Fungal Metabolism of Sorbic Acid

The metabolism of sorbic acid (S A) by 11 species of Mucor and 14 other fungi in addition to Mucor sp. A–73 were studied. All species of Mucor metabolized SA to trans-4-hexenol (4–HEL) without exception. The metabolic pathway was investigated precisely by using the resting cells of Mucor sp. A–73. The reduction of SA to 4–HEL by the fungus demanded glucose. The reduction of SA to sorbic alcohol (SAL) was catalyzed by the inducible enzymic system and that of SAL to 4–HEL by the constitutive enzyme. In the phosphate buffer, a part of 4–HEL produced from SA was further transformed into mono-trans-4-hexenyl phosphate by Mucor sp. A–73.

The reductive action of the fungus upon several trans-2-alkenoic acids was also examined.     

Purification and characterization of an alcohol dehydrogenase from 1,2-propanediol-grownDesulfovibrio strain HDv

The sulfate-reducing bacterimDesulfovibrio strain HDv (DSM 6830) grew faster on (S)- and on (R, S)-1,2-propanediol (μ_max 0.053 h⁻) than on (R)-propanediol (0.017 h⁻¹) and ethanol (0.027 h⁻¹). From (R, S)-1,2-propanediol-grown cells, an alcohol dehydrogenase was purified. The enzyme was oxygen-labile, NAD-dependent, and decameric; the subunit mol. mass was 48 kDa. The N-terminal amino acid sequence indicated similarity to alcohol dehydrogenases belonging to family III of NAD-dependent alcohol dehydrogenases, the first 21 N-terminal amino acids being identical to those of theDesulfovibrio gigas alcohol dehydrogenase. Best substrates were ethanol and propanol (K _m of 0.48 and 0.33 mM, respectively). (R, S)-1,2-Propanediol was a relatively poor substrate for the enzyme, but activities in cell extracts were high enough to account for the growth rate. The enzyme showed a preference for (S)-1,2-propanediol over (R)-1,2-propanediol. Antibodies raised against the alcohol dehydrogenase ofD. gigas showed cross-reactivity with the alcohol dehydrogenase ofDesulfovibrio strain HDv and with cell extracts of six other ethanol-grown sulfate-reducing bacteria.     

Anaerobic growth ability and alcohol fermentation activity of microscopic fungi

The method proposed in this study was used to isolate fungi grown under anaerobic conditions and to reveal distinctions in their abundance and species composition in different habitats. The ability of micromycetes of different taxa to grow under anaerobic conditions and ensure alcohol fermentation was determined for a representative sample (344 strains belonging to more than 60 species). The group of fungi growing under anaerobic conditions included species with high, moderate, and low fermentation activity. The ability for anaerobic growth and fermentation depended on the taxonomic affiliation of fungi. In some cases, the expression of these characteristics depended on the habitat from which the strain was isolated. The maximum level of ethanol accumulation in culture liquid (1.2–4.7%) was detected for Absidia spinosa, Aspergillus sp. of group flavus, Aspergillus terreus, Acremonium sp., Mucor circinelloides, Mucor sp., Fusarium oxysporum, F. solani, F. sambucinum, Rhizopus arrhizus var. arrhizus, Trichoderma atroviride, and Trichoderma sp.     

TADH,the thermostable alcohol dehydrogenase from <Emphasis Type="Italic">Thermus</Emphasis> sp. ATN1: a versatile new biocatalyst for organic synthesis

The alcohol dehydrogenase from Thermus sp. ATN1 (TADH) was characterized biochemically with respect to its potential as a biocatalyst for organic synthesis. TADH is a NAD(H)-dependent enzyme and shows a very broad substrate spectrum producing exclusively the (S)-enantiomer in high enantiomeric excess (>99%) during asymmetric reduction of ketones. TADH is active in the presence of 10% (v/v) water-miscible solvents like 2-propanol or acetone, which permits the use of these solvents as sacrificial substrates in substrate-coupled cofactor regeneration approaches. Furthermore, the presence of a second phase of a water-insoluble solvent like hexane or octane had only minor effects on the enzyme, which retained 80% of its activity, allowing the use of these solvents in aqueous/organic mixtures to increase the availability of low-water soluble substrates. A further activity of TADH, the production of carboxylic acids by dismutation of aldehydes, was investigated. This reaction usually proceeds without net change of the NAD⁺/NADH concentration, leading to equimolar amounts of alcohol and carboxylic acid. When applying cofactor regeneration at high pH, however, the ratio of acid to alcohol could be changed, and full conversion to the carboxylic acid was achieved.     

