Metabolic Engineering Of Saccharomyces Cerevisiae For Producing Malate Through Oxaloacetate Reductive Pathway

Four-carbon dicarboxylic acids are important food and beverage additives.Furthermore,to be the important chemical building blocks,they are precursors of many significant chemical compounds such as butanediol,tetrahydrofuran,butyl ketone and so on.Producing these compounds by microbiological fermentation had good specificity with mild production conditions and low environment pollutions.The rapid development of synthetic biology and fermentation technology offered this production manner good prospects.Saccharomyces cerevisiae is a good candidate for large-scale four-carbon dicarboxylic acids production because of its robustness,low pH tolerance,and quick use of glucose.Although S.cerevisiae prefer to product ethanol in glucose fermentation,it can produce four-carbon dicarboxylic acids though TCA cycle and glyoxylate pathway,et al.In this thesis,a pathway was established in S.cerevisiae to produce malate,which is one of the four-carbon dicarboxylic acids,via phosphoenolpyruvate(PEP)and oxaloacetic acid.We enhanced the production and efflux of malate by overexpressed gene cMDH3,which encoding the malate dehydrogenase that relocated in cytoplasm,and four-carbon dicarboxylic acid transporter gene SpMAE1.Then we increased the metabolic flux from PEP to malate by overexpression genes ppc(cloned from Escherichia coli,encoding the phosphoenolpyruvate carboxylase),pck(cloned from E.coli,encoding the phosphoenolpyruvate carboxykinase),PCK1(cloned from S.cerevisiae,encoding the phosphoenolpyruvate carboxykinase)and its mutants PCK1E23K.These genetic operations significantly improved the malate synthesis ability of strain.In the batch fermentation of 2%glucose,the total succinate and malate improved from 0.22g/L to 0.60g/L(malate 0.24g/L,succinate 0.36g/L)in 48 h,and reach to 1.07g/L(malate 0.43g/L,succinate 0.64g/L)in 72h.Basing on that,we further weakened pyruvate kinase(Pykp)activity from 7.13 U/mg protein to 0.55 U/mg by removing upstream activator sequences of PYK1 promoter and replacing Pykp by low activity mutation.The result engineering strain YMAT23 produced 0.31 g/L malate at 18h in 2%glucose fermentation.The optimiztion of feed batch fermentation will be done in the subsequent work.