Characterization of a novel ω-transaminase from a Triassic salt mine metagenome

Abstract

Chiral amines are valuable building blocks for the pharmaceutical industry, and are increasingly synthesized by transaminase-mediated (TAm) synthesis. Currently available TAms, primarily isolated from the genomes of cultured mesophilic bacteria, often suffer from a number of drawbacks, including poor substrate range and an inability to tolerate the harsh conditions often demanded by industrial processes. These characteristics have, in part, driven the search for novel biocatalysts from both metagenomic sources and extreme environments. Herein, we report the isolation and characterization of an ω-TAm from a metagenome of a Triassic salt mine in Kilroot, N. Ireland, an extremely hypersaline environment formed circa 220-250 mya. The gene sequence was identified based on homology with existing bacterial TAms, synthesized within a pET28a(+) plasmid and expressed in E. coli BL21 DE3 cells. The resultant 49 kDa protein accepted (S)-methylbenzylamine (MBA) as amino donor and had a specific activity of 0.54 U/mg using α-ketoglutarate (ΑKG) as substrate. Molecular modeling and substrate docking indicated the presence of key residues, conserved in a number of other TAms. Despite the hypersaline environment from which it was isolated, the enzyme displayed low halotolerance, highlighting that not all biocatalysts will demonstrate the extreme characteristics associated with their source environment. This study does however reinforce the viability of mining metagenomic datasets as a means of discovering novel and functional biocatalysts, and adds to a currently scant list of such examples in the field of TAms.

Transaminases for industrial biocatalysis: novel enzyme discovery

“…Recent metagenomic mining from a hypersaline environment also yielded a functional TAm, KMG-TAm4 (Kelly et al 2018b). In this case, however, the enzyme produced did not exhibit a halophilic or halotolerant activity profile, as had been observed in previous enzymes characterized from cultured microbes from the same source (Kelly et al 2017(Kelly et al , 2019a.…”

“…This is due largely to the search criteria used in enzyme discovery, with similarity to Class III TAms (which are S-selective) sought in studies by the Hailes and Ward. However, in the studies by Ferrandi et al (2017) and Kelly et al (2018b), PLP fold type IV enzymes were used as query sequences, but in either case, no functional R-selective TAms were uncovered. While this is a fairly small sample size and S-selective enzymes are more abundant naturally, it does perhaps highlight the need to use enhanced search techniques in the quest for novel R-selective enzymes from metagenomes, such as key motif-based searches which proved so successful with cultured microorganisms.…”

Transaminases for Green Chemistry: Recent Progress and Future Prospects

“…They were able to clone and express successfully 29 sequences in E. coli of the 36 fulllength non-redundant class-III Tam sequences. Another work on metagenomic mining was carried out in the hypersaline environment which yielded a functional Tam [41]. The Tams were obtained using a sequencedriven approach and were found to be S-selective.…”

A first insight into the Polish Bochnia Salt Mine metagenome

“…If this observation is confirmed by further biological analysis, it will be possible to identify sequences of genomes of the oldest halophilic microorganisms discovered so far (Schreder-Gomes et al 2022 ). Some of the isolated microbes also encoded potentially valuable enzymes, such as a novel ω-transaminase (Kelly et al 2018 ).…”