The Construction of a New Integrative Vector with a New Selective Marker of Copper Resistance for Glycerol Producer Candida glycerinogenes
Candida glycerinogenes WL2002-5 has been used for industrial-scale fermentation of glycerol and may be a promising genetic host due to its tolerance to high osmotic pressure and fast growth. It resists many kinds of drugs, such as G418/hygromycin/cycloheximide. In previous studies, only Zeocin was used as a drug-resistant marker. But Zeocin is so expensive that it largely limits the genetic and molecular study. Here, we constructed a eukaryotic integrative vector pGAPZU, based on pGAPZB, to gain a new selectable marker of copper resistance for this strain. The results showed that the CUP1 gene of Saccharomyces cerevisiae elevated copper resistance of C. glycerinogenes. The C. glycerinogenes transformed with recombinant vector pGUC, obtained from introducing CUP1 gene into plasmid pGAPZU, could resist 21 mM copper, while the minimum inhibitory concentration (MIC) of wild type was 18 mM in solid YEPD medium. With copper resistance as a selective marker, research cost was largely reduced from 114.0 $/L with Zeocin as selective marker to 0.1 $/L. The new expression vector pGUC and selective marker of copper resistance gene establish a good foundation for further study on this industrial strain.
KeywordsPolymerase Chain Reaction Amplification URA3 Gene Copper Resistance Autonomous Replication Sequence Polymerase Chain Reaction Experiment
This study was supported by the National High Technology Research and Development Program of China (863 Program, No. 2009AA02Z210) and the 111 Project (No. 111-2-06).
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