Cloning and characterization of three fatty alcohol oxidase genes from Candida tropicalis strain ATCC 20336.

Candida tropicalis (ATCC 20336) converts fatty acids to long-chain dicarboxylic acids via a pathway that includes among other reactions the oxidation of omega-hydroxy fatty acids to omega-aldehydes by a fatty alcohol oxidase (FAO). Three FAO genes (one gene designated FAO1 and two putative allelic genes designated FAO2a and FAO2b), have been cloned and sequenced from this strain. A comparison of the DNA sequence homology and derived amino acid sequence homology between these three genes and previously published Candida FAO genes indicates that FAO1 and FAO2 are distinct genes. Both genes were individually cloned and expressed in Escherichia coli. The substrate specificity and K(m) values for the recombinant FAO1 and FAO2 were significantly different. Particularly striking is the fact that FAO1 oxidizes omega-hydroxy fatty acids but not 2-alkanols, whereas FAO2 oxidizes 2-alkanols but not omega-hydroxy fatty acids. Analysis of extracts of strain H5343 during growth on fatty acids indicated that only FAO1 was highly induced under these conditions. FAO2 contains one CTG codon, which codes for serine (amino acid 177) in C. tropicalis but codes for leucine in E. coli. An FAO2a construct, with a TCG codon (codes for serine in E. coli) substituted for the CTG codon, was prepared and expressed in E. coli. Neither the substrate specificity nor the K(m) values for the FAO2a variant with a serine at position 177 were radically different from those of the variant with a leucine at that position.

Identification and characterization of the CYP52 family of Candida tropicalis ATCC 20336, important for the conversion of fatty acids and alkanes to alpha,omega-dicarboxylic acids.

Candida tropicalis ATCC 20336 excretes alpha,omega-dicarboxylic acids as a by-product when cultured on n-alkanes or fatty acids as the carbon source. Previously, a beta-oxidation-blocked derivative of ATCC 20336 was constructed which showed a dramatic increase in the production of dicarboxylic acids. This paper describes the next steps in strain improvement, which were directed toward the isolation and characterization of genes encoding the omega-hydroxylase enzymes catalyzing the first step in the omega-oxidation pathway. Cytochrome P450 monooxygenase (CYP) and the accompanying NADPH cytochrome P450 reductase (NCP) constitute the hydroxylase complex responsible for the first and rate-limiting step of omega-oxidation of n-alkanes and fatty acids. 10 members of the alkane-inducible P450 gene family (CYP52) of C. tropicalis ATCC20336 as well as the accompanying NCP were cloned and sequenced. The 10 CYP genes represent four unique genes with their putative alleles and two unique genes for which no allelic variant was identified. Of the 10 genes, CYP52A13 and CYP52A14 showed the highest levels of mRNA induction, as determined by quantitative competitive reverse transcription-PCR during fermentation with pure oleic fatty acid (27-fold increase), pure octadecane (32-fold increase), and a mixed fatty acid feed, Emersol 267 (54-fold increase). The allelic pair CYP52A17 and CYP52A18 was also induced under all three conditions but to a lesser extent. Moderate induction of CYP52A12 was observed. These results identify the CYP52 and NCP genes as being involved in alpha,omega-dicarboxylic acid production by C. tropicalis and provide the foundation for biocatalyst improvement.