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Functional Cooperation of the Glycine Synthase-Reductase And Functional cooperation of the glycine synthase- reductase and Wood–Ljungdahl pathways for autotrophic growth of Clostridium drakei Yoseb Songa,b, Jin Soo Leea,b, Jongoh Shina,b, Gyu Min Leec, Sangrak Jina,b, Seulgi Kanga,b, Jung-Kul Leed, Dong Rip Kime, Eun Yeol Leef, Sun Chang Kima,b,g, Suhyung Choa,b, Donghyuk Kimc,1, and Byung-Kwan Choa,b,g,1 aDepartment of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), 34141 Daejeon, Republic of Korea; bKAIST Institute for the BioCentury, Korea Advanced Institute of Science and Technology, 34141 Daejeon, Republic of Korea; cSchool of Energy and Chemical Engineering, Ulsan National Institute of Science and Technology, 44919 Ulsan, Republic of Korea; dDepartment of Chemical Engineering, Konkuk University, 05029 Seoul, Republic of Korea; eDepartment of Mechanical Engineering, Hanyang University, 04763 Seoul, Republic of Korea; fDepartment of Chemical Engineering, Kyung Hee University, 17104 Seoul, Gyeonggi-do, Republic of Korea; and gIntelligent Synthetic Biology Center, 34141 Daejeon, Republic of Korea Edited by Largus T. Angenent, University of Tübingen, Tübingen, Germany, and accepted by Editorial Board Member Carl F. Nathan February 11, 2020 (received for review July 17, 2019) Among CO2-fixing metabolic pathways in nature, the linear Wood– functional role of the pathway in the presence of intact WLP has Ljungdahl pathway (WLP) in phylogenetically diverse acetate- remained uncertain. forming acetogens comprises the most energetically efficient path- In this study, we elucidated the role of the GSRP and RGP in T way, requires the least number of reactions, and converts CO2 to pure-cultured Clostridium drakei SL1 during autotrophic growth. formate and then into acetyl-CoA. Despite two genes encoding gly- Initially, assembly of the C. drakei genome revealed the coexis- cine synthase being well-conserved in WLP gene clusters, the func- tence of the GSRP, RGP, and WLP, which were then utilized for tional role of glycine synthase under autotrophic growth conditions reconstruction of a genome-scale metabolic model (GEM) to has remained uncertain. Here, using the reconstructed genome- scale metabolic model iSL771 based on the completed genome se- predict metabolic flux through the core carbon pathways. Sub- quence, transcriptomics, 13C isotope-based metabolite-tracing ex- sequently, transcriptome analysis revealed the transcriptional ac- periments, biochemical assays, and heterologous expression of the tivation of genes encoding the pathways; this was then validated by SYSTEMS BIOLOGY 13 pathway in another acetogen, we discovered that the WLP and the using C isotope-based metabolite-tracing experiments, bio- glycine synthase pathway are functionally interconnected to fix CO2, chemical assays, and genetic engineering. Based on the integration subsequently converting CO2 into acetyl-CoA, acetyl-phosphate, and of the data, we concluded that C. drakei coutilizes the GSRP, serine. Moreover, the functional cooperation of the pathways en- hances CO2 consumption and cellular growth rates via bypassing Significance reducing power required reactions for cellular metabolism during autotrophic growth of acetogens. Despite sharing the first four reactions, coutilization of the Wood–Ljungdahl pathway (WLP) with the glycine synthase- CO fixation | acetogen | Wood–Ljungdahl pathway | systems biology | 2 reductase pathway (GSRP) and reductive glycine pathway glycine synthase-reductase pathway (RGP) to fix C1 compounds has remained unknown. In this study, using Clostridium drakei, we elucidated the role of the – he linear Wood Ljungdahl pathway (WLP) in anaerobic ace- GSRP and RGP in the presence of the WLP, via a genome-scale Ttogens is considered the most energetically efficient pathway to metabolic model, RNA-seq, 13C isotope-based metabolite- convert CO2 to formate and then into acetyl-CoA. With this ad- tracing experiments, biochemical assays, and heterologous vantage, acetogens are considered to be the most promising in- expression. Overall, the data suggested the pathways are dustrial platform to produce biofuels and chemical commodities functional under autotrophic conditions. Along with the WLP, – through synthesis gas fermentation (1 4). Although gene compo- GSRP and RGP convert CO2 to glycine and then to acetyl- sition and arrangement of the WLP vary among acetogens, the phosphate and serine, which then obtain ATP by producing WLP-coding genes are well-conserved, along with two genes acetate and operate with limited reducing power. This is a encoding a partial glycine synthase: the glycine cleavage system H unique coutilization of the pathways under autotrophic condi- protein (gcvH) and dihydrolipoyl dehydrogenase (lpdA) genes (5– tions in acetogens. 