University) for modelling the structures. The authors thank Prof. William Metcalf (University of Illinois, USA) for providing the strains and plasmid used in this study. The authors acknowledge the Aalto University Raw Materials Research Infrastructures and Bioeconomy facilities. 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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-4694130","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":330306150,"identity":"32d7b3d9-47b6-429b-b6d0-0f0d444f5505","order_by":0,"name":"Tejas Somvanshi","email":"","orcid":"","institution":"Aalto University","correspondingAuthor":false,"prefix":"","firstName":"Tejas","middleName":"","lastName":"Somvanshi","suffix":""},{"id":330306151,"identity":"747c4bac-c9b0-4624-a614-f3f1052311a9","order_by":1,"name":"Jichen Bao","email":"","orcid":"","institution":"Aalto University","correspondingAuthor":false,"prefix":"","firstName":"Jichen","middleName":"","lastName":"Bao","suffix":""},{"id":330306152,"identity":"3dd1096a-4f92-46c6-b1b7-31a1231f3b81","order_by":2,"name":"Silvan Scheller","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABA0lEQVRIie3OsUoDQRCA4VmujaRdQe9eYUJa4V5lloBpEixsUljcsrBXSdoVX0I7ywkHqQ7SXiFyh7WwraDiHRELZWNrsX81DHzMAMRi/7EkKfaDBMEtQNqvhk06ChPxTYAJYAogdL+ahgn8IKr4IkGQlcLK1wfIx7eGmVZP83W5KVsPeBIiWAl7fF2Dco9bYqovl65WWjvA4GOYCINHFgjlAllZWhastBnBW5BkRpjJu4Uc5YVn9UHzbNcNJHwFKqGf+yviTi6AVUGEjTpMcCCnVirXnCPTlib3TadvHIZJtq5482LP8rGbda2/oizdzdj7FebBx/bJX9f/ALFYLBY72Ccjwlqx4ob9YwAAAABJRU5ErkJggg==","orcid":"","institution":"Aalto University","correspondingAuthor":true,"prefix":"","firstName":"Silvan","middleName":"","lastName":"Scheller","suffix":""}],"badges":[],"createdAt":"2024-07-05 20:30:48","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4694130/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4694130/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":61441794,"identity":"1cc451e8-8fb5-44f2-a970-876efa27d88f","added_by":"auto","created_at":"2024-07-30 20:22:02","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":38044,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHypothesized pathway for the conversion of ethanol to ethane\u003c/strong\u003e. The metabolism follows the terminal part of the methylotrophic methanogenesis pathway, but the reactions proceed with an ethyl group instead of a methyl group. Methanol:5-hydroxybenzimidazolylcobamide methyltransferase (MtaB) is the methyl donor module that activates methanol/ethanol and methylates/ethylates the corrinoid protein MtaC. \u003cem\u003eCo\u003c/em\u003e-methyl-5-hydroxybenzimidazolylcobamide:2-mercaptoethanesulfonate methyltransferase (MtaA) is the methyl acceptor module that transfers the methyl/ethyl group to coenzyme M. Methyl-CoM/ethyl-CoM is then reduced to methane/ethane by methyl-CoM reductase (Mcr) forming a heterodisulfide of coenzyme M and coenzyme B (HDS). In classical methanogenesis the HDS is recycled using H\u003csub\u003e2\u003c/sub\u003e or carbon disproportionation. During ethane production from ethanol, accumulation of HDS and lack of free CoM prevents continuous ethane formation.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4694130/v1/955b868c25c536a8a1d75567.jpg"},{"id":61441512,"identity":"60b5df30-2833-4ea4-8af8-ed3b49504a99","added_by":"auto","created_at":"2024-07-30 20:14:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":187695,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e ethane formation in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eM. acetivorans\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e WWM73 and WWM13 growing on TMA in presence of ethanol. \u003c/strong\u003e\u003cem\u003eM. acetivorans\u003c/em\u003e WWM73, expressing all methanol-specific methyltransferases shows trace ethane production from ethanol when grown on TMA. No ethane is detected from \u003cem\u003eM. acetivorans\u003c/em\u003e WWM13 which lacks all 3 methanol-specific methyltransferases.\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4694130/v1/d4e9bb3dce8be081852c883d.png"},{"id":61441509,"identity":"fbc173e3-b9ec-4374-b4ee-9c2911595bf4","added_by":"auto","created_at":"2024-07-30 20:14:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":78763,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e ethane formation in plasmid-free \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eM. acetivorans\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e WWM73. \u003c/strong\u003eX-axis labels denote the substrates available in the growth media. The ethane levels were measured after 1824 h (76 days) of incubation. The reduced ethane level when grown on TMA is the result of suppressed expression of methanol-specific methyltransferases.