Cheese whey as a renewable substrate for microbial lipid and biomass production by Zygomycetes

Three Zygomycetes, Mortierella isabellina, Thamnidium elegans and Mucor sp., were tested for their ability of producing biomass and lipid‐containing γ‐linolenic acid (GLA) during their cultivation on cheese whey. M. isabellina consumed all of the available lactose and a significant amount of the available protein. On the contrary, the two other fungi seemed incapable of consuming lactose after protein exhaustion. In the second series of experiments, for M. isabellina a supplementary quantity of lactose was added into the medium in order to increase the C/N ratio and hence to increase the production of fat. In the case of T. elegans and Mucor sp., a supplementary quantity of ammonium sulfate was added in order to favor the consumption of lactose and the production of biomass. Indeed, enhancement of lipid production was observed for M. isabellina and biomass production for T. elegans and Mucor sp.. Fatty acid analysis of the microbial lipid showed a composition that presented non‐negligible changes in relation with the age of the culture and the C/N molar ratio of the medium. Further analysis of the fat showed that the quantity of neutral lipids was the more abundant. The fatty acid composition of neutral lipids resembled to that of total lipids. Phospholipids were the more unsaturated fraction for Mucor sp. and M. isabellina. GLA was synthesized in all trials but its concentration presented differences related with the utilized strains and the fermentation time. Growth of M. isabellina on lactose‐supplemented whey resulted in a maximum GLA production of 301 mg/L.     

Improved production of biosurfactant by a <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> mutant using vegetable oil refinery wastes

Biosurfactant production by Pseudomonas aeruginosa EBN-8 mutant was studied in shake flasks on separate wastes from canola, soybean and corn oil refineries. Of the substrates tested, canola oil refinery waste (COD=20 g l⁻¹) supplemented with sodium nitrate (at COD/N=20) showed the best microbial growth (4.50 g l⁻¹) and rhamnolipid production (8.50 g l⁻¹), at 10 d of incubation with the specific growth rate of 0.316 h⁻¹ and specific product yield of 0.597 g g⁻¹ h. Its cell-free supernatant showed the critical micelle dilution (CMD) of 150 and surface tension (ST) of 28.5 mN m⁻¹.     

Characterization of enzymes involved in the ethanol production of <Emphasis Type="Italic">Moorella</Emphasis> sp. HUC22-1

Since the thermophilic bacterium Moorella sp. HUC22-1 produces 120 mM acetate and 5.2 mM ethanol from H₂–CO₂, several candidate genes, which were predicted to code for three alcohol dehydrogenases (AdhA, B, C) and one acetaldehyde dehydrogenase (Aldh), were cloned from HUC22-1. The cloned genes were subcloned into a His-tagged expression vector and expressed in Escherichia coli. Recombinant AdhA and B were both dependent on NADP(H) but independent of NAD(H), and their reduction activities from aldehyde to alcohol were higher than their oxidation activities. In contrast with AdhA and B, no activity of AdhC was observed in either reaction. On the other hand, Aldh was active toward both NADP(H) and NAD(H). The enzyme activity of Aldh was directed toward the thioester cleavage and the thioester condensation. When 50 μg of AdhA and 50 μg Aldh were added to the buffer solution (pH 8.0) containing NADPH, NADH and acetyl-CoA at 60°C, 1.6 mM ethanol was produced from 3 mM acetyl-CoA after 90 min. Expression analysis of the mRNAs revealed that the expression level of aldh was threefold higher in the H₂–CO₂ culture than that in the fructose culture, but levels of adhA, B and C were decreased.     

