Construction Of Pyruvate-producing Corynebacterium Glutamicum Based On Metabolic Engineering Strategy

Traditional microbial fermentation uses starch materials such as corn and potato for fermentation production.With the increasing trend of population in recent years,the demand for grain is also increasing.However,the constraints of production conditions make it more difficult to further increase grain yield,and the starch sugar from grain is in short supply.Lignocellulose is the most abundant renewable resource in nature.Glucose and xylose in its hydrolysates have the potential to be used as fermentation substrates.Considering the long-term development,the development and utilization of lignocellulose can not only promote the production with low raw material cost,but also optimize and reduce the resource burden.Pyruvate,a key intermediate of cell metabolism,is a synthetic precursor of various amino acids,fats,alkaloids and terpenes.It has been widely used in food,medicine,chemical industry and other fields,with a large market gap.At present,the main production strains of pyruvate are yeast at home and abroad.Due to the narrow substrate spectrum of yeast,and the low tolerance to sugar and by-products,there is some defects in mass production.Therefore,it is important to construct an excellent strain that could produce pyruvate from glucose and xylose of lignocellulose.In this work,the wild Corynebacterium glutamicum ATCC13032 was used as the research object by metabolic engineering strategy.Through the reconstruction of xylose metabolic pathway,the truncation of by-product synthesis pathway and the regulation of cofactor balances,the efficient production of pyruvate using glucose and xylose was realized.The main conclusions are as follows:(1)By reconstructing xylose metabolic pathways,Corynebacterium glutamicum ATCC13032 can be used to synthesize pyruvate from xylose.Xylose gene xly A from Xanthomonas campestris was introduced into E.coli-C.glutamicum shuttle vector p JYW-4.Xylose gene xly A,xylokinase gene xly B,transketolase gene tkt A and transaldolase gene tal were introduced into E.coli-C.glutamicum shuttle vector p JYW-4.Then transformed into C.glutamicum ATCC13032 for fermentation.when single expression of xly A,ATCC13032 can synthesize pyruvate from glucose and xylose.When xly A and xly B,or xly B-tkt A-tal were co-expressed,the pyruvate production could be significantly increased.After 72 h fermentation,the pyruvate yield of YLY/p JYW-4-xly A-xly B-tkt A-tal increased to 2.77 g/L,which was 77.12%higher than the control group,and the yield was 0.195 g/g.(2)By truncating the by-product synthesis pathway,pyruvate was efficiently synthesized by YLY/p JYW-4-xly A-xly B-tkt A-tal of recombinant bacteria.The L-alanine transaminase ala T and pyruvate quinone oxidoreductase gene pqo were separately knocked out and double-knocked based on the Cre/lox P gene knockout system..The mutant strains YLY-1/p JYW-4-xly A-xly B-tkt A-tal?YLY-2/p JYW-4-xly A-xly B-tkt A-tal?and YLY-3/p JYW-4-xly A-xly B-tkt A-tal were constructed.After 72 h of fermentation,the yield of pyruvate increased by 81.20%and 102.21%,and the yield of L-alanine and acetic acid decreased by 29.2%and 18%,respectively,when ala T or pqo were deletion separately.When ala T and pqo were deletion together,pyruvate production increased by 263.40%to15.59 g/L,L-alanine and acetic acid decreased by 30.1%and 20.56%.(3)The overexpression of NADH oxidase regulates the balance of NAD~+/NADH,controls the carbon metabolic flow,and further improves the pyruvate production efficiency of C.glutamicum ATCC13032.NADH oxidase gene nox E from Lactobacillus brevis CD0817,Bacillus subtilis 168 and Streptococcus pneumoniae R6 was constitutively expressed in YLY-3/p JYW-4-xly A-xly B-tkt A-tal.The work found that the excessive expression of NADH oxidase in YLY-3/p JYW-4-xly A-xly B-tkt A-tal-nox E the NAD~+/NADH ratio increased by 278.28%and 68.25%,and the pyruvate production was 22.02g/L.The yield increasing by 424.29%and 41.24%,respectively,compared with ATCC13032 and YLY-3/p JYW-4-xly A-xly B-tkt A-tal.However,YLY-3/p JYW-4-xly A-xly B-tkt A-tal-nox E1 and YLY-3/p JYW-4-xly A-xly B-tkt A-tal-nox E2 could not successfully express the NADH oxidase encoded by nox E1 and nox E2.They compared with the control YLY-3/p JYW-4-xly A-xly B-tkt A-tal,the enzyme activity and pyruvate production levels were not improved.