Study On The Characteristics Of Nutrition And Metabolism Of Acetogen Clostridium Bovifaecis

Acetogens are a diverse group of anaerobes that using acetyl-Co A pathway as the terminal electron accepting process for synthesis of acetyl-Co A and cell carbon from CO2.They are widely distributed in natural and artificial anaerobic environments,showing a metabolic diversity for utilizing a wide range of different substrates.In this study,a novel acetogen Clostridium bovifaecis isolated by our laboratory was used,it is confirmed that gene encoding formate dehydrogenase,catalyzing the first step of the methyl branch of acetyl-Co A pathway,was lack in its genome.Acetogenic utilization of glucose occurred only in the presence of formate and exogeneous CO2.Comparative genomic studies between C.bovifaecis and other acetogens were carried out basing on pan-genome analysis.The evolution relationship of acetyl-Co A pathway-related genes and its corelation with their habitats was analyzed.The unique feature of formatedependent acetogenesis provides an insight into the metabolic mechanism and ecological role of acetogens.The main results are as follows:(1)The complete genome of C.bovifaecis strain BXX(accession number of Gen Bank is CP046522.1)contained a total of 3837 coding genes.A total of 11 gene islands were predicted,suggesting that some genes might be obtained from the environment during the evolution process.It is shown that almost all the genes encoding enzymes of the WL pathway were contained,but only the gene encoding formate dehydrogenase which is the first step of methyl branch was lack.The absence of the gene encoding formate dehydrogenase was verified by PCR,RT-PCR and enzyme activity detection at DNA,RNA and protein level,respectively.Carbohydratedegrading enzyme genes including complete genes of glycolysis pathway and pyruvate decarboxylation and other glucose-related enzyme genes in subsequent fermentation were also detected,which is consistent with the glucose metabolism ability of C.bovifaecis.In addition,C.bovifaecis strain BXX contains genes encoding biological nitrogenase and alanine metabolism pathway,suggesting that it may have the potential of nitrogen fixation and alanine metabolism.(2)The growth of C.bovifaecis depends on the presence of yeast extract.0.5 g/L of yeast extract concentration can support the growth of C.bovifaecis and the amount of acetate and ethanol produced is the lowest under this concentration,which has insignificent effect on the subsequent fermentation experiments.C.bovifaecis displayed poor metabolic capabilities of autotrophic acetogenesis even with supplemental formate,utilized glucose acetogenically only in the presense of formate and exogenous CO2.When glucose concentrations are 11.5,23 and 46 mmol/L,C.bovifaecis converted 1 molecule of glucose to 2.75 molecule acetyl-Co A in the presence of formate and exogenous CO2,which fits the theoretical glucose/product ratio 1:3 of homoacetogenesis.Ethanol was the main product when the concentration of glucose was 11.5 and 23 mmol/L,and acetate was the main product when the concentration of glucose was 46 mmol/L,which may be related to different glucose metabolism pathways under different glucose concentrations.C.bovifaecis reduced acetylene to ethylene,indicating that it had the ability of nitrogen fixation.(3)Pan-genome analysis of 35 strains of acetogens and 10 strains of Clostridium genus bacteria showed that the functions of core genome mainly related to translation,post-translational modification,reproduction,carbohydrate metabolism,cell wall synthesis and so on,and the functions of accessory genome mainly related amino acid metabolism,transcription,signal transduction,energy metabolism,carbohydrate metabolism and so on.Compared with acetogens from non-digestive tract,the core genomes,genes encoding enzymes involved in acetyl-Co A pathway and gene encoding formate dehydrogenase of 14 strains of acetogens from digestive tract were clustered in phylogenetic tree,which indicated closest genetic relationship.Gene diversity and positive pressure analysis showed that the gene encoding formate dehydrogenase of acetoegns had a tend of gradual degeneration in digestive tract habitats.C.bovifaecis has no obvious growth advantage compared with Escherichia coli using glucose as substrate in the presence of formate and exogenous CO₂.