\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4694130/v1/c6031ee49169ed661a04462d.png"},{"id":61441511,"identity":"5dd11f8c-9944-422e-964c-b23690a2d408","added_by":"auto","created_at":"2024-07-30 20:14:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":230963,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003ein vivo \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eEthane formation capability of MtaABC chimeras. \u003c/strong\u003e\u0026nbsp;Ethane production was measured at the end of 312 h (13 days) of incubation. The chimera combination that produced the highest amount of ethane is MtaA1B2C3.\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4694130/v1/0c55f89bb1e9d314b11a1675.png"},{"id":61630175,"identity":"35b89fd5-c194-400c-930b-d493347b9650","added_by":"auto","created_at":"2024-08-02 07:46:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1236199,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4694130/v1/da0e5c3d-ed0d-449e-a8b0-6e36ceceee52.pdf"},{"id":61441513,"identity":"10b836ad-e55d-423e-9893-20ed86a94aaf","added_by":"auto","created_at":"2024-07-30 20:14:03","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2948524,"visible":true,"origin":"","legend":"","description":"","filename":"AVLSI240705.docx","url":"https://assets-eu.researchsquare.com/files/rs-4694130/v1/764e487bc4988f666d16dce5.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Corrinoid-dependent ethyl-transfer catalyzed by the methanol:coenzyme M methyl transferase from Methanosarcina acetivorans ","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDescribed as the \u0026lsquo;nature\u0026rsquo;s most beautiful cofactor\u0026rsquo;, corrinoids are catalytic centers of non-\u003cem\u003eS\u003c/em\u003e-adenosylmethionine dependent methyltransferases (Matthews \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Corrinoid-dependent methyltransferases are present in all 3 domains of life and are part of the energy metabolism of many anaerobic organisms (Matthews et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). The cobalt centre of the cobalamin acts as a shuttle for the methyl groups, cycling between its Co(I) and its Me-Co(III) state. Occasionally the Co(I) is oxidized to an inactive Co(II) state which requires reductive activation either dependent on adenosylmethionine or ATP to return to catalytically active Co(I) state (Matthews et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn all methanogens and in anaerobic methanotrophic archaea, corrinoid dependent methyltransferases catalyze transfer of methyl group in at least one of the following catabolic reactions: (i) between tetrahydromethanopterin and coenzyme M in most methanogenesis pathways and in anaerobic methanotrophy (ii) from acetate to tetrahydromethanopterin in aceticlastic methanogenesis (iii) from methylated substrates to coenzyme M in methylotrophic methanogenesis and (iv) from a methylated corrinoid to homocysteine for methionine synthesis (Matthews et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAlthough considered exclusive to methyl groups, methyltransferases have been hypothesized to also convert ethyl groups. Notably in bacteria where ethionine production and secretion has been studied (Loerch and Mallette \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1963\u003c/span\u003e) and in biogenic ethane formation. While most ethane is formed by thermogenic or abiotic processes, evidence for biogenic ethane has been reported for anoxic slurries (Belay and Daniels \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1987\u003c/span\u003e; Oremland et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1988\u003c/span\u003e; Hinrichs et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Xie et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Methanogens were identified as the source of biogenic ethane from ethanol when pure cultures of \u003cem\u003eMethanosarcina barkeri\u003c/em\u003e strains showed trace ethane formation in an ethanol-supplemented medium. While the metabolic pathway responsible for this conversion has not been elucidated, it was hypothesized that methanol-specific methylotrophic methyltransferase