Modification Of Pyruvate Bypass Synthesis Pathway Of Saccharomyces Cerevisiae And Its Application In The Synthesis Of Poplar

Chrysin（Chrysin）is a group of flavonoids widely found in propolis,honey,momordica,pear,passionflower peel,walnut membrane and walnut kernel,as well as Chrysin.Abicin and its derivatives,such as baicalin and Hanhuang,have been widely used in the fields of antitumor drugs and immunological preparations.At present,there have been studies on the production of flavonoids such as aspicin,baicalin and dihydroquercetin using Escherichia coli or saccharomyces cerevisiae.However,the synthetic aspicin production of saccharomyces cerevisiae is still at a relatively low level.In this study,the yeast chassis cells were used as the starting strain to study the modification of pyruvate bypass synthesis pathway in yeast cells and its role in improving albuminin synthesis.In order to improve the yield of albuminin in strain W303-WJ,Pan K,ACS and ACC1 were cloned and expressed according to the supply and synthesis regulation of coenzyme A,acetyl Coenzyme A and malonyl Coenzyme A in the albuminin synthesis pathway,and the integrated expression vectors of the above enzyme genes were constructed separately or with different combinations:Carriers PHBM368-ACS^{s e l 6 4 1 p},PHBM368-MPank1Co A,PHBM368-ACC1,PHBM368-ACS^{s e l 6 4 1 p}-ACS^{s e l 6 4 1 p},PHBM368-m Pank1Co A-ACS^{s e l 6 4 1 p},p HBM368-m Pan K1Co A-ACC1 plasmid,The exogenous gene was homologous recombined into Saccharomyces cerevisiae W303 by r DNA recombinant tag on the plasmid The r DNA site obtained integrated expression of m Pan K1Co A,ACS^{s e L6 4 1 P},ACC1,ACS^{s e l 6 4 1 p}-ACS^{s e l 6 4 1 p},m Pank1Co A-ACS^{s e l 6 4 1 p},m Pan K1Co A-ACC1recombinant strains L11,L12,L13,L14,L15,L16.Six recombinant saccharomyces cerevisiae strains were used to draw the growth curve by detecting OD600 of fermentation liquid,and the PH,biomass,reducing sugar consumption and alcohol content of fermentation liquid were measured at the same time.The acetyl-co A content of yeast strains was detected by kit method,and the metabolite albuminin content was detected by high performance liquid chromatography,and the optimal strain with the comprehensive yield of albuminin and acetyl-co A was screened.Furthermore,single factor tests were conducted on the initial glucose concentration,initial peptone concentration and initial calcium pantothenate concentration of the medium by using the optimal strain,and the optimal ratio of interaction between the three factors was obtained by response surface experiments.The results showed that six plasmids were successfully introduced into the W303-WJ r DNA site of Saccharomyces cerevisiae.The results of fermentation growth curve showed that the six strains L11-L16 showed good growth characteristics,and the fermentation OD600 and biomass of strains L14,L15 and L16 were significantly higher than those of the original strains.The results indicated that the introduction of ACS alone or combined introduction of ACS-Pank and Pan K-ACC1 could significantly promote the growth of saccharomyces cerevisiae.The glucose consumption rate of the six strains L11-L16 increased slightly,and the ethanol production decreased to a certain extent.Among them,the alcohol content of the strains L11 and L15 was lower,indicating that the introduction of ACS and the simultaneous introduction of ACS-Pank significantly increased the ethanol consumption during fermentation.The content of acetyl co A in fermentation broth of strain L11,L12,L14,L15 and L16 was significantly increased by 0.62 times,0.64 times,1.9 times,2.34 times and 1.11 times,respectively.The results indicated that the introduction of ACS and Pan K genes could promote the production of acetyl-co A.After 84 h of fermentation,the yield of allicin in fermentation broth of strain L11-L16increased significantly,by 0.33 times,0.37 times,0.43 times,0.58 times,0.8 times and 0.73 times,respectively.After 84 h of fermentation,the albumin production level of ACC1-overexpressing strain L13 was significantly higher than that of strain L11 and L12.The strain（L15）combined with ACS-Pan K was higher than the strain（L14 and L16）combined with ACS-ACS and Pank-ACC1.By single factor experiment,the optimum fermentation conditions were determined as 6%glucose,1.5%peptone and 0.1%calcium pantothenate.The optimal fermentation conditions were selected by response surface experiment:glucose supplemental level:8.292%,peptone supplemental level:1.528%,calcium pantothenate supplemental level:0.2673%.The results showed that the yield of poplar in L15 strain was the highest after genome modification and fermentation medium optimization.Under the above fermentation conditions,the concentration of aspinin was 15.724 mg/L.