Use of a Doehlert factorial design to investigate the effects of pH and aeration on the accumulation of lactones by Yarrowia lipolytica.

AIMS: To detect rate-limiting steps in the production of lactones by studying the combined effect of pH and aeration on their accumulation. METHODS AND RESULTS: A Doehlert experimental design was chosen to evaluate the accumulation of four lactones in the pH (3.5-7.3) and K(L)a (4.1 h(-1) to 26 h(-1)) experimental domain. The accumulation of gamma-decalactone was higher at pH around 5 and increased at low aeration reaching 496 mg l(-1) at pH 6.35 and K(L)a 4.5 h(-1). The specific accumulation increased at low aeration. The 3-hydroxy-gamma-decalactone accumulation was higher at low pH and high aeration conditions: 660 mg l(-1) at pH 4.4 and 26 h(-1). For dec-2-en-4-olide and dec-3-en-4-olide, lower amounts were reached (104 mg l(-1) and 66 mg l(-1), respectively). CONCLUSIONS: Although the accumulation of the four lactones should be related to catalytic steps requiring oxygen, the accumulation of gamma-decalactone was higher in low aeration conditions whereas the one of 3-hydroxy-gamma-decalactone was promoted for high aeration. Decenolides accumulate independently of pH or aeration. SIGNIFICANCE AND IMPACT OF THE STUDY: This study gives new insights into the catabolism of lipids, such as the role of co-factor regulation and the fact that the 3-hydroxylactone dehydration step is insensitive to pH or aeration.

Diversity of the surface properties of Lactococci and consequences on adhesion to food components.

Bacteria possess surface properties, related to their charge, hydrophobicity and Lewis acid/base characteristics, that are involved in the attachment processes of microorganisms to surfaces. Fermentation bulks and food matrixes are complex heterogeneous media containing various components with different physicochemical characteristics. The aim of the present study was to investigate whether (i) bacteria present in a food matrix, interacted physicochemically at their surface level with the other constituents and (ii) the diversity of bacterial surface properties could result in a diversity of microbial adhesion to components and thus in a diversity of tolerance to toxic compounds. The surface properties of 20 lactic acid bacteria were characterized by the MATS method showing their relatively hydrophilic and various basic characteristics. The results obtained from a set of representative strains showed that (i) the strains with higher affinity for apolar solvents adsorbed more to lipids and hydrophobic compounds, (ii) the more the strains adsorbed to a toxic solvent, the less they were tolerant to this solvent. A diversity of bacterial surface properties was observed for the strains in the same species showing the importance of choosing bacteria according to their surface properties in function of technological objectives.

Yeast as an efficient biocatalyst for the production of lipid-derived flavours and fragrances.

Responding to consumer' demand for natural products, biotechnology is constantly seeking new biocatalysts. In the field of hydrophobic substrate degradation, some yeast species known some years ago as non-conventional, have acquired their right to be considered as good biocatalysts. These Candida, Yarrowia, Sporobolomyces ... are now used for themselves or for their lipases in processes to produce flavours and fragrances. In this paper we present some examples of use of these biocatalysts to generate high-value compounds and discuss the new trends related to progress in the development of molecular tools or the mastering of the redox characteristics of the medium.

Catabolism of hydroxyacids and biotechnological production of lactones by Yarrowia lipolytica.

The gamma- and delta-lactones of less than 12 carbons constitute a group of compounds of great interest to the flavour industry. It is possible to produce some of these lactones through biotechnology. For instance, gamma-decalactone can be obtained by biotransformation of methyl ricinoleate. Among the organisms used for this bioproduction, Yarrowia lipolytica is a yeast of choice. It is well adapted to growth on hydrophobic substrates, thanks to its efficient and numerous lipases, cytochrome P450, acyl-CoA oxidases and its ability to produce biosurfactants. Furthermore, genetic tools have been developed for its study. This review deals with the production of lactones by Y. lipolytica with special emphasis on the biotransformation of methyl ricinoleate to gamma-decalactone. When appropriate, information from the lipid metabolism of other yeast species is presented.