system (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) is involved, given that significant ethane production was only seen in methanogens capable of utilizing methanol and when grown using methanol (Belay and Daniels \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1988\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHere we focus on the methanol: coenzyme M methyltransferase (Mta) from \u003cem\u003eMethanosarcina acetivorans\u003c/em\u003e, since this organism is a versatile, genetically tractable model methanogen capable of producing methane from methylated substrates such as methanol and methylamines (Sowers et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1984\u003c/span\u003e; Metcalf et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Nayak and Metcalf \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Lyu and Liu \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Zhu et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Bao et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The methanol-specific methyltransferase is a 3-subunit protein complex (MtaABC) which catalyses methyl transfer from methanol to coenzyme M (CoM) forming methyl-CoM. \u003cem\u003eM. acetivorans\u003c/em\u003e encodes 3 isoforms of MtaCB (\u003cem\u003emtaCB1- ma_0455\u0026ndash;0456, mtaCB2- ma_4391\u0026ndash;4392, mtaCB3- ma_1616\u0026ndash;1617\u003c/em\u003e), each with varying activity towards methanol and expression pattern (Pritchett and Metcalf \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Bose et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Methyl coenzyme M reductase (Mcr) reduces the methyl-CoM to methane. Mcr has shown promiscuity towards ethyl-CoM (Goenrich et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Scheller et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) and homologs of Mcr have been identified in other archaea catalyzing the reversible oxidation of non-methane alkanes (Laso-P\u0026eacute;rez et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Chen et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Hahn et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Zehnle et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn this study, we tested the microbial pathway of ethane production in \u003cem\u003eM. acetivorans\u003c/em\u003e, showing differential promiscuity between isoforms towards ethanol conversion. We engineered chimeras between the individual Mta subunits to enhance the reactivity of the corrinoids towards ethane production from ethanol.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e \u003cb\u003eMicrobiological methods.\u003c/b\u003e Lysogenic broth containing 50 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e ampicillin was used for plasmid construction in \u003cem\u003eE. coli\u003c/em\u003e NEB5α. \u003cem\u003eM. acetivorans\u003c/em\u003e was cultured in a high-salt medium tailored to the specific requirements of the experiment (Sowers et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). The optical density of the cultures was tracked using Eppendorf BioPhotometer plus spectrophotometer. Plasmid construction was carried out according to standard protocols. Liposome-mediated methods and polyethylene glycol-mediated methods were used to transform \u003cem\u003eM. acetivorans\u003c/em\u003e (Metcalf et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1997\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eResting cell suspension assays.\u003c/b\u003e 30 ml of mid-exponential phase cells grown in 60 mM methanol or 50 mM trimethylamine (TMA) with headspace 50% N\u003csub\u003e2\u003c/sub\u003e/ 20% CO\u003csub\u003e2\u003c/sub\u003e/ 30% of 1% H\u003csub\u003e2\u003c/sub\u003eS in N\u003csub\u003e2\u003c/sub\u003e at atmospheric pressure were centrifuged, washed twice and then resuspended in 6 ml of HS media with 33.6 \u0026micro;l of 99.99% ethanol (final concentration 96 mM) and 2 \u0026micro;l puromycin to block protein synthesis. The master mix was then divided to 3 Balch tubes (2 ml each), and the headspace was replaced by 50% N\u003csub\u003e2\u003c/sub\u003e/ 20% CO\u003csub\u003e2\u003c/sub\u003e/ 30% of 1% H\u003csub\u003e2\u003c/sub\u003eS in N\u003csub\u003e2\u003c/sub\u003e at atmospheric pressure.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGC-FID quantification of methane and ethane.