Highly efficient synthesis of chiral alcohols with a novel NADH-dependent reductase from Streptomyces coelicolor

Wang, Limin; Li, Chun-Xiu; Ni, Yan; Zhang, Jie; Liu, Xiang; Xu, Jian‐He

doi:10.1016/j.biortech.2011.04.046

Cited by 135 publications

(54 citation statements)

References 35 publications

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“…The selection of reported enzymes as probe and the choice of candidate proteins in database with moderate identity are the two key factors for the successful identification of new enzymes. The sequence of several known ethyl 4-chloro-3-oxobutanate (COBE) reductases was regarded as probes to search for novel enzymes in GenBank, resulting in an excellent reductase from Streptomyces coelicolor out of ten candidates for the synthesis of optically pure ethyl (S)-4-chloro-3-hydroxybutanoate, a chiral building blocks for HMG-CoA reductase inhibitors, with a stunning high productivity of 609 g L −1 day −1 [49].…”

Section: Genome Database Miningmentioning

confidence: 99%

Bioreductive preparation of ACE inhibitors precursor (R)-2-hydroxy-4-phenylbutanoate esters: Recent advances and future perspectives

2015

Bioresour. Bioprocess.

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Optically active (R)-2-hydroxy-4-phenylbutanoate esters ((R)-HPBE) are key precursors for the production of angiotension-converting enzyme (ACE) inhibitors, which are important prescriptive drugs for preventing the formation of angiotensin II and lowering the blood pressure. The biocatalytic asymmetric reduction of ethyl 2-oxo-4-phenylbutanoate (OPBE) to (R)-HPBE with carbonyl reductases has several advantageous attributes, including high enantioselectivity, mild reaction condition, high catalytic efficiency, and environmental benignity. An increasing number of OPBE reductases have been discovered owing to the drastic achievements in genomics, screening and evolution technologies, and process engineering. The potential of (R)-HPBE production process has also been intensively evaluated. This review covers recent progress on the bioreductive preparation of (R)-HPBE, especially on various screening approaches for the identification of OPBE reductases and their characterization.

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Section: Genome Database Miningmentioning

confidence: 99%

Bioreductive preparation of ACE inhibitors precursor (R)-2-hydroxy-4-phenylbutanoate esters: Recent advances and future perspectives

2015

Bioresour. Bioprocess.

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“…Gene mining, a method through which homologous proteins are searched from a gene database using known enzymes as the template, is a promising approach (Wang et al 2011). Through this method, various carbonyl reductases and AKRs exhibiting catalytic activities towards various carbonyl substrates have been discovered from the genome of Candida parapsilosis (Guo et al 2014;Nie et al 2011).…”

Section: Introductionmentioning

confidence: 99%

Gene mining-based identification of aldo–keto reductases for highly stereoselective reduction of bulky ketones

Liang

Nie

et al. 2018

Bioresour. Bioprocess.

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Background: Aldo-keto reductase (AKR) or alcohol dehydrogenases (ADH)-mediated stereoselective reduction of prochiral carbonyl compounds is an efficient way of preparing single enantiomers of chiral alcohols. However, steric hindrance of substrate affects the catalytic performance of enzymes. The present study aims to discover and identify AKRs/ADHs capable of catalyzing highly stereoselective reduction of sterically hindered ketones. Results:Five AKRs from different microorganisms (CaCR, ScCR, KmCR, CPR-C1, and CPR-C2) were identified through gene mining, and overexpressed in recombinant Escherichia coli BL21(DE3). The specific activity and stereoselectivity of the AKRs were further evaluated towards various ketoesters and heterocyclic ketones, which are sterically bulky and are valuable in industrial applications. Each purified enzyme exhibited catalytic activity to one or more of the tested substrates. Among the enzymes, ScCR showed a broader substrate spectrum compared to the others. Regarding K m values related to substrate association, we also provided insights into the specificity and preference of certain enzymes. Consequently, enantiopure (R)-methyl mandelate, ethyl (R)-mandelate, ethyl (R)-2-hydroxy-4-phenylbutyrate, and (S)-N-benzyl-3-pyrrolidinol (> 99%e.e.) were obtained through the identified AKRs. Conclusion:The stereospecific AKRs were obtained through gene mining, which possesses the potential for application in the preparation of important optically active alcohols.

