Chirality of the γ-lactones formed by Fusarium poae INRA 45

A filamentous fungus, Fusarium poae INRA 45, was grown in two liquid Czapek-type media (a Czapek medium, and a yeast extract plus casaminoacids-enriched Czapek medium). Eight γ-lactones, i.e., γ-penta-, γ-octa-, γ-nona-, γ-deca-, and γ-dodecalactone, (6Z)-γ- and (6E)-γ-dodecenolactones, and a di-unsaturated γ-dodecalactone, were tentatively, or conclusively, identified by capillary gas chromatography (GC) and coupled GC-MS, in the volatile fractions of the cultures. Kinetics of the formation of γ-decalactone, (6Z)-γ- and (6E)-γ-dodecenolactones, (three major lactones), and γ-dodecalactone (a minor lactone) were quantitatively studied by computerized GC integration. Variations of the (R/S) ratios of the lactone enantiomers were quantitatively studied by computerized multidimensional gas chromatography (MDGC) integration. The levels of the lactones were superior in the enriched Czapek-type medium to those of the Czapek medium, but the compositions of these media did not influence significantly the (R/S) ratios of these lactones. Formation of the enantiomers is discussed in terms of metabolism of potential precursors. © 1993 Wiley-Liss, Inc.     

Enantiodifferentiation of four γ-lactones produced by Penicillium Roqueforti

Identification and enantiodifferentiation of γ-lactones produced during the bioconversion of soy bean fatty acids by Penicillium roqueforti spores in the presence of an exogenous lipase was performed using gas chromatography-mass spectrometry (GC-MS) in selected ion monitoring (SIM) mode. It was shown that 4-dodecanolide and 4-hexanolide were first produced with an enantiomeric excess (99%) in favor of the (R) form, whereas an enantiomeric excess in favor of the (S) form (94%) is found for (6Z)-dodecen-4-olide, the major lactone produced by the fungus. If incubation was continued, mixtures of both enantiomers were found, more particularly for 4-decanolide (R/S:40/60), which was produced only after 120 hr of incubation. The results obtained can be explained by the stereoselective hydroxylation by a 10-hydratase and/or a 10-lipoxygenase of the unsaturated fatty acid precursors, oleic and linoleic acids, and by competition between different pathways. The results point out the limitations of chiral GC analysis as a criterion for the natural origin of relevant lactones, and the complexity of mechanisms involved in γ-lactone formation by microorganisms. Chirality 10:786–790, 1998. © 1998 Wiley-Liss, Inc.     

<Emphasis Type="Italic">Sporidiobolus johnsonii</Emphasis> and <Emphasis Type="Italic">Sporidiobolus salmonicolor</Emphasis> revisited

The relationship between Sporidiobolus johnsonii and S. salmonicolor was investigated using rDNA sequence data. Two statistically well-supported clades were obtained. One clade included the type strain of S. johnsonii and the other included the type strain of S. salmonicolor. However, some mating strains of S. salmonicolor were found in the S. johnsonii group. These strains belonged to mating type A2 and were sexually compatible with mating type A1 strains from the S. salmonicolor group. DNA–DNA reassociation values were high within each clade and moderate between the two clades. In the re-investigation of teliospore germination, we observed that the basidia of S. salmonicolor were two-celled. In S. johnsonii, basidia were not formed and teliospore germination resulted in direct formation of yeast cells. We hypothesize that the S. johnsonii clade is becoming genetically isolated from the S. salmonicolor group and that a speciation process is presently going on. We suspect that the observed sexual compatibility between strains of the S. johnsonii and S. salmonicolor groups and the possible genetic flow between the two species has little biological relevance because distinct phenotypes have been fixed in the two taxa and intermediate (hybrid) sequences for LSU and ITS rDNAs have not been detected. An erratum to this article can be found at     

Influence of Water Potential on γ-Decalactone Production by the Yeast Sporidiobolus salmonicolor

The influence of water potential on γ-decalactone production by the yeast Sporidiobolus salmonicolor cultivated in a liquid medium was evaluated by gas-chromatographic analysis. Modifications in water potential led to a number of variations in the aroma production. Maximum extracellular production occurred at water activity (a_w) with a value of 0.99. Further analyses revealed an important phenomenon of cellular accumulation of aroma for a_w values between 0.97 and 0.99.     

