A Consensus Sequence for Long-chain Fatty-acid Alcohol Oxidases from Candida Identifies a Family of Genes Involved in Lipid ω-Oxidation in Yeast with Homologues in Plants and Bacteria

Abstract

The yeast Candida cloacae is capable of growing on alkanes and fatty acids as sole carbon sources. Transfer of cultures from a glucose medium to one containing oleic acid induced seven proteins of M r 102,000, 73,000, 61,000, 54,000, and 46,000 and two in the region of M r 45,000 and repressed a protein of M r 64,000. The induction of the M r 73,000 protein reached a 7-fold maximum 24 h after induction. The protein was confirmed by its enzyme activity to be a long-chain fatty-acid alcohol oxidase (LC-FAO) and purified to homogeneity from microsomes by a rapid procedure involving hydrophobic chromatography. An internal peptide of 30 amino acids was sequenced. A 1100-base pair cDNA fragment containing the LC-FAO peptide coding sequence was used to isolate a single exon genomic clone containing the full-length coding sequence of an LC-FAO (fao1). The fao1 gene product was expressed in Escherichia coli and was translated as a functional long-chain alcohol oxidase, which was present in the membrane fraction. In addition, full-length coding sequences for a Candida tropicalis LC-FAO (faoT) and a second C. cloacae LC-FAO (fao2) were isolated. The DNA sequences obtained had open reading frames of 2094 (fao1), 2091 (fao2), and 2112 (faoT) base pairs. The derived amino acid sequences of fao2 and faoT showed 89.4 and 76.2% similarities to fao1. The fao1 gene is much more highly induced on alkane than is fao2. Although this study describes the first known DNA sequences encoding LC-FAOs from any source, there are unassigned Arabidopsis sequences and an unassigned Mycobacterium sequence in the GenBank TM Data Bank that show strong homology to the described LC-FAO sequences. The conservation of sequence between yeast, plants, and bacteria suggests that an as yet undescribed family of long-chain fattyacid oxidases exists in both eukaryotes and prokaryotes.

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

“…The inner pH of the cell, as well as aeration, both affect the induction of yeast CYP monooxygenase and CYP reductase [10]. Studies show that a higher dissolved oxygen (DO) level can remarkably improve P450 activity [99,100]. This level was kept at 80% saturation for LCDCA production from C. ropiclais [14].…”

Molecular Characterization of the Fatty Alcohol Oxidation Pathway for Wax-Ester Mobilization in Germinated Jojoba Seeds

“…Both ScFAO and ScFADH contain predicted membrane-spanning domains, and CcFAO1 and HsALDH3A2 are known to be membrane associated (Kelson et al, 1997;Vanhanen et al, 2000). Furthermore, ScFAO and ScFADH also contain putative di-Lys motif ER retention signals at their C termini (Supplemental Figs.…”

“…The deduced ScFAO protein has a predicted mass of 77 kD and, based on ClustalW2 amino acid sequence alignment (Larkin et al, 2007), is approximately 24% identical to C. cloacae FAO1 (Vanhanen et al, 2000) and approximately 52% identical to Arabidopsis FAO3 (At3g23410; Cheng et al, 2004;Supplemental Fig. S1).…”

“…A TBLASTN search of this database identified several ESTs with similar sequences to alcohol oxidases and aldehyde dehydrogenases. However, two ESTs were selected for further analysis based on their greater similarity to the well-characterized archetypal FAO1 and ALDH3A2 proteins from C. cloacae and humans, respectively (De Laurenzi et al, 1996;Vanhanen et al, 2000). Gene-specific primers were designed against these EST sequences and were used to obtain full-length complementary DNAs (cDNAs) by 39-and 59-RACE.…”

“…For instance, FAO was first cloned in the yeast Candida cloacae (Vanhanen et al, 2000), and functional homologs have since been characterized in Arabidopsis and Lotus japonicus (Cheng et al, 2004;Zhao et al, 2008), although their precise physiological role(s) remains to be determined. FADH has been studied extensively in humans (Homo sapiens), where mutations in HsALDH3A2 are causal for the neurological disorder Sjögren-Larsson syndrome (De Laurenzi et al, 1996).…”