\u003c/b\u003e The T\u003csub\u003e0\u003c/sub\u003e sample was measured with GC-FID right after setting up the resting cell suspension and the tubes were incubated at 37˚C. Samples were taken periodically. The methane and ethane concentrations in the headspace gas samples were measured using a GC-FID (Agilent HP 6890 Gas Chromatograph, Hewlett-Packard), equipped with an HP-AL/KCL column (length, 50 m; diameter, 0.32 mm, thickness, 8 \u0026micro;m). The headspace gas samples were injected with a Gastight 1700 SampleLock Syringe (100 \u0026micro;L, PN81056) (Hamilton). A calibration curve was generated using methane standards. The ratio of the peak areas generated by methane and ethane for the same concentration each was used to quantify ethane. The detection limit for ethane was 3 nmol.\u003c/p\u003e \u003cp\u003e \u003cb\u003ein vivo\u003c/b\u003e \u003cb\u003emethane and ethane production.\u003c/b\u003e The cultures were grown in Hungate tubes. 4.5 ml cultures were set for the \u003cem\u003eM. acetivorans\u003c/em\u003e WWM73 background test whereas 3 ml for the cultures comparing WWM73 to WWM13 and cultures expressing engineered Mta chimeras. The headspace was replaced by 70% N\u003csub\u003e2\u003c/sub\u003e/ 20% CO\u003csub\u003e2\u003c/sub\u003e/ 10% of 1% H\u003csub\u003e2\u003c/sub\u003eS in N\u003csub\u003e2\u003c/sub\u003e at atmospheric pressure. The substrates added for both \u003cem\u003ein vivo\u003c/em\u003e tests were 120 mM methanol or 50 mM TMA with 580 mM ethanol supplemented where necessary. For the experiments with engineered chimeras of Mta subunits, the growth media consisted of 150 mM methanol and ethanol with puromycin.\u003c/p\u003e \u003cp\u003e \u003cb\u003eConstruction of strains expressing engineered chimeras of MtaABC subunits.\u003c/b\u003e The plasmids pMSJ5 and pMSJ6 were derived from pM000 (Zhu et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). pMSJ5 contains the expression cassette P\u003csub\u003emtr_Mb fusaro\u003c/sub\u003e-\u003cem\u003emtaA1\u003c/em\u003e-T\u003csub\u003efpo_Mb 227\u003c/sub\u003e, while pMSJ6 hosts P\u003csub\u003emtr_Mb fusaro\u003c/sub\u003e-\u003cem\u003emtaA2\u003c/em\u003e-T\u003csub\u003efpo_Mb 227\u003c/sub\u003e. All chimeric plasmids containing \u003cem\u003emtaA1\u003c/em\u003e were constructed based on pMSJ5. Briefly, the genes \u003cem\u003emtaC\u003c/em\u003e and \u003cem\u003emtaB\u003c/em\u003e were amplified using specific primers (Suppl Table. S2). \u003cem\u003emtaC\u003c/em\u003e was then fused to the P\u003csub\u003emcr\u0026minus;tetO1\u003c/sub\u003e promoter via overlap PCR, while \u003cem\u003emtaB\u003c/em\u003e was joined with T\u003csub\u003emcr_Mb fusaro\u003c/sub\u003e. These two cassettes were assembled into SbfI-linearized pMSJ5 using the Gibson assembly method. Similarly, all chimeric plasmids containing \u003cem\u003emtaA2\u003c/em\u003e were constructed based on pMSJ6 using the same methods described above. The resulting plasmids were transformed into \u003cem\u003eM. acetivorans\u003c/em\u003e WWM73.\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cp\u003e \u003cb\u003eEthane production in resting cell suspensions and\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e. Resting cell suspension of \u003cem\u003eM. acetivorans\u003c/em\u003e WWM1 (containing all 3 methyltransferases) grown in methanol catalysed the conversion of ethanol to 28\u0026thinsp;\u0026plusmn;\u0026thinsp;11 nmol of ethane (n\u0026thinsp;=\u0026thinsp;3) in 118 hours of incubation. Because growth on TMA supresses expression of methanol-specific methyltransferases (Bose et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), M. \u003cem\u003eacetivorans\u003c/em\u003e WWM1 cells grown in TMA showed no detectable ethane production from ethanol in 118 hours. \u003cem\u003eM. acetivorans\u003c/em\u003e WWM13, a triple \u003cem\u003emtaCB\u003c/em\u003e mutant, also failed to show ethane production in presence of ethanol (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA similar experiment was repeated with \u003cem\u003eM. acetivorans\u003c/em\u003e WWM73 and WWM13 grown in TMA to meaure \u003cem\u003ein vivo\u003c/em\u003e ethane production (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). \u003cem\u003eM. acetivorans\u003c/em\u003e WWM13 did not show any ethane production after incubation for 31 days. Whereas \u003cem\u003eM. acetivorans\u003c/em\u003e WWM73 showed \u003cem\u003ein vivo\u003c/em\u003e ethane production, albeit reduced, that was absent in cell suspensions. This \u003cem\u003ein vivo\u003c/em\u003e ethane production compared to its absence in resting cell suspension is likely due to the extended incubation period (744 hrs) and higher ethanol concentration (580 mM) along with absence of protein synthesis inhibitor in the \u003cem\u003ein vivo\u003c/em\u003e studies. Necessity of \u003cem\u003emtaCB\u003c/em\u003e shows that the promiscuity of methanol-specific methyltransferases indeed leads to formation of ethane in \u003cem\u003eM. acetivorans\u003c/em\u003e, and putatively also in all other methanol utilizing methanogens.