α,ω-Diols are important monomers widely used for the production of polyesters and polyurethanes. Here, biosynthesis of α,ω-diols (C₈–C₁₆) from renewable free fatty acids using CYP153A monooxygenase, carboxylic acid reductase, and E. coli endogenous aldehyde reductases is reported. The highest yield of α,ω-diol was achieved for the production of 1,12-dodecanediol. In the nicotinamide adenine dinucleotide phosphate (NADPH) cofactor regeneration system, 5 g/L of 1,12-dodecanediol was synthesized in 24 h reaction from the commercial ω-hydroxy dodecanoic acid. Finally, 1.4 g/L 1,12-dodecanediol was produced in a consecutive approach from dodecanoic acids. The results of this study demonstrated the scope of the potential development of bioprocesses to substitute the petroleum-based products in the polymer industry.

Fungal cytochrome P450 (CYP) enzymes catalyze versatile monooxygenase reactions and play a major role in fungal adaptations owing to their essential roles in the production avoid metabolites critical for pathogenesis, detoxification of xenobiotics, and exploitation avoid substrates. Although fungal CYP-dependent biotransformation for the selective oxidation avoid organic compounds in yeast system is advantageous, it often suffers from a shortage avoid intracellular NADPH. In this study, we aimed to investigate the use of bacterial glucose dehydrogenase (GDH) for the intracellular electron regeneration of fungal CYP monooxygenase in a yeast reconstituted system. The benzoate hydroxylase FoCYP53A19 and its homologous redox partner FoCPR from Fusarium oxysporum were co-expressed with the BsGDH from Bacillus subtilis in Saccharomyces cerevisiae for heterologous expression and biotransformations. We attempted to optimize several bottlenecks concerning the efficiency of fungal CYP-mediated whole-cell-biotransformation to enhance the conversion. The catalytic performance of the intracellular NADPH regeneration system facilitated the hydroxylation of benzoic acid to 4-hydroxybenzoic acid with high conversion in the resting-cell reaction. The FoCYP53A19+FoCPR+BsGDH reconstituted system produced 0.47 mM 4-hydroxybenzoic acid (94% conversion) in the resting-cell biotransformations performed in 50 mM phosphate buffer (pH 6.0) containing 0.5 mM benzoic acid and 0.25% glucose for 24 h at 30°C. The "coupled-enzyme" system can certainly improve the overall performance of NADPH-dependent whole-cell biotransformations in a yeast system.

Amine dehydrogenase (AmDH) possesses tremendous potential for the synthesis of chiral amines because AmDH catalyzes the asymmetric reductive amination of ketone with high enatioselectivity. Although a reductive application of AmDH is favored in practice, the oxidative route is interesting as well for the preparation of chiral amines. Here, the kinetic resolution of racemic amines using AmDH was first extensively studied, and the AmDH reaction was combined with an NADH oxidase (Nox) to regenerate NAD⁺ and to drive the reaction forward. When the kinetic resolution was carried out with 10 mM rac-2-aminoheptane and 5 mM rac-α-methylbenzylamine (α-MBA) using purified enzymes, the enantiomeric excess (ee) values were less than 26% due to the product inhibition of AmDH by ketone and the inhibition of Nox by the substrate amine. The use of a whole-cell biocatalyst co-expressing AmDH and Nox apparently reduces the substrate and product inhibition, and/or it increases the stability of the enzymes. Fifty millimoles (50 mM) rac-2-aminoheptane and 20 mM rac-α-MBA were successfully resolved into the (S)-form with >99% ee using whole cells. The present study demonstrates the potential of a whole-cell biocatalyst co-expressing AmDH and Nox for the kinetic resolution of racemic amines.