Jean-Marc Belin

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

ABSTRACT Some microorganisms can transform methyl ricinoleate into γ-decalactone, a valuable aroma compound, but yields of the bioconversion are low due to (i) incomplete conversion of ricinoleate (C18) to the C10 precursor of γ-decalactone, (ii) accumulation of other lactones (3-hydroxy-γ-decalactone and 2- and 3-decen-4-olide), and (iii) γ-decalactone reconsumption. We evaluated acyl coenzyme A (acyl-CoA) oxidase activity (encoded by the POX1 throughPOX5 genes) in Yarrowia lipolytica in lactone accumulation and γ-decalactone reconsumption inPOX mutants. Mutants with no acyl-CoA oxidase activity could not reconsume γ-decalactone, and mutants with a disruption ofpox3, which encodes the short-chain acyl-CoA oxidase, reconsumed it more slowly. 3-Hydroxy-γ-decalactone accumulation during transformation of methyl ricinoleate suggests that, in wild-type strains, β-oxidation is controlled by 3-hydroxyacyl-CoA dehydrogenase. In mutants with low acyl-CoA oxidase activity, however, the acyl-CoA oxidase controls the β-oxidation flux. We also identified mutant strains that produced 26 times more γ-decalactone than the wild-type parents.

Production of volatile compounds in model milk and cheese media by eight strains of Geotrichum candidum Link

Eight strains of Geotrichum candidum isolated from cheese were compared for aroma production. Twenty-four components, including alcohols, methyl ketones, fatty acids and esters, were identified by gas–liquid chromatography and gas chromatography–mass spectrometry of the volatile compounds produced by G. candidum cultures. Strains could be differentiated by their ability to produce dimethyldisulphide, phenol, phenylethanol and methyl ketones.

Production of volatile compounds in liquid cultures by six strains of coryneform bacteria

SummaryThe aromatic profiles of four strains of Brevibacterium linens, one strain of Brevibacterium sp. and one strain of Microbacterium sp. were determined with some pure cultures of these microorganisms in standard trypcase soy liquid medium, which enabled four of these six strains to produce flavour compounds of ripened cheese. Thirty-two flavour compounds were identified by gas chromatography and mass spectrometry. The identified flavour compounds included the following: fatty acids, alcohols, methylketones, sulphur compounds, aromatic compounds and a pyrazine. Some important differences were found among the six strains studied. The four strains of B. linens had similar flavour profiles. Their typical flavour was probably due to dimethyltrisulphide. The two other strains did not appear to produce this compound. Three strains produced significant amounts of the floral aromas phenylethanol and phenylpropanone.

Involvement of Acyl Coenzyme A Oxidase Isozymes in Biotransformation of Methyl Ricinoleate into γ-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 throughPOX5) (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 Δpox3, which produced 220 mg of γ-decalactone per liter after 24 h. The Δpox2 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 biotransformation. The Δpox2 Δpox3 Δpox5 triple-disrupted strain produced and consumed lactone very slowly. On the contrary, the Δpox2 Δpox3 Δpox4 Δpox5 multidisrupted strain did not grow or biotransform methyl ricinoleate into γ-decalactone, demonstrating that Aox4p is essential for the biotransformation.

Cloning and Characterization of the Peroxisomal Acyl CoA Oxidase ACO3 Gene from the Alkane-utilizing Yeast Yarrowia 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.

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) by playing on genetic factors.

Production of 6-pentyl-α-pyrone byTrichoderma harzianum from 18∶n fatty acid methyl esters

Biosynthesis of 6-pentyl-α-pyrone byTrichoderma harzianum in two different media was evaluated. Best yields were found in nitrogen deficient medium (C/N=60). Limited growth seems to favour the production of this lactone. When fungal cells, precultured in low nitrogen medium, were incubated on methyl ricinoleate (10 g/l, C/N=60) an increase in 6-pentyl-α-pyrone production was observed in comparison with the media containing methyl oleate or methyl linoleate.

Apple pomace: An enzyme system for producing aroma compounds from polyunsaturated fatty acids

SummaryThis paper reports on the possibility of obtaining C-6 (hexanal) and C-10 (2,4-dedadienal) volatile aldehydes by degradation of linoleic acid (C18∶2 Δ 9–12) under the action of the intrinsic enzyme systems found in apple pomace. More aroma compounds are produced by micronization of the pomace and by adding SO2 (60 ppm) and vitamin C (500 ppm), thereby synergistically counteracting oxidation of phenolic compounds, which is a limiting factor in bioconversion.

Medium-size droplets of methyl ricinoleate are reduced by cell-surface activity in the γ-decalactone production by Yarrowia lipolytica

. Size of methyl ricinoleate droplets during biotransformation into γ‐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 µm whereas it was 0·7 µm in homogenized and shaken medium. But as soon as yeast cells were inoculated, both diameters became similar at about 0·7 µm 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.

Biogeneration of 1-octen-3-ol by lipoxygenase and hydroperoxide lyase activities of Agaricus bisporus

Abstract The biogeneration of 1-octen-3-ol by lipoxygenase and associated hydroperoxide lyase activities of a homogenate of the cultivated mushroom Agaricus bisporus was investigated using linoleic acid as a substrate. The optimal substrate and protein concentrations were 1.5 mM and 1.5 mg ml −1 , respectively. The V max value for the enzymic coupled reaction lipoxygenase–hydroperoxide lyase was 6 μg of 1-octen-3-ol ml −1 min −1 , with a K m value of 0.3×10 −3 M. The highest enzymic activity was observed in the presence of pure oxygen (22.9 μg of 1-octen-3-ol ml −1 ), with a bioconversion yield of 36%. The production of 380 μg 1-octen-3-ol per g of mushroom homogenate was demonstrated using an oxygenated reaction medium of 1-l containing 0.5% polyvinylpyrrolidone.