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eResting cell suspension assay for ethane formation capability of \u003cem\u003eM. acetivorans\u003c/em\u003e WWM1 and WWM13.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStrain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGenotype\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGrowth media\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAccumulated ethane after 118 h incubation (in nmol)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWWM1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003e∆hpt\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMethanol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eYes (28\u0026thinsp;\u0026plusmn;\u0026thinsp;11 nmol)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWWM1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003e∆hpt\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTMA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNo (Not detectable)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWWM13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003e∆hpt ∆mtaCB1 ∆mtaCB2 ∆mtaCB3\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTMA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNo (Not detectable)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eTesting the substrate promiscuity of Mta isoforms in resting cell suspensions.\u003c/b\u003e Because \u003cem\u003eM. acetivorans\u003c/em\u003e encodes 3 different isozymes of MtaCB, we tested the promiscuity of each isozyme towards ethanol-to-ethane conversion. \u003cem\u003eM. acetivorans\u003c/em\u003e mutant strains containing only one isozyme were used from a previous study (Pritchett and Metcalf \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). All three isozymes showed ethane formation, but with different rate normalized to OD. The ethane formation rate was significantly higher in mutants expressing only MtaCB3 and MtaCB2 (WWM9 and WWM7 respectively) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The mutant expressing only MtaCB3 isozyme also had the lowest methanol conversion rate whereas MtaCB1 had the highest, as shown in a previous study (Pritchett and Metcalf \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eResting cell suspension assay to measure the rate of ethane production in strains expressing only one MtaCB isoform\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStrain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGenotype\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRate of ethane production (nmol h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e OD\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWWM5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003e∆hpt ∆mtaCB2 ∆mtaCB3\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWWM7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003e∆hpt ∆mtaCB1 ∆mtaCB3\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWWM9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003e∆hpt ∆mtaCB1 ∆mtaCB2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eTesting strains that express engineered chimeras of MtaCB for their capabilities to produce ethane.