Dehydrogenases are widely employed as biocatalysts for the production of optically pure chemicals under mild conditions. Most dehydrogenases are nicotinamide cofactor (NADPH or NADH)-dependent oxidoreductases. 7β-Hydroxysteroid dehydrogenase (7β-HSDH) is a key enzyme for the biochemical synthesis of ursodeoxycholic acid (UDCA). To date, all reported 7β-HSDHs are strictly NADPH-dependent enzymes. However, compared with NADPH, NADH is much more economical, making it the preferential cofactor for synthetic applications of dehydrogenases. In this work, a recombinant 7β-HSDH originating from Ruminococcus torques was rationally engineered to alter its cofactor dependence using a strategy referred to as Cofactor Specificity Reversal: Small-and-Smart Library Design (CSR-SaSLiD), which is based on structural information and conservative sequence alignment. We rationally designed a small-and-smart library containing only five mutants that enabled the quick identification of target variants. Compared with the wild type, the resultant mutant, G39D, showed a 953 000-fold switch in cofactor specificity from NADPH to NADH, and another mutant, G39D/T17A, resulted in 223-fold enhanced activity with NADH. The structural mechanism regarding the effect of mutation on the reversal of cofactor preference and improvement of catalytic activity was elucidated with the aid of molecular dynamics simulation. Furthermore, it was confirmed that the CSR-SaSLiD strategy can be extended to other 7β-HSDHs. This work provides an efficient approach to altering cofactor preference and subsequently recovering the enzymatic activity of dehydrogenases for cost-effective biotechnical applications.

Abstract12α‐Hydroxysteroid dehydrogenase (12α‐HSDH) has the potential to convert cheap and readily available cholic acid (CA) to 12‐oxochenodeoxycholic acid (12‐oxo‐CDCA), a key precursor for chemoenzymatic synthesis of the therapeutic bile acid ursodeoxycholic acid (UDCA). In this work, a native nicotinamide adenine dinucleotide (NAD+)‐dependent 12α‐hydroxysteroid dehydrogenase (Rr12α‐HSDH) from Rhodococcus ruber was identified using a structure‐guided genome mining (SSGM) approach, which is based on the structure of cofactor pocket and the conserved nicotinamide cofactor binding motif alignment. Rr12α‐HSDH was heterologously overexpressed in Escherichia coli BL21 (DE3), purified and characterized. The purified Rr12α‐HSDH showed a high oxidative activity of 290 U mg−1protein toward CA, with a catalytic efficiency (kcat/KM) of 5.10×103 mM−1 s−1. In a preparative biotransformation (100 mL), CA (200 mM, 80 g L−1) was efficiently converted to 12‐oxo‐CDCA in 1 h, with a 85% isolated yield and a space‐time yield (STY) of up to 1632 g L−1 d−1. Furthermore, Rr12α‐HSDH was shown to be able to catalyze the oxidation of other 12α‐hydroxysteroids at high substrate loads (up to 200 mM), giving the corresponding 12‐oxo‐hydroxysteroids in 71%–85% yields, indicating the great potential of Rr12α‐HSDH as a promising biocatalyst for the synthesis of various therapeutic bile acids.magnified image

Background β-Nicotinamide mononucleotide (NMN) is the direct precursor of nicotinamide coenzymes such as NAD+ and NADP+, which are widely applied in industrial biocatalysis especially involving cofactor-dependent oxidoreductases. Moreover, NMN is a promising candidate for medical uses since it is considered to be beneficial for improving health of aged people who usually suffer from an insufficient level of NAD+. To date, various methods have been developed for the synthesis of NMN. Chemical phosphorylation of nicotinamide riboside (NR) to NMN depends on excessive phosphine oxychloride and delicate temperature control, while fermentation of NMN is limited by low product titers, making it unsuitable for industrial-scale NMN production. As a result, the more efficient synthesis process of NMN is still challenging. Aim This work attempted to construct an eco-friendly and cost-effective biocatalytic process for transforming the chemically synthesized NR into the highly value-added NMN. Results A new nicotinamide riboside kinase (Klm-NRK) was identified from Kluyveromyces marxianus. The specific activity of purified Klm-NRK was 7.9 U·mg–1 protein, ranking the highest record among the reported NRKs. The optimal pH of Klm-NRK was 7.0 in potassium phosphate buffer. The purified Klm-NRK retained a half activity after 7.29 h at 50 °C. The catalytic efficiencies (kcat/KM) toward ATP and nicotinamide riboside (NR) were 57.4 s−1·mM−1 and 84.4 s−1·mM−1, respectively. In the presence of an external ATP regeneration system (AcK/AcP), as much as 100 g·L–1 of NR could be completely phosphorylated to NMN in 8 h with Klm-NRK, achieving a molar isolation yield of 84.2% and a space–time yield of 281 g·L−1·day−1. These inspiring results indicated that Klm-NRK is a promising biocatalyst which provides an efficient approach for the bio-manufacturing of NMN in a high titer. Graphical Abstract

Oxidoreductases represent one group of the most important biocatalysts for synthesis of various chiral synthons. However, their practical application was hindered by the expensive nicotinamide cofactors used.