Cloning and characterization of the peroxisomal acyl CoA oxidase ACO3 gene from the alkane‐utilizing yeast Yarrowia lipolytica

Abstract

The ACO3 gene, which encodes one of the acyl-CoA oxidase isoenzymes, was isolated from the alkane-utilizing yeast Yarrowia lipolytica as a 10 kb genomic fragment. It was sequenced and found to encode a 701-amino acid protein very similar to other ACOs, 67.5% identical to Y. lipolytica Aco1p and about 40% identical to S. cerevisiae Pox1p. Haploid strains with a disrupted allele were able to grow on fatty acids. The levels of acyl-CoA oxidase activity in the ACO3 deleted strain, in an ACO1 deleted strain and in the wild-type strain, suggested that ACO3 encodes a short chain acyl-CoA oxidase isoenzyme. This narrow substrate spectrum was confirmed by expression of Aco3p in E. coli.

Role of β-Oxidation Enzymes in γ-Decalactone Production by the Yeast Yarrowia lipolytica

“…We used Y. lipolytica W29 (ATCC 20460) or derived mutants that were disrupted in genes coding for one or more Aox (POX) ( Table 1) (23,24).…”

Catabolism of hydroxyacids and biotechnological production of lactones by Yarrowia lipolytica

“…Interest in this enzyme family came from our first experiments: disruption of pox1 resulted in an increased b-oxidation activity but a decreased production of gdecalactone, whereas overexpression of the thiolase did not modify the production . Since then, the Aox family has been better characterised, using functional genetics Wang et al 1998;1999a, 1999b. This enabled us to show that two Aox exhibited a high activity and a chain-length specificity, one being long-chain-specific (Aox2) and the other short-chain-specific (Aox3).…”

Involvement of Acyl Coenzyme A Oxidase Isozymes in Biotransformation of Methyl Ricinoleate into γ-Decalactone by Yarrowia lipolytica

“…Their constructions have been described elsewhere (21,22). All strains were cultured for 48 h on malt extract agar (Difco) at 27°C and used to inoculate a 500-ml baffled Erlenmeyer flask containing 200 ml of glucose medium (15 g of glucose/liter, 2.5 g of ammonium chloride/liter, 0.1 g of yeast extract/liter, 2.1 g of monopotassium phosphate/liter, 4.51 g of disodium phosphate/liter, 0.2 g of magnesium sulfate/liter, 0.1 g of sodium chloride/liter, 9.14 mg of iron (II) sulfate heptahydrate/liter, 0.5 mg of zinc chloride/liter, 1.56 mg of copper sulfate/liter) to an optical density at 600 nm (OD 600 ) of 0.25 (6 ϫ 10 6 cells/ml).…”

“…1, which represents the effect of gene disruption on the activity of the remaining enzymes in the cell extracts depending on substrate chain length (21,22). As shown in Table 1, a POX monodisruption had no significant effect on the growth characteristics of the mutant strains.…”

“…Acyl-CoA oxidase activity profiles of mutant strains as a function of substrate chain length. Activity was measured independently with C 6 -CoA, C 10 -CoA, and C 14 -CoA substrates and was standardized for protein concentration (21). The strains presented here are the wild type, ⌬pox2, ⌬pox3, ⌬pox2 ⌬pox3, and ⌬pox2 ⌬pox3 ⌬pox5.…”