Degradation of methamidophos by <Emphasis Type="Italic">Hyphomicrobium</Emphasis> species MAP-1 and the biochemical degradation pathway

Methamidophos is one of the most widely used organophosphorus insecticides usually detectable in the environment. A facultative methylotroph, Hyphomicrobium sp. MAP-1, capable of high efficiently degrading methamidophos, was isolated from methamidophos-contaminated soil in China. It was found that the addition of methanol significantly promoted the growth of strain MAP-1 and enhanced its degradation of methamidophos. Further, this strain could utilize methamidophos as its sole carbon, nitrogen and phosphorus source for growth and could completely degrade 3,000 mg l⁻¹ methamidophos in 84 h under optimal conditions (pH 7.0, 30°C). The enzyme responsible for methamidophos degradation was mainly located on the cell inner membrane (90.4%). During methamidophos degradation, three metabolites were detected and identified based on tandem mass spectrometry (MS/MS) and gas chromatography-mass spectrometry (GC–MS) analysis. Using this information, a biochemical degradation pathway of methamidophos by Hyphomicrobium sp. MAP-1 was proposed for the first time. Methamidophos is first cleaved at the P–N bond to form O,S-dimethyl hydrogen thiophosphate and NH₃. Subsequently, O,S-dimethyl hydrogen thiophosphate is hydrolyzed at the P–O bond to release –OCH₃ and form S-methyl dihydrogen thiophosphate. O,S-dimethyl hydrogen thiophosphate can also be hydrolyzed at the P–S bond to release –SCH₃ and form methyl dihydrogen phosphate. Finally, S-methyl dihydrogen thiophosphate and methyl dihydrogen phosphate are likely transformed into phosphoric acid.     

<i>In vitro</i> evaluation of antifungal activity of Agave (<i>Agave scabra</i>, Salm Dyck) extracts against post-harvest mushrooms

The agricultural sector, and particularly the horticultural production, has a singular importance in agriculture, considering that it ranks second on agricultural products, nationally and worldwide. Fungal diseases are one of the major causes of vegetable loss during storage, reducing their nutritional value, quality and sale price. Vegetables are usually exposed to diverse treatments with chemical products before storage; as a result, fungal populations develop an increased resistance over time becoming more difficult to control. Because of this, research efforts toward finding more suitable chemicals to control fungal diseases are needed. Natural extracts may be an alternative solve this problem. In the present investigation the fungicidal activity of aqueous and ethanol extracts of Agave scabra was evaluated on the growth of Botrytis cinerea, Mucor sp., Aspergillus niger, Fusarium sp. and Penicillium sp., whose strains were isolated from potato and tomato. To assess their effects, the agar-dilution and agar-well techniques were performed. The ethanol extract was more effective against Botrytis cinerea and Mucor sp. when the agar-well method was used. However, when using the agar-dilution method the ethanol extract of Agave scabra inhibited the growth of Botrytis cinerea, Mucor sp. and Penicillium sp.     

Fluctuations of plant pathogens in sugar factory's waste in Kerman province of Iran

Sugar beet roots and the attached soil are transferred to Bardsir factory for processing, and the roots are washed by water pressure. Muddy water flows as waste into canals and are used for irrigation of sugar beet farms. The possibility of occurrence and fluctuations of the pathogenic organisms in irrigation was studied during 2006 and 2007. The samples were collected from the wastewater once a week. Isolation of pathogens was done by four methods: (a) Citrus leaf baiting method and on PARPH selective medium, and common culture media such as: PDA, CMA, MA, NA and WA; (b) Irrigation of sugar beet seedling with wastewater; (c) Bioassay with healthy sugarbeet roots; (d) Cysts of nematode were extracted from waste by sieves. Many different pathogens were isolated from wastewater such as: Fusarium oxysporum, Fusarium solani, Rhizopus stolonifer, Penicillium digitatum, Aspergillus niger, Pythium aphanidermatum, Pythium akanoganese, Phytophthora cryptogea, Phytophthora drechsleri, Mucor sp., Rhizoctonia solani, Geotrichum sp., Erwinia carotovora and cysts of Heterodera schachtii. Pathogencity tests were done for each pathogen. During the first period of processing, the recovery of pythiaceae and Erwinia carotovora was in high level, and in the last period of processing the recovery of Penicillium digitatum, Aspergillus niger, Mucor sp. and Geotrichum sp. showed high levels. The recovery of Alternaria alternata, Rhizoctonia solani and Fusarium solani were stable but the cysts of H. schachtii were variable during the processing period. These pathogens were isolated from sugar beet farms irrigated with wastewater.     

