Evaluation of acyl coenzyme A oxidase (Aox) isozyme function in the n- alkane-assimilating yeast Yarrowia lipolytica
ABSTRACT We have identified five acyl coenzyme A (CoA) oxidase isozymes (Aox1 through Aox5) in the n -alkane-assimilating yeast Yarrowia lipolytica , encoded by the POX1 through POX5 genes. The physiological function of these oxidases has been investigated by gene disruption. Single, double, triple, and quadruple disruptants were constructed. Global Aox activity was determined as a function of time after induction and of substrate chain length. Single null mutations did not affect growth but affected the chain length preference of acyl-CoA oxidase activity, as evidenced by a chain length specificity for Aox2 and Aox3. Aox2 was shown to be a long-chain acyl-CoA oxidase and Aox3 was found to …
Involvement of acyl coenzyme A oxidase isozymes in biotransformation of methyl ricinoleate into gamma-decalactone by Yarrowia lipolytica.
ABSTRACT We reported previously on the function of acyl coenzyme A (acyl-CoA) oxidase isozymes in the yeast Yarrowia lipolytica by investigating strains disrupted in one or several acyl-CoA oxidase-encoding genes ( POX1 through POX5 ) (H. Wang et al., J. Bacteriol. 181:5140–5148, 1999). Here, these mutants were studied for lactone production. Monodisrupted strains produced similar levels of lactone as the wild-type strain (50 mg/liter) except for Δ pox3 , which produced 220 mg of γ-decalactone per liter after 24 h. The Δ pox2 Δpox3 double-disrupted strain, although slightly affected in growth, produced about 150 mg of lactone per liter, indicating that Aox2p was not essential for the biotra…
Optimization of Yarrowia lipolytica’s β-oxidation pathway for γ-decalactone production
The yeast Yarrowia lipolytica growing on methyl ricinoleate produces various lactones, γ-decalactone, the worthy aroma compound, 3-hydroxy-γ-decalactone without sensorial properties and two decenolides of various interest. Unfortunately, these three latter lactones are produced at high levels by this yeast, decreasing yields and complicating the extraction of γ-decalactone. In this study, the production of γ-decalactone was increased through a genetic engineering of the strain and the accumulation of the three other lactones was lowered. Theses results show that it is possible to improve the mastering of the complex β-oxidation pathway (the metabolic pathway involved in these bioconversions…
Effect of acyl-CoA oxidase activity on the accumulation of gamma-decalactone by the yeast Yarrowia lipolytica: a factorial approach.
International audience; beta-Oxidation is a cyclic pathway involved in the degradation of lipids. In yeast, it occurs in peroxisomes and the first step is catalyzed by an acyl-CoA oxidase (Aoxp). The yeast Yarrowia lipolytica possesses several genes (POX) coding for Aoxps. This study is based on the factorial analysis of results obtained with the many POX derivative strains that have been constructed previously. The effect of interactions between Aoxps on the acyl-CoA oxidase (Aox) activity was important even at the second order. We then investigated the effect of Aox activity on growth and lactone production. Aox activity was correlated with acidification of the medium by cells and with ce…
Predicted secondary structure of hydroperoxide lyase from green bell pepper cloned in the yeast Yarrowia lipolytica
International audience; Fatty acid hydroperoxide lyase (HPL) is a member of the cytochrome P450 family acting on fatty acid hydroperoxides in many organisms. The active green bell pepper HPL, cloned and expressed in the yeast Yarrowia lipolytica, was purified by immobilized metal-ion affinity chromatography (IMAC) in the presence of 2% of Triton X-100R. The secondary structure prediction by bioinformatics servers of HPL was realized by ANTHEPROT software, using the GOR, DPM and Predator methods. The theoretical results which are average values obtained from three different calculation methods showed 33% α-helix, 18% β-sheet, 7% turn and 42% coil. On the other hand, the secondary structure a…
Medium-size droplets of methyl ricinoleate are reduced by cell-surface activity in the gamma-decalactone production by Yarrowia lipolytica.
