Peiyuan Yao

Efficient Biosynthesis of Ethyl (R)‐3‐Hydroxyglutarate through a One‐Pot Bienzymatic Cascade of Halohydrin Dehalogenase and Nitrilase

An effective one‐pot bienzymatic synthesis of ethyl (R)‐3‐hydroxyglutarate (EHG) from ethyl (S)‐4‐chloro‐3‐hydroxybutyrate (ECHB) was achieved by using recombinant Escherichia coli cells expressing separately or co‐expressing a mutant halohydrin dehalogenase gene from Agrobacterium radiobacter AD1 and a nitrilase gene from Arabidopsis thaliana. The activity of nitrilase was inhibited by high concentration of ECHB and NaCN. Consequently, the one‐pot one‐step process was implemented by fed‐batch of ECHB and NaCN with high accumulative product concentration (up to 0.9 mol L−1). The biotransformation of ECHB to EHG was successfully achieved at 1.2 mol L−1 substrate concentration by a one‐pot two‐step process. As such, this one‐pot bienzymatic transformation should be useful in synthesizing these important optical pure β‐hydroxycarboxylic acids.

Synthesis of α,β-unsaturated esters via a chemo-enzymatic chain elongation approach by combining carboxylic acid reduction and Wittig reaction.

Summary α,β-Unsaturated esters are versatile building blocks for organic synthesis and of significant importance for industrial applications. A great variety of synthetic methods have been developed, and quite a number of them use aldehydes as precursors. Herein we report a chemo-enzymatic chain elongation approach to access α,β-unsaturated esters by combining an enzymatic carboxylic acid reduction and Wittig reaction. Recently, we have found that Mycobacterium sp. was able to reduce phenylacetic acid (1a) to 2-phenyl-1-ethanol (1c) and two sequences in the Mycobacterium sp. genome had high identity with the carboxylic acid reductase (CAR) gene from Nocardia iowensis. These two putative CAR genes were cloned, overexpressed in E. coli and one of two proteins could reduce 1a. The recombinant CAR was purified and characterized. The enzyme exhibited high activity toward a variety of aromatic and aliphatic carboxylic acids, including ibuprofen. The Mycobacterium CAR catalyzed carboxylic acid reduction to give aldehydes, followed by a Wittig reaction to afford the products α,β-unsaturated esters with extension of two carbon atoms, demonstrating a new chemo-enzymatic method for the synthesis of these important compounds.

Enzymatic Synthesis of a Key Intermediate for Rosuvastatin by Nitrilase-Catalyzed Hydrolysis of Ethyl (R)-4-Cyano-3-hydroxybutyate at High Substrate Concentration

An enzymatic method for the synthesis of ethyl (R)‐3‐hydroxyglutarate from ethyl (R)‐4‐cyano‐3‐hydroxybutyate was developed by using free and immobilized recombinant Escherichia coli BL21(DE3)pLysS harboring a nitrilase gene from Arabidopsis thaliana (AtNIT2). The hydrolysis of ethyl (R)‐4‐cyano‐3‐hydroxybutyate proceeded with the freely suspended cells of the biocatalyst under the optimized conditions of 1.5 mol L−1 (235.5 g L−1) substrate concentration and 6.0 wt % loading of wet cells at pH 8.0 and 25 °C, with 100 % conversion obtained in 4.5 h. Furthermore, immobilization of the whole cells enhanced their substrate tolerance, stability, and reusability. Under the optimized conditions (100 mmol L−1 tris(hydroxymethyl)aminomethane hydrochloride buffer, pH 8.0, 25 °C), the immobilized biocatalyst could be reused for up to 16 batches, with a biocatalyst productivity of 55.6 g gwet cells−1 and a space‐time productivity of 625.5 g L−1 d−1. These results demonstrated that the immobilized whole cells might be used as a biocatalyst in the industrial production of ethyl (R)‐3‐hydroxyglutarate, a key intermediate for the synthesis of rosuvastatin.

