| Cyanidin-3-O-glucoside,a type of anthocyanins,is water-soluble natural pigments widely existed in plants,which have many biological and pharmacological activities to human body,including antioxidation,hypoglycemic,and prevention human from cardiovascular diseases.At present,the supply of anthocyanins in the market mainly relies on the extraction of plant materials,which has problems such as low purity,high extraction cost,unstable structure,and ecological damage.Furthermore,it is also easily affected by productivity and environmental conditions.Given the rapid growth and easy cultivation of microorganisms,constructing a genetically engineered microbial cell factory for anthocyanin biosynthesis is a highly potential,more convenient and controllable production pathway.In order to explore the microbial fermentation synthesis of anthocyanins and realize its application in the food industry,this study takes Saccharomyces cerevisiae,a food safety grade yeast,as the research object.Based on molecular biology,a genetic engineering strain that can synthesize C3G was constructed,thus realizing the heterologous biosynthesis of C3G in Saccharomyces cerevisiae.The metabolic engineering,genetic engineering and fermentation engineering were carried out to improve the production of C3G in genetically engineered Saccharomyces cerevisiae from the aspects of increasing the supply of precursors,removing speed limit steps,balancing metabolic flux,etc.Finally,the genetically engineered Saccharomyces cerevisiae was applied to ferment Chinese bayberry wine to verify its antioxidant activity and investigate its application value.It has certain practical significance for solving the problem of color deterioration in bayberry fermented wine.The main research conclusions of this article are as follows:（1）Construction of biosynthetic pathway of anthocyanin precursor naringenin and optimization of its yieldThe genetically engineered Saccharomyces cerevisiae YJN1 capable of de novo synthesis of naringenin was successfully constructed,and its initial naringenin yield was 4.71 mg/L;Aro3,Aro4 and Aro7 were knocked out,and the mutant Aro4K229L and Aro7G141S that were not inhibited by tyrosine feedback were overexpressed respectively.The key enzyme genes of the bypass pathway,Aro10 and PDC5,were knocked out,PHA2 was weakened,and the transketolase TKL1 and prephenic acid dehydrogenase Tyr C that were not inhibited by tyrosine feedback were overexpressed,increasing the yield of naringenin to 45.34 mg/L;The global regulatory factor YPL062W was knocked out and ACC1 was overexpressed to increase the accumulation of malonyl-Co A,increasing the production of naringenin to 56.84 mg/L;Naringenin synthase genes TAL,4CL,CHS,CHI were overexpressed making the yield of naringenin reach 97.62 mg/L.（2）Construction of anthocyanin de novo synthesis pathwayDFR and ANS can be expressed in Saccharomyces cerevisiae.By adjusting the gene copy number of DFR and ANS,the highest yield of cyanidin was 5.78 mg/L;The complete pathway of anthocyanin de novo synthesis was successfully constructed after the removal of 3GT transport peptide,and the corresponding genetic recombination yeast strain YJC3 could synthesize 0.54 mg/L C3G;The fusion expression of ANS and tr3GT could greatly increase the production of C3G.The yield of the recombinant yeast strain YJC4 constructed by fusion expression of the protein was 1.76 mg/L;Overexpression of PGM could affect C3G production,and the production of C3G in strain YJC5 overexpressing PGM ultimately reached 1.92 mg/L;Knocking out the endogenous degrading enzyme EXG1 could reduce the degradation of anthocyanins and increase the yield of anthocyanins,ultimately reaching 2.31 mg/L.（3）Fermentation optimization of anthocyanin biosynthesisThe effects of different carbon sources,cofactors and surfactant Tween 80 on anthocyanin biosynthesis in the culture medium were investigated.The anthocyanin biosynthesis pathway was constructed into two kinds of Saccharomyces cerevisiae through a co-culture strategy,the burden of cell metabolism was alleviated,and the biomass and anthocyanin production were increased.The combination of 1%sucrose and 1%glycerol had the highest cost performance ratio,and the anthocyanin yield was5.02 mg/L;The addition of cofactor ferrous ions had a significant impact on the biosynthesis of anthocyanins（p<0.05）,with the highest yield of anthocyanins reaching5.77 mg/L at a concentration of 0.4 m M.The utility of surfactant Tween 80 could effectively improve the efficiency of anthocyanin biosynthesis.The optimal concentration is 8 g/L,and the anthocyanin yield reached 6.12 mg/L;The application of co-culture strategy was conducive to the growth of Saccharomyces cerevisiae cells,could reduce the metabolic pressure caused by gene manipulation,and finally the anthocyanin yield was increased to 7.85 mg/L.（4）Optimizing anthocyanin production through two stage dynamic regulationThe two-stage pH regulation strategy was conducive to the biosynthesis of anthocyanins in Saccharomyces cerevisiae by using the pH control fermentation.By comparing the regulation of different pH values,it was found that the regulation effect was the best when pH is 2,and the anthocyanin yield reached 7.36 mg/L,which was20.26%higher than that of the group without metabolic transformation;Based on proteomic analysis using TMT labeling,we identified a total of 74 differentially expressed proteins in the pH control group,of which 8 were significantly upregulated and 66 were significantly downregulated.These differentially expressed proteins are mainly related to oxidative phosphorylation and other processes.It was speculated that pH regulation caused the stress response of Saccharomyces cerevisiae,promoted cell growth and metabolic process.And it was conducive to anthocyanin biosynthetic process and its stability in the culture medium,ultimately increased anthocyanin production.（5）Application of genetically engineered Saccharomyces cerevisiae in bayberry fermented wineCompared with the ordinary Saccharomyces cerevisiae group,the fermentation capacity of genetically engineered Saccharomyces cerevisiae was roughly equivalent,but the color of fermented wine was more abundant,specifically,the content of soluble solids and residual sugar were higher.Besides,the alcohol content was lower than the control,the titratable acid content was higher,the red color was stronger,the color saturation was higher strong glossiness;the contents of total phenols and total flavonoids in the experimental group were higher than that in the control group.The phenolic substances included gallic acid,protocatechuic acid,myricetin,isoquercitrin,quercitrin,and cyanidin-3-O-glucoside,and the contents were higher than that of the control group;The genetic engineering yeast group also increased the content of naringenin,taxifolin,eriodictyol,and aromadendrin;The experimental group of genetically engineered yeast showed better ABTS free radical scavenging ability,DPPH free radical scavenging ability,and FARP antioxidant ability compared to the control group.The sensory evaluation results showed that the difference between the two groups of bayberry fermented wine was mainly reflected in its color. |