Heterothallism in the basidiomycetous yeast genusSporidiobolus Nyland

The sexual speciesSporidiobolus salmonicolor andSporidiobolus pararoseus spp. nov. are described as heterothallic species in the genusSporidiobolus, a genus consisting of two homothallic species,Sporidiobolus johnsonii andSporidiobolus ruinenii. The four species are compared and a key to the genus is presented. As a taxonomic correctionSporobolomyces pararoseus nom. nud. is reported as a synonym ofSporobolomyces shibatanus.     

The Effect of Zn(II) Ions and Reactive Oxygen on the Uptake of Zinc and Production of Carotenoids by Selected Red Yeasts

Three strains of red yeast Rhodosporidium kratochvilovae, Rhodotorula glutinis and Sporidiobolus salmonicolor were studied for their responses to the presence metal stress, oxidative stress and a combination of these stress factors. For all yeast strains, the production of β‐carotene increased in stress conditions. The combination of H₂O₂ and Zn²⁺ significantly activated the pathways for the production of torularhodin in the strain R. glutinis (from 250 to 470 μg g^?1 DCW) as well as β‐carotene (from 360 to 1100 μg g^?1 DCW) and torulene (from 100 to 360 μg g^?1 DCW) in Sp. salmonicolor. Strains of R. glutinis and Rh. kratochvilovae bound the majority of Zn(II) ions to the fibrillar part of the cell walls, whereas the strain Sp. salmonicolor bound them to both extracellular polymers and the fibrillar part of the cell walls. A decrease in the ability of yeasts to tolerate higher concentrations of Zn(II) in the presence of free radicals (hydrogen peroxide) was also found.     

Screening for microorganism with cell wall lytic activity to produce protoplast-forming enzymes

Several different bacteria and fungi capable of degrading yeast cell walls were isolated in the course of a screening programme. One Streptomyces and one Acremonium strain were found to degrade yeast cell walls extremely well. Both isolates produced enzymes in liquid culture that could be used for protoplasting of Sporobolomyces salmonicolor (DSM 70851) and Rhodotorula rubra (DSM 70403). This fact is quite remarkable as, so far, S. salmonicolor could not be protoplasted by commercially available enzymes. Correspondence to: W. Kaul     

Asymmetric synthesis of (S)-3-chloro-1-phenyl-1-propanol using Saccharomyces cerevisiae reductase with high enantioselectivity

3-Chloro-1-phenyl-1-propanol is used as a chiral intermediate in the synthesis of antidepressant drugs. Various microbial reductases were expressed in Escherichia coli, and their activities toward 3-chloro-1-phenyl-1-propanone were evaluated. The yeast reductase YOL151W (GenBank locus tag) exhibited the highest level of activity and exclusively generated the (S)-alcohol. Recombinant YOL151W was purified by Ni-nitrilotriacetic acid (Ni-NTA) and desalting column chromatography. It displayed an optimal temperature and pH of 40°C and 7.5–8.0, respectively. The glucose dehydrogenase coupling reaction was introduced as an NADPH regeneration system. NaOH solution was occasionally added to maintain the reaction solution pH within the range of 7.0–7.5. By using this reaction system, the substrate (30 mM) could be completely converted to the (S)-alcohol product with an enantiomeric excess value of 100%. A homology model of YOL151W was constructed based on the structure of Sporobolomyces salmonicolor carbonyl reductase (Protein Data Bank ID: 1Y1P). A docking model of YOL151W with NADPH and 3-chloro-1-phenyl-1-propanone was then constructed, which showed that the cofactor and substrate bound tightly to the active site of the enzyme in the lowest free energy state and explained how the (S)-alcohol was produced exclusively in the reduction process.     

<Emphasis Type="Italic">Sporidiobolus johnsonii</Emphasis> and <Emphasis Type="Italic">Sporidiobolus salmonicolor</Emphasis> revisited

The relationship between Sporidiobolus johnsonii and S. salmonicolor was investigated using rDNA sequence data. Two statistically well-supported clades were obtained. One clade included the type strain of S. johnsonii and the other included the type strain of S. salmonicolor. However, some mating strains of S. salmonicolor were found in the S. johnsonii group. These strains belonged to mating type A2 and were sexually compatible with mating type A1 strains from the S. salmonicolor group. DNA–DNA reassociation values were high within each clade and moderate between the two clades. In the re-investigation of teliospore germination, we observed that the basidia of S. salmonicolor were two-celled. In S. johnsonii, basidia were not formed and teliospore germination resulted in direct formation of yeast cells. We hypothesize that the S. johnsonii clade is becoming genetically isolated from the S. salmonicolor group and that a speciation process is presently going on. We suspect that the observed sexual compatibility between strains of the S. johnsonii and S. salmonicolor groups and the possible genetic flow between the two species has little biological relevance because distinct phenotypes have been fixed in the two taxa and intermediate (hybrid) sequences for LSU and ITS rDNAs have not been detected.     

