A strategy to identify a ketoreductase that preferentially synthesizes pharmaceutically relevant (S)-alcohols using whole-cell biotransformation

Saiful F Haq,Sunilkumar Sukumaran,S Ramaswamy,Kannan Thanukrishnan,Nagakumar Bharatham,Santanu Datta,Shalaka Samant,Nainesh Katagihallimath,Imran Hassan,Anirudh P Shanbhag,Subbulakshmi Karthikeyan,Abhishek Ashok

doi:10.1186/s12934-018-1036-2

Abstract

IntroductionChemical industries are constantly in search of an expeditious and environmentally benign method for producing chiral synthons. Ketoreductases have been used as catalysts for enantioselective conversion of desired prochiral ketones to their corresponding alcohol. We chose reported promiscuous ketoreductases belonging to different protein families and expressed them in E. coli to evaluate their ability as whole-cell catalysts for obtaining chiral alcohol intermediates of pharmaceutical importance. Apart from establishing a method to produce high value (S)-specific alcohols that have not been evaluated before, we propose an in silico analysis procedure to predict product chirality.ResultsSix enzymes originating from Sulfolobus sulfotaricus, Zygosaccharomyces rouxii, Hansenula polymorpha, Corynebacterium sp. ST-10, Synechococcus sp. PCC 7942 and Bacillus sp. ECU0013 with reported efficient activity for dissimilar substrates are compared here to arrive at an optimal enzyme for the method. Whole–cell catalysis of ketone intermediates for drugs like Aprepitant, Sitagliptin and Dolastatin using E. coli over-expressing these enzymes yielded (S)-specific chiral alcohols. We explain this chiral specificity for the best-performing enzyme, i.e., Z. rouxii ketoreductase using in silico modelling and MD simulations. This rationale was applied to five additional ketones that are used in the synthesis of Crizotinib, MA-20565 (an antifungal agent), Sulopenem, Rivastigmine, Talampanel and Barnidipine and predicted the yield of (S) enantiomers. Experimental evaluation matched the in silico analysis wherein ~ 95% (S)-specific alcohol with a chemical yield of 23–79% was obtained through biotransformation. Further, the cofactor re-cycling was optimized by switching the carbon source from glucose to sorbitol that improved the chemical yield to 85–99%.ConclusionsHere, we present a strategy to synthesize pharmaceutically relevant chiral alcohols by ketoreductases using a cofactor balanced whole-cell catalysis scheme that is useful for the industry. Based on the results obtained in these trials, Zygosaccharomyces rouxii ketoreductase was identified as a proficient enzyme to obtain (S)-specific alcohols from their respective ketones. The whole–cell catalyst when combined with nutrient modulation of using sorbitol as a carbon source helped obtain high enantiomeric and chemical yield.

Highlights

Chemical industries are constantly in search of an expeditious and environmentally benign method for producing chiral synthons
Whole–cell catalysis of ketone intermediates for drugs like Aprepitant, Sitagliptin and Dolastatin using E. coli over-expressing these enzymes yielded (S)-specific chiral alcohols. We explain this chiral specificity for the best-performing enzyme, i.e., Z. rouxii ketoreductase using in silico modelling and molecular dynamics (MD) simulations
Here, we present a strategy to synthesize pharmaceutically relevant chiral alcohols by ketoreductases using a cofactor balanced whole-cell catalysis scheme that is useful for the industry

Summary

Introduction

Chemical industries are constantly in search of an expeditious and environmentally benign method for producing chiral synthons. In contrast to chemical methods that often do not comply with the principles of green chemistry, biocatalysis offers numerous advantages, such as mild and environmentally benign conditions and remarkable chemo-, regio- and stereoselectivity [4, 5]. It often facilitates the circumvention of arduous syntheses routes that require multiple protection and deprotection steps [6, 7]. The use of whole-cells offers the advantage of being a simple and low-cost catalyst preparation compared to purified enzymes that are generally expensive due to the need of protein purification, their diminished activity under process conditions, insufficient stability and vulnerability to the substrate and product inhibitions [9]. To identify an efficient enzyme for the process of ketone reduction, we considered two super families of ketoreductases that can convert a broad range of prochiral ketones namely, the medium-chain (MDR) and shortchain (SDR) dehydrogenases/reductases

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