Evaluation Of Synthetic Metabolism Of Crabtree Negative Saccharomyces Cerevisiae

Saccharomyces cerevisiae is an important type of safe microorganism in microbial industrial production,and is often used as a cell factory for compound synthesis.Due to the intrinsic Crabtree effect of S.cerevisiae during the long-term evolution,and ethanol is the main product of S.cerevisiae.Ethanol production will result in wasting of carbon flux and lead to the low yield of target compounds in Crabtree positive S.cerevisiae.In order to increase the yield of the target product in S.cerevisiae and reduce the loss of carbon flux,the pyruvate decarboxylase gene was knocked out to eliminate the production of ethanol in S.cerevisiae.However,this type of pyruvate decarboxylase-deficient strains cannot grow in a medium that uses glucose as a carbon source.Therefore,the introduction of a new acetyl-Co A synthesis pathway combined with laboratory adaptive evolution and reverse metabolic engineering has restored the growth of the strain in glucose medium.This Crabtree negative S.cerevisiae has a maximal specific growth rate at 0.218 h^-1.This strain has no ethanol production and has the potential to synthesize the target product with high yield.We used the Crabtree negative S.cerevisiae strain as the starting strain and constructed strains that produce different target products through synthetic biology methods to verify the advantages of Crabtree negative S.cerevisiae strains.Pyruvate,malonyl-Co A,and mevalonate are three key metabolic nodes in S.cerevisiae.These three compounds are the key precursors of many important industrial compounds.Terpenoids and fatty acids that have important uses in industry and biofuels can be synthesized from metabolic nodes.Overexpressing the endogenous gene,increased the production of 2,3-butanediol which is derived product from pyruvate;modifying the mevalonate pathway by introducing exogenous pathway obtained lycopene producing strain;introduce foreign genes,enhance the expression of key downstream enzymes of malonyl-Co A,and obtain 3-hydroxypropionic acid synthesizing strains;optimizing the fatty acid synthesis pathway and strengthening the precursor supply enhanced fatty aicds production.Compared with wild type strain,it showed that the Crabtree negative strain exhibited significant advantanges in biosynthisizing above compounds.The unit OD₆₀₀ titer of 2,3-butanediol,lycopene,3-hydroxypropionic acid and fatty acids in Crabtree negative S.cerevisiae is increased by 6.6,3.5,1.2 and 2.4 folds respectively compared with the wild-type.Among them,the yield of 2,3-butanediol,lycopene,3-hydroxypropionic acid and fatty acids from glucose in Crabtree negative S.cerevisiae were increased by 4.3,1.8,0.2,and 1.5 folds compared with the wild type.Although this Crabtree negative S.cerevisiae has a slower growth rate than the wild-type strain,the advantages of higher titer and yield in producing these compounds indicated the great potential as a platform strain for industrial production of other target compounds.