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CO2-Fixing One-Carbon Metabolism in a Cellulose-Degrading Bacterium

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CO2-Fixing One-Carbon Metabolism in a Cellulose-Degrading Bacterium CO2-fixing one-carbon metabolism in a cellulose- degrading bacterium Clostridium thermocellum Wei Xionga, Paul P. Linb, Lauren Magnussona, Lisa Warnerc, James C. Liaob,d, Pin-Ching Manessa,1, and Katherine J. Choua,1 aBiosciences Center, National Renewable Energy Laboratory, Golden, CO 80401; bDepartment of Chemical and Biomolecular Engineering, University of California, Los Angeles, CA 90095; cNational Bioenergy Center, National Renewable Energy Laboratory, Golden, CO 80401; and dAcademia Sinica, Taipei City, Taiwan 115 Edited by Lonnie O. Ingram, University of Florida, Gainesville, FL, and approved September 23, 2016 (received for review April 6, 2016) Clostridium thermocellum can ferment cellulosic biomass to for- proposed pathway involving CO2 utilization in C. thermocellum is mate and other end products, including CO2. This organism lacks the malate shunt and its variant pathway (7, 14), in which CO2 is formate dehydrogenase (Fdh), which catalyzes the reduction of first incorporated by phosphoenolpyruvate carboxykinase (PEPCK) 13 CO2 to formate. However, feeding the bacterium C-bicarbonate to form oxaloacetate and then is released either by malic enzyme or and cellobiose followed by NMR analysis showed the production via oxaloacetate decarboxylase complex, yielding pyruvate. How- of 13C-formate in C. thermocellum culture, indicating the presence ever, other pathways capable of carbon fixation may also exist but of an uncharacterized pathway capable of converting CO2 to for- remain uncharacterized. mate. Combining genomic and experimental data, we demon- In recent years, isotopic tracer experiments followed by mass strated that the conversion of CO2 to formate serves as a CO2 spectrometry (MS) (15, 16) or NMR measurements (17) have entry point into the reductive one-carbon (C1) metabolism, and emerged as powerful tools for metabolic analysis. This methodol- internalizes CO2 via two biochemical reactions: the reversed pyru- ogy traces the transit of isotopic atoms throughout the biochemical vate:ferredoxin oxidoreductase (rPFOR), which incorporates CO2 network and sheds light on active reactions as well as molecular using acetyl-CoA as a substrate and generates pyruvate, and py- fluxes in vivo. Coupled with classic genetic manipulations, this ruvate-formate lyase (PFL) converting pyruvate to formate and technology has been successfully applied to provide insights into acetyl-CoA. We analyzed the labeling patterns of proteinogenic novel metabolic pathways in bacteria (18), yet it has not been used MICROBIOLOGY amino acids in individual deletions of all five putative PFOR mu- to study C. thermocellum. tants and in a PFL deletion mutant. We identified two enzymes In several species of Clostridium, the one-carbon (C1) metabolism acting as rPFOR, confirmed the dual activities of rPFOR and PFL is initiated from CO2 reduction to become formate via formate crucial for CO2 uptake, and provided physical evidence of a distinct dehydrogenase (Fdh) (19). This pathway provides C1 units for many “ ” in vivo rPFOR-PFL shunt to reduce CO2 to formate while circum- crucial reactions, including amino acids and purine biosynthesis. venting the lack of Fdh. Such a pathway precedes CO2 fixation via Formate serves as a precursor to the folate biosynthesis pathway that the reductive C1 metabolic pathway in C. thermocellum. These forms the “methyl” branch of the Wood–Ljungdahl pathway, yield- C. thermocel- findings demonstrated the metabolic versatility of ing acetyl-CoA from CO2 (19). However, C. thermocellum lacks the lum, which is thought of as primarily a cellulosic heterotroph but is first key enzyme, Fdh, based on genome information. In the pre- shown here to be endowed with the ability to fix CO2 as well. sent study, we investigate CO2-fixing pathway(s) in C. thermocellum involving C1 metabolism by using 13C-tracer experiments. We Clostridium thermocellum | pyruvate:ferredoxin oxidoreducase | formate | 13 C-isotopic tracing | one-carbon metabolism Significance he gram-positive Clostridium thermocellum is a thermophilic High carbon yield in the bioengineering of heterotrophic and strict anaerobic bacterium. It has gained a great amount T bacteria is hindered by carbon loss to CO2 production. We of interest due to its cellulolytic abilities. By taking advantage provide evidence showing Clostridium thermocellum, a cellulose- of an extracellular cellulase system called the cellulosome (1), degrading bacterium and a model consolidated bioprocessing C. thermocellum can depolymerize cellulose into soluble oligo- organism, can fix CO2 while growing predominantly on cellobi- saccharides. The latter are further transported into