Identification of novel transaminases from a 12‐aminododecanoic acid‐metabolizing Pseudomonas strain

Matthew Wilding,Susan J Dorrian,Colin Scott,Ellen F A Walsh

doi:10.1111/1751-7915.12278

Matthew Wilding, Susan J Dorrian + Show 2 more

Open Access

https://doi.org/10.1111/1751-7915.12278

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Abstract

A Pseudomonas species [Pseudomonas sp. strain amino alkanoate catabolism (AAC)] was identified that has the capacity to use 12-aminododecanoic acid, the constituent building block of homo-nylon-12, as a sole nitrogen source. Growth of Pseudomonas sp. strain AAC could also be supported using a range of additional ω-amino alkanoates. This metabolic function was shown to be most probably dependent upon one or more transaminases (TAs). Fourteen genes encoding putative TAs were identified from the genome of Pseudomonas sp. AAC. Each of the 14 genes was cloned, 11 of which were successfully expressed in Escherichia coli and tested for activity against 12-aminododecanoic acid. In addition, physiological functions were proposed for 9 of the 14 TAs. Of the 14 proteins, activity was demonstrated in 9, and of note, 3 TAs were shown to be able to catalyse the transfer of the ω-amine from 12-aminododecanoic acid to pyruvate. Based on this study, three enzymes have been identified that are promising biocatalysts for the production of nylon and related polymers.

Highlights

There has been a substantial increase in the impact that biocatalysis has had on the chemical manufacturing industry, to the extent that for some reaction chemistries, biocatalysis is the preferred option
Many of the commercial successes of biocatalytic processes have been in the manufacture of fine chemicals and pharmaceuticals, reboxetine, pregabalin and sitagliptin, for example (Martinez et al, 2008; Savile et al, 2010; Hayes et al, 2011; Bornscheuer et al, 2012)
Non-physiological promiscuous catalytic activities are relatively common (Copley, 2003; Khersonsky et al, 2006), and it is possible that an appropriate promiscuous function could be used in the biocatalytic synthesis of 12-aminododecanoic acid, or as a starting point for directed evolution (Turner, 2009)

Summary

A Pseudomonas species [Pseudomonas sp. strain amino alkanoate catabolism (AAC)] was identified that has the capacity to use 12-aminododecanoic acid, the constituent building block of homonylon-12, as a sole nitrogen source. Strain amino alkanoate catabolism (AAC)] was identified that has the capacity to use 12-aminododecanoic acid, the constituent building block of homonylon-12, as a sole nitrogen source. Strain AAC could be supported using a range of additional ω-amino alkanoates. This metabolic function was shown to be most probably dependent upon one or more transaminases (TAs). Fourteen genes encoding putative TAs were identified from the genome of Pseudomonas sp. Physiological functions were proposed for 9 of the 14 TAs. Of the 14 proteins, activity was demonstrated in 9, and of note, 3 TAs were shown to be able to catalyse the transfer of the ω-amine from 12-aminododecanoic acid to pyruvate. Three enzymes have been identified that are promising biocatalysts for the production of nylon and related polymers

Introduction

Results and discussion

Experimental procedures