Construction and characterization of a Yarrowia lipolytica mutant lacking genes encoding cytochromes P450 subfamily 52

• Published in: Fungal genetics and biology 2012-01, Vol.49 (1), p.58-64
• Main Authors: Takai, Hiroshi, Iwama, Ryo, Kobayashi, Satoshi, Horiuchi, Hiroyuki, Fukuda, Ryouichi, Ohta, Akinori
• Format: Article
• Language: English
• Subjects: alkanes; Alkanes - metabolism; cytochrome P-450; Cytochrome P-450 Enzyme System - biosynthesis; Cytochrome P-450 Enzyme System - deficiency; cytochrome P450ALK; Gene Deletion; Gene Expression Profiling; Gene Expression Regulation, Fungal; genes; hydroxylation; mutants; n-alkane; oxidation; Oxidation-Reduction; substrate specificity; transcriptional activation; Yarrowia - enzymology; Yarrowia - genetics; Yarrowia - growth & development; Yarrowia - metabolism; Yarrowia lipolytica; yeasts
• ISSN: 1087-1845; 1096-0937
• DOI: 10.1016/j.fgb.2011.11.003

► A Yarrowia lipolytica mutant deleted in 12 P450ALK genes (Δalk1-12) was constructed. ► Δalk1-12 did not grow on n-alkanes, but grew on their metabolites. ► Δalk1-12 failed to convert n-dodecane to n-dodecanoic acid. ► P450ALKs are indispensable for n-alkane oxidation. ► Transcriptional activation of ALK1 by n-alkane was independent of P450ALKs. The initial hydroxylation of n-alkane is catalyzed by cytochrome P450ALK of the CYP52 family in the n-alkane-assimilating yeast Yarrowia lipolytica. A mutant with a deletion of all 12 genes, ALK1 to ALK12, which are deduced to encode cytochromes P450 of the CYP52 family in Y. lipolytica, was successfully constructed. This deletion mutant, Δalk1-12, completely lost the ability to grow on n-alkanes of 10–16 carbons. In contrast, Δalk1-12 grew on the metabolite of n-dodecane, i.e., n-dodecanol, n-dodecanal, or n-dodecanoic acid, as well as the wild-type strain. In addition, production of n-dodecanoic acid was not observed when Δalk1-12 was incubated in the presence of n-dodecane. These results indicate the essential roles of P450ALKs in the oxidation of n-alkane. Δalk1-12 will be valuable as a host strain to express an individual ALK gene to elucidate the molecular function and substrate specificity of each P450ALK. Transcriptional activation of the ALK1 promoter by n-alkanes was observed in Δalk1-12 as in the wild-type strain, implying that n-alkanes per se, but not their metabolites, trigger n-alkane-induced transcriptional activation in Y. lipolytica.