Nitrile hydratase (NHase, EC 4.2.1.84) is one type of metalloenzyme participating in the biotransformation of nitriles into amides. Given its catalytic specificity in amide production and eco-friendliness, NHase has overwhelmed its chemical counterpart during the past few decades. However, unclear catalytic mechanism, low thermostablity, and narrow substrate specificity limit the further application of NHase. During the past few years, numerous studies on the theoretical and industrial aspects of NHase have advanced the development of this green catalyst. This review critically focuses on NHase research from recent years, including the natural distribution, gene types, posttranslational modifications, expression, proposed catalytic mechanism, biochemical properties, and potential applications of NHase. The developments of NHase described here are not only useful for further application of NHase, but also beneficial for the development of the fields of biocatalysis and biotransformation.

Efficient transcription termination relying on intrinsic terminators is critical to maintain cell fitness by avoiding unwanted read-through in bacteria. Natural intrinsic terminator (NIT) typically appears in mRNA as a hairpin followed by approximately eight conserved uridines (U-tract) at the 3′ terminus. Owing to their simple structure, small size, and protein independence, assorted NITs have been redesigned as robust tools to construct gene circuits. However, most NITs exert functions to adapt to their physiological requirements rather than the demand for building synthetic gene circuits, rendering uncertain working performance when they are constructed intact in synthetic gene circuits. Here, rather than modifying NITs, we established a datadriven and in silico-assisted (DISA) design framework to forward engineer minimal intrinsic terminators (MITs). By comprehensively analyzing 75 natural intrinsic terminators from Bacillus subtilis, we revealed that two pivotal features, the length of the U-tract and the thermodynamics of the terminator hairpin, were involved in the sequence−activity relationship (SAR) of termination efficiency (TE). As per the SAR, we leveraged DISA to fabricate an array of MITs composed of in silico-assisted designed minimal hairpins and fixed U-tracts. Most of these MITs exhibited high TE in diverse Gram-positive and Gram-negative bacteria. In contrast, the TEs of the NITs were highly varied in different hosts. Moreover, TEs of MITs were flexibly tuned over a wide range by modulating the length of the U-tract. Overall, these results demonstrate an efficient framework to forward design functional and broad host-range terminators independent of tedious and iterative screening of mutagenesis libraries of natural terminators.

Protein evolution has significantly enhanced the development of life science. However, it is difficult to achieve in vitro evolution of some special proteins because of difficulties with heterologous expression, purification, and function detection. To achieve protein evolution via in situ mutation in vivo, we developed a base editor by fusing nCas with a cytidine deaminase in Bacillus subtilis through genome integration. The base editor introduced a cytidine-to-thymidine mutation of approximately 100% across a 5 nt editable window, which was much higher than those of other base editors. The editable window was expanded to 8 nt by extending the length of sgRNA, and conversion efficiency could be regulated by changing culture conditions, which was suitable for constructing a mutant protein library efficiently in vivo. As proof-of-concept, the Sec-translocase complex and bacitracin-resistance-related protein BceB were successfully evolved in vivo using the base editor. A Sec mutant with 3.6-fold translocation efficiency and the BceB mutants with different sensitivity to bacitracin were obtained. As the construction of the base editor does not rely on any additional or host-dependent factors, such base editors (BEs) may be readily constructed and applicable to a wide range of bacteria for protein evolution via in situ mutation.

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Optically pure compounds are important in the synthesis of fine chemicals. Using directed evolution of enzymes to obtain biocatalysts that can selectively produce high-value chiral chemicals is often time-, money-, and resource-intensive; traditional semi-rational designs based on structural data and docking experiments are still limited due to the lack of accurate selection of hot-spot residues. In this study, through ligand-protein collision counts based on steered molecular dynamics simulation, we accurately identified four residues related to improving nitrile hydratase stereoselectivity toward rac-mandelonitrile (MAN). All the four selected residues had numerous collisions with rac-MAN. Five mutants significantly shifting stereoselectivity towards (S)-MAN were obtained from site-saturation mutagenesis, one of them, at position βPhe37, exhibiting efficient production of (S)-MAN with 96.8% ee , was isolated and further analyzed. The increased pulling force observed during SMD simulation was found to be in good coincidence with the formation of hydrogen bonds between (R)-MAN and residue βHis37. (R)-MAN had to break these barriers to enter the active site of nitrile hydratase and S selectivity was thus improved. The results indicated that combining steered molecular dynamics simulation with a traditional semi-rational design significantly reduced the select range of hot-spot residues for the evolution of NHase stereoselectivity, which could serve as an alternative for the modulation of enzyme stereoselectivity.

The transition metal (iron or cobalt) is a mandatory part that constitutes the catalytic center of nitrile hydratase (NHase). The incorporation of the cobalt ion into cobalt‐containing NHase (Co‐NHase) was reported to depend on self‐subunit swapping and the activator of the Co‐NHase acts as a self‐subunit swapping chaperone for subunit exchange. Here we discovered that the activator acting as a metallochaperone transferred the cobalt ion into subunit‐fused Co‐NHase. We successfully isolated two activators, P14K and NhlE, which were the activators of NHases from Pseudomonas putida NRRL‐18668 and the activator of low‐molecular‐mass NHase from Rhodococcus rhodochrous J1, respectively. Cobalt content determination demonstrated that NhlE and P14K were two cobalt‐containing proteins. Substitution of the amino acids involved in the C‐terminus of the activators affected the activity of the two NHases, indicating that the potential cobalt‐binding sites might be located at the flexible C‐terminal region. The cobalt‐free NHases could be activated by either of the two activators, and both the two activators activated their cognate NHase more efficiently than did the noncognate ones. This study provided insights into the maturation of subunit‐fused NHases and confirmed the metallochaperone function of the self‐subunit swapping chaperone.

Programmable dual-base editor: a propellent for evolving bacterial chassis.