Cloning and characterization of an n-alkane-inducible cytochrome P450 gene essential for n-decane assimilation by Yarrowia lipolytica.
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.
Iida
T
T
Department of Biotechnology, University of Tokyo, Japan.
Ohta
A
A
Takagi
M
M
D000473:Alkanes / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D018274:Electroporation; D004790:Enzyme Induction; D017353:Gene Deletion; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D004718:Saccharomycetales / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D016415:Sequence Alignment
D000473:Alkanes; D012333:RNA, Messenger; C024713:n-tetradecane; D003577:Cytochrome P-450 Enzyme System; C012867:decane