Breeding Of Ethyl Lactate-producing Saccharomyces Cerevisiae Strains

Ethyl lactate is an important aroma substance in Baijiu,which affects it's quality and style.Ethyl lactate in wine is generally produced by esterification of lactic acid and ethanol,while lactic acid is derived from lactic acid bacteria mixed in the fermentation system.The application of clean and wine mechanization production process will greatly reduce the chance of lactic acid bacteria in the environment being mixed into the fermentation system,resulting in low ethyl lactate content and a decline in flavor quality,which will cause to lose its unique style.Allowing the main brewer'syeast-Saccharomyces cerevisiae,to produce lactic acid then esterification with ethanol to produce ethyl lactate is a solution.By using metabolic engineering methods,this study aimed at building three enzyme catalytic pathways in Saccharomyces cerevisiae,in which the production of L-lactate is catalyzed by lactate dehydrogenase from pyruvate and transfers into lactoyl CoA catalyzed by propionyl-CoA transferase(Pct),and lactoyl CoA reacting with ethanol under the catalysis of alcohol acyltransferase(AAT)to produce ethyl lactate.Combined with the metabolic engineering strategy,improving the copy number of Pets,strengthen the synthesis of lactyl-CoA,which designed to increase the yield of ethyl lactate.The main contents and results were as follows:The introduction of lactic acid metabolic pathway laid the material foundation for the biosynthesis of ethyl lactate.This study introduced the lactate dehydrogenase gene ldhLl of Lactobacillus plantarum WCFSll.The yields of L-lactate and ethyl lactate of the modified strain a(L)were 12 g/L and 140 mg/L,respectively.It was necessary to further convert free lactic acid into ethyl lactate.Pets can convert lactic acid into lactyl-CoA and then catalyzed by AAT to produce ethyl lactate.In this study,Clostridium propionicum Pctcp,Ralstonia eutropha H16 PctRe,Megasphaera elsdenii PctMe,and Moorella thermoacetica PctMt,were introduced,all of which were constructed into different Pct expression boxes.Different AAT expression boxes were constructed,which consist of the Saccharomyces cerevisiae ATFl,EHT1 and EEB1,strawberry SAAT and VAAT,Actinidia eriantha AeAT9,melon Mel2,respectively.By liquid fermentation of corn hydrolysate,this study screened the highest yield of ethyl lactate and ethyl acetate modified strain named a(L)-PctCp-AeAT9.the production of ethyl lactate and ethyl acetate were about 68.6%and 35 times of the parental strain a(L),respectively.The strain ?(L)-PctMt-VAAT with a significant increase in ethyl lactate and a smaller increase in ethyl acetate was also screened.Compared with the strain a(L),it was increased by 45.4%and 7.5 times,respectively.The results showed that the introduction of the Pct metabolic pathway succeeded in increasing the content of lactyl-CoA,and the resulting lactyl-CoA was then catalyzed by alcohol acylases such as AeAT9 and VAAT to produce ethyl lactate.Pets can catalyze the formation of lactyl-CoA,but its catalytic activity is limited.Compared to the parental strains ?(L)-Pctcp-AeAT9 and a(L)-PctMt-VAAT,adding a copy number of Pct,the strain a(L)-dPctcp-AeAT9 and ?(L)-dPctMt-VAAT producted ethyl lactate increasing by 18.1%and 22.6%,respectively.In addition,one copy number of Pct were then added,which the yield of ethyl lactate was increased by 37.2%and 36.9%of the triple Pets strain a(L)-tPctcp-AeAT9 and a(L)-tPctMt-VAAT.It can conclude that the increase of Pct copy number could strengthen the synthesis of lactyl-CoA,which led to more lactic acid convert to lactyl-CoA,and then the production of ethyl lactate was improved.