Effects of <Emphasis Type="Italic">dapA</Emphasis> gene deletion on coronatine biosynthesis in <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">glycinea</Emphasis> PG4180

Coronatine (COR) is a structural and functional analogue of jasmonic acid that might be employed in agriculture to elicit plant resistance against various aggressors. However, the yield of COR is low both in chemosynthesis and biosynthesis, so broad investigation of COR is difficult. Coronatine combines two distinct components: coronafacic acid (CFA) and coronamic acid (CMA). Synthesis of both CMA and CFA is involved in l-isoleucine metabolism, so the objective of this work was to investigate if COR production can be improved by regulating amino acid biosynthesis in P. syringae pv. glycinea. Inhibition of dihydrodipicolinate synthase was achieved by removing the dapA gene via homologous recombination, which resulted in a COR yield by the dapA ⁻ mutant of about 1.5-fold greater than the wild strain. Thus, regulation of amino acid metabolism is a feasible way to increase COR production, which could be a more effective method than adding substrates into culture medium.     

Cyanuric acid biodegradation by a mixed bacterial culture of <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> and <Emphasis Type="Italic">Acinetobacter</Emphasis> sp. in a packed bed biofilm reactor

Cyanuric acid (1,3,5-triazine-2,4,6-triol [OOOT]) is a common biodegradation byproduct of triazinic herbicides, frequently accumulated in soils or water when supplementary carbon sources are absent. A binary bacterial culture able to degrade OOOT was selected through a continuous selection process accomplished in a chemostat fed with a mineral salt (MS) medium containing cyanuric acid as the sole carbon and nitrogen source. By sequence comparison of their 16S rDNA amplicons, bacterial strains were identified as Agrobacterium tumefaciens, and Acinetobacter sp. When the binary culture immobilized in a packed bed reactor (PBR) was fed with MS medium containing OOOT (50 mg L⁻¹), its removal efficiencies were about 95%; when it was fed with OOOT plus glucose (120 mg L⁻¹) as a supplementary carbon source, its removal efficiencies were closer to 100%. From sessile cells, attached to PBR porous support, or free cells present in the outflowing medium, DNA was extracted and used for Random Amplification of Polymorphic DNA analysis. Electrophoretic patterns obtained were compared to those of pure bacterial strains, a clear predominance of A. tumefaciens in PBR was observed. Although in continuous suspended cell culture, a stable binary community could be maintained, the attachment capability of A. tumefaciens represented a selective advantage over Acinetobacter sp. in the biofilm reactor, favoring its predominance in the porous stone support.     

Influence of growth regulators and elicitors on cell growth and α-tocopherol and pigment productions in cell cultures of <Emphasis Type="Italic">Carthamus tinctorius</Emphasis> L.

The present study examined the effects of plant growth hormones, incubation period, biotic (Trametes versicolor, Mucor sp., Penicillium notatum, Rhizopus stolonifer, and Fusarium oxysporum) and abiotic (NaCl, MgSO₄, FeSO₄, ZnSO₄, and FeCl₃) elicitors on cell growth and α-tocopherol and pigment (red and yellow) productions in Carthamus tinctorius cell cultures. The cell growth and α-tocopherol and pigment contents improved significantly on Murashige and Skoog (MS) liquid medium containing 50.0 μM α-naphthalene acetic acid (NAA) and 2.5 μM 6-Benzyladenine (BA) at 28 days of incubation period. Incorporation of T. versicolor (50 mg l⁻¹) significantly enhanced the production of α-tocopherol (12.7-fold) and red pigment (4.24-fold). Similarly, supplementation of 30 mg l⁻¹ T. versicolor (7.54-fold) and 70 mg l⁻¹ Mucor sp. (7.40-fold) significantly increased the production of yellow pigment. Among abiotic elicitors, NaCl (50–70 mg l⁻¹) and MgSO₄ (10–30 mg l⁻¹) significantly improved production of α-tocopherol (1.24-fold) and red pigment (20-fold), whereas yellow pigment content increased considerably by all the abiotic elicitor treatments. Taken together, the present study reports improved productions of α-tocopherol and the pigment as a stress response of safflower cell cultures exposed to these elicitors.     

Asymmetric bioreduction of p-haloacetophenones by Mucor

Abstract

A number of p-haloacetophenones were asymmetrically bioreduced to their corresponding (S)-alcohols by Mucor sp. CG10 with good conversion and excellent enantioselectivity. The results showed that the electronic effects of the halogen substituent (X-group) affected the conversion of the substrates and the enantioselectivity of the reaction. The trend observed was as the X-group at the para-position became more electron donating from F, to Cl, Br and I, the conversion of substrates decreased, while the enantioselectivity increased.     

