Functional And Utilization Of Sucrose Metabolic System For Streptococcus Zooepidemicus

Streptococcus zooepidemicus is the ideal strain for industrial production of hyaluronic acid.Sucrose is one of the most economic substrate to produce hyaluronic acid.However,the genes function of sucrose metabolism have not been systematically studied.In this paper,Streptococcus zooepidemicus ATCC39920 as the research object,I mainly studied the related gene function and applications of sucrose metabolism,and built the engineering strain which could produce HA efficiently by utilizing sucrose.The main results are as follows:(1)Two of the genes among sucrose metabolism of Streptococcus zooepidemicus ATCC35246 were found in S.Zooepidemicus ATCC39920 genome through sequences BLAST in NCBI:scrA and scrB,encoding sucrose-specific phosphotransferase system(PTS)and sucrose-6-phosphotransferase.(2)Functional characterization of the two genes scrA and scrB were studied.The phenotype and shake flask fermentation indicated that both genes scrA and scrB were required and unique for sucrose metabolism;accumulation of sucrose-6-phosphate will produce cytotoxic because of the lack of scrB.(3)The growth phenotype analysis showed that the excessive expression of scrB to speed up the bacterium utilization of sucrose,promoted the growth of bacteria,and was beneficial to the HA synthesis;while excessive expression of scrA could cause raising the level of sucrose-6-phosphate intracellular to inhibit the growth of bacterium and was adverse to the formation of HA.(4)Biomass and HA concentration analysis of S.zooepidimicus WT,△fruA,△fruK and △scrK in shake flask fermentation exhibited that it did not affect glucose metabolism of cell without fruA,fruK and scrK gene;it had little impact on carbon direction of flow to delect scrK gene;the lack of fruA and fruK gene guided the carbon direction of flow to cell growth and HA yield.(5)In this paper,I constructed recombination strains:ScrB/OP,△fruA-ScrB/OP and△fruK-ScrB/OP to improve the conversion efficiency of sucrose to HA.Fermentation analysis of the above mutant strains displayed an engineering strain △fruK-ScrB/OP:with higher conversion efficiency of sucrose to HA.The HA concentration was 5.4 g/L which was higher 42%than that of WT,the production of byproduct LA was only about 65%and the consumption of sucrose was about 84%of WT.