Metabolic Engineering Of Saccharomyces Cerevisiae For Production Of Glucaric Acid

Glucaric acid?GA?,one kind of natural organic acid,has been widely used in the fields of food,chemical and medicine industries.This study aimed to produce glucaric acid by metabolic engineering of Saccharomyces cerevisiae.Strategies were applied including constructing a synthetic glucaric acid biosynthetic pathway,overexpression of genes in myo-inositol biosynthesis pathway,weakening the competing glycolytic pathway and constructing MIOX mutation libraries.The main results were described as follows.?1?Construction of a glucaric acid biosynthetic pathway in S.cerevisiae.As S.cerevisiae does not naturally produce glucaric acid,it was necessary to establish a synthetic metabolic pathway.Coexpression of the genes encoding myo-inositol oxygenase?MIOX?from mice and uronate dehydrogenase?Udh?from Pseudomonas putida led to a glucaric acid production of28.28±3.15 mg?L^-1.To further enhance the production,genes in myo-inositol biosynthesis pathway,including INO1,INM1 and INM2,were overexpressed respectively.Upregulation myo-inositol-1-phosphate synthase?INO1?resulted in a production of 107.51±10.87 mg?L^-1with 2.8-fold increasement.While no significant differences were detected after overexpression of INM1 and INM2.No increased GA production was achieved when continuing enhancing the expression of INO1,indicating a moderate level overexpression of INO1 can enhance the GA production.?2?Downregulating the competing pathway by knocking out PFK1/PFK2.Glucose-6-phosphate was tuned into inositol phosphate pathway by traditional gene knock-out strategy.The PFK1-deleted and PFK2-deleted strains were found to produce GA 230.22±10.75 mg?L^-1and 178.99±9.21 mg?L^-1,respectively,with no influence on the growth of recombinant strains,indicating an increased GA production was achieved by downregulating the glycolytic pathway.Also,?35?PFK1 mutation's higher titer level indicated that knocking out of PFK1 played a better role in improving the GA production.?3?GA production was further improved by MIOX mutation.To further increase GA production,it was necessary to improve MIOX activity as it was rate limiting in GA biosynthetic pathway.A high throughput screening strategy was employed to identify MIOX mutants with higher activities.Mutated enzymes K59V/R60A and R171S both displayed higher enzyme activities compared with that of wild-type enzyme.After introducing the mutated enzymes K59V/R60A and R171S into the recombinant strain,GA production was further improved to 254.63±12.44 mg?L^-1 and 300±9.82 mg?L^-1,respectively.?4?Optimization of cultivation conditions in shake flasks and fed-batch production in a3 L fermenter.The results showed that the best initial concentration of glucose was 40 g·L^-1.The optimum initial pH,liquid volume and the optimum inoculation amount were 7.0,50 m L and 15%,respectively.After optimization,the glucaric acid production was improved to360.48±10.20 mg·L^-1 in shake flasks.The optimized conditions were used for fed-batch production.After 108 h of fermentation,the production of glucaric acid reached758.91±5.43 mg·L^-1.