An (R)‐selective transaminase RbTA with excellent stereoselectivity (>99% ee) in the asymmetric amination of hydroxy ketones was identified. Biochemical characterization showed that RbTA exhibited the highest activity toward 4‐hydroxy‐2‐butanone among reported enzymes, and that it has broad substrate specificity, including for aliphatic, aromatic, and alicyclic ketones. Crystallization of RbTA were performed, as were molecular docking and mutagenesis studies. Residue Tyr125 plays a key role in substrate recognition by forming a hydrogen bond with hydroxy ketone. The applicability of the enzyme was determined in preparative‐scale synthesis of (R)‐3‐amino‐1‐butanol, demonstrating the potential of RbTA as a green biocatalyst for production of value‐added chiral hydroxy amines. This study provides an efficient tool for enzymatic synthesis of chiral hydroxy amines, as well as structural insight into substrate recognition by transaminases in the asymmetric amination of hydroxy ketones.

Transaminases show potential for the industrial synthesis of important pharmaceutical ingredients. However, these naturally occurring enzymes show poor activity toward bulky N-heterocyclic compounds. To produce a catalyst with enhanced catalytic efficiency, this study redesigned an (R)-selective transaminase from Rhodobacter sp. 140A (RbTA). Key residues for substrate binding were identified by molecular docking and molecular dynamics simulations. A “simplified amino acid alphabet,” consisting of amino acids of different sizes (Phe, Asn, Val, and Ala), was then used to fine-tune the substrate-binding pocket by producing a small but smart variant library. Residue Y125 was found to be critical for substrate binding, and variant RbTAM1(Y125A), exhibiting a remarkable activity enhancement, was obtained. Through combined mutation, the most active variant, RbTAM2(Y125A/I6A/L7A/L158V), was constructed, exhibiting 1064-fold greater catalytic efficiency (k cat/K m) toward substrate N-Boc-3-piperidone (7a) than the wild-type enzyme. This variant also exhibited significantly improved activity (4–110-fold) toward a series of cyclic and bulky heterocyclic ketones. Structure-guided analysis of variant Y125A and molecular simulations revealed that the introduction of residue A125 enlarged the substrate-binding pocket volume and enabled additional hydrophobic interactions with the substrate, facilitating binding in a more favorable conformation for catalysis. The activity of variant RbTAM2 was verified in the gram-scale synthesis of chiral N-heterocyclic amine (R)-1-Boc-3-piperidinamine (7b), achieving 99% conversion and a space-time yield of 222 g L–1 d–1.

Genome engineering of Rhodococcus opacus PD630, an important microorganism used for the bioconversion of lignin, is currently dependent on inefficient homologous recombination. Although a CRISPR interference procedure for gene repression has previously been developed for R. opacus PD630, a CRISPR/Cas9 system for gene knockout has yet to be reported for the strain. In this study, we found that the cytotoxicity of Cas9 and the deficiency in pathways for repairing DNA double-strand breaks (DSBs) were the major causes of the failure of conventional CRISPR/Cas9 technologies in R. opacus , even when augmented with the recombinases Che9c60 and Che9c61. We successfully developed an efficient single-stranded DNA (ssDNA) recombineering system coupled with CRISPR/Cas9 counter-selection, which facilitated rapid and scarless editing of the R. opacus genome. A two-plasmid system, comprising Cas9 driven by a weak Rhodococcus promoter Pniami, designed to prevent cytotoxicity, and a single-guide RNA (sgRNA) under the control of a strong constitutive promoter, was proven to be appropriate with respect to cleavage function. A novel recombinase, Rr RecT derived from a Rhodococcus ruber prophage, was identified for the first time, which facilitated recombination of short ssDNA donors (40–80 nt) targeted to the lagging strand and enabled us to obtain a recombination efficiency up to 10 3 -fold higher than that of endogenous pathways. Finally, by incorporating Rr RecT and Cas9 into a single plasmid and then co-transforming cells with sgRNA plasmids and short ssDNA donors, we efficiently achieved gene disruption and base mutation in R. opacus , with editing efficiencies ranging from 22 % to 100 %. Simultaneous disruption of double genes was also confirmed, although at a lower efficiency. This effective genome editing tool will accelerate the engineering of R. opacus metabolism.