Enhanced production of D-psicose from D-fructose by a redox-driven multi-enzyme cascade system.

D-Psicose, a new-generation sugar substitute, has been enzymatically synthesized through D-fructose isomerization. However, isomerization often causes low yields due to unfavorable thermodynamic equilibria, which limited its further industrial application. In this study, we present a redox-driven multi-enzyme cascade, two-step biotransformation system to produce D-psicose from D-fructose. Compared to D-fructose isomerization, this method has a maximized theoretical conversion rate of 100%. D-Psicose-3-epimerase from Clostridiales (CBDPE), ribitol 2-dehydrogenase from Providencia alcalifaciens (PRDH), and formate dehydrogenase from Starkeya (SFDH) were co-expressed in Escherichia coli in the first step to produce D-allitol from D-fructose. Afterward, NADH oxidase from Streptococcus pyogenes (SPNOX), and ribitol 2-dehydrogenase from Rubrivivax sp. (RSRDH) were co-expressed in E. coli to oxidize D-allitol into D-psicose in the second step. The two-step biotransformation system was optimized to maximize the D-fructose-to-D-psicose conversion rate (up to 90%), corresponding to a concentration of 450 mM. This study suggests that this redox-driven multi-enzyme cascade strategy through a sugar-to-alcohol-to-sugar pathway has the advantage of great application for enhanced production of D-psicose and other rare sugars.

Protein Engineering and Homologous Expression of Serratia marcescens Lipase for Efficient Synthesis of a Pharmaceutically Relevant Chiral Epoxyester.

The lipase isolated from Serratia marcescens (LipA) is a useful biocatalyst for kinetic resolution of a pharmaceutically relevant epoxyester, (±)-3-(4'-methoxyphenyl) glycidic acid methyl ester [(±)-MPGM], to afford optically pure (-)-MPGM, a key intermediate for the synthesis of diltiazem hydrochloride. Two mutants, LipAL315S and LipAS271F, were identified from the combinatorial saturation mutation library of 14 amino acid residues lining the substrate-binding pocket. LipAL315S, LipAS271F, and their combination LipAL315S/S271F showed 2.6-, 2.2-, and 4.6-fold improvements in their specific activities towards para-nitrophenyl butyrate (pNPB), respectively. Among these positive mutants, LipAS271F displayed a 3.5-fold higher specific activity towards the pharmaco substrate (±)-MPGM. Kinetic study showed that the improvement in catalytic efficiency of LipAS271F against (±)-MPGM was mainly resulted from the enhanced affinity between substrate and enzyme, as indicated by the decrease of K m. Furthermore, to address the insoluble expression issue in Escherichia coli, the homologous expression of LipA gene in S. marcescens was achieved by introducing it into an expression vector pUC18, resulting in ca. 20-fold higher lipase production. The significantly improved volumeric production and specific activity of S. marcescens lipase make it very attractive as a new-generation biocatalyst for more efficient and economical manufacturing of (-)-MPGM.

Engineering 7ß-Hydroxysteroid Dehydrogenase for Enhanced Ursodeoxycholic Acid Production by Multiobjective Directed Evolution.

Ursodeoxycholic acid (UDCA) is the main active ingredient of natural bear bile powder with multiple pharmacological functions. 7ß-Hydroxysteroid dehydrogenase (HSDH) is a key biocatalyst for the synthesis of UDCA. However, all the 7ß-HSDHs reported commonly suffer from poor activity and thermostability, resulting in limited productivity of UDCA. In this study, a multiobjective directed evolution (MODE) strategy was proposed and applied to improve the activity, thermostability, and pH optimum of a 7ß-HSDH. The best variant (V3-1) showed a specific activity 5.5-fold higher than and a half-life 3-fold longer than those of the wild type. In addition, the pH optimum of the variant was shifted to a weakly alkaline value. In the cascade reaction, the productivity of UDCA with V3-1 increased to 942 g L-1 day-1, in contrast to 141 g L-1 day-1 with the wild type. Therefore, this study provides a useful strategy for improving the catalytic efficiency of a key enzyme that significantly facilitated the bioproduction of UDCA.