International audience; Size of methyl ricinoleate droplets during biotransformation into gamma-decalactone by Yarrowia lipolytica was measured in both homogenized and non-homogenized media. In non-homogenized but shaken medium, droplets had an average volume surface diameter d32 of 2.5 microm whereas it was 0.7 microm in homogenized and shaken medium. But as soon as yeast cells were inoculated, both diameters became similar at about 0.7 microm and did not vary significantly until the end of the culture. The growth of Y. lipolytica in both media was very similar except for the lag phase which was lowered in homogenized medium conditions.
Fatty acid hydroperoxide lyase of green bell pepper: cloning in Yarrowia lipolytica and biogenesis of volatile aldehydes
Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; International audience; Fatty acid hydroperoxide lyase (HPO lyase) is a cytochrome P450 acting on fatty acid hydroperoxides in many organisms. The expression of green bell pepper HPO lyase in the yeast Yarrowia lipolytica is described for the first time. HPO lyase activity from yeast extract and whole yeast cells is measured and aldehydes production from yeast extract and whole yeast cells is compared. 1200 U/L reaction medium were obtained after 96 h of culture on olive oil rich me…
Secondary structure conformation of hydroperoxide lyase from green bell pepper, cloned in Yarrowia lipolytica, and its activity in selected media
International audience; Circular dichroism (CD) spectroscopy of secondary structure conformation of the purified green bell pepper hydroperoxide lyase (HPL), cloned in the yeast Yarrowia lipolytica, was investigated. The CD spectra of HPL in iso-octane, obtained at 60 °C, in the presence of the reducing agent dithiothreitol showed dramatic increase in α-helix content. The enzyme conformation remained unchanged over a range of pH values of 5.0–7.0. Using 13-hydroperoxide of linoleic acid (13-HPOD) as substrate, the biocatalysis of HPL in organic solvent media, including chloroform, dichloromethane, hexane, iso-octane, octane and toluene, was investigated. The results indicated an increase in…
Cloning and characterization of the peroxisomal acyl CoA oxidaseACO3 gene from the alkane-utilizing yeastYarrowia lipolytica
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.
Modification génétique de la levure Yarrowia lipolytica pour maîtriser la β-oxydation
Les biotechnologies font regulierement appel a la β-oxydation pour produire des composes a haute valeur ajoutee ou depolluer des effluents industriels. Cette voie metabolique est difficile a maitriser car elle implique un nombre reduit d'enzymes pour une voie tres longue. En particulier, la production d'intermediaires reactionnels est delicate car les enzymes de la voie de reconsommation sont souvent les memes que celles impliquees dans la production. Ainsi, la production de γ-decalactone a partir de ricinoleate de methyle chez Y. lipolytica implique la β-oxydation (Endrizzi et al., 1996). Cette levure est un bon modele pour etudier le metabolisme des acides gras car elle possede cinq acyl-…
Peroxisomal beta-oxidation activities and gamma-decalactone production by the yeast Yarrowia lipolytica.
International audience; gamma-Decalactone is a peachy aroma compound resulting from the peroxisomal beta-oxidation of ricinoleic acid by yeasts. The expression levels of acyl-CoA oxidase (gene deletion) and 3-ketoacyl-CoA thiolase activities (gene amplification on replicative plasmids) were modified in the yeast Yarrowia lipolytica. The effects of these modifications on beta-oxidation were measured. Overexpression of thiolase activity did not have any effect on the overall beta-oxidation activity. The disruption of one of the acyl-CoA oxidase genes resulted in an enhanced activity. The enhancement led to an increase of overall beta-oxidation activity but reduced the gamma-decalactone produc…
Characterization of Purified Green Bell Pepper Hydroperoxide Lyase Expressed by Yarrowia Lipolytica: Radicals Detection during Catalysis
International audience; The optimization of the expression of recombinant 6-His-tagged HPO lyase in Yarrowia lipolytica is described: 1800U/L of culture was detected at 24h of culture on a medium containing olive oil as the sole carbon source. The enzyme was purified by IMAC and showed an optimal pH at 5.5, an optimal temperature at 20^\circC and a Km value of 9μM with 13-HPOD substrate. The participation of radicals during the catalysis of purified bell pepper fruit hydroperoxide lyase has been observed by electron paramagnetic resonance spectroscopy and the yet unidentified radical species might be an alkyl or alkoxyl radical linked to the enzyme.