Research And Application Of Integrative Expression Vectors For Osmoregulation In Candida Glycerinogenes

Candida glycerinogenes, an osmotolerant yeast with the highest glycerol productivity, provides an attractive eukaryotic expression platform. Compared with many other yeast strains, C. glycerinogenes has a high tolerance to glucose, high glycerol yield and high growth rate. Despite its industrial importance, the potential of C. glycerinogenes as a host for heterologous gene expression has not been considered due to the lack of an efficient genetic transformation system. These potential applications require efficient genetic engineering strategy to increase the production capability of target compounds in C. glycerinogenes. The vectors described in this study offer a convenient tool for creating a range of recombinant yeast strains based on various species and enable a comparative assessment of such yeasts for osmoregulated by high osmotic pressure to convert xylose. In this study, we do the following work in the target of developing a conversion system used for C. glycerinogenes:(1) To obtain the promoter for integrative vector, a genomic fragment from C. glycerinogenes containing the CgGAP gene encoding a glyceraldehyde-3-phosphate dehydrogenase homologous to GAP genes in other yeasts using degenerate primers was cloned and characterized with inverse PCR. Six stress response elements were found in the upstream region of the CgGAP gene. Promoter studies in S. cerevisiae using green fluorescent protein(gfp) gene as a reporter showed that the GAP promoter is osmoregulated by high osmotic pressure in S. cerevisiae cel s grown on glucose.(2) C. glycerinogenes 5.8S rRNA was cloned as integrative site for the integrative vectors, the integrative vectors pURGAP-gfp and pURGPD-gfp harbouring phleomycinresistance coding sequence with 2A peptides and GFP coding sequence wit h CgGAP, CgGPD promoter, respectively, were constructed. These vectors can be integrated into the 5.8S rRNA site of C. glycerinogenes WL2002-5. The vectors described in this study offer a convenient tool for creating a range of recombinant yeast strains based on various species and enable a comparative assessment of such yeasts for identification of the optimal host.(3) The regeneration of C. glycerinogenes protoplasts is a major step following genetic manipulations. An investigation of protoplast formation and cytology was made to gain further insight into the loss of protoplast viability in osmotically stabilized support media. Protoplasts with the highest regeneration frequency was isolated, using lysozyme dissolved in 1 mol?L-1 sorbitol osmoticum. The commercial enzyme preparations, osmotic stabilisers, and microorganism age were effective in raising the protoplast yield. Regeneration of colonies from protoplasts was maximal(15%) when protoplasts were supplemented with 1 mol?L-1 sorbitol as osmotic stabilizer. An improved regeneration medium and protocol which supported higher and more consistent levels of regeneration of C. glycerinogenes protoplasts resulted from these experiments.(4) The type of promoter used in the initiating vector affects the GFP gene expression and decides the role in the regulation of GFP expression. The recombinant plasmid pURPp GAP-gfp with the promoter PpGAP based on the sequence of Pichia pastoris GAP gene and the plasmid p URSc GAP-gfp with the promoter ScGAP from Saccharomyces cerevisiae were constructed. Expressions of gfp at different levels were conducted using different promoters by osmotic stress containing NaC l, glucose, KCl, sorbitol or glycerol for the recombinant strains. The results showed that gfp was functionally expressed under the control of CgGAP promoter in C. glycerinogenes. The green fluorescence was less intense at low osmotic stress, but was much more intense when the osmotic stress increased by NaC l and glucose. The expression efficiency differed when the recombinant strain was under varying osmotic stress, but CgGAP promoter was induced remarkably by high osmotic stress. The integrative expression system constructed in this study was not only regulated by osmotic pressure but also by different osmotic pressure inducing agents(carbon source as glucose, NaCl and other salts or other organic matter as glycerol), which will provide a convenient practical application compared with widely used single chemical inducer expression system.(5) To verify the recombinant vectors constructed was suit for C. glycerinogenes, we compared D-xylose utilization in C. glycerinogenes transformants in D-xylose metabolism. Compared to C. glycerinogenes strain, glycerol production from xylose was increased by 103.2%. C. glycerinogenes expressing XYL1 from Schefferomyces stipitis can produce xylitol. Xylitol production by the recombinant strains was evaluated using a xylitol fermentation medium with glucose and NaC l as osmoregulater. The recombinant strains could produce 66.1 g?L-1 xylitol. The XYL1-overexpressed mutant produced xylitol from D-xylose using glycerol as a co-substrate for cell growth and NAD(P)H regeneration: 100 g?L-1 D-xylose was completely converted into xylitol when at least 20 g?L-1 glycerol was used as a co-substrate. XYL1 overexpressed mutant grown on glycose as co-substrate accumulated xylitol increased by 62.3% from xylose. There results indicated that the molecular manipulation system succeeded in improving the ability of utilization of pentose-xylose for industrial yeast. This will offer a convenient tool for creating a range of recombinant yeast strains and enable a comparative assessment of such yeasts for identification of the optimal host.