Molecular characterization of carotenogenic yeasts from aquatic environments in Patagonia, Argentina

Fifteen aquatic environments (lakes, lagoons and rivers) of glacial origin in the northern Andean Patagonia (Argentina) were surveyed for the occurrence of red yeasts. Subsurface water samples were filtered and used for colony counting and yeast isolation. A preliminary quantitative analysis indicated that total yeast counts ranged between 0 and 250 cells l⁻¹. A polyphasic approach including physiological and molecular methods was used for the identification of 64 carotenogenic yeast strains. The molecular characterisation of the isolates was based on the mini/microsatellite-primed PCR technique (MSP-PCR) employing the (GTG)₅ and the M13 primers. Comparison of representative fingerprints of each group with those of the type strains of pigmented yeasts allowed the expeditious identification of 87.5% isolates. The sequence analysis of the D1/D2 domains of the 26S rDNA was employed to confirm identifications and in the characterization of the unidentified MSP-PCR groups. Teleomorphic yeast species were detected by performing sexual compatibility assays. The isolates corresponded to 6 genera and 15 yeast species, including four new yeast species of the genera Cryptococcus (1), Rhodotorula (1) and Sporobolomyces (2). Rhodotorula mucilaginosa was found in the majority of the samples and represented ca. 50% of the total number of isolates. However, this yeast was not detected in aquatic environments with very low anthropic influence. Other frequent yeast isolates were teleomorphic yeast species of Rhodosporidium babjevae, R. kratochvilovae and Sporidiobolus salmonicolor. This study represents the first report on red yeast occurrence and biodiversity in northwestern Patagonia. This revised version was published online in June 2006 with corrections to the Cover Date.     

Enzymatic production of ethyl (R )-4-chloro-3-hydroxybutanoate: asymmetric reduction of ethyl 4-chloro-3-oxobutanoate by an Escherichia coli transformant expressing the aldehyde reductase gene from yeast

The asymmetric reduction of ethyl 4-chloro-3-oxobutanoate (COBE) to ethyl (R)-4-chloro-3-hydroxybutanoate (CHBE) using Escherichia coli JM109 (pKAR) cells expressing the aldehyde reductase gene from Sporobolomyces salmonicolor AKU4429 as a catalyst was studied. The reduction required NADP⁺, glucose and glucose dehydrogenase for NADPH regeneration. In an aqueous system, the substrate was unstable, and inhibition of the reaction by the substrate was also observed. Efficient conversion of COBE to (R)-CHBE with a satisfactory enantiomeric excess (ee) was attained on incubation with transformant cells in an n-butyl acetate/water two-phase system containing the above NADPH-regeneration system. Under the optimized conditions, with the periodical addition of COBE, glucose and glucose dehydrogenase, the (R)-CHBE yield reached 1530 mM (255 mg/ml) in the organic phase, with a molar conversion yield of 91.1% and an optical purity of 91% ee. The calculated turnover of NADP⁺, based on the amounts of NADP⁺ added and CHBE formed, was about 5100 mol/mol. Received: 26 May 1997 / Received revision: 16 July 1997 / Accepted: 29 August 1997     

Production, Identification, and Toxicity of (gamma)-Decalactone and 4-Hydroxydecanoic Acid from Sporidiobolus spp

During the bioconversion of ricinoleic acid to (gamma)-decalactone under controlled pH conditions, Sporidiobolus salmonicolor produced only the lactone form, while Sporidiobolus ruinenii produced both the lactone form and a precursor. By using gas chromatography-mass spectrometry and gas chromatography-Fourier transform infrared analysis techniques, the precursor was identified as 4-hydroxydecanoic acid. The levels of production in the presence of high concentrations of ricinoleic acid methyl ester differed in the two Sporidiobolus species. This difference was due on the one hand to the high sensitivity of S. salmonicolor to the lactone and on the other hand to the high level of 4-hydroxydecanoic acid produced by S. ruinenii. 4-Hydroxydecanoic acid is much less toxic to the microorganisms than the lactone. In contrast to S. ruinenii, S. salmonicolor is not able to catabolize 4-hydroxydecanoic acid.     

