Biocatalysis has become an important aspect of modern organic synthesis, both in academia and across the chemical and pharmaceutical industries. Its success has been largely due to a rapid expansion of the range of chemical reactions accessible, made possible by advanced tools for enzyme discovery coupled with high-throughput laboratory evolution techniques for biocatalyst optimization. A wide range of tailor-made enzymes with high efficiencies and selectivities can now be produced quickly and on a gram to kilogram scale, with dedicated databases and search tools aimed at making these biocatalysts accessible to a broader scientific community. This Primer discusses the current state-of-the-art methodology in the field, including route design, enzyme discovery, protein engineering and the implementation of biocatalysis in industry. We highlight recent advances, such as de novo design and directed evolution, and discuss parameters that make a good reproducible biocatalytic process for industry. The general concepts will be illustrated by recent examples of applications in academia and industry, including the development of multistep enzyme cascades.

The nitrogen–nitrogen bond is a core feature of diverse functional groups like hydrazines, nitrosamines, diazos, and pyrazoles.

The formation of a N−N bond is a unique biochemical transformation, and nature employs diverse biosynthetic strategies to activate nitrogen for bond formation. Among molecules that contain a N−N bond, biosynthetic routes to diazeniumdiolates remain enigmatic. We here report the biosynthetic pathway for the diazeniumdiolate‐containing amino acid l‐alanosine. Our work reveals that the two nitrogen atoms in the diazeniumdiolate of l‐alanosine arise from glutamic acid and aspartic acid, and we clarify the early steps of the biosynthetic pathway by using both in vitro and in vivo approaches. Our work demonstrates a peptidyl‐carrier‐protein‐based mechanism for activation of the precursor l‐diaminopropionate, and we also show that nitric oxide can participate in non‐enzymatic diazeniumdiolate formation. Furthermore, we demonstrate that the gene alnA, which encodes a fusion protein with an N‐terminal cupin domain and a C‐terminal AraC‐like DNA‐binding domain, is required for alanosine biosynthesis.

Piperazic acid (Piz) is a nonproteinogenic amino acid possessing a rare nitrogen–nitrogen bond. However, little is known about how Piz is incorporated into nonribosomal peptides, including whether adenylation domains specific to Piz exist. In this study, we show that free piperazic acid is directly adenylated and then incorporated into the incarnatapeptin nonribosomal peptides through isotopic incorporation studies. We also use in vitro reconstitution to demonstrate adenylation of free piperazic acid with a three-domain nonribosomal peptide synthetase from the incarnatapeptin gene cluster. We furthermore use bioinformatics and site-directed mutagenesis to outline consensus sequences for the adenylation of piperazic acid, which can now be used for the prediction of gene clusters linked to piperazic-acid-containing peptides. Finally, we discover a fusion protein of a piperazate synthase and an adenylation domain, highlighting the close biosynthetic relationship of piperazic acid formation and its adenylation. Altogether, our work demonstrates the evolution of biosynthetic systems for the activation of free piperazic acid through adenylation, a pathway we suggest is likely to be employed in the majority of pathways to piperazic-acid-containing peptides.

The antibiotic streptothricin (ST) possesses an amino sugar bound to an L-␤-lysine (␤-Lys) residue via a peptide bond. The peptide bond formation has been shown to be catalyzed by a nonribosomal peptide synthetase (NRPS) during ST biosynthesis. The focus of this study is the closely related ST analogue BD-12, which carries a glycine-derived side chain rather than a ␤-Lys residue. Here, in Streptomyces luteocolor NBRC13826, we describe our biosynthetic studies of BD-12, which revealed that the peptide bond between the amino sugar and the glycine residue is catalyzed by a Fem-like enzyme (Orf11) in a tRNA-dependent manner rather than by an NRPS. Although there have been several reports of peptide bond-forming tRNA-dependent enzymes, to our knowledge, Orf11 is the first enzyme that can accept an amino sugar as a substrate. Our findings clearly demonstrate that the structural diversity of the side chains of ST-type compounds in nature is generated in an unusual manner via two distinct peptide bond-forming mechanisms. Moreover, the identification and functional analysis of Orf11 resulted in not only the production of new ST-related compounds, but also the provision of new insights into the structure-activity relationship of the ST-related antibiotics. (1), STs with an oligo(␤-Lys) consisting of two to seven residues have been identified (Fig. 1). ST-F inhibits protein biosynthesis in prokaryotic cells (2), and STs carrying the longer oligo(␤-Lys) side chains show higher levels of antibacterial activity. Moreover, STs strongly inhibit the growth of eukaryotes, such as yeasts (3-5), fungi (6), protozoa (7), insects (8), plants (9), and mammals (10-13). Although STs have been used effectively as selective agents for recombinant DNA work in some of these organisms, STs are not currently used therapeutically due to their inherent toxicity. In addition to the STs, it has been reported that Streptomyces strains produce ST analogues that possess a glycinederived side chain rather than the ␤-Lys residue: 15), citromycin (16,17), glycinothricin (18), A-269A (19), and A-269A= (19) (Fig. 1). These analogues display potent antibacterial activities, although their molecular targets remain unclear. Also, like the STs, the ST analogues are not used clinically due to their toxicity.We previously identified the ST-biosynthetic gene cluster (accession no. AB684619) in Streptomyces rochei NBRC 12908 (Fig. 2) and elucidated the biosynthetic mechanisms of the oligo(␤-Lys) side chains (20). Nonribosomal peptide synthetases (NRPSs) are known to catalyze the assembly of a myriad of structurally complex peptide natural products (21). However, in the previous study, we identified three unique stand-alone NRPSs among the ST-biosynthetic enzymes and showed that they assembled the structurally simple peptide oligo(␤-Lys) (Fig. 3A). The biosynthesis is initiated by adenylation of ␤-Lys in Orf5 (stand-alone adenylation [A] domain), and the resulting L-␤-lysyl-O-AMP is loaded