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“…In our previous work (16), a novel NADH-dependent reductase, Streptomyces coelicolor CR1 (ScCR1), was discovered by genome data mining, which could efficiently convert COBE to (S)-CHBE with high enantiomeric excess (ee) (Ͼ99%) using 2-propanol as a cosubstrate (Fig. 1).…”

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confidence: 99%

“…By process optimization and scale-up, the specific production could reach levels as high as 36.8 g product/g biocatalyst using lyophilized recombinant Escherichia coli cells expressing ScCR1 as biocatalyst, the highest value reported so far (17). However, the specific activity of ScCR1 toward COBE was relatively low (38.8 U/mg protein); therefore, high biocatalyst loading (e.g., 20 g/liter) was required, and significant emulsion was formed during downstream processing, which would in turn increase the cost of the bioprocess (16). Meanwhile, ScCR1 shows a thermostability (T 50 15 , where T 50 15 is defined as the temperature at which 50% of initial enzyme activity is lost following a heat treatment for 15 min) of 47.4°C and a substrate tolerance (C 50 15 , where C 50 15 is defined as the concentration at which 50% of initial enzyme activity is lost following incubation for 15 min) of 34 mM.…”

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confidence: 99%

Engineering Streptomyces coelicolor Carbonyl Reductase for Efficient Atorvastatin Precursor Synthesis

Zhang

Kong

et al. 2017

Appl Environ Microbiol

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Streptomyces coelicolor CR1 (ScCR1) has been shown to be a promising biocatalyst for the synthesis of an atorvastatin precursor, ethyl-(S)-4-chloro-3-hydroxybutyrate [(S)-CHBE]. However, limitations of ScCR1 observed for practical application include low activity and poor stability. In this work, protein engineering was employed to improve the catalytic efficiency and stability of ScCR1. First, the crystal structure of ScCR1 complexed with NADH and cosubstrate 2-propanol was solved, and the specific activity of ScCR1 was increased from 38.8 U/mg to 168 U/mg (ScCR1 I158V/P168S ) by structure-guided engineering. Second, directed evolution was performed to improve the stability using ScCR1 I158V/P168S as a template, affording a triple mutant, ScCR1 A60T/I158V/P168S , whose thermostability (T 50 15 , defined as the temperature at which 50% of initial enzyme activity is lost following a heat treatment for 15 min) and substrate tolerance (C 50 15 , defined as the concentration at which 50% of initial enzyme activity is lost following incubation for 15 min) were 6.2°C and 4.7-fold higher than those of the wild-type enzyme. Interestingly, the specific activity of the triple mutant was further increased to 260 U/mg. Protein modeling and docking analysis shed light on the origin of the improved activity and stability. In the asymmetric reduction of ethyl-4-chloro-3-oxobutyrate (COBE) on a 300-ml scale, 100 g/liter COBE could be completely converted by only 2 g/liter of lyophilized ScCR1 A60T/I158V/P168S within 9 h, affording an excellent enantiomeric excess (ee) of Ͼ99% and a space-time yield of 255 g liter Ϫ1 day Ϫ1 . These results suggest high efficiency of the protein engineering strategy and good potential of the resulting variant for efficient synthesis of the atorvastatin precursor.IMPORTANCE Application of the carbonyl reductase ScCR1 in asymmetrically synthesizing (S)-CHBE, a key precursor for the blockbuster drug Lipitor, from COBE has been hindered by its low catalytic activity and poor thermostability and substrate tolerance. In this work, protein engineering was employed to improve the catalytic efficiency and stability of ScCR1. The catalytic efficiency, thermostability, and substrate tolerance of ScCR1 were significantly improved by structure-guided engineering and directed evolution. The engineered ScCR1 may serve as a promising biocatalyst for the biosynthesis of (S)-CHBE, and the protein engineering strategy adopted in this work would serve as a useful approach for future engineering of other reductases toward potential application in organic synthesis.

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Highly efficient synthesis of chiral alcohols with a novel NADH-dependent reductase from Streptomyces coelicolor

Cited by 135 publications

References 35 publications

Bioreductive preparation of ACE inhibitors precursor (R)-2-hydroxy-4-phenylbutanoate esters: Recent advances and future perspectives

Bioreductive preparation of ACE inhibitors precursor (R)-2-hydroxy-4-phenylbutanoate esters: Recent advances and future perspectives

Gene mining-based identification of aldo–keto reductases for highly stereoselective reduction of bulky ketones

Engineering Streptomyces coelicolor Carbonyl Reductase for Efficient Atorvastatin Precursor Synthesis

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