Cascade catalysis – strategies and challenges en route to preparative synthetic biology

Chiral sulfinyl compounds, sulfoxides, sulfoximines, sulfinamides, and other derivatives, play an important role in asymmetric synthesis as versatile auxiliaries, ligands, and catalysts. They are also recognized as pharmacophores found in already marketed and well-sold drugs (e.g., esomeprazole) and used in drug design. This review is devoted to the modern methods of preparation of sulfinyl derivatives in enantiopure or enantiomerically enriched form. Selected new approaches leading to racemic products for which the asymmetric variant can be developed in the future are mentioned as well.

Modern Stereoselective Synthesis of Chiral Sulfinyl Compounds.

Chiral organic sulfoxides (COSs) are important compounds that act as chiral auxiliaries in numerous asymmetric reactions and as intermediates in chiral drug synthesis. In addition to their optical resolution, stereoselective oxidation of sulfides can be used for COS production. This reaction is facilitated by oxygenases and peroxidases from various microbial resources. To meet the current demand for esomeprazole, a proton pump inhibitor used in the treatment of gastric-acid-related disorders, and the (S)-isomer of an organic sulfoxide compound, omeprazole, a successful biotechnological production method using a Baeyer-Villiger monooxygenase (BVMO), was developed. In this review, we summarize the recent advancements in COS production using biocatalysts, including enzyme identification, protein engineering, and process development.

Biotechnological production of chiral organic sulfoxides: current state and perspectives

External flavoprotein monooxygenases comprise a group of flavin-dependent oxidoreductases that catalyze the insertion of one atom of molecular oxygen into an organic substrate and the second atom is reduced to water. These enzymes are involved in a great number of metabolic pathways both in prokaryotes and eukaryotes. Flavoprotein monooxygenases have attracted the attention of researchers for several decades and the advent of recombinant DNA technology caused a great progress in the field. These enzymes are subjected to detailed biochemical and structural characterization and some of them are also regarded as appealing oxidative biocatalysts for the production of fine chemicals and valuable intermediates toward active pharmaceutical ingredients due to their high chemo-, stereo- and regioselectivity. Here, we review the most representative reactions catalyzed both in vivo and in vitro by prototype flavoprotein monooxygenases, highlighting the strategies employed to produce them recombinantly, to enhance the yield of soluble proteins, and to improve cofactor regeneration in order to obtain versatile biocatalysts. Although we describe the most outstanding features of flavoprotein monooxygenases, we mainly focus on enzymes that were cloned, expressed and used for biocatalysis during the last years.

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Flavoprotein monooxygenases for oxidative biocatalysis: recombinant expression in microbial hosts and applications

Cloning and characterization of a NADH-dependent aldo-keto reductase from a newly isolated Kluyveromyces lactis XP1461.

A novel aldo–keto reductase gene, Tm1743, from Thermotoga maritima was overexpressed in Escherichia coli. The enzyme displayed the highest activity at 90 °C and at pH 9. It retained 63 % of its activity after 15 h at 85 °C. The enzyme also could tolerate (up to 10 % v/v) acetonitrile, ethanol and 2-propanol with slightly increased activities. Methanol, DMSO and acetone decreased activity slightly. Furthermore, Tm1743 exhibited broad substrate specificity towards various keto esters, ketones and aldehydes, with relative activities ranging from 2 to 460 % compared to the control. Its optimum substrate, 2,2,2-trifluoroacetophenone, was asymmetrically reduced in a coupled NADPH-regeneration system with an enantioselectivity of 99.8 % and a conversion of 98 %.

Characterization of an aldo–keto reductase from Thermotoga maritima with high thermostability and a broad substrate spectrum

A formate dehydrogenase gene (fdh) originated from Candida boidinii was co-expressed in E. coli BL21 (DE3) with the cyclohexanone monooxygenase gene (chmo) cloned from Acinetobacter calcoaceticus NCIMB 9871. The co-expression system was then used as a whole-cell biocatalyst to synthesize chiral phenyl methyl sulfoxide (PMSO) from thioanisole (PMS) and the reaction conditions were investigated. When the initial concentration of PMS was 20 mM, the specific productivity of PMSO in this system was 2.07 μmol g−1 cw min−1 (cw: wet cell weight) and the ee value for the R-sulfoxide was 99 %. In contrast, when chmo was the only gene expressed in E. coli, the specific productivity of PMSO was 0.053 μmol g−1 cw min−1 with no exact enantioselectivity. Further determination of NADPH concentration in the whole-cell catalysts suggested that co-expression of fdh with chmo significantly improved NADPH supply. Thus, this whole-cell biocatalyst system is highly advantageous for the synthesis of optically pure R-sulfoxide.

Development of a whole-cell biocatalyst with NADPH regeneration system for biosulfoxidation

Recombinant heat resisting aldehyde ketoreductase gene, coding enzyme, vector, engineering bacteria and application

The invention discloses a penicillin G acylation enzyme mutant, and application thereof in synthesis of cephalosporin antibiotics. An amino acid sequence of the penicillin G acylation enzyme mutant is shown in SEQ ID NO.1; a nucleotide sequence of an encoding gene is shown in SEQ ID NO.2. The invention also provides application of the penicillin G acylation enzyme mutant in synthesis of cefaclor. The invention provides a novel penicillin acylation enzyme mutant. Compared with the wild penicillin acylation enzyme mutant, the penicillin G acylation enzyme mutant has higher activity in synthesis of cephalosporin antibiotics, such as cefprozil, cefaclor or cefadroxil; especially when the penicillin G acylation enzyme mutant is used for catalyzing 7-ACCA and phenylglycine methyl ester to synthesize the cefaclor, the vitality is improved to 29.8U/mg from 1.2U/mg in the past, the synthesis and hydrolysis activity is almost consistent with that of the wild penicillin acylation enzyme mutant, the synthesis and hydrolysis ratio can be up to 1.7, and the yield of the cefaclor can be up to 71.5%.

Penicillin G acylation enzyme mutant, and application thereof in synthesis of cephalosporin antibiotics

The invention discloses a recombined heat-resisting short-chain dehydrogenase gene, a codase, a carrier, an engineering bacterium and an application. The recombined heat-resisting short-chain dehydrogenase gene has above 90% homology with a nucleotide sequence shown by SEQ ID No.1. The recombined heat-resisting short-chain dehydrogenase gene related to the invention has high activity and excellent heat stability; a chiral alcohol compound is efficiently and high-selectively prepared; a coenzyme NADPH (Nicotinamide Adenine Dinucleotide Phosphate) or NADH (Nicotinamide Adenine Dinucleotide) circulating regenerating system is constructed under the condition of GDH (Glucose Dehydrogenase) or hydrogenlyase; and the consumption of the coenzyme in a reaction process is greatly reduced. According to the method provided by the invention, the production cost is greatly lowered, the reaction condition is mild, the reaction is environment-friendly, the operation is simple and convenient, and the method has an excellent industrial application prospect in the aspect of synthesizing a drug chiral intermediate.

Zhou Shuo

Papers

Characterization of an aldo–keto reductase from Thermotoga maritima with high thermostability and a broad substrate spectrum

Development of a whole-cell biocatalyst with NADPH regeneration system for biosulfoxidation

Recombinant heat resisting aldehyde ketoreductase gene, coding enzyme, vector, engineering bacteria and application

Penicillin G acylation enzyme mutant, and application thereof in synthesis of cephalosporin antibiotics

Recombined heat-resisting short-chain dehydrogenase gene, codase, carrier, engineering bacterium and application