Mechanisms underlying the toxicity of lactone aroma compounds towards the producing yeast cells.

AIMS: To study the fundamental mechanisms of toxicity of the fruity aroma compound gamma-decalactone, that lead to alterations in cell viability during its biotechnological production by yeast cells; Yarrowia lipolytica that is able to produce high amounts of this metabolite was used here as a model. METHODS AND RESULTS: Lactone concentrations above 150 mg l-1 inhibited cell growth, depolarized the living cells and increased membrane fluidity. Infrared spectroscopic measurements revealed that the introduction of the lactone into model phospholipid bilayers, decreased the phase transition temperature. Moreover, the H+-ATPase activity in membrane preparations was strongly affected by the presence of the lactone. On the other hand, only a slight decrease in the intracellular pH occurred. CONCLUSIONS: We propose that the toxic effects of gamma-decalactone on yeast may be initially linked to a strong interaction of the compound with cell membrane lipids and components. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings may enable the elaboration of strategies to improve yeast cell viability during the process of lactones bioproduction.

Purification of hydroperoxide lyase from green bell pepper (Capsicum annuum L.) fruits for the generation of C6-aldehydes in vitro.

The aim of this work was to compare the efficiency of different extracts of hydroperoxide lyase from green bell peppers in producing aldehydes: a crude extract, a chloroplastic fraction, and a purified enzyme were investigated. From a crude extract, the HPO lyase was purified by ion-exchange chromatography with a 22.3-fold increase in purification factor. Analysis by SDS-PAGE electrophoresis under denaturating conditions showed only one protein with a molecular weight of 55 kDa, whereas size-exclusion chromatography indicated a molecular weight of 170 kDa. A maximum of 7500 mg of aldehydes per g of protein was obtained with the purified enzyme within 20 min of bioconversion compared to 392 and 88 mg of aldehydes per g of protein within 50 and 60 min, respectively, for the chloroplast fraction and the crude extract.

Role of beta-oxidation enzymes in gamma-decalactone production by the yeast Yarrowia lipolytica.

Some microorganisms can transform methyl ricinoleate into gamma-decalactone, a valuable aroma compound, but yields of the bioconversion are low due to (i) incomplete conversion of ricinoleate (C(18)) to the C(10) precursor of gamma-decalactone, (ii) accumulation of other lactones (3-hydroxy-gamma-decalactone and 2- and 3-decen-4-olide), and (iii) gamma-decalactone reconsumption. We evaluated acyl coenzyme A (acyl-CoA) oxidase activity (encoded by the POX1 through POX5 genes) in Yarrowia lipolytica in lactone accumulation and gamma-decalactone reconsumption in POX mutants. Mutants with no acyl-CoA oxidase activity could not reconsume gamma-decalactone, and mutants with a disruption of pox3, which encodes the short-chain acyl-CoA oxidase, reconsumed it more slowly. 3-Hydroxy-gamma-decalactone accumulation during transformation of methyl ricinoleate suggests that, in wild-type strains, beta-oxidation is controlled by 3-hydroxyacyl-CoA dehydrogenase. In mutants with low acyl-CoA oxidase activity, however, the acyl-CoA oxidase controls the beta-oxidation flux. We also identified mutant strains that produced 26 times more gamma-decalactone than the wild-type parents.

Intracellular pH-dependent efflux of the fluorescent probe pyranine in the yeast Yarrowia lipolytica.

8-Hydroxypyrene-1,3,6-trisulfonic acid (pyranine) can be used as a vital intracellular pH (pH(i)) indicator. In the yeast Yarrowia lipolytica, a partial efflux of the probe was detected by using the pH-independent wavelength of 415 nm. A simplified correction of the fluorescent signals was applied, enabling to show for this species a good near-neutral pH(i) maintenance capacity in a pH 3.9 medium. Octanoic acid, which is known to have toxic effects on yeast, decreased the pH(i) and increased the 260-nm-absorbing compounds leakage. However, this acid inhibited the fluorescent probe efflux linearly with its concentration suggesting a pH(i)-dependent efflux of pyranine from cells.