γ-decalactone production by Yarrowia lipolytica and Lindnera saturnus in crude glycerol

Flavor compounds are commonly obtained from chemical synthesis or extracted from plants. These sources have disadvantages, such as racemic mixture generation, more steps to yield the final product, low yield, and high cost, making the microbial fermentation an alternative and potential way to obtain flavor compounds. The most important lactone for flavor application is γ-decalactone, which has an aroma of peach and can be obtained by ricinoleic acid biotransformation through yeast peroxisomal β-oxidation. The aim of this work was to use crude glycerol, a residual biodiesel industry, for the production of bioaroma from two different yeasts. Yarrowia lipolytica CCMA 0357 and Lindnera saturnus CCMA 0243 were grown at different concentrations (10, 20, and 30% w/v) of substrates (castor oil and crude glycerol) for γ-decalactone production. L. saturnus CCMA 0243 produced higher concentration of y-decalactone (5.8?g/L) in crude glycerol, whereas Y. lipolytica CCMA 0357 showed a maximum production in castor oil (3.5?g/L). Crude glycerol showed better results for γ-decalactone production when compared to castor oil. L. saturnus CCMA 0243 has been shown to have a high potential for γ-decalactone production from crude glycerol.     

Microbial production of chiral pantothenonitrile through stereospecific reduction of 2′-ketopantothenonitrile

Summary A novel enzymatic process for the synthesis of chiral pantothenonitrile through the asymmetric reduction of 2-ketopantothenonitrile is described.Sporidiobolus salmonicolor AKU 4440 was found to convert 2-ketopantothenonitrile (50 mg/ml) to D-(+)-pantothenonitrile (93.6% e.e.), with a molar yield of 95.6%. In a similar manner, conversion to L-(–)-pantothenonitrile occurred on incubation with washed cells ofRhodotorula glutinis AKU 4845 as the catalyst.     

Bioorganic Research of Galactites tomentosa Moench. Honey Extracts: Enantiomeric Purity of Chiral Marker 3‐Phenyllactic Acid

Thistle (Galactites tomentosa Moench.) honey organic extracts were obtained by headspace solid‐phase microextraction (HS‐SPME) and ultrasonic solvent extraction (USE) and analyzed by gas chromatography (GC‐FID and GC‐MS) for the first time. Most abundant headspace compounds were terpenes, particularly linalool derivatives (hotrienol was predominant with a range of 38.6–57.5%). 3‐Phenyllactic acid dominated in the solvent extracts (77.4–86.4%) followed by minor percentages of other shikimate pathway derivatives. After determination of an adequate enantioseparation protocol on Chirallica PST‐4 column, the honey solvent extracts were analyzed by high‐performance liquid chromatography (HPLC). The chiral analysis revealed high enantiomeric excess (>95%) of (–)‐3‐phenyllactic acid in all samples. Therefore, previous findings of chemical markers of thistle honey were extended, providing new potential for advanced chemical fingerprinting (optical pure chemical marker). Chirality 26:405–410, 2014. © 2014 Wiley Periodicals, Inc.     

Peroxisomal β-oxidation activities and γ-decalactone production by the yeast Yarrowia lipolytica

γ-Decalactone is a peachy aroma compound resulting from the peroxisomal β-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 β-oxidation were measured. Overexpression of thiolase activity did not have any effect on the overall β-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 β-oxidation activity but reduced the γ-decalactone production rates. This seemed to indicate a non-rate-limiting role for β-oxidation in the biotransformation of ricinoleic acid to γ-decalactone by the yeast Yarrowia lipolytica. All strains produced and then consumed γ-decalactone. We checked the ability of the different strains to consume γ-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 β-oxidation is certainly involved in γ-decalactone catabolism by the yeast Y.␣lipolytica. The observed production rates probably depend on an equilibrium between production and consumption of the lactone. Received: 13 June 1997 / Received revision: 2 October 1997 / Accepted: 14 October 1997     

