Iida T, Ohta A, Takagi M
Department of Biotechnology, University of Tokyo, Japan.
Yeast. 1998 Nov;14(15):1387-97. doi: 10.1002/(SICI)1097-0061(199811)14:15<1387::AID-YEA333>3.0.CO;2-M.
A gene encoding cytochrome P450 involved in n-alkane utilization was cloned from an n-alkane assimilating yeast, Yarrowia lipolytica CX161-1B. The RT-PCR was performed on the mRNA prepared from the cells grown on n-alkane as a template using degenerated PCR primers designed for the conserved amino acid sequences of the CYP52 family. The RT-PCR amplified fragment was then used as a probe to isolate genes coding for P450 of the CYP52 family from the genomic DNA library of the strain CX161-1B. The nucleotide sequence of one of the positive clones was determined. An open reading frame which had the same nucleotide sequence as the RT-PCR-amplified fragment was identified. It was of 523 amino acid residues, 60.2 kDa in molecular mass, and had 30-45% sequence identity with the other members of the CYP52 family of Candida species so far analysed. The expression of the P450 gene that was named as YlALK1 was induced by n-tetradecane and repressed by glycerol. A YlALK1 gene disruptant did not grow well on n-decane, but grew on longer-chain n-alkanes such as hexadecane as a sole carbon source. Introduction of YlALK1 on a plasmid to the disruptant restored the decane assimilation. These results suggest that the YlALK1 gene product is the major P450A1k to metabolize short-chain n-alkanes such as decane and dodecane in Y. lipolytica.
从一株同化正构烷烃的解脂耶氏酵母Yarrowia lipolytica CX161-1B中克隆到一个参与正构烷烃利用的细胞色素P450编码基因。以在正构烷烃上生长的细胞制备的mRNA为模板，使用针对CYP52家族保守氨基酸序列设计的简并PCR引物进行逆转录聚合酶链反应（RT-PCR）。然后将RT-PCR扩增片段用作探针，从菌株CX161-1B的基因组DNA文库中分离编码CYP52家族P450的基因。测定了其中一个阳性克隆的核苷酸序列。鉴定出一个与RT-PCR扩增片段具有相同核苷酸序列的开放阅读框。它由523个氨基酸残基组成，分子量为60.2 kDa，与迄今为止分析的念珠菌属CYP52家族的其他成员具有30%-45%的序列同一性。命名为YlALK1的P450基因的表达受正十四烷诱导，受甘油抑制。YlALK1基因破坏株在正癸烷上生长不佳，但在较长链的正构烷烃如十六烷作为唯一碳源时能够生长。将携带YlALK1基因的质粒导入破坏株可恢复癸烷同化能力。这些结果表明，YlALK1基因产物是解脂耶氏酵母中代谢短链正构烷烃如癸烷和十二烷的主要P450A1k。
