Biotransformation of electron-poor alkenes by yeasts: asymmetric reduction of (4S)-(+)-carvone by yeast enoate reductases

Within the framework of a large-scale screening carried out on 146 yeasts of environmental origin, 16 strains (11% of the total) exhibited the ability to biotransform (4S)-(+)-carvone. Such positive yeasts, belonging to 14 species of 6 genera (Candida, Cryptococcus, Hanseniaspora, Kluyveromyces, Pichia andSaccharomyces), were thus used under different physiological state (growing, resting and lyophilised cells). Yields (expressed as% of biotransformation) varied from 0.14 to 30.04%, in dependence of both the strain and the physiological state of the cells. Products obtained from reduction of (4S)-(+)-carvone were1S,4S- and 1R,4S-dihydrocarvone, (1S,2S,4S)-, (1S,2R,4S)- and (1R,2S,4S)-dihydrocarveol. Only traces of (1R,2R,4S)-dihydrocarveol were observed in a few strains. As far as the stereoselectivity of the biocatalysis, with the sole exception of a few strains, the use of yeasts determined the prevalent accumulation of 1S,4S-isomers [(1S,4S)-dihydrocarvone + (1S,2S,4S)-dihydrocarveol + (1S,2R,4S)-dihydrocarveol].The addition of glucose (acting as auxiliary substrate for cofactor-recycling system) to lyophilised yeast cells determined a considerable increase of biocatalytic activity: in particular, two strains showed a surprising increase of the% of biotransformation of (4S)-(+)-carvone (to values >98%)

Biotransformation of Acyclic Monoterpenoids by Debaryomyces sp., Kluyveromyces sp. and Pichia sp. Strains of Environmental Origin

Sixty yeast strains, which belong to 32 species of the genera Debaryomyces, Kluyveromyces and Pichia, and which were isolated from plant-, soil- or insect-associated habitats, were screened for their ability to biotransform the acyclic monoterpenes geraniol and nerol. The aptitude to convert both compounds (from 2.6 to 30.6 and from 2.7 to 29.1 %/g cell DW, = dry weight), resp.) was apparently a broad distributed character in such yeasts. Depending upon the substrate used, the production of linalool, alfa-terpineol, β-myrcene, D-limonene, (E)-β-ocimene, (Z)-β-ocimene or carene was observed. Linalool was the main product obtained from geraniol, whereas linalool and alfa-terpineol were the main products obtained through the conversion of nerol. Yet, differently from nerol, the aptitude to exhibit high bioconversion yields of geraniol to linalool was an apparently genus-related character, whereas the ability to produce other monoterpenes was a both genus- and habitat-related character. The possible pathways of bioconversion of geraniol or nerol to their derivative were proposed/discussed

Psychrophilics yeasts: new biocatalytic agents for enoate reductase activities

Owing to widespread diffusion of areas characterized by permanent low temperatures (e.g. deep-sea waters, mountains and polar regions), psychrophilic microorganisms have the largest terrestrial distribution. Adaptation of microbial life at low temperatures requires some changes, mainly at the level of cell membrane components (including transport systems), proteins and nucleic acids.Psychrophilic organisms produce enzymes functioning in coldenvironment and simply denaturing at high temperature. Suchcold-active enzymes have a more flexible structure to undergo the conformational changes necessary for catalysis with a lower energy demand. Owing to their high catalytic activity at low temperatures, low thermostability and unusual specificities, cold-active enzymes offer novel opportunities for biotechnological exploitation.The enormous potential of biocatalysts for the production of enantiopure products of industrial interest remains to beexploited, especially in the area of enantioselective alkene reductions, due to their marked regio- and stereo-selectivity. One of the emerging reductive chemistry is the asymmetric reductionof alpha,beta-unsaturated carbonyl compounds through microbialwhole-cells rich in enoate reductase (ER), a flavin-dependentenzyme belonging to the ‘old yellow enzyme’ (OYE) family.Even though massive screenings for yeasts as reducing catalystshave been carried out so far, there is still a need for new biocatalysts able to performing highly stereoselective reactions. Consequently, the potential of yeast biocatalysis appears to be still far from being fully explored. In this study we report the results of a screening on facultative psychrophilic yeasts (belonging to the species Cryptococcus gilvescens and Cryptococcus gastricus) for new cold-adapted enoate reductases using (4S)-(+)-carvone as a model substrate. The preliminary results obtained using growing cells demonstrate that the ERs activities are apparently expressed to a greater extent inpsychrophilic yeasts with respect to the mesophilic ones. In particular, C. gastricus exhibited the better biocatalytic performances.The results obtained by using other catalytic forms (resting orlyophilized cells) will be presented