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Steroidal hormone pharmaceuticals are the second largest class of medicines after antibiotics. At present, the initial materials of the steroidal industry have shifted from sapogenins, which were extracted from plants of the genus Dioscore to phytosterols. As a byproduct of soybean oil production, phytosterols are readily available and of low prices. Androstenedione (AD), androstadiendione (ADD), 9α-hydroxy-androstenedione (9α-OH-AD) and a series of key intermediates used in the synthesis of steroidal pharmaceuticals can be produced from phytosterols by microbial transformation. Nevertheless, due to the long metabolic pathways, the byproducts and the complex regulation, traditional microbial screening, mutagenizing methods and the oil-water biphasic transformation systems are no longer suitable for current industrial production. A new generation strains for the production of key steroidal pharmaceutical intermediates have been constructed and an intelligent production process has been jointly developed by us and Zhejiang Xianju Junye Pharmaceutical Co. Ltd.. Taking these products and processes as an example, this article reviews the improvement of strains for the production of steroidal pharmaceutical intermediates and the development of biotransformation process on an industrial scale. With the development of synthetic biology, it is expected to develop a new generation of intermediates which are more suitable for the synthesis of steroidal medicines. Moreover, de novo biosynthesis the steroidal active pharmaceutical ingredients from glucose is also expected. The application of these new-generation strains constructed by biotechnology (BT) in modern factories based on informatization and intelligent technology (IT) will be more efficient and greener, and create remarkable social and economic values.

Production of chiral amines and unnatural amino-acid using ω-transaminase can be achieved by kinetic resolution and asymmetric synthesis, thus ω-transaminase is of great importance in the synthesis of pharmaceutical intermediates. By genomic data mining, a putative ω-transaminase gene hbp was found in Burkholderia phytofirmans PsJN. The gene was cloned and over-expressed in Escherichia coli BL21 (DE3). The recombinant enzyme (HBP) was purified by Ni-NTA column and its catalytic properties and substrate profile were studied. HBP showed high relative activity (33.80 U/mg) and enantioselectivity toward β-phenylalanine (β-Phe). The optimal reaction temperature and pH were 40 ℃ and 8.0-8.5, respectively. We also established a simpler and more effective method to detect the deamination reaction of β-Phe by UV absorption method using microplate reader, and demonstrated the thermodynamic property of this reaction. The substrate profiling showed that HBP was specific to β-Phe and its derivatives as the amino donor. HBP catalyzed the resolution of rac-β-Phe and its derivatives, the products (R)-amino acids were obtained with about 50% conversions and 99% ee.

In industrial application of NAD(P)H-dependent dehydrogenases, NAD(H) has the advantages over NADP(H) in higher stability, lower price and wider recycling system. Recently, a meso-2,6-diaminopimelate dehydrogenase from Symbiobacterium thermophilum (StDAPDH) has been found to be a useful biocatalyst for the production of D-amino acids, but it requires NADP(H) as co-enzyme. To switch the co-enzyme specificity from NADP(H) to NAD(H), we studied the effect of Y76 on the co-enzyme specificity of StDAPDH, because the crystal structural analysis indicated that residue Y76 is near the adenine ring. The mutation of Y76 exerted significant effect on the co-enzyme specificity. Furthermore, the double mutant R35S/R36V significantly lowered the specific activity toward NADP+, and the combination of R35S/R36V with some of the Y76 mutants resulted in mutant enzymes favorable NAD+ over NADP+. This study should provide useful guidance for the further development of highly active NAD(+)-dependent StDAPDH by enzyme engineering.

In this study, a fast carbonyl reductases colorimetric screening method for discovering stereoselective carbonyl reductases was established by combining the reverse alcohol oxidation with the azoreductase-catalyzed reduction of azo dye. When azo dye (Orange I , 4-(4-hydroxy-1-naphthylazo) benzenesulfonic acid) and azoreductase (AzoB) were added into the reaction system of alcohol oxidation catalyzed by carbonyl reductase, the produced NAD(P)H served as electron donor for the azoreductase to reduce the azo dye, resulting the color fade. Hence, the carbonyl reductases can be screened by the obvious color change. When chiral alcohol was used as the substrate, the activity and stereoselectivity of carbonyl reductases can be screened at the same time.

A N-acetylneuraminate lyase gene (shnal) from Staphylococcus hominis was cloned into pET-28a and expressed in Escherichia coli BL21 (DE3) host cells. The recombinant enzyme was purified and characterized. It is a homotetrameric enzyme with the optimum pH at 8.0 for the cleavage direction and the optimum pH and temperature were 7.5 and 45 degrees C for the synthetic direction. The activity of ShNAL is stable when incubated at 45 degrees C for 2 h but decreased rapidly over 50 degrees C. ShNAL showed high stability in a wide range pH from 5.0 to 10.0 with the residual activity being > 70% when the enzyme was incubated in different buffers at 4 degrees C for 24 h. Its K(m) towards N-acetylneuraminic acid, pyruvate and ManNAc were (4.0 +/- 0.2) mmol/L, (35.1 +/- 3.2) mmol/L and (131.7 +/- 12.1) mmol/L, respectively. The k(cat)/K(m) value of Neu5Ac, ManNAc, and Pyr for ShNAL were 1.9 L/(mmol x s), 0.08 L/(mmol x s) and 0.08 L/(mmol x s), respectively.

Carbonyl reductases catalyze carbonyl compounds to chiral alcohols that are important building blocks in fine chemical industry. To study carbonyl reductase from Pichia pastoris GS115 (ppcr), we discovered a new gene (ppcr) encoding an NADPH-dependent carbonyl reductase by genomic data mining. It was amplified by PCR from the genomic DNA, and expressed in Escherichia coli BL21 (DE3). The recombinant protein was purified to homogeneity. The optimum temperature was 37 degrees C and the optimum pH of PPCR was 6.0. PPCR was stable below 45 degrees C. The Km and k(cat) value of the enzyme for ethyl 3-methyl-2-oxobutanoate were 9.48 mmol/L and 0.12 s, respectively. The enzyme had broad substrate specificity and high enantioselectivity. It catalyzed the reduction of aldehydes, a-ketoesters, beta-ketoesters and aryl ketones to give the corresponding alcohols with >97% ee with only a few exceptions, showing its application potential in the synthesis of chiral alcohols.