Gao-Wei Zheng

New opportunities for biocatalysis: driving the synthesis of chiral chemicals.

Various biocatalytic methods have been developed for the synthesis of chiral chemicals, which have made their synthesis more environmentally friendly and product-specific. New opportunities for biocatalysis, including new scientific developments in genomics and protein engineering technologies, novel process developments and the increased availability of useful enzymes, offer many possibilities for the manufacture of new chiral compounds and deliver greener and economically competitive processes. In this review, new opportunities for biocatalysis in the preparation of chiral molecules are outlined and highlighted.

Efficient Synthesis of a Chiral Precursor for Angiotensin-Converting Enzyme (ACE) Inhibitors in High Space-Time Yield by a New Reductase without External Cofactors

A new reductase, CgKR2, with the ability to reduce ethyl 2-oxo-4-phenylbutyrate (OPBE) to ethyl (R)-2-hydroxy-4-phenylbutyrate ((R)-HPBE), an important chiral precursor for angiotensin-converting enzyme (ACE) inhibitors, was discovered. For the first time, (R)-HPBE with >99% ee was produced via bioreduction of OPBE at 1 M without external addition of cofactors. The space-time yield (700 g·L(-1)·d(-1)) was 27 times higher than the highest record.

An efficient bioprocess for enzymatic production of l-menthol with high ratio of substrate to catalyst using whole cells of recombinant E. coli

A gene encoding an esterase of Bacillus subtilis ECU0554 previously isolated from soil was cloned and overexpressed in Escherichia coli BL21. The recombinant esterase (recBsE) showed the best enantioselectivity (E>100) towards DL-menthyl acetate, in contrast to DL-menthyl esters propionate and butyrate. A high ratio of substrate to catalyst (S/C-ratio, ≥50) was achieved in the kinetic resolution of DL-menthyl acetate by using whole cells of recombinant E. coli BL21. Some key parameters of the biocatalytic process, including amount of cosolvent, catalyst loading and substrate loading, were optimized. Compared with the process catalyzed by wild-type whole cells of B. subtilis ECU0554, the second-generation bioprocess using whole cells of recombinant E. coli BL21 afforded a 40-fold improvement in S/C-ratio and a 75-fold improvement in the volumetric productivity per biocatalyst loading. Moreover, the substrate loading was increased up to 200 g L(-1) (∼1 M), the biocatalyst loading was reduced to 2.5 g L(-1) and the space-time yield was improved from 54 g L(-1) d(-1) to 202 g L(-1) d(-1).

Highly selective and controllable synthesis of arylhydroxylamines by the reduction of nitroarenes with an electron-withdrawing group using a new nitroreductase BaNTR1

A new bacterial nitroreductase has been identified and used as a biocatalyst for the controllable reduction of a variety of nitroarenes with an electron-withdrawing group to the corresponding N-arylhydroxylamines under mild reaction conditions with excellent selectivity (>99%). This method therefore represents a green and efficient method for the synthesis of arylhydroxylamines.

Engineering of a novel carbonyl reductase with coenzyme regeneration in E. coli for efficient biosynthesis of enantiopure chiral alcohols

The novel anti-Prelog stereospecific carbonyl reductase from Acetobacter sp. CCTCC M209061 was successfully expressed in E. coli combined with glucose dehydrogenase (GDH) to construct an efficient whole-cell biocatalyst with coenzyme NADH regeneration. The enzymatic activity of GAcCR (AcCR with a GST tag) reached 304.9U/g-dcw, even 9 folds higher than that of wild strain, and the activity of GDH for NADH regeneration recorded 46.0U/mg-protein in the recombinant E. coli. As a whole-cell biocatalyst, the recombinant E. coli BL21(DE3)pLysS (pETDuet-gaccr-gdh) possessed a broad substrate spectrum for kinds of carbonyl compounds with encouraging yield and stereoselectivity. Besides, the asymmetric reduction of ethyl 4-chloroacetoacetate (COBE) to optically pure ethyl 4-chloro-3-hydroxybutyrate (CHBE) catalyzed by the whole-cell biocatalyst was systematically investigated. Under the optimal reaction conditions, the optical purity of CHBE was over 99% e.e. for (S)-enantiomer, and the initial rate and product yield reached 8.04μmol/min and 99.4%, respectively. Moreover, the space-time yield was almost 20 folds higher than that catalyzed by the wild strain. Therefore, a new, high efficiency biocatalyst for asymmetric reductions was constructed successfully, and the enantioselective reduction of prochiral compounds using the biocatalyst was a promising approach for obtaining enantiopure chiral alcohols.

