产甘油假丝酵母TRP1基因的克隆与功能分析

Cloning and Functional Analysis of the Candida glycerinogenes TRP1 Gene

摘要:

产甘油假丝酵母(Candida glycerinogenes) WL2002-5 是我国发酵甘油生产菌种, 具有高产甘油和耐高渗透压的优良性能。本文采用遗传互补的方法从产甘油假丝酵母基因文库中克隆了TRP1基因(CgTRP1)。序列分析显示, 该基因编码区全长735 bp, 编码的磷酸核糖氨基苯甲酸同分异构酶(CgPRAI)氨基酸序列与其他酵母来源的PRAI蛋白同源性在32.9%~49.2%之间。功能互补实验显示, CgTRP1基因在高拷贝情况下可以互补酿酒酵母trp1基因功能但在低拷贝情况下只能部分互补酿酒酵母trp1基因功能, 是一条功能明确、结构完整的酵母新基因。在CgTRP1 基因下游发现另一蛋白编码基因, 编码的氨基酸序列与酵母无机焦磷酸酶有很高的相似性。

Abstract:

Candida glycerinogenes WL2002-5 is an osmotolerant yeast used for the commercial production of glycerol. TRP1 gene of Candida glycerinogenes (CgTRP1), encoding phosphoribosyl anthranilate isomerase(PRA1) was cloned by complementation of the trp1 mutation of Saccharomyces cerevisiae W303-1A. Sequence analysis revealed the presence of a 735 bp open reading frame(ORF) encoding 244 amino acids protein, which shares 32.9 %~49.2% amino acids sequence similarity to PRAI proteins from other species of Saccharomycetales. Functional analysis reveal, high copy number of CgTRP1 can complement the trp1 mu-tation of S. cerevisiae completely, but low copy number of CgTRP1 can only partially complement the trp1 mutation. Downstream from CgTRP1 was found a gene highly similar to IPP1 gene which encoding inor-ganic pyrophosphatase. The nucleotides have been deposited in the GenBank database under accession No. EU078909.