Nonribosomal Peptides Produced by Minimal and Engineered Synthetases with Terminal Reductase Domains

Abstract

Nonribosomal peptide synthetases (NRPSs) use terminal reductase domains for 2-electron reduction of the enzyme-bound thioester releasing the generated peptides as C-terminal aldehydes. Herein, we reveal the biosynthesis of a pyrazine that originates from an aldehyde-generating minimal NRPS termed ATRed in entomopathogenic Xenorhabdus indica. Reductase domains were also investigated in terms of NRPS engineering and, although no general applicable approach was deduced, we show that they can indeed be used for the production of similar natural and unnatural pyrazinones.

Pyrazines: Synthesis and Industrial Application of these Valuable Flavor and Fragrance Compounds

“…In 2018, Turner et al reported a chemo-enzymatic synthesis of substituted pyrazines using an amino transaminase (S-selective, ATA-113, Codexis [24] ) in the presence of a suitable amine donor, which mediated the key amination of the 1,2-diketone precursor to -aminoketones that underwent oxidative dimerization to the final product (Figure 4). [25] In the case of pyrazines, the chirality S. marcescens 3B2 l-threonine → aminoacetone 2,5-dimethylpyrazine 3 3-ethyl-2,5-dimethylpyrazine 5a [31] HqlA NRPS from P. herquei in E. coli l-thyrosine 4,4′-(pyrazine-2,5-diyl-bis(methylene))diphenol 15 [32] ATRed NRPS from X. indica l-phenylalanine 4,4′-(pyrazine-2,5-diyl-bis(methylene))diphenyl [33] SBW25 from P. fluorescens in E. coli 4-aminophenyl alanine 2,5-dimethyl-3,6-bis(4-aminobenzyl)pyrazine 16 [34] B. subtilis l-threonine, d-glucose 2-ethyl-3,5(3,6)-dimethyl pyrazine 5a/b [35] of the amine group is irrelevant for the synthesis of the aromatic heterocycle core. All reactions were carried out at room temperature with isopropyl amine as the amine donor.…”

“…A similar approach was followed recently by Tietze et al with an NRPS from Xenorhabdu indica to reduce l-phenylalanine to the amino-aldehyde to yield the corresponding pyrazine. [33] Another approach was recently published (2020), in which an enzymatic cascade from Pseudomonas fluorescens SBW25 was introduced into E. coli to produce reactive aminoketone species, which can immediately dimerize to yield pyrazines. The gene sequence included the synthesis of the natural -amino acid 4-aminophenylalanine and several other genes for the transfor-mation to non-natural symmetric 2,5-dimethyl-3,6-bis(4-aminobenzyl)pyrazine 16.…”

“…with an NRPS from Xenorhabdu indica to reduce l ‐phenylalanine to the amino‐aldehyde to yield the corresponding pyrazine. [ 33 ]…”

Discovery and biosynthesis of guanipiperazine from a NRPS-like pathway

“…These enzymes are functionally diverse in primary and secondary metabolism. Depending the type of downstream releasing domains, NRPS-like enzymes catalyse a broad range of reactions, including but not limited to Dieckmann cyclization, [6,7] reduction, [8,9] and Claisen condensation. [10] The carboxylic acid reductases (CARs) belong to the singlemodule NRPS-like enzymes, and comprise an N-terminal adenylation domain, which is fused to a C-terminal reductase (R) domain via a T domain.…”

The inherent flexibility of type I non-ribosomal peptide synthetase multienzymes drives their catalytic activities

“…This prospect exists since the discovery of the modular organization of NRPSs [61]. To date, a straightforward strategy to re-engineer NRPS assembly lines to produce artificial peptides has been difficult to establish, although some successful reports of re-engineering were published [62][63][64][65]. Classical strategies using substitutions of A, C-A, PCP-C-A units or entire modules yielded only a small amount of synthesized peptide [59,60].…”