Identification and Characterization of the CYP52 Family of Candida tropicalis ATCC 20336, Important for the Conversion of Fatty Acids and Alkanes to α,ω-Dicarboxylic Acids

Abstract

Candida tropicalis ATCC 20336 excretes ␣,-dicarboxylic acids as a by-product when cultured on n-alkanes or fatty acids as the carbon source. Previously, a ␤-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 -hydroxylase enzymes catalyzing the first step in the -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 -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 ␣,-dicarboxylic acid production by C. tropicalis and provide the foundation for biocatalyst improvement.␣,-Dicarboxylic acids (␣,-diacids) are versatile chemical intermediates useful as raw materials for the preparation of perfumes, polymers, adhesives, and macrolide antibiotics. Currently, only three diacids, nonanedioic (azelaic), decanedioic (sebacic), and dodecanedioic, are available at a quantity and cost acceptable for commercial applications. Long-chain diacids with more than 12 carbons offer potential advantages over shorter-chain diacids, but their limited commercial availability and high price have prevented widespread growth in many of these applications.Several species of yeasts belonging to the genus Candida excrete ␣,-diacids as a by-product when cultured on n-alkanes or fatty acids as the carbon source (22). One such yeast, Candida tropicalis ATCC 20336, is the subject of this paper.In Candida spp., n-alkanes and fatty acids are metabolized by enzymes present in the ␤-oxidation and -oxidation pathways. For alkanes, the first reaction occurs in the -oxidation pathway with the formation of the corresponding alcohol. This reaction is catalyzed by a cytochrome P450 hydroxylase complex, which consists of a cytochrome P450 monooxygenase (the CYP protein) and the accompanying NADPH cytochrome P450 reductase (NCP). A terminal carboxy function is ultimately formed from two additional oxidation steps, catalyzed by a fatty alcohol oxidase and a fatty aldehyde dehydrogenase. Fatty acid fo...

Functional expression and characterization of cytochrome P450 52A21 from Candida albicans

“…CYP52 enzymes have been found in soil yeasts such as Candida tropicalis and Candida maltosa. These P450 enzymes are commonly responsible for the first and rate-limiting step in the ω-oxidation of n-alkanes and/or fatty acids [10][11][12][13]. The CYP52 from C. albicans was previously identified and was shown in vivo to possibly play a role in multidrug resistance, although it was not involved in azole drug resistance [14].…”

CYP153A6, a Soluble P450 Oxygenase Catalyzing Terminal-Alkane Hydroxylation

“…They contain enzyme systems like the diiron-and copper-containing methane monooxygenases found in many methanotrophs, such as Methylococcus capsulatus (Bath) (24), integral membrane-bound diiron AlkB proteins present in many ␣, ␤, and ␥-proteobacteria and high-GϩC-content gram-positive bacteria (40), and membranebound heme-containing cytochrome P450 enzymes (CYP) found in many alkane-degrading yeasts and fungi (3,9,36).…”

Combinatorial Engineering of Yarrowia lipolytica as a Promising Cell Biorefinery Platform for the de novo Production of Multi-Purpose Long Chain Dicarboxylic Acids

“…A whole cell biocatalyst is a cheaper option than the isolated or immobilized enzyme [106]. The pathogenic yeast, C. tropicalis, has been commonly engineered for whole cell biotransformation of aliphatic feedstock to LCDCAs [14,25]. However, Y. lipolytica has safe status, and has been categorized as a GRAS microorganism by the FDA (Food and Drug Administration) [56,88].…”

“…Despite some chemical based-production of LCDCA at large scale, the high price of the LCDCAs, and their limited commercial availability are still major constraints in expanding the spectrum of their application [25]. Therefore, researchers should use various renewable resources for the sustainable production of these multi-purpose building blocks.…”