A Novel (R)-Imine Reductase fromPaenibacillus lactisfor Asymmetric Reduction of 3 H-Indoles

A novel (R)‐imine reductase (PlRIR) from Paenibacillus lactis was heterologously overexpressed in Escherichia coli, purified and characterized. The purified PlRIR exhibited relatively high catalytic efficiency (kcat/Km=1.58 s−1 mm−1) towards 2,3,3‐trimethylindolenine. A panel of 3H‐indoles and 3H‐indole iodides were reduced by PlRIR to yield the corresponding products with good‐to‐excellent enantioselectivity (66–98 % ee). In addition, PlRIR also possesses good activities toward other types of imines such as pyrroline, tetrahydropyridine, and dihydroisoquinoline, indicating a reasonably broad substrate acceptance. In a 100 mg scale preparative reaction, 100 mm 2,3,3‐trimethylindolenine was converted efficiently to afford (R)‐2,3,3‐trimethylindoline with 96 % ee and 81 % yield.

Recent progress on deep eutectic solvents in biocatalysis

Deep eutectic solvents (DESs) are eutectic mixtures of salts and hydrogen bond donors with melting points low enough to be used as solvents. DESs have proved to be a good alternative to traditional organic solvents and ionic liquids (ILs) in many biocatalytic processes. Apart from the benign characteristics similar to those of ILs (e.g., low volatility, low inflammability and low melting point), DESs have their unique merits of easy preparation and low cost owing to their renewable and available raw materials. To better apply such solvents in green and sustainable chemistry, this review firstly describes some basic properties, mainly the toxicity and biodegradability of DESs. Secondly, it presents several valuable applications of DES as solvent/co-solvent in biocatalytic reactions, such as lipase-catalyzed transesterification and ester hydrolysis reactions. The roles, serving as extractive reagent for an enzymatic product and pretreatment solvent of enzymatic biomass hydrolysis, are also discussed. Further understanding how DESs affect biocatalytic reaction will facilitate the design of novel solvents and contribute to the discovery of new reactions in these solvents.

Green access to chiral Vince lactam in a buffer-free aqueous system using a newly identified substrate-tolerant (−)-γ-lactamase

A novel non-heme chloroperoxidase (SvGL) with promiscuous (−)-γ-lactamase activity towards Vince lactam was identified from Streptomyces viridochromogenes by genome data-mining. SvGL possesses high activity and excellent thermal stability and enantioselectivity. Furthermore, it is able to tolerate extremely high substrate concentrations (4.0 M, 436.5 g L−1). Using the newly discovered (−)-γ-lactamase as a biocatalyst, an efficient and environmentally benign process for the production of (+)-γ-lactam was developed. The process allowed an enantioselective resolution of 436.5 g L−1 racemic γ-lactam with only 0.2 g L−1 lyophilized cell-free extract, affording an extremely high substrate/catalyst ratio of 2183 (g g−1), a space-time yield of 458 g L−1 d−1, and a very low E factor (environmental factor) of 5.7 (kg waste per kg product) even when the process water is included.

Stepwise and combinatorial optimization of enantioselectivity for the asymmetric hydrolysis of 1-(3’,4’-methylenedioxyphenyl)ethyl acetate under use of a cold-adapted Bacillus amyloliquefaciens esterase

Optically pure 1-(3’,4’-methylenedioxyphenyl) ethanol is a key chiral intermediate for the synthesis of Steganacin and Salmeterol. A para-nitrobenzyl esterase cloned from Bacillus amyloliquefaciens (BAE) was employed to hydrolyze 1-(3’,4’-methylenedioxyphenyl) ethyl ester for the production of (R)-1-(3’,4’-methylenedioxyphenyl)ethanol. Initially, a moderate enantioselectivity (E = 35) only was obtained at 30°C. Some reaction conditions such as reaction temperature and additive approach were investigated in order to improve the enantioselectivity of the BAEcatalyzed reaction.. As a result, the enantioselectivity was improved significantly to 140 under addition of Tween-80 and a decreasing reaction temperature to 0°C. The result was confirmed in a decagram-scale preparative bioresolution also. The optimized enzymatic hydrolysis conditions provide a more effective process for the (R)-1-(3’,4’-methylenedioxyphenyl) ethanol bioproduction.

Identification of an Imine Reductase for Asymmetric Reduction of Bulky Dihydroisoquinolines

A new imine reductase from Stackebrandtia nassauensis (SnIR) was identified, which displayed over 25- to 1400-fold greater catalytic efficiency for 1-methyl-3,4-dihydroisoquinoline (1-Me DHIQ) compared to other imine reductases reported. Subsequently, an efficient SnIR-catalyzed process was developed by simply optimizing the amount of cosolvent, and up to 15 g L-1 1-Me DHIQ was converted completely without a feeding strategy. Furthermore, the reaction proceeded well for a panel of dihydroisoquinolines, affording the corresponding tetrahydroisoquinolines (mostly in S-configuration) in good yields (up to 81%) and with moderate to excellent enantioselectivities (up to 99% ee).