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

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.

Involvement of acyl coenzyme A oxidase isozymes in biotransformation of methyl ricinoleate into gamma-decalactone by Yarrowia lipolytica.

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 Deltapox3, which produced 220 mg of gamma-decalactone per liter after 24 h. The Deltapox2 Deltapox3 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 Deltapox2 Deltapox3 Deltapox5 triple-disrupted strain produced and consumed lactone very slowly. On the contrary, the Deltapox2 Deltapox3 Deltapox4 Deltapox5 multidisrupted strain did not grow or biotransform methyl ricinoleate into gamma-decalactone, demonstrating that Aox4p is essential for the biotransformation.

Cloning, sequencing, and characterization of five genes coding for acyl-CoA oxidase isozymes in the yeast Yarrowia lipolytica.

The Acyl-CoA oxidase (AOX) isozymes catalyze the first steps of peroxisomal beta-oxidation, which is important for the degradation of fatty acids. Using conserved blocks in previously identified yeast POX genes encoding AOXs, the authors have shown that five POX genes are present in the yeast Yarrowia lipolytica. These genes show approx 63% identity among themselves, and 42% identity with the POX genes from other yeasts. Mono-disrupted Y. lipolytica strains were constructed using a variation of the sticky-end polymerase chain reaction method. AOX activity in the mono-disrupted strains revealed that a long-chain oxidase is encoded by the POX2 gene and a short-chain oxidase by the POX3 gene.

Evaluation of acyl coenzyme A oxidase (Aox) isozyme function in the n-alkane-assimilating yeast Yarrowia lipolytica.

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 be active against short-chain fatty acids, whereas Aox5 was active against molecules of all chain lengths. Mutations in Aox4 and Aox5 resulted in an increase in total Aox activity. The growth of mutant strains was analyzed. In the presence of POX1 only, strains did not grow on fatty acids, whereas POX4 alone elicited partial growth, and the growth of the double POX2-POX3-deleted mutant was normal excepted on plates containing oleic acid as the carbon source. The amounts of Aox protein detected by Western blotting paralleled the Aox activity levels, demonstrating the regulation of Aox in cells according to the POX genotype.

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.

Peroxisomal beta-oxidation activities and gamma-decalactone production by the yeast Yarrowia lipolytica.

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 production rates. This seemed to indicate a non-rate-limiting role for beta-oxidation in the biotransformation of ricinoleic acid to gamma-decalactone by the yeast Yarrowia lipolytica. All strains produced and then consumed gamma-decalactone. We checked the ability of the different strains to consume gamma-decalactone in a medium containing the lactone as sole carbon source. The consumption of the strain overexpressing acyl-CoA oxidase activity was higher than that of the wild-type strain. We concluded that peroxisomal beta-oxidation is certainly involved in gamma-decalactone catabolism by the yeast Y. lipolytica. The observed production rates probably depend on an equilibrium between production and consumption of the lactone.

Utilization of an auxotrophic strain of the yeast Yarrowia lipolytica to improve gamma-decalactone production yields.

gamma-Decalactone is an aroma compound with a pleasant peachy odour. Most industrial processes use the bioconversion of ricinoleic acid by yeasts to produce gamma-decalactone. Peroxisomal beta-oxidation activity is responsible for the bioconversion. Some yeasts, Yarrowia lipolytica in particular, grow during the bioconversion, yielding a low bioconversion rate. Auxotrophy for uracil of a genetically engineered Y. lipolytica strain was used to prevent growth in the bioconversion medium. beta-Oxidation activities and gamma-decalactone production of the auxotrophic strain were measured and compared with a wild-type strain in media supplemented or not. Induction of beta-oxidation was observed in the non-supplemented medium, although to a lesser extent than in supplemented medium. Aroma productivity of the auxotrophic strain in the supplemented medium was similar to that observed for the wild-type strain in both media. However, in the non-supplemented medium the productivity of the auxotrophic strain was 10-20-fold higher.

