Asymmetric reduction of α, β-unsaturated ketone to (R) allylic alcohol byCandida chilensis

Abstract

A pilot scale whole cell process was developed for the enantioselective 1,2-reduction of prochiral alpha,beta-unsaturated ketone to (R) allylic alcohol using Candida chilensis. Initial development showed high enantiomeric excess (EE > 95%) but low product yield (10%). Process development, using a combination of statistically designed screening and optimization experiments, improved the desired alcohol yield to 90%. The fermentation growth stage, particularly medium composition and growth pH, had a significant impact on the bioconversion while process characterization identified diverse challenges including the presence of multiple enzymes, substrate/product toxicity, and biphasic cellular morphology. Manipulating the fermentation media allowed control of the whole cell morphology to a predominantly unicellular broth, away from the viscous pseudohyphae, which were detrimental to the bioconversion. The activity of a competing enzyme, which produced the undesired saturated ketone and (R) saturated alcohol, was minimized to < or =5% by controlling the reaction pH, temperature, substrate concentration, and biomass level. Despite the toxicity effects limiting the volumetric productivity, a reproducible and scaleable process was demonstrated at pilot scale with high enantioselectivity (EE > 95%) and overall yield greater than 80%. This was the preferred route compared to a partially purified process using ultra centrifugation, which led to improved volumetric productivity but reduced yield (g/day). The whole cell approach proved to be a valuable alternative to chemical reduction routes, as an intermediate step for the asymmetric synthesis of an integrin receptor antagonist for the inhibition of bone resorption and treatment of osteoporosis.

Comparison of Three Enoate Reductases and their Potential Use for Biotransformations

“…Their development towards large-scale use would be improved with the establishment of a high-throughput screening system, which already exists for ketoreductases. [36] Regarding the specificity of these enzymes, tests with selected products of the ER reaction revealed that, while the carbonyl group was not reduced, additionally present double bonds could be further reduced and form unwanted side products.…”

“…The latter employ the same cofactor, NAD(P)H, to reduce the carbonyl function, which can lower selectivity and produce additional impurities. [36,37] The use of isolated enzymes can circumvent this problem but still requires a cofactor regeneration system. Importantly, both ER and the regenerating enzyme have to be active at the same pH range.…”

“…Another advantage is that isolated enzyme systems show higher productivity and are less tedious to optimize than wholecell systems. [36,37] Lastly, the expression and purification of his-tagged, isolated enzymes is straightforward, does not seem to influence activity very much, and allows easy assessment of new activities and reactions without interference from side reactions.…”

Characterization of an Allylic/Benzyl Alcohol Dehydrogenase from Yokenella sp. Strain WZY002, an Organism Potentially Useful for the Synthesis of α,β-Unsaturated Alcohols from Allylic Aldehydes and Ketones

“…Even so, the whole-cell catalyzed reduction of ␣,␤-unsaturated carbonyl compounds can be complex and hampered by competing enzymatic reactions (8). Thus, in the asymmetric bioreduction of citral to the ␣,␤-saturated aldehyde citronellal, the undesirable by-products nerol, geraniol, and citronellol were formed due to the action of competing alcohol dehydrogenases and citral lyase activity (9); in stereoselective biocatalytic reduction of ␣-ionone by Glomerella cingulata, (6S,9R)-␣-ionol was produced, some of which was subsequently hydrogenated by enoate reductase to form (6S,9R)-7,8-dyhidro-␣-ionol (10); and with use of ␣,␤-unsaturated aldehydes as the substrate, the aldehyde oxidation catalyzed by aldehyde dehydrogenase usually led to the formation of undesirable ␣,␤-unsaturated carboxylic acids (11,12).…”

Purification, Characterization, Gene Cloning, and Expression of a Novel Alcohol Dehydrogenase with Anti-Prelog Stereospecificity from Candida parapsilosis

“…They display different physical and enzymatic properties, show a wide variety of substrate specificities, and are mainly classified into three superfamilies, zinc-dependent alcohol dehydrogenases, short-chain dehydrogenase/reductases (SDRs), and aldo-keto reductases, based on their catalytic properties and sequence information (8,14,32). Among the various enzyme sources, Candida species are attractive as highly stereospecific oxidoreductase donors (4,13,15,16,25,30). In many cases, the stereospecific oxidoreductases from strains of the genus Candida are dissimilar in structure and classified in different superfamilies (15,16,38).…”