Cloning, Expression And Application Of Key Enzyme Genes For Glycerol Synthesis From Candida Glycerinogenes And Saccharomyces Cerevisiae

Glycerol, not only the important biological function material, but also the industrialalcohol compounds for three-carbon platform, had wide applications in defense, medicine,food and other industries. In recent years, glycerol as a raw material compounds has beenpaid more attention to the production of high value-added, such as1,3-propanediol,3-hydroxy acid, epoxy chloropropane, polyhydroxyalkanoate etc.. Among which,1,3-propanediol is worldwidkly recognized as the12most important industrial materials. It is aninternational hotspot that1,3-propanediol has the potential to be polymerized withterephthalic acid to form a new type of polyester material polytrimethylene terephthalate(PTT). This study is based on the Candida glycerinogenes stored by jiangnan University andcloned glycerol synthesis key enzyme gene, expressed the genes in different hosts. Then theglycerol synthesis pathway was converted to explore the application in1,3-propanediolbiosynthesis in Klebsiella pneumoniae.This research cloned the CgGPD gene encoding glycerol3-phosphate dehydrogenasefrom C.glycerinogenes and ScGPP2gene encoding glycerol3-phosphatase fromSaccharomyces cerevisiae to build a glycerol synthesis pathway. On the basis ofbioinformatic analysis, built the plasmids of pEtac-CgGPD，pEtac-ScGPP2and pEtac-CgGPD-tac-ScGPP2.To investigate the glycerol pathway, the plasmids were transformed into E.coli JM109.To determine the effects of expression of CgGPD and ScGPP2in E.coli, the SDS-PAGE andenzyme activity were analyzed. The results confirmed that the glycerol synthesis pathwaywas successfully introduced in E.coli.On the basis of above research, the glycerol synthesis pathway was constructed in the1,3-propanediol production host K. pneumoniae. SDS-PAGE and enzyme activities analysisconfirmed that recombinant K. pneumoniae possessed a metabolic pathway of glucose to1,3-PDO, and could be utilized to produce1,3-PDO from glucose instead of glycerol.In the fermentration process, the results confirmed that the certain glycerol accumulationcould activate1,3-PDO synthesis pathway. Moreover, adding the amount of glycerol couldquick start the1,3-PDO synthesis pathway which could shorten the fermentation cycle and save costing. These results have important implications for further studies involving use ofone strain for bioconversion of glucose to1,3-PDO.Analyzing the carbon flow metabolism of the recombinant and wild strain confirmedthat the introduction of glycerol synthesis pathway built a bridge between glucose andglycerol, which could successfully convert glucose to1,3-PDO in K. pneumoniae. From theresults, a potential process route to product1,3-PDO was provided by the insertion ofglycerol synthesis pathway in K. pneumoniae.