Genome shuffling of <Emphasis Type="Italic">Streptomyces</Emphasis> sp. U121 for improved production of hydroxycitric acid

(2S, 3R)-Hydroxycitric acid (HCA) from Hibiscus subdariffa inhibits pancreatic α-amylase and intestine α-glucosidase, leading to reduction of carbohydrate metabolism. In our previous study, Streptomyces sp. U121 was identified as a producer of (2S, 3R)-HCA [Hida et al. (2005) Bioscience, Biotechnology, and Biochemistry 69:1555–1561]. Here, we applied genome shuffling of Streptomyces sp. U121 to achieve rapid improvement of HCA production. The initial mutant population was generated by nitrosoguanidine treatment of the spores, and an improved population producing fivefold more HCA over wild type was obtained by three rounds of genome shuffling. For efficient screening of the mutant library, trans-epoxyaconitic acid (EAA), an antibiotic analog of HCA, was utilized. EAA inhibited the regeneration of nonfused protoplasts, resulting in selective screening of shuffled strains. Mutant strains with enhanced EAA resistance exhibited significantly higher HCA production in liquid media. Furthermore, the best mutant showed increased cell growth in flask culture, as well as increased HCA production.     

Descriptive morphology of the eggs,larvae, and juveniles of two cyprinid fishes belonging to the <Emphasis Type="Italic">Zacco temminckii</Emphasis> species' group

 Embryonic, larval, and juvenile development of two cyprinid species belonging to the Zacco temminckii species' group, Z. temminckii (Temminck and Schlegel) and Zacco sp. (type A), are described and compared with each other from laboratory-reared and wild specimens. The eggs of both species were closely similar except in diameter [1.92–2.20 mm in Z. temminckii vs. 1.60–1.75 mm in Z. sp. (type A)], being demersal, almost spherical in shape, transparent and unpigmented, with a pale yellow yolk, and no oil globule. Hatching occurred 40–53 h after fertilization in Z. temminckii and after 47–60 h in Z. sp. (type A). The newly hatched larvae of both species [4.9–5.3 mm in body length (BL) in Z. temminckii and 3.5–4.8 mm BL in Z. sp. (type A)] also resembled each other, having a large transparent pear-shaped yolk and lacking body pigmentation. Myomere counts of Z. temminckii and Z. sp. (type A) larvae and juveniles were 24–27 + 14–17 = 41–42 and 23–27 + 14–17 = 40–41, respectively. The yolk was completely absorbed at 8.3 mm BL in Z. temminckii and at 6.6 mm BL in Z. sp. (type A). Notochord flexion was initiated and completed at 7.8 mm BL and 8.2 mm BL in Z. temminckii and at 6.3 mm BL and 6.6 mm BL in Z. sp. (type A), respectively. Aggregate numbers of all fin rays were completed at 17 mm BL in Z. temminckii and 13 mm BL in Z. sp. (type A). Although the morphology of larvae and juveniles of both species was very similar, differences in body length of each developmental stage, the duration and process of disappearance of the adipose finfold, the anal fin ray counts, and pigmentation on the lateral body surface were clearly recognized. Received: August 10, 2001 / Revised: March 14, 2002 / Accepted: March 27, 2002     

Photoautotrophic Production of Lipids by Some <Emphasis Type="Italic">Chlorella</Emphasis> Strains

The microalgae Chlorella protothecoides UTEX 25, Chlorella sp. TISTR 8991, and Chlorella sp. TISTR 8990 were compared for use in the production of biomass and lipids under photoautotrophic conditions. Chlorella sp. TISTR 8990 was shown to be potentially suitable for lipid production at 30°C in a culture medium that contained only inorganic salts. For Chlorella sp. TISTR 8990 in optimal conditions in a stirred tank photobioreactor, the lipid productivity was 2.3 mg L⁻¹ h⁻¹ and after 14 days the biomass contained more than 30% lipids by dry weight. To attain this, the nitrogen was provided as KNO₃ at an initial concentration of 2.05 g L⁻¹ and chelated ferric iron was added at a concentration of 1.2 × 10⁻⁵ mol L⁻¹ on the ninth day. Under the same conditions in culture tubes (36 mm outer diameter), the biomass productivity was 2.8-fold greater than in the photobioreactor (0.125 m in diameter), but the lipid productivity was only 1.2-fold higher. Thus, the average low-light level in the photobioreactor actually increased the biomass specific lipid production compared to the culture tubes. A light-limited growth model closely agreed with the experimental profiles of biomass production, nitrogen consumption, and lipid production in the photobioreactor.     

