热带假丝酵母1230细胞色素P450基因CYPA14与CYPA16的克隆和表达

细胞色素P450(CYP)是一种单加氧酶,在热带假丝酵母(Candidatropicalis)ω-氧化过程中发挥关键作用。通过对来源不同的P450基因进行同源性分析,首先克隆到热带假丝酵母1230中P450基因的部分序列,再利用基因组步行法克隆其未知序列,结果分别获得了两个P450同工酶基因CYPA14和CYPA16的完整序列。经PCR方法证实,二者在染色体上的位置相邻,其读码框分别编码522和540个氨基酸残基的肽链。经NCBIBLAST搜索比较后发现,二者与热带假丝酵母ATCC20336中的P450成员CYP52A14和CYP52A16分别编码的序列几乎完全一致,与热带假丝酵母ATCC750中的P450成员CYP52A2和CYP52A1也具有较高的相似性。同时,对经诱变后的几株二元酸生产菌株的CYPA14与CYPA16也进行了克隆和序列比较,发现部分序列中的个别氨基酸残基发生了突变。CYPA14和CYPA16均在酿酒酵母中获得了有效表达,其中CYPA16的P450表达含量高于CYPA14,后者有部分表达产物发生了变性。

Cloning and expression of P450 genes CYPA14 and CYPA16 from Candida tropicalis 1230

Cytochrome P450 (CYP) as a monooxygenase catalyzes the limiting-rate reaction in the oxidation pathway of Candida tropicalis (C. tropicalis). Two members of P450 gene CYPA16 and CYPA14 were isolated from C. tropicalis 1230 by cassette PCR method, based on cloning of the P450 partial gene. They are tandemly arranged on the chromosome, encoding the gene products of 522 and 540 amino acid residues respectively. The deduced amino acid sequences of CYPA14 and CYPA16 are identical to those of CYP52A14 and CYP52A16 respectively,and highly similar to those of CYP52A2 and CYP52A1 from C. tropicalis ATCC 750 respectively. Simultaneously, CYPA14 and CYPA16 from several dicarboxylic-acid-producing strains were also cloned, and some site-mutations were found in their sequence. CYPA14 and CYPA16 were expressed in the Saccharomyces cerevisiae strain. It was shown that the recombinant P450 content of CYPA14 was lower than that of CYPA16, and part of them were denatured. Comparing to C. tropicalis 1230, no significant changes were observed in recombinant P450 contents of CYPA14 and CYPA16 from every dicarboxylic-acid-producing strain.