Construction Of Saccharomyces Cerevisiae Engineered Strains Overexpressing Heterologous 2,3-butanediol Pathway Key Enzyme Genes

2,3-butanediol(2,3-BD)is a kind of extremely valuable chemical material and novel fuel which can be widely used in chemical,food,medicine,aerospace and many other fields.However,the main strains that are Klebsiella and Bacillus of microbial production of 2,3-BD are potentially pathogenic bacteria without mass production value.Thus the large-scale production of 2,3-BD by GRAS microbiologies that is Saccharomyces cerevisiae at present is desirable.In this paper,Saccharomyces cerevisiae is utilized as the original strain to constructe the engineering strain by over-expression of exogenous genes of key enzymes in 2,3-BD pathway which is helpful to improve the production of 2,3-BD,providing security of the strain yielding2,3-BD.Firstly,the features of the strains were tested to screen the most potential strain of 2,3-BD production.Growth curves,substrate and product tolerances,intermediate levels,and the fermentation performance of S.cerevisiae W5,WBG1,WBG3,RHZ7 strains were determined.The results showed that W5 strains grow quickly with benign toleraces of 7% ethanol,2% acetic acid and8% lactic acid and higher intermediate pyruvate and acetoin contents that were 1.7 g/L and 0.276 g/L respectively.It also had higher ethanol yield(0.430 ± 0.021 g/g)and carbon source utilization(100%of glucose fermentation rate 24 h),so it is the ideal original strain.Furthermore,the highest ethanol yield of W5 strains could reach to 0.479 ± 0.02 g/g and the peak pyruvate kinase enzyme,alcohol dehydrogenase enzyme and fructose phosphate kinase enzyme activities were achieved at 12 h(26.81 U/mg prot),24 h(484.91 U/mg prot)and 24 h(10.49 U/mg prot)respectively and the trend of the three kinds of enzyme activities were the same as the changing of ethanol accumulation trend indicating well performance and capacity of alcohol fermentation of the W5 strain.The production of acetic acid was stimulated while the production of glycerol and ethanol was inhibited overall performance of acetic acid stress.The W5 strains had the ability to produce 2,3-BD with a yielding of 0.216±0.002 g/L and productivity of 0.0054±0.0007 g/g;Compared with the results of two Bacillus subtilis,the strain CICC10026 is a good heterologous gene template source strain of2,3-BD.Due to the 2,3-BD ynthesis pathway in Saccharomyces cerevisiae is not complete,only alpha acetolactate synthase/alpha ALS and 2,3-BD dehydrogenase/BDH are contained without alpha acetolactate decarboxylase/alpha ALDC.Therefore,the exogenous alpha acetyl lactic acid synthase gene(alsS)and alpha acetyl lactic acid decarboxylase(alsD)gene in 2,3-BD pathway were converted into S.cerevisiae by means of homologous recombination to realize efficient expression of 2,3-BD in S.cerevisiae by setting up the network access.The genome DNA of Bacillus subtilis CICC10026 was used as the template to amplify alsD and alsS genes fragments by PCR which were than connected to the expression vector pYXAK-xyl1 to build the expressing recombinant plasmid pYXAK-alsD and pYXAB-alsS with selection markers of G418 and Blasticidin respectively.Lithium acetate was applied to convert the expression plasmids into W5 strains and heterologous expression of engineering strains HSFW5-01 was obtained through resistance screening and the effect of heterologous gene expression was determined.Fermentation results showed that 2,3-BD production ability of the engineering strain HSFW5-01 was significantly higher than the original strain with the maximal yield of 2.05±0.04 g/L,9.49 times more than the original strains(0.216±0.002 g/L).While the conversion rate of HSFW5-01 was 0.098±0.001 g/g,increased by 171.5% of W5(0.0054 ± 0.0007 g/g).As contrast of W5 strains,the fluorescent quantitative PCR method was applied to detect the mRNA expression levels of the purpose genes in HSFW5-01.The results showed that expression of the two genes did not detected in original strain while alsD and alsS in HSFW5-01 had higher expression with the ? CT value of 8.93 and 5.51,respectively,the difference was extremely significant(p < 0.01).Protein expression differences of genes in engineering strains and original strain was detected by SDS-PAGE,finding that clear bands of HSFW5-01 in 28.5 kDa(alsD)and 61.2 kDa(alsS)appeared while the original strains did not appear the two bands,the difference of grey value analysis were very significant(p < 0.01).The study will provide some referential value to learn more about the connections between the expression levels of the three key enzymes in 2,3-BD synthesis route and the yield of 2,3-BD as well as lay the foundation of the industrialization of 2,3-BD production in some degree.