\u003c/b\u003e The different ethane production rates of mutant strains containing only one isozyme of MtaCB motivated us to construct and test all possible combinations of individual subunits. The engineered chimeras of \u003cem\u003emtaCBs\u003c/em\u003e were expressed in \u003cem\u003eM. acetivorans\u003c/em\u003e WWM73 from a plasmid. The \u003cem\u003ein vivo\u003c/em\u003e ethane production capability of the plasmid-free \u003cem\u003eM. acetivorans\u003c/em\u003e WWM73 grown in methanol and TMA was tested as a background (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe engineered chimeras included MtaA subunits (mtaA subunits \u003cem\u003ema_0855\u003c/em\u003e \u0026ndash; \u003cem\u003emtaA1\u003c/em\u003e or \u003cem\u003ema_4379 \u0026ndash; mtaA2\u003c/em\u003e) to ensure that the ethane formation rate would not be limited due to the lack availability of MtaA. A total of 18 chimeras of different combinations of MtaABC isoforms were compared for the total amount of methane and ethane formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Compared to the control, the MtaA1B2C3 construct showed the highest ethane production, almost two times relative to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Even if the construction of the chimera of MtaA1B2C3 enhanced ethane production, there is only trace levels of ethane produced. The final ethane concentrations are not only dependent on the ethyltransferase activity and ethanol concentrations (Belay \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1995\u003c/span\u003e), but also on the incubation period. Our construction of the chimeras shows that the MtaB and MtaC subunits can work with each other even if originated from different operons and by mixing and matching, the overall substrate promiscuity of the enzyme can be increased.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMtaA1 is a pyrrolysine-containing methyltransferase that was shown to play an important role in methylotrophic methanogenesis in \u003cem\u003epylT\u003c/em\u003e deletion strain of \u003cem\u003eM. acetivorans\u003c/em\u003e (O\u0026rsquo;Donoghue et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In the top six of the 18 combinations regarding ethyl-group transfer, MtaA1 showed up five times, which indicates that MtaA1 is more promiscuous to transfer ethyl group from MtaC to CoM.\u003c/p\u003e \u003cp\u003eThe higher substrate promiscuity of mixed Mta combinations could be due to the formation of more flexible Mta complexes, which could form larger catalytic pockets with enhanced accessibility for ethyl groups. We modelled the structures in AplhaFold (SI Text). Our estimations of the catalytic pockets\u0026rsquo; volumes in the isozymes and the chimeras relying on AlphaFold-predicted structures (Varadi et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022\u003c/span\u003e)​​ and the MtaBC crystal structure from \u003cem\u003eM. barkeri\u003c/em\u003e (Hagemeier et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2006\u003c/span\u003e)​ show differences between 40 to 200 \u0026Aring;\u003csup\u003e3\u003c/sup\u003e between each other (Suppl Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e; Suppl Table. S1). However, the volume near the cobalt core of the cobalamin cofactor where the catalytic zinc would be hosted seems consistent among the tested predicted structures. Given that methyltransferases rely on a wide molecular motion for catalysis (Hagemeier et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), structural studies showing different movement states instead of static predicted structures would be warranted to understand the difference in the catalytic activity of the isozymes and the chimeras.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eLack of ethane production from ethanol in \u003cem\u003eM. acetivorans\u003c/em\u003e WWM13 shows that the ethanol-to-ethane metabolism in \u003cem\u003eM. acetivorans\u003c/em\u003e follows the terminal portion of methylotrophic methanogenesis via substrate promiscuity for transferring an ethyl group. The three isozymes of the multisubunit methanol-specific methyltransferase showed distinct levels of substrate promiscuity where strains containing only MtaCB2 and MtaCB3 had a higher rate of ethane production. Synthetic chimera combining individual subunits from different operons, MtaA1B2C3, showed increased ethane production from ethanol. Our work highlights that ethyl transfer reactions via corrinoid-dependent enzymes need to be considered as additional reactions in metabolic networks. In the metabolism of non-methane alkane-degrading archaea, where the alkane is oxidized to alkyl-CoM as the first step, this alkyl group needs to be further transferred via steps that are currently considered unknown (Wegener et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This transfer in some archaea may rely on a corrinoid protein that has evolved from transferring methyl-groups towards catalyzing a longer alkyl group.