Growth-inhibiting effects of <Emphasis Type="Italic">Paeonia lactiflora</Emphasis> root steam distillate constituents and structurally related compounds on human intestinal bacteria

The growth-inhibiting activities of Paeonia lactiflora (Paeoniaceae) root steam distillate constituents and structurally related compounds against nine harmful intestinal bacteria and eight lactic acid-producing bacteria were compared with those of two antibiotics, amoxicillin and tetracycline. Thymol, α-terpinolene, (−)-perilla alcohol and (1R)-(−)-myrtenol exhibited high to extremely high levels of growth inhibition of all the harmful bacteria, whereas thymol and α-terpinolene (except for Lactobacillus casei ATCC 393) inhibited the growth of all the beneficial bacteria (MIC, both 0.08–0.62 mg mL⁻¹). Tetracycline and amoxicillin exhibited extremely high level of growth inhibition of all the test bacteria (MIC, <0.00002–0.001 mg mL⁻¹). 1,8-Cineole, geraniol, (−)-borneol, (1S,2S,5S)-(−)-myrtanol, nerol, (S)-(−)-β-citronellol and (±)-lavandulol also exhibited inhibitory activity but with differing specificity and levels of activity. Structure–activity relationship indicates that structural characteristics, such as geometric isomerism, degrees of saturation, types of functional groups and types of carbon skeleton, appear to play a role in determining the growth-inhibiting activity of monoterpenoids. Global efforts to reduce the level of antibiotics justify further studies on naturally occurring P. lactiflora root-derived materials as potential preventive agents against various diseases caused by harmful intestinal bacteria such as clostridia.     

Occurrence of itraconazole-tolerant micromycetes in the soil and food products

Unexpected pathogens from the environment represent considerable risk for humans with impaired health. We examined the occurrence of itraconazole tolerant micromycetes in soil and in maize products. Five concentrations of itraconazole (2.5–12.5 μg/mL) selected according to known treatment schedules for human patients were incorporated into Sabouraud agar with chloramphenicol and Rose Bengal and diluted samples were inoculated onto the agar surface. After 7-d growth at 22°C colonies ofAlternaria sp.,Aspergillus clavatus. A. glaucus group,A. flavus. A, fumigatus, A. niger group,A. ochraceus group,A. ochraceus, Chœtomium sp.,Cladosporium cladosporioides. Cylindrocarpon sp.Doratomyces sp.,Fusarium sp.,F. moniliforme. F. oxysporum. F. solani, F. subglutinans. Marianaea elegans, Mortierella sp.,Mucor sp.,Myrothecium sp.,Penicillium sp.,Rhizopus sp.,Scopulariopsis brevicaulis. Sepedonium sp.,Stachybotrys chartarum. Stemphylium sp.,Torula humicola andTrichoderma viride were isolated.     

Chemoenzymatic resolution of racemic Wieland–Miescher and Hajos–Parrish ketones

Enantiospecific microbial reduction of bicyclic ketones was described. Racemic Wieland–Miescher (1) and Hajos–Parrish (2) ketones were used as substrates. In a 4-h biotransformation of Hajos–Parrish ketone (2) using the strain of Didymosphaeria igniaria an optically pure ketone (R)-2 was obtained, whereas the (S)-2 ketone underwent reduction to (4aS,5S)-4 alcohol with 100% of enantiomeric excess and with over 60% of diastereoisomeric excess. Jones oxidation of the alcohol obtained in the biotransformation gave an optically pure ketone (S)-2. Enzymatic system of Coryneum betulinum reduced the (R)-2 ketone to (4aR,5S)-4 alcohol with a high enantiomerical purity in a 6-day reaction. Wieland-Miescher (1) ketone was transformed by these microorganisms in an analogous way, but the reaction times were longer.