The Key Laboratory of Industrial Biotechnology, Ministry of Education, Research Centre of Industrial Microorganisms, School of Biotechnology, Jiangnan University, Wuxi 214122, China.
Curr Microbiol. 2012 Apr;64(4):357-64. doi: 10.1007/s00284-011-0075-2. Epub 2012 Jan 12.
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.
产甘油假丝酵母 WL2002-5 已被用于工业规模的甘油发酵，由于其耐受高渗透压和快速生长的能力，可能成为一种有前途的遗传宿主。它抵抗多种药物，如 G418/潮霉素/环己酰亚胺。在以前的研究中，只有 Zeocin 被用作耐药标记物。但是 Zeocin 非常昂贵，这在很大程度上限制了遗传和分子研究。在这里，我们基于 pGAPZB 构建了一个真核整合载体 pGAPZU，以获得该菌株的新的铜抗性选择标记物。结果表明，酿酒酵母的 CUP1 基因提高了产甘油假丝酵母的铜抗性。通过将 CUP1 基因引入质粒 pGAPZU 获得的重组载体 pGUC 转化的 C. glycerinogenes 能够抵抗 21 mM 的铜，而在固体 YEPD 培养基中野生型的最小抑菌浓度 (MIC) 为 18 mM。以铜抗性作为选择标记物，研究成本从使用 Zeocin 作为选择标记物时的 114.0 美元/升大幅降低至 0.1 美元/升。新的表达载体 pGUC 和铜抗性基因的选择标记为进一步研究该工业菌株奠定了良好的基础。