the cells and ose, a cellulose-derived disaccharide. By adding 13C-bicarbonate fermented through its glycolytic pathway to pyruvate, the pre- to the bacterial culture and tracking 13C-labeled metabolites, we cursor to an array of fermentation products (e.g., H2, formate, discovered active reductive one-carbon (C1) metabolism in this lactate, acetate, ethanol, secreted amino acids) (2, 3). This ca- bacterium. We further identified critical enzymes responsible for pability makes C. thermocellum an attractive candidate for con- fixing CO2 and channeling the fixed carbon to the C1 metabolic solidated bioprocessing of lignocellulosic biomass, a process pathway. Our findings pave the way to future engineering of this configuration that directly converts plant biomass into biofuels bacterium to use cellulose and CO2 simultaneously as a means to and chemicals without separate additions of enzymes (4). To date, improve microbial carbon efficiency that is constrained by theo- there have been numerous works describing the molecular and retical limitation and to reduce CO2 in the environment. genetic details of the cellulolytic system and fermentation path- ways in C. thermocellum (5–9), whereas other metabolic charac- Author contributions: W.X., P.P.L., L.M., L.W., J.C.L., P.-C.M., and K.J.C. designed research; W.X., P.P.L., L.M., L.W., and K.J.C. performed research; W.X., P.P.L., L.M., L.W., J.C.L., P.-C.M., teristics of this species have not been adequately understood. and K.J.C. analyzed data; and W.X., P.P.L., L.M., L.W., J.C.L., P.-C.M., and K.J.C. wrote Currently, few details regarding inorganic carbon utilization the paper. are known for C. thermocellum. However, several investigators The authors declare no conflict of interest. routinely add bicarbonate, a dissolved form of CO2,intotheme- This article is a PNAS Direct Submission. – dium to promote C. thermocellum growth (6, 10 12), implying its Freely available online through the PNAS open access option. ability to use CO . This observation raises the questions of how 1 2 To whom correspondence may be addressed. Email: [email protected] or pinching. CO2 plays a role in promoting C. thermocellum growth (13), and [email protected]. whether CO2 is incorporated into the metabolism. There is limited This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. information on pathways relating to CO2 uptake thus far. One 1073/pnas.1605482113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1605482113 PNAS Early Edition | 1of6 Downloaded by guest on September 27, 2021 measure the isotopomers of fermentation products and proteino- media and did not change in relative intensity from sample genic amino acids using NMR and GC/MS, and uncover a route to sample. C. thermocellum employs to fix CO2 when grown in a primarily heterotrophic mode supplemented with sodium bicarbonate. Putative Reductive C1 Metabolism Based on Genome Annotation. The above labeling experiments provided evidence of a significant CO2-to- Results formate reduction pathway that remains to be characterized. To Effects of Bicarbonate on C. thermocellum Growth and Metabolism. identify genes that are putatively responsible for this conversion, To examine C. thermocellum’s ability to use inorganic carbon, we we reconstructed the C1 metabolic pathway de novo, based compared cell growth and metabolic product formation during on the genomic information of C. thermocellum DSM1313 strain cellobiose fermentation with and without bicarbonate supple- (20). Its genome sequence suggests that C. thermocellum is ca- mentation (20 mM). To eliminate any potential buffering effect pable of converting formate to methionine, serine, and pyruvate exerted by bicarbonate, fermentation was performed in batch via the methyl branch of the Wood–Ljungdahl pathway (Fig. 2). mode with pH controlled at 7.0 throughout. Upon consumption However, it lacks Fdh, the enzyme microbes typically use to of all cellobiose, cultures with added bicarbonate yielded ∼40% convert CO2 to formate (Fig. 2). more cell biomass than in its absence (Table 1). Without ac- Lacking the fdh gene, the only other gene encoding formate counting for the excreted amino acids that have been observed in production found in C. thermocellum genome is pyruvate-formate overflow metabolism (3), this result increased the possibility that lyase (PFL), catalyzing the conversion of pyruvate and CoA to extra total carbon output (i.e., cell biomass, lactate) could emerge acetyl-CoA and formate (21). We thereby hypothesize that a by- from incorporation of the inorganic carbon (bicarbonate) as sug- pass consisting of two reactions catalyzed by pyruvate:ferredoxin gested by the calculated apparent carbon efficiency (65.7% with- oxidoreductase (PFOR) in the reversed direction and PFL may out bicarbonate and 75.5% with bicarbonate; Table 1). We also realize the
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