The Construction Study Of Integrative Vectors For Candida Glycerinogenes WL2002-5

Candida glycerinogenes WL2002-5, originally isolated from nature by our lab, is widely used in industrial production of glycerol. Compared with many other yeast strains, it has several advantages for industrial applications, such as tolerance to high concentrations of glucose, high growth rate and high glycerol productivity. This strain may be a promising host to produce 3-hydroxypropioic, 1,3-propanediol and other compounds. However, few molecular studies on this strain were done and there are few suitable transforming vectors for this strain. In this study, we constructed several integrative vectors to better utilize this industrial strain as the basis to construct a three-carbon-compound biotransformation platform. The results are as follows:1. An universal integrative vector pGAPZU of a low molecular weight was constructed for C. glycerinogenes by inserting a 1.0 kb URA3 gene into plasmid pGAPZB. Based on the backbone of plasmid pGAPZB, pGAPZU had a 1.0 kb URA3 gene as an integration site and Zeocin-resistance as a selective marker. After electroporation transformation, Zeocin-resistance colonies were selected. The result proved that plasmid pGAPZU was introduced into host cells.2. Another integrative vector pGUGGA containing two strong GAP promoters and a green fluorescent protein (GFP) encoding gene was constructed for C. glycerinogenes, based on the backbone of plasmid pGAPZB. pGUGGA had an URA3 gene as an integration site, Zeocin-resistance as a selective marker and an independently expressed GFP encoding gene as a reporter gene. After digesting with BstXI, linearized pGUGGA was introduced into C. glycerinogenes through electroporation and then Zeocin-resistant colonies were selected. Diagnostic polymerase chain reaction (PCR) confirmed that plasmid pGUGGA was integrated into the URA3 site of yeast genome. SDS-PAGE and fluorescence microscopy experiments confirmed that GFP was functionally expressed. The results also showed that the vector transformed into this strain could be as big as 5.9 kb. pGUGGA plasmid provided a valuable way to co-express two or more target genes in C. glycerinogenes. The transformants constructed here were URA3 negative, so they could be used for molecular modifications and genetic studies for C. glycerinogenes with URA3 as a selective marker.3. A new selective drug-resistance marker was obtained. Previous studies showed that Zeocin was one of the few drugs that could inhibit wild C. glycerinogenes, but it is so expensive that its application in molecular biology was limited. After studying over 300 vectors, nine commonly used drug resistance markers were selected. The inhibitory effects of Zeocin, Cu²⁺, G418 and Hygromycin B on C. glycerinogenes were further assayed in YEPD medium. The minimum concentrations of Cu²⁺, G418, Hygromycin B, Zeocin in YEPD plates to completely inhibit the growth of wild C. glycerinogenes were 18 mmol/L, 400μg/mL, 500μg/mL and 100μg/mL, respectively. Combined drugs of G418 and Zeocin (1:1) could reduce experiment cost. The results showed that C. glycerinogenes was sensitive to Cu²⁺ and Zeocin. Cu²⁺ was a cheap selective marker for C. glycerinogenes.4. An eukaryotic integrative vector pGUC was constructed by inserting a CUP1 gene from Saccharomyces cerevisiae into plasmid pGAPZU, to gain a new selectable marker of Cu²⁺ resistance for C. glycerinogenes. After digesting with HindIII, linearized pGUC was introduced into this industrial strain through electroporation and then Zeocin resistant colonies were selected. Diagnostic PCR showed that pGUC was integrated into the URA3 gene in the host genome. The resistance of transformed and wild C. glycerinogenes to Cu²⁺ was assayed in YEPD medium. Transformed C. glycerinogenes could resist 21 mmol/L Cu²⁺ while the minimum inhibitory concentration of Cu²⁺ to the wild was 18 mmol/L in the same medium. The results showed that the CUP1 gene increased Cu²⁺ resistance of C. glycerinogenes and 21 mmol/L Cu²⁺ could be used to select Cu²⁺-resistant transformants. The new expression vector pGUC with a selective marker of Cu²⁺ resistance established a good foundation for further studies on this industrial strain at a low research cost.