Cloning and characterization of the peroxisomal acyl CoA oxidaseACO3 gene from the alkane-utilizing yeastYarrowia 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. © 1998 John Wiley & Sons, Ltd.

Optimization of Yarrowia lipolytica’s β-oxidation pathway for γ-decalactone production

“…Using mutant strains disrupted for one or several pox genes enabled us to evaluate the activity of the different enzymes. Aox1 exhibits no detectable activity in the conditions we tested, Aox2 and Aox3 are longand short-chain specific, respectively and Aox4 and Aox5 exhibit a weak activity on a wide range of substrates [8]. We have observed in previous works the important role of the acyl-CoA oxidase in the pathway, Aox3 (short-chain specific) being responsible for continuation of oxidation after the C10 level [9] and for lactone reconsumption [10].…”

“…This shows that the metabolic pathway of the modified strain is altered by the disruption of several Aox encoding genes even if, in a previous study [8] we have shown that Aox2 (the remaining and long-chain specific enzyme) was very active. The low Aox activity in the mutant strain could result from the lack of several Aox important in the enzyme structure formation.…”

“…The strain used in this study is Y. lipolytica W29 MatA (ATCC 20460, CLIB89) or its derived mutant disrupted for genes coding for acyl-CoA oxidases (POX) [8]: strain MTLY35, MatA pox2∆ pox3∆ pox5∆ (disrupted for the genes coding for Aox2, Aox3 and Aox5); strain JMY185, MatA pox2∆ pox3∆ pox5∆ POX2 amplified, corresponds to an Ura + transformant of strain MTLY35 with the plasmid JMP3POX2. The plasmid JMP3POX2 was constructed by insertion of the POX2 gene into the defective vector JMP3 (this vector contained the ura3d4 defective allele which allows multicopy integration of the plasmid into the genome upon Ura selection) [11].…”

Genetic engineering of the β-oxidation pathway in the yeast Yarrowia lipolytica to increase the production of aroma compounds

“…The strain used in this study is Y. lipolytica W29 MatA (ATCC 20460, CLIB89) or its derived mutant disrupted for genes coding for acyl-CoA oxidases (POX) [8]. To introduce the uracil auxotrophy into the mutant strain MTL37, the ura3-41 allele from the H222 ura3-41 strain [15] was introduced by transformation and selection of Ura− cells on YNB-5FOA medium as described previously [16] giving rise to strain MTLY40.…”

“…1) [1]. Y. lipolytica possesses a five-member family of acyl-CoA oxidases (Aox1p to 5 encoded by POX1 to 5), the enzymes catalysing the first step of ␤-oxidation, some of which are long-chain specific (Aox2p) [8] or short-chain specific (Aox3p) [9,10], the specificity resulting from only a small number of amino-acids (see Mlíčková et al in this volume, [11]). The short-chain spe-cific enzyme is involved in the degradation of the lactone ( Fig.…”