Involvement of carnitine acyltransferases in peroxisomal fatty acid metabolism by the yeast Pichia guilliermondii.

This article provides information about peroxisomal fatty acid metabolism in the yeast Pichia guilliermondii. The existence of inducible mitochondrial carnitine palmitoyltransferase and peroxisomal carnitine octanoyl-transferase activities was demonstrated after culture of this yeast in a medium containing methyl oleate. The subcellular sites and induction patterns were studied. The inhibition of carnitine octanoyl- and palmitoyl-transferases by chlorpromazine to a large extent prevented the otherwise observed metabolism-dependent inactivation of thiolase by 2-bromofatty acids in vivo. We concluded that the metabolism of long- and medium-chain fatty acids in the peroxisome of this yeast involved carnitine intermediates.

Production of lactones and peroxisomal beta-oxidation in yeasts.

Among aroma compounds interesting for the food industry, lactones may be produced by biotechnological means using yeasts. These microorganisms are able to synthesize lactones de novo or by biotransformation of fatty acids with higher yields. Obtained lactone concentrations are compatible with industrial production, although detailed metabolic pathways have not been completely elucidated. The biotransformation of ricinoleic acid into gamma-decalactone is taken here as an example to better understand the uptake of hydroxy fatty acids by yeasts and the different pathways of fatty acid degradation. The localization of ricinoleic acid beta-oxidation in peroxisomes is demonstrated. Then the regulation of the biotransformation is described, particularly the induction of peroxisome proliferation and peroxisomal beta-oxidation and its regulation at the genome level. The nature of the biotransformation product is then discussed (4-hydroxydecanoic acid or gamma-decalactone), because the localization and the mechanisms of the lactonization are still not properly known. Lactone production may also be limited by the degradation of this aroma compound by the yeasts which produced it. Thus, different possible ways of modification and degradation of gamma-decalactone are described.

Bioconversion of methyl ricinoleate to 4-hydroxy-decanoic acid and to gamma-decalactone by yeasts of the genus Candida.

The capacity of several strains of yeasts to do the bioconversion of methyl ricinoleate into gamma-decalactone, was studied in a medium containing this methylic ester of fatty acid as sole carbon source. Amongst the strains which are able to do this bioconversion, two types of behaviour are observed: some of the strains produce gamma-decalactone during all the incubation in bioconversion medium while others produce this aroma compound very quickly and then consume it fast too. The tested strains produce at the same time gamma-decalactone and the corresponding acid form (4-hydroxy-decanoic acid), and this, in variable proportions.

Metabolism of Linoleic Acid or Mevalonate and 6-Pentyl-alpha-Pyrone Biosynthesis by Trichoderma Species.

The understanding of the biosynthetic pathway of 6-pentyl-alpha-pyrone in Trichoderma species was achieved by using labelled linoleic acid or mevalonate as a tracer. Incubation of growing cultures of Trichoderma harzianum and T. viride with [U-C]linoleic acid or [5-C]sodium mevalonate revealed that both fungal strains were able to incorporate these labelled compounds (50 and 15%, respectively). Most intracellular radioactivity was found in the neutral lipid fraction. At the initial time of incubation, the radioactivity from [C]linoleic acid was incorporated into 6-pentyl-alpha-pyrone more rapidly than that from [C]mevalonate. No radioactivity incorporation was detected in 6-pentyl-alpha-pyrone when fungal cultures were incubated with [1-C]linoleic acid. These results suggested that beta-oxidation of linoleic acid was a probable main step in the biosynthetic pathway of 6-pentyl-alpha-pyrone in Trichoderma species.

Quantitative separation of Trichoderma lipid classes on a bonded phase column.

Bond Elut aminopropyl columns were used to purify the different lipid classes of Trichoderma harzianum and Trichoderma viride. This methodology permitted good separation of the fungal lipid classes in less time than traditional techniques. The incorporation of [1 (14)C]linoleic acid into neutral lipids, free fatty acids and phospholipids was quantified for both strains. The fatty acid profile of the different lipid classes of these fungal strains was determined as a function of culture time.