Co-Expression Of Glycerol 3-Phosphate Dehydrogenase And Glycerol 3-Phosphatase Genes Of Saccharomyces Cerevisiae In Escherichia Coli

A novel metabolic pathway for fermenting glucose to glycerol is constructed in E.coli, which is not a natural glycerol producer. The recombinant microorganism can produce glycerol, which could lead to a new pathway for directly converting glucose to 1,3-propanediol.First, the genes encoding glycerol 3-phosphate dehydrogenase (GPD1) and glycerol 3-phospatase (HOR2) were amplified from total DNA of Saccharomyces cerevisiae using PCR, respectively. The genes of gpd1 and hor2 were separately inserted into expression vector pSE380 and then transformed into E.coli JM109. So the recombinant plasmids pSE-gpdl and pSE-hor2 can be used to construct recombinant plasmid with two cassettes and recombinant plasmid with polycistron.Expression cassette with gpdl or hor2 was amplified from the recombinant plasmids pSE-gpd1 or pSE-hor2 using PCR. The two expression cassettes were inserted into plasmid pGEM-3zf (p) and transformed into E.coli JM109. The recombinant E.coli JM109 was incubated in Minimal media 71% glucose shake-flasks at 37℃, with vigorous shaking for 24h, after which they were sampled for HPLC of supernatant. The recombinant E.coli JM109 showed production of glycerol was 1.18g/L after 24h.Then, the gene of gpdl and the gene of hor2 with RBS were amplified from pSE-gpd1 and pSE-hor2 using PCR. Then gpdl and hor2 with RBS were inserted into expression vector pSE380. The recombinant plasmid was transformed into E.coli JM109. The recombinantE.coli JM109 was incubated in Minimal media/1% glucose shake-flasks at 37℃, with vigorous shaking for 24h, after which they were sampled for HPLC of supernatant. The recombinant E.coli JM109 showed production of glycerol was 3.79g/L. The recombinant plasmid was transformed into E.coli BL21. The recombinant E.coli BL21 was fermented in the LB/glucose media at 37℃. The maximal concentration of glycerol was 22.64g/L at 28h.In this research, a novel metabolic pathway is successfully constructed in E.coli for converting glucose to glycerol directly by introducing S. cerevisiae genes encoding GPD1 and HOR2 into E.coli. The recombinant E.coli with polycistron is superior to whose with two expression cassettes in the production of glycerol. It is reported for the first time in China.