New product identification in the sterol metabolism by an industrial strain Mycobacterium neoaurum NRRL B-3805

&NA; Mycobacterium neoaurum NRRL B‐3805 metabolizes sterols to produce androst‐4‐en‐3,17‐dione (AD) as the main product, and androsta‐1,4‐dien‐3,17‐dione, 9&agr;‐hydroxy androst‐4‐en‐3,17‐dione and 22‐hydroxy‐23,24‐bisnorchol‐4‐en‐3‐one have been identified as by‐products. In this study, a new by‐product was isolated from the metabolites of sterols and identified as methyl 3‐oxo‐23,24‐bisnorchol‐4‐en‐22‐oate (BNC methyl ester), which was proposed to be produced via the esterification of BNC catalyzed by an O‐methyltransferase using S‐adenosyl‐L‐methionine as the methyl group donor. These results might open a new dimension for improvement of the efficiency of microbial AD production by eliminating this by‐product via genetic manipulation of the strain.

Biochemical characterization and substrate profiling of a reversible 2,3-dihydroxybenzoic acid decarboxylase for biocatalytic Kolbe-Schmitt reaction.

Reversible benzoic acid decarboxylases are versatile biocatalysts by taking advantage of both decarboxylation and carboxylation reactions, especially for the biocatalytic Kolbe-Schmitt reaction. In the course of developing a benzoic acid decarboxylase tool-box, a putative benzoic acid decarboxylase gene from Fusarium oxysporum was heterologously over-expressed in Escherichia coli, the recombinant protein was purified and characterized. The purified enzyme exhibited relatively high catalytic efficiencies for the decarboxylation of 2, 3-dihydroxybenzoic acid and carboxylation of catechol (kcat/Km = 2.03 × 102 and 1.88 mM-1 min-1, respectively), and thus characterized as 2, 3-dihydroxybenzoic acid decarboxylase (2, 3-DHBD_Fo). The enzyme also catalyzed the decarboxylation of various substituted salicylic acids with different groups at varied positions except 5-position and the carboxylation of phenol and the substituted phenols. In a preparative reaction, catechol was carboxylated into 2, 3-dihydroxybenoic acid with 95% conversion by adding dodecyldimethylbenzylammonium chloride into the reaction system, and the product was isolated in 72% yield. These results demonstrate that 2, 3-DHBD_Fo is a valuable addition to the benzoic acid decarboxylase tool-box with potential practical applications.

Accessing d-Valine Synthesis by Improved Variants of Bacterial Cyclohexylamine Oxidase

Chemoenzymatic deracemization was applied to prepare d‐valine from racemic valine ethyl ester or l‐valine ethyl ester in high yield (up to 95 %) with excellent optical purity (>99 % ee) by employing a newly evolved cyclohexylamine oxidase (CHAO) variant Y321I/M226T exhibiting catalytic efficiency that was 30 times higher than that of the wildtype CHAO. Interestingly, CHAO and its variants showed opposite enantioselectivity for valine ethyl ester and phenylalanine ethyl ester.

Manipulating the stereoselectivity of a thermostable alcohol dehydrogenase by directed evolution for efficient asymmetric synthesis of arylpropanols

Abstract Chiral arylpropanols are valuable components in important pharmaceuticals and fragrances, which is the motivation for previous attempts to prepare these building blocks enantioselectively in asymmetric processes using either enzymes or transition metal catalysts. Thus far, enzymes used in kinetic resolution proved to be best, but several problems prevented ecologically and economically viable processes from being developed. In the present study, directed evolution was applied to the thermostable alcohol dehydrogenase TbSADH in the successful quest to obtain mutants that are effective in the dynamic reductive kinetic resolution (DYRKR) of racemic arylpropanals. Using rac-2-phenyl-1-propanal in a model reaction, (S)- and (R)-selective mutants were evolved which catalyzed DYRKR of this racemic substrate with formation of the respective (S)- and (R)-alcohols in essentially enantiomerically pure form. This was achieved on the basis of an unconventional form of iterative saturation mutagenesis (ISM) at randomization sites lining the binding pocket using a reduced amino acid alphabet. The best mutants were also effective in the DYRKR of several other structurally related racemic aldehydes.

Recent progress and challenge in the research of chemo-enzymatic transformation

Chemo-enzymatic transformation can give full play to the advantages of chemical synthesis and biosynthesis, respectively, and better meet the demands for efficient synthetic technology to address the issues of health, environment, energy, and security that we face today. The recent progress of chemo-enzymatic transformation has been reviewed in this paper based on the modes of transformations, and the characteristics and advantages of each modes have been discussed. Furthermore, the challenges and prospects in this field have also been discussed.