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJ.B., T.S., and S.S. conceptualized the study. J.B. and T.S. performed the experiments and analyzed the data. S.S supervised the experiments. T.S., J.B., and S.S, wrote the manuscript. All authors read and approved the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThis research has received financial support from the NovoNordisk foundation (Grant no NNF19OC0054329 to S.S.) and the Research Council of Finland (Grant no 329510 to S.S.). The authors thank Maxime Laird (Aalto University) for modelling the structures. The authors thank Prof. William Metcalf (University of Illinois, USA) for providing the strains and plasmid used in this study. The authors acknowledge the Aalto University Raw Materials Research Infrastructures and Bioeconomy facilities.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBao J, Somvanshi T, Tian Y, et al (2024) Nature AND Nurture: Enabling formate-dependent growth in Methanosarcina acetivorans. bioRxiv 2024.01.08.574737. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1101/2024.01.08.574737\u003c/span\u003e\u003cspan address=\"10.1101/2024.01.08.574737\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBelay N (1995) Demonstration and physiological characterization of two novel properties in methanogens: Ethane formation from ethanol and assimilatory nitrate reduction\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBelay N, Daniels L (1987) Production of Ethane, Ethylene, and Acetylene from Halogenated Hydrocarbons by Methanogenic Bacteria. 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Front Microbiol 14:1\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fmicb.2023.1235616\u003c/span\u003e\u003cspan address=\"10.3389/fmicb.2023.1235616\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Ethane, Promiscuity, ethyl-transfer, Methyltransferases, Methanosarcina, Corrinoids","lastPublishedDoi":"10.21203/rs.3.rs-4694130/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4694130/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCorrinoid-dependent methyltransferases catalyze methyl-group transfer reactions in all domains of life. These enzymes are generally considered exclusive for C1-substrates (methyl-groups). However, in \u003cem\u003eMethanosarcina\u003c/em\u003e trace ethane production from ethanol has been demonstrated \u003cem\u003ein vivo\u003c/em\u003e, which led to the hypothesis that corrinoid-dependent methanol specific methyltransferases are promiscuous towards also accepting ethyl-groups.\u003c/p\u003e \u003cp\u003eHere we show that the conversion of ethanol to trace amounts of ethane in \u003cem\u003eMethanosarcina acetivorans\u003c/em\u003e proceeds via the known methanol-to-methane metabolism, involving the methanol:5-hydroxybenzimidazolylcobamide methyltransferase (MtaB) and a corrinoid-containing methyl-accepting protein (MtaC), but via transfer of ethyl groups instead of methyl groups. We demonstrate that all three isozymes of the methanol specific MtaB subunit and the corrinoid protein MtaC of \u003cem\u003eM. acetivorans\u003c/em\u003e are promiscuous towards accepting ethanol, granting the microbe capacity of ethane production via promiscuity downstream in \u003cem\u003eCo\u003c/em\u003e-methyl-5-hydroxybenzimidazolylcobamide:2-mercaptoethanesulfonate methyltransferase (MtaA) and methyl-coenzyme M reductase (Mcr). We assessed the ethyl-group transfer efficiency of each of the three isozymes and engineered chimeras that combine 2 different MtaA subunits with the 3 isoforms of MtaCB together to increase the ethane production capability of \u003cem\u003eM. acetivorans\u003c/em\u003e. Demonstrating that corrinoid-dependent coenzyme M methyltransferases can catalyze transfer of higher alkyl groups extends the pool of reactions to be considered in metabolic networks.\u003c/p\u003e","manuscriptTitle":"Corrinoid-dependent ethyl-transfer catalyzed by the methanol:coenzyme M methyl transferase from Methanosarcina acetivorans ","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-30 20:13:58","doi":"10.21203/rs.3.rs-4694130/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"377598f7-5663-4935-af00-eb1310ce3ba9","owner":[],"postedDate":"July 30th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-06T14:38:41+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-30 20:13:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4694130","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4694130","identity":"rs-4694130","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}