Research On The Synthesis Of Hyaluronic Acid By Modification Lactic Acid Pathway In Streptococcus Zooepidemicus

Hyaluronic acid(HA)is a acid polysaccharide which is significantly applied in pharmacy and cosmetic etc.Streptococcus zooepidemicus is used for microbial production of hyaluronan on an industrial scale.In the process of synthesis Hyaluronic acid,about 75%of the carbon source were converted to lactic acid from pyruvic catalyzed by lactate dehydrogenase LDH,resulting in a great waste of carbon source.In this paper,Streptococcus equi subsp zooepidemicus ATCC39920 was used as the object of study.We knocked out the lactic acid dehydrogenase gene ldh,then expression NADH oxidase gene nox and vitreoscilla hemoglobin genes vgb,which could improve the oxygen uptake capacity,to research the effect of the lactic acid metabolic pathway in Streptococcus zooepidemicus synthesis hyaluronic acid.The main research results are as follows:By using the scarless delection method,the ldh gene was knocked out and the △ldh strain was named as S58.The fermentation results shows that Aldh strain can no longer synthesis lactate,and biomass dclined 45%,Hyaluronic acid production is 1.28 g/L,only 37.7%of the wild strain,HA molecular weight decreased to 1.44 × 106Da compare to the wild strains of 2.13 × 1V6 Da.The rate of sugar consumption was reduced,the lactate precursors pyruvic acid accumulation to 17 g/L.The carbon flux invoved in the synthesis of HA was 4.2%,which is lower than the wild type by 6.9%.As the ldh gene knocked out,the balance of intracellular oxidative/reductive state was broken,this inhibit the cell growth and the synthesis of HA.Then we build NADH oxidase gene expression vector pLH278,electroporational transformed it into △ldh got a strain named S273.Compared to the Aldh strain,the NOX activity increased by three times in S273.The rate of growth and sucrose consumption was increased,the yield of hyaluronic acid increased to 1.53 g/L.The HA molecular weight of hyaluronic acid increased to 1.86×106Da compared to △ldh strains.The carbon sources used for HA synthesis is 5.2%,that is higher than the△ldh strain which is 4.2%only.The accumulation of pyruvate decreased to 15 g/L.Inorder to increase the capability of cells to trap O2,which is the NADH oxidase catalyzed substrate,the co-expression vector pLH317 was constructed,it contains NADH oxidase gene nox and Vitreoscilla hemoglobin gene vgb.Then electroporational transformed into Aldh get a strain marked S301.Compared to the Aldh strain,the NOX activity increased two times and molecular weight increased to 1.80 × 106 Da,there is no significant difference of other characters between Aldh strain and S3 01 strain.We explored the effects of lactic acid metabolism in Streptococcus zooepidemicus on HA synthesis for the first time,and explored the effcets of nox and vgb gene over expression on the reduction force supply in Streptcoccus zooepidemicus,lay a theoretical basis to increase HA yield and synthesiz the certain molecular HA through regulate the carbon metabolic flux in Streptococcus zooepidemicus...