Chromatographic profiles of tryptophan and kynurenine enantiomers derivatized with (S)‐4‐(3‐isothiocyanatopyrrolidin‐1‐yl)‐7‐(N,N‐dimethylaminosulfonyl)‐2,1,3‐benzoxadiazole using LC–MS/MS on a triazole‐bonded column

d ‐ and l ‐Tryptophan (Trp) and d ‐ and l ‐kynurenine (KYN) were derivatized with a chiral reagent, (S )‐4‐(3‐isothiocyanatopyrrolidin‐1‐yl)‐7‐(N,N‐dimethylaminosulfonyl)‐2,1,3‐benzoxadiazole (DBD‐PyNCS), and were separated enantiomerically by high‐performance liquid chromatography (HPLC) equipped with a triazole‐bonded column (Cosmosil HILIC) using tandem mass spectrometric (MS/MS) detection. Effects of column temperature, salt (HCO₂NH₄) concentration, and pH of the mobile phase in the enantiomeric separation, followed by MS detection of (S )‐DBD‐PyNCS‐d ,l ‐Trp and ‐d ,l ‐KYN, were investigated. The mobile phase consisting of CH₃CN/10 mM ammonium formate in H₂O (pH 5.0) (90/10) with a column temperature of 50–60 °C gave satisfactory resolution (R s) and mass‐spectrometric detection. The enantiomeric separation of d ,l ‐Trp and d ,l ‐KYN produced R s values of 2.22 and 2.13, and separation factors (α) of 1.08 and 1.08, for the Trp and KYN enantiomers, respectively. The proposed LC–MS/MS method provided excellent detection sensitivity of both enantiomers of Trp and KYN (5.1–19 nM).     

Production and Characterization of an Exopolysaccharide by Yeast

Antarctic yeast strains were investigated for exopolysaccharide biosynthesis and the Sporobolomyces salmonicolor AL₁ strain was selected. It was studied for exopolysaccharide biosynthesis on different carbon and nitrogen sources. The investigations showed that sucrose and ammonium sulphate were suitable culture medium components for polymer biosynthesis. Exopolysaccharide formation by the yeast strain was accompanied by a decrease in the culture medium pH value from the initial pH 5.3 to pH 1.7–2.0. During the biosynthetic process, the dynamic viscosity of the culture broth increased to the maximum value of 15.37 mPas and the polysaccharide yield reached 5.63 g/l on a culture medium containing 5.00% sucrose and 0.25% ammonium sulphate at a temperature of 22 °C for 120 h. The crude polysaccharide obtained from Sp. salmonicolor AL₁ featured high purity (90.16% of carbon content) and consisted of glucose (54.1%), mannose (42.6%) and fucose (3.3%). Pure mannan containing 98.6% of mannose was isolated from it.     

Lactonohydrolase-catalyzed optical resolution of pantoyl lactone: selection of a potent enzyme producer and optimization of culture and reaction conditions for practical resolution

A fungal lactonohydrolase catalyzes the stereospecific hydrolysis of the intramolecular ester bond of d-pantoyl lactone and is useful for optical resolution of racemic pantoyl lactone. High activity of this stereospecific hydrolysis reaction was found in several filamentous fungi belonging to the genera Fusarium, Gibberella and Cylindrocarpon through the screening in a variety of microorganisms. Fusarium oxysporum AKU 3702 showed high productivity of the enzyme and the cells containing the enzyme could be used repeatedly for this hydrolysis reaction. On incubation with the mycelia of this fungus, which had been cultivated in 3% glycerol, 0.5% Polypepton, 0.5% yeast extract and 0.5% corn steep liquor, pH 6.0, 46.0% of the racemic pantoyl lactone (700 mg/ml) was hydrolyzed and the optical purity of the pantoic acid formed was 96% enantiomeric excess for the d-isomer.     

Metabolism of ricinoleic acid into gamma-decalactone: beta-oxidation and long chain acyl intermediates of ricinoleic acid in the genus Sporidiobolus sp

In order to study differences in gamma-decalactone production in yeast, four species of Sporidiobolus were cultivated with 5% of methyl ricinoleate as the lactone substrate. In vivo studies showed different time courses of intermediates of ricinoleic acid breakdown between the four species. In vitro studies of the beta-oxidation system were conducted with crude cell extracts of Sporidiobolus spp. and with ricinoleyl-CoA (RCoA) as substrate. The beta-oxidation was detected by measuring acyl-CoA oxidase, 3-hydroxyacyl-CoA dehydrogenase activities, and acetyl-CoA production. The time courses of the CoA esters resulting from RCoA breakdown by crude extract of Sporidiobolus spp. permit the proposal of different metabolic models in the yeast. These models explained the differences observed during in vivo studies.