8). The glycine synthase pathway was initially proposed for the Author contributions: Y.S., S.C., D.K., and B.-K.C. designed research; Y.S., J.S.L., J.S., S.J., utilization of CO2 under autotrophic growth conditions (2). While S.K., and S.C. performed research; Y.S., G.M.L., J.-K.L., D.R.K., E.Y.L., D.K., and B.-K.C. the two genes are well-conserved in the gene cluster, other genes in analyzed data; and Y.S., S.C.K., S.C., D.K., and B.-K.C. wrote the paper. the glycine synthase pathway are missing in many acetogen ge- The authors declare no competing interest. nomes, which raises questions regarding a potential functional role This article is a PNAS Direct Submission. L.T.A. is a guest editor invited by the of these enzymes under autotrophic growth conditions. Following Editorial Board. synthesis, glycine can be reduced to acetyl-phosphate (acetyl-P), This open access article is distributed under Creative Commons Attribution-NonCommercial- which is likely to be converted into acetate by acetate kinase NoDerivatives License 4.0 (CC BY-NC-ND). (ackA), thereby producing one ATP, termed the glycine synthase- Data deposition: Associated data reported in this paper have been deposited in the Gene reductase pathway (GSRP). Alternatively, serine hydroxymethyl- Expression Omnibus (GEO) database, https://www.ncbi.nlm.nih.gov/geo (accession no. transferase (SHMT) converts the produced glycine to serine, GSE118519). The complete genome sequence of C. drakei SL1T has been deposited in – the DNA DataBank of Japan/European Molecular Biology Laboratory/GenBank (DDBJ/ which then becomes transformed to pyruvate and biomass (9 11). EMBL/GenBank) database (accession no. CP020953). Recently, an artificial metabolic pathway constructed with glycine 1To whom correspondence may be addressed. Email: [email protected] or bcho@ synthase and SHMT, termed the reductive glycine pathway kaist.ac.kr. (RGP), has shown the capability of fixing CO2 using alternative This article contains supporting information online at https://www.pnas.org/lookup/suppl/ electron donors (12, 13). Despite sharing common reactions and doi:10.1073/pnas.1912289117/-/DCSupplemental. the presence of genes encoding a partial glycine synthase, the www.pnas.org/cgi/doi/10.1073/pnas.1912289117 PNAS Latest Articles | 1of8 Downloaded by guest on September 23, 2021 RGP, and WLP to fix CO2, and then converts it into acetyl-CoA gene cluster, and one CO dehydrogenase (CODH) gene cluster or acetate during autotrophic growth. (Fig. 1A and Dataset S3) (15, 16). The third FDH (B9W14_20090) was located downstream of hydrogenase- and NAD(P)H-coding Results genes, indicating a similar genomic composition to that found in Genes and Metabolic Pathways for Carbon Fixation in the C. drakei Clostridium autoethanogenum, which was reported to reduce for- Genome. The C. drakei genome was assembled completely, mate using charged ferredoxin (Fd) and NAD(P)H (17). A single resulting in a 5.7-Mbp complete genome with 29.7% GC content WLP gene cluster consisting of 15 genes associated with the car- (SI Appendix,Figs.S1andS2and Datasets S1 and S2)(14). bonyl and the methyl branches was identified, which is identical to Following the assembly, we identified 5,144 genes, which were the gene composition in the Clostridium species (Fig. 1B;seeSI composed of 5,024 coding sequences, 30 ribosomal RNAs, and Appendix, Text S3 for details) (6, 7). Energy conservation systems 90 transfer RNAs (Dataset S3;seeSI Appendix, Text S2 for play essential roles in the autotrophic growth of acetogens by details). The C. drakei genome encodes three formate dehydro- orchestrating the required reduction power for the WLP reactions genase (FDH) genes, one WLP gene cluster, one ATP synthase and generating ATP (18, 19). In the C. drakei genome, hydrogenase A Absent Score 25% 75% acsVgcvHacsBacsEacsCacsDcooClpdAmetFmetVfolD fchAfhs acsFacsA hytE1hytAhytE2hytDhytBhytC fdhA rnfC rnfD rnfE rnfGrnfA rnfB atpI atpA1atpC atpB1atpDatpA2atpGatpB2atpE T. primitia Ac. arabaticum 0 Th. phaeum M. thermoacetica Thr. kivui C. aceticum P. difficile A. woodii E. limosum C. autoethanogenum C. ljungdahlii C. scatologenes C. carboxidivorans C. drakei Hydrogenase FDH RNF complex ATP synthesis Wood-Ljungdahl pathway B 1 kb E-value Methyl Carbonyl GLYS GLYR 10E-5 0 fhs folDmetFlpdA acsA acsDacsCacsEacsBgcvHacsV gcvT gcvPA grdX grdB grdI grdT trxA grdD fhcAmetV cooC gcvH gcvPB grdE grdA grdH trxB grdC grdR C. drakei C. carboxidivorans C. scatologenes C. ljungdahlii C. autoethanogenum E. limosum A. woodii C. difficile C. aceticum Thr. kivui M. thermoacetica Th. phaeum Ac. arabaticum T. primitia Fig. 1. Comparative autotrophic metabolic pathway analysis of C. drakei.(A) Comparison of proteins associated with the Wood–Ljungdahl pathway and energy conservation
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