Metagenomic discovery of a novel transaminase for valorization of monoaromatic compounds

Abstract

The profitability of next-generation biorefineries is acutely contingent on the discovery and utilization of biocatalysts that can valorize lignin. To this end, the metabolic catalogues of diverse microbiota have been mined previously using functional metagenomics in order to identify biocatalysts that can selectively degrade lignin into monoaromatic compounds. Herein, we have further improved the valorization factor of biorefining by deploying functional metagenomics toward the identification of a novel transaminase that can selectively functionalize lignin-derived monoaromatics to produce value-added feedstocks for pharmaceutical synthesis.We implemented a high-throughput colorimetric assay using o-xylylenediamine as the amino donor and successfully identified a transaminase that utilizes the canonical cofactor, pyridoxal 5 0 -phosphate, to aminate as many as 14 monoaromatic aldehydes and ketones. We subsequently identified the optimal conditions for enzyme activity towards the most favoured amino acceptor, benzaldehyde, including temperature, pH and choice of co-solvent. We also evaluated the specificity of the enzyme towards a variety of amino donors, as well as the optimal concentration of the most favoured amino donor. Significantly, the novel enzyme is markedly smaller than typical transaminases, and it is stably expressed in E. coli without any modifications to its amino acid sequence. Finally, we developed and implemented a computational methodology to assess the activity of the novel transaminase. The methodology is generalizable for assessing any transaminase and facilitates in silico screening of enzyme-substrate combinations in order to develop efficient biocatalytic routes to value-added amines. The computational pipeline is an ideal complement to metagenomics and opens new possibilities for biocatalyst discovery.

The identification and use of robust transaminases from a domestic drain metagenome

“…Previous work has employed metagenomic approaches in the search for novel transaminases, using either a functional based32 or a sequence-based approach 33,34. In this work, transaminases have been retrieved from a domestic household drain metagenome.…”

Transaminases for industrial biocatalysis: novel enzyme discovery

“…Using a similar approach of fosmid library generation and colorimetric screening using o-xylylenediamine as amino donor, Pawar and co-workers (Pawar et al 2018) discovered a (S)-selective TAm with a high degree of novelty. pRT15-TA was < 30% similar to Vf-TAm and Cv-TAm, as well as being 15% shorter.…”

“…2, with pRT15-TA occupying a position on its own branch, with considerable evolutionary distance from all other characterized TAms. Despite these differences in sequence, pRT15-TA was stably expressed in E. coli and capable of accepting a number of ketone and amine substrates, as well as being the first TAm to be discovered using a functional metagenomics approach (Pawar et al 2018).…”

“…As with other discovery methodologies discussed, functional metagenomics is not without limitations. While colorimetric screens such as those employed by Coscolín et al (2019) and Pawar et al (2018) increase screening efficiency considerably, expression and screening of clones is not trivial and can be time consuming and resource heavy. Furthermore, not all enzymes can be easily expressed in established E. coli-based systems, such as those from extremophiles, whose characteristics can be very desirable.…”

Screening and Comparative Characterization of Microorganisms from Iranian Soil Samples Showing ω-Transaminase Activity toward a Plethora of Substrates

“…Chiral amines as building blocks are prevalent in pharmaceuticals and chemical industries [1]. For instance, chiral amines are present in roughly 40% of FDA approved pharmaceuticals [2,3]. Furthermore, chiral β-amino acids (β-aas) have attracted significant attention recently [4].…”