Functional Analysis Of The CgGPD Gene Promoter Of Candida Glycerinogenes In Saccharomyces Cerevisiae

Osmotolerant yeast, Candida glycerinogenes WL2002-5, is an excellent glycerol producer and has been used for industrial scale fermentation. Because of the lack of an effective genetic manipulation tool, several attempts to study its mechanim of glycerol synthesis was not achieved. It was reported that glycerol-3-phosphate dehydrogenase was the key enzyme for glycerol synthesis in C. glycerinogenes, in order to improve the glycerol yield, understand the underlying molecular mechanisms of glycerol synthesis, 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 was only 54.15%. So studying it's molecular mechanisms would be important for explaining the reason of C. glycerinogenes possesing the property of high glycerol concentration, yield and recovery yield. At the same time, we found that the expression of CgGPD gene was induced by the osmotic stress. In order to determine whether the CgGPD promoter of C. glycerinogenes (PCggpd) was also induced by the high osmotic stress, we amplified the 950-bp promoter of CgGPD from C. glycerinogenes by PCR. It was a new promoter because of the nucleotide homology between PCggpd and the promoter of GPD1 gene from S. cerevisiae was only 28.75%.The objective of this study was cloning and functional analysis of the promoter of CgGPD gene from C. glycerinogenes. The PCggpd and the gene gfp encoding green fluorescent protein were cloned, and then two fragments were tandemly ligated into the expression vector pYX212-zeocin, the plasmid pYX212-zeocin-PCggpd-gfp was constructed, then transformed into S. cerevisiae W303-1A by electroporation. Green fluorescent of recombinant strain S. cerevisiae W303-1A-GFP was examined by using fluorescent microscopy when the recombinant strains were cultured in the YEPD medium containing different NaCl, KCl, glucose, mannitol and sorbitol concentrations, respectively. The results showed that the gene gfp was functionally expressed under the control of PCggpd in S. cerevisiae. Furthermore, the expression of the gene gfp at different level was conducted by the different osmotic stress for the recombinant strain. The green fluorescence was less intensive when the osmotic stress was low for culturing the recombinant strain, but it became much more intensive when the osmotic stress increased. The expression efficiency of PCggpd was different when the PCggpd was under different osmotic stress, the PCggpd could be induced remarkably by the high osmotic stress. This may be one of the most important reasons of C. glycerinogenes possesing the property of high glycerol concentration, yield and recovery yield. The functional analysisof PCggpd will facilitate further studies on the mechanism of glycerol synthesis from C. glycerinogenes WL2002-5 under osmotic stress conditions.In order to utilize the PCggpd in the direction of resistance breeding, expression of heterologous proteins, we cloned the gene amy encodingα-amylase from the Bacillus subtilis genomic DNA by PCR. The expression vector pYX212-zeocin harboring the PCggpd and gene amy was constructed, in which amy expression was controlled by the promoter of CgGPD. The recombinant plasmid was transformed into S.cerevisiae W303-1A by electroporation and transformants were selected. We studied the differential expression of the gene amy when the recombinant strain were cultured in YEPD medium and the NaCl-induced medium, while theα-amylase activity of recombinant strain was detected. This study will be an important basis for the application of the promoter of CgGPD gene from C.glycerinogenes.