Metabolic Engineering And Fermentation Optimization For Improved Production Of Glucaric Acid In Saccharomyces Cerevisiae

Glucaric acid is an important naturally occurring dibasic acid.It has high practical application value in the fields of medical care and chemical industry,so it is regarded as "top value-added chemicals from biomass".The traditional industrial production of glucaric acid mainly uses chemical oxidation method.However,due to its low yield,high cost and environmental pollution disadvantages of this method,synthetic biological method of glucaric acid production has been attracted more attention in recent years.The glucaric acid producing recombinant strain Bga-3（integrating the exogenous genes miox and udh into the delta locus of BY474 opi1 ? strain）,which was constructed by our laboratory in the previously,can produce glucaric acid with a maximum yield of 6.01 g·L^-1 from glucose and inositol as carbon sources.In this study,the expression and activity of key enzymes in the glucaric acid synthesis pathway,and the transport efficiency of inositol in yeast cells are all very low,which all limit the production of glucaric acid.To solve these problems,this research is carried out mainly in the following three aspects:（1）Exploring the effect of magnesium ion on the growth and glucaric acid synthesis through fermentation optimization.In the present study,we found that MgCl₂ could increase the glucaric acid production and the cell growth rate of the Bga-3 strain harboring the glucaric acid synthesis pathway.Finally,10.6 g·L^-1 glucaric acid was achieved through fed-batch fermentation in a 5 L bioreactor with 100 m M MgCl₂.In addition,in fed-batch fermentation with 100 m M MgCl₂,about 5.4 g·L^-1 of inositol was transformed into cells,which was 1.54g·L^-1 more than the 3.6 g·L^-1 inositol reported in our previous study.（2）In order to investigate the underlying mechanisms associated with the increased glucaric acid production by magnesium ion,we performed RNA-Seq analysis during the glucaric acid production by the engineered yeast strain,Bga-3.From this genome-wide transcriptional analysis,we found that most of genes involved in the TCA cycle,gluconeogenesis and inositol synthesis pathway were all upregulated.Further more,we confirmed that overexpression of inm1 and pck1 genes could promote the glucaric acid productivity.In addition,we found that the activity of Miox a key enzyme in the glucaric acid synthesis pathway,could also be increased by MgCl₂ and thus to promote the biosynthesis of glucaric acid.（3）Metabolic modification of key nodes in gluconic acid synthesis pathway to accumulate glucaric acid efficiently.The following three strategies are used to increase the production of glucaric acid: the over-expression of inositol transporter Itr1,fusion expression of inositol oxygenase and glucuronate dehydrogenase,and weakening expression of glucose 6-phosphate dehydrogenase gene zwf1.The results showed that over-expression of the inositol transporter Itr1 increased the production of glucaric acid by 26% compared with the starting strain Bga-3under shake flask fermentation conditions.The expression of Miox-Udh fusion protein increased the production of glucaric acid by 40% compared with the Bga-3 strain.On this basis,after weakening the expression of the glucose 6-phosphate dehydrogenase gene zwf1,the yield of glucaric acid reached 5.5 g·L^-1,which was 60% higher than that of the Bga-3strain under the same fermentation conditions.In a 5 L fermentor,the highest yield of glucaric acid by this strain is 10.85 g·L^-1,which is 80% higher than that of the Bga-3 strain in the fermentor.It can be concluded that the application of the above-mentioned metabolic modification strategy has significantly improved the efficiency and yield of glucaric acid pathways,and provided an important theoretical basis for the study of the synthesis of other compounds in Saccharomyces cerevisiae through metabolic engineering methods.