Yang Fan, Zhang Sufang, Tang Wei, Zhao Zongbao K
Division of Biotechnology, Dalian Institute of Chemical Physics, CAS, Dalian 116023, People's Republic of China.
Yeast. 2008 Sep;25(9):623-30. doi: 10.1002/yea.1607.
Oleaginous yeast Rhodosporidium toruloides is an excellent microbial lipid producer of great industrial potential, yet there is no effective genetic tool for rationally engineering this microorganism. To develop a marker recycling system, the orotidine-5'-monophosphate (OMP) decarboxylase gene of R. toruloides (RtURA3) was isolated using methods of degenerate polymerase chain reaction (PCR) together with rapid amplification of cDNA ends. The results showed that RtURA3 contains four extrons and three introns, and that the encoded polypeptide holds a sequence of 279 amino acid residues with significant homology to those of OMP decarboxylases from other yeasts. A shuttle vector pYES2/CT-RtURA3 was constructed via site-specific insertion of RtURA3 into the commercial vector pYES2/CT. Transformation of the shuttle vector into Saccharomyces cerevisiae BY4741, a URA3-deficient yeast strain, ensured the viability of the strain on synthetic dextrose agar plate without uracil, suggesting that the isolated RtURA3 was functionally equivalent to the URA3 gene from S. cerevisiae.
产油酵母红冬孢酵母是一种具有巨大工业潜力的优秀微生物脂质生产者，但目前尚无有效的遗传工具对该微生物进行合理改造。为了开发一种标记回收系统，利用简并聚合酶链反应（PCR）方法并结合cDNA末端快速扩增技术，分离了红冬孢酵母的乳清苷-5'-单磷酸（OMP）脱羧酶基因（RtURA3）。结果表明，RtURA3包含四个外显子和三个内含子，其编码的多肽含有279个氨基酸残基序列，与其他酵母的OMP脱羧酶具有显著同源性。通过将RtURA3位点特异性插入商业载体pYES2/CT，构建了穿梭载体pYES2/CT-RtURA3。将该穿梭载体转化到URA3缺陷型酵母菌株酿酒酵母BY4741中，确保了该菌株在不含尿嘧啶的合成葡萄糖琼脂平板上的活力，这表明分离得到的RtURA3在功能上等同于酿酒酵母的URA3基因。
