CO₂ Fixation Mechanism Of Formate Dehydrogenase-Lacking Wood-Ljungdahl Pathway In The Acetogen Clostridium Bovifaecis

Carbon dioxide（CO₂）is one of the greenhouse gases that contribute to global warming,and wood-Ljungdahl pathway（WLP）is the oldest CO₂ fixation pathway on the Earth and plays an important role in the global biosynthesis of acetate.Formate dehydrogenase（FDH）catalyzes the conversion of CO₂ to formate as the first step of the methyl branch in WLP.FDH-lacking WLP has recently been found to distribute widely in gastrointestinal acetogens.However,the metabolic mechanisms and ecological roles of FDH-lacking WLP in the formate-rich environments are unclear.In this study,FDH-lacking acetogen Clostridium bovifaecis was used as the model bacterium.The carbon and energy metabolic pathways of formate co-fixation under heterotrophic conditions in Clostridium bovifaecis were analyzed,and the effects of fermentative bacterial co-culture or nitrate on formate co-fixation under heterotrophic conditions in Clostridium bovifaecis were also studied.The carbon fixation metabolic pathways of Clostridium bovifaecis in the metabolism of formate were investigated.The main results are as follows:（1）The metabolic pathway of CO₂ fixation in C.bovifaecis under heterotrophic conditions was investigated based on transcriptomic approach.The genes encoding enzymes involved in glycolysis（formate C-acetyltransferase,ethanol dehydrogenase,aldehyde ferredoxin oxidoreductase,etc.）,electron transport（[Fe Fe]hydrogenase,NADH dehydrogenase and NADH-quinone oxidoreductase）and methyl-accepting chemotaxis protein（MCP）were significantly up-regulated（1-6.43 folds）in the 46 mmol/L glucose incubations in comparison with the 11.5 mmol/L glucose,suggesting the response of glycolysis to higher concentrations of glucose in C.bovifaecis and the change of fermentation product from ethanol to acetate.The expression of genes encoding enzymes involved in the synthesis of vitamin B12 and histidine were significantly up-regulated（3.35-6.39 folds）,suggesting that vitamin B12 and histidine synthesis was increased in C.bovifaecis under higher glucose concentration.In the presence of formate+CO₂,genes involved in glycolysis and NADH-quinone oxidoreductase were significantly up-regulated,as well as some genes encoding methyl branching of the WLP and CO dehydrogenase/acetyl-Co A synthase,but genes encoding formate C-acetyltransferase were significantly down-regulated.（2）The main product in the glucose+formate+CO₂ incubations was ethanol with the concentrations of 5.80 and 1.66 mmol/L for 10 and 30 mmol/L nitrate,respectively,which were significantly lower than that of the controls with no nitrate（7.13 mmol/L）.In addition,the consumptions of glucose were significantly decreased in the presence of nitrate.The consumption of formate decreased with the increase of nitrate concentration and were zero at the nitrate concentration of 30 mmol/L.30 mmol/L of nitrate inhibited the CO₂ fixation of formate dehydrogenase-lacking WLP,and 10 mmol/L of nitrate negatively affected the glycolysis in C.bovifaecis.（3）In the coculture of C.bovifaecis and intestinal fermentive bacteria Clostridium butyricum,C.butyricum increased by 3 orders of magnitude and C.bovifaecis increased by 1order of magnitude（glucose）or remained the same level（glucose+formate+CO₂）,and the consumption of glucose and the production of ethanol or acetate by C.butyricum were significantly higher than that of C.bovifaecis,indicating that C.butyricum was grown better than C.bovifaecis in the co-culture system.Compared with the pure cultures,the concentration of C.butyricum in the coculture was 1 order of magnitude higher（glucose）or the same level（glucose+formate+CO₂）,the concentration of C.bovifaecis in the coculture was 1 order of magnitude lower,accompanied by the decrease of glucose consumption and acetate or ethanol production.It is indicated there was amensalism relationship between C.butyricum and C.bovifaecis,which favored C.butyricum and inhibited C.bovifaecis,but the growth advantages of C.butyricum in the coculture were weakened under the glucose+formate+CO₂ substrate.（4）C.bovifaecis converted 11.5-69 mmol/L formate to acetate with a molar ratio of 2.4-2.6:1 as the sole carbon source,which fits the stoichiometry of acetogenesis.PCR and reverse transcription PCR results showed that C.bovifaecis contained genes encoding anaerobic ribonucleoside triphosphate reductase（nrd D and nrd G）which were highly transcripted during the formate metabolism.The results show that C.bovifaecis converted 1 mol of formate into 1mol of CO₂ which is required for the carbonyl branch of WLP via ribonucleoside triphosphate reductase,then another mol of formate and the mol of CO₂ was synthesized 1 mol acetyl-Co A via WLP,and finally the acetyl-Co A was further converted to acetate.The reducing substance L-cysteine in the medium provide electrons for WLP.The results aim to elucidate the CO₂ fixation mechanism of FDH-lacking WLP,which is important for exploring the environmental effect and ecological roles of WLP,and is helpful for the biosynthesis of high-value organic acids and the reduction of greenhouse gas emissions.