Expression Of Key Enzymes For Glycerol Synthesis From Yeasts In Escherichia Coli

There is a metabolic pathway of glucose to glycerol waidly in Yeast. Studies of Saccharomyces cerevisiae have showed that glycerol is produced by reduction of the glycolytic intermediate dihydroxyacetone phosphate to glycerol 3-phosphate (G3P) followed by a dephosphorylation of G3P to glycerol. The first step is catalyzed by NAD-dependent glycerol 3-phosphate dehydrogenase (GPD), which is encoded by the two isogenes GPD1 and GPD2. The second step is catalyzed by Glycerol-3-phosphate (GPP), which is encoded by the two isogenes GPP1 and GPP2, while the GPD1 and GPP2 are induced at high osmolarity. Osmotolerant yeast, Candida glycerinogenes WL2002-5, is an excellent glycerol producer. We cloned and characterized a 4900-bp genomic fragment containing the CgGPD gene encoding a glycerol-3-phosphate dehydrogenase homologous to GPD genes in other yeasts using degenerate primers in conjunction with inverse PCR. It was regarded as a new gene because of the nucleotide homology between CgGPD and GPD1 gene from Saccharomyces cerevisiae is only 54.15%. There is no some reports about characterition about this enzyme.In this study, we cloned gene CgGPD1 and ScGPD1 from C. glycerinogenes and S.cerevisiae respectively. Then, we constructed the plasmid pET-28a-CgGPD1 and pET-28a-ScGPD1, transformed into Escherichia coli BL21. The CgGPD1 and ScGPD1 were expressed, purified and characterized respectively, contrasted with each other. The SDS-PAGE showed that CgGPD1 and ScGPD1 have a samiliar molecular mass. The CgGPD1 showed optimal activity at pH 6.2 and 25℃, however the CgGPD1 was 6.8 and 37℃. The CgGPD1 showed stability between pH3.5-7.5 and -20℃. The ScGPD1 showed stability between pH3.5-7.5 and -20℃. Both of CgGPD1 and ScGPD1 were activated by Mg2+ and depressed by Zn2+. The study showed that glycerol products changed by added Mg2+ and Zn2+ in C.glycerinogenes culture respectively. Km values obtained were 0.55mmol/L and 0.75mmol/L for DHAP and Vmax were 1.06umol/(mg/min) and 0.51umol/(mg/min) respectively of CgGPD1 and ScGPD1.This study we cloned gene ScGPP2 from S.cerevisiae and constructed a new strain pET28a-ScGPP2/BL21. The ScGPP2 was expressed, purified and characterized. Its molecular weight was determined to be 31KDa by SDS-PAGE. The enzyme showed optimal activity at pH 4.5 and 55℃and showed stability between pH5.5-7.5 and 4℃and -20℃. The ScGPP2 was activated by iro Fe3+ and Mg2+ , however, was inhibited strongly by Zn2+, Mn2 + and Sn2 +. The Km value was 0.55mmol/L and Vmax was 0.30μmol/(mg·min) for Glycerol-3-phosphate. The transformation ability of ScGPP2 from glycerol-3-phosphate to glycerol is proved by the stady of pET28a -ScGPP2/BL21 whole cells biocatalytic.It is reported that Escherichia coli can not transform glucose to glycerol. In this paper, we cloned gene CgGPD1 and ScGPP2 from C. glycerinogenes and S.cerevisiae respectively and constructed a new plasmid pEtac-CgGPD1-ScGPP2. Then the recombinant plasmid was translated into E.coli BL21 and transformants were selected. SDS-PAGE showed that CgGPD1 and ScGPP2 expressed obviously. The new E.coli strain was cultured in LB and 1%glucose mediun while the CgGPD1 and ScGPP2 special activity of recombinant strain were detected. The glycerol product was also detected and achieved 1.56g/L.