Discovery of Novel Transaminases from Halophilic Microorganisms

  • Stephen Kelly

Student thesis: Doctoral ThesisDoctor of Philosophy

Abstract

Chiral amines are valuable intermediates in the pharmaceutical industry, but their synthesis by chemical means has a number of drawbacks, including the need for toxic transition metal catalysts, volatile organic compounds (VOCs), and a lack of stereoselectivity in a single step. Increasingly, biocatalysis has been viewed as a ‘green’ and economically viable to their production, with a number of enzymes, including hydrolases, lyases, oxidases and transaminases (TAms).TAms are PLP-dependent enzymes which catalyse the transfer of an amine group from an amine donor to a ketone or aldehyde, resulting in a chiral amine. Their use has been successfully applied to the synthesis of chiral amines for active pharmaceutical ingredient (API) synthesis, most notably with the antidiabetic drug, sitagliptin. However, the application of TAms derived from mesophilic organisms has been limited by their inability to retain function in the harsh reaction conditions often demanded by industrial processes. This has driven the search for novel enzymes from extreme environments, the extremozymes, capable of retaining function in conditions which would render mesophilic enzymes ineffective.Halophiles are one such group of extremophile, adapted to survive at high salinity. Their enzymes also exhibit a number of other useful properties, such as thermotolerance, alkalitolerance, and tolerance to the presence of organic solvent. As such, they represent a viable source of robust enzymes which could be exploited for use in biocatalysis.In this thesis, work has focused on the discovery of novel TAms from an ancient hypersaline environment, Kilroot salt mine, formed during the Triassic period (circa220-250 mya). As well as its extreme nature, Kilroot salt mine has remained relatively undisturbed during this time, representing a vastly untapped resource of biocatalyst sequences from a unique microbiome. Sequence analysis of a cultured isolate libraryrevealed the presence of a number of putative TAm-coding genes. Sequences annotated as belonging to industrially relevant PLP fold types I and IV were aligned and genes were selected for cloning and expression using Escherichia coli BL21 DE3 cells as an expression host.Several active TAms were produced, with enzyme Ad2-TAm, isolated from the genome of the halophilic bacterium, Halomonas sp. CSM-2, showing a number of industrially desirable properties. As well as an ability to accept a range of structurally diverse aldehyde and ketone substrates, Ad2-TAm performed over a range of pH values (optimum pH 9.0), in the presence of organic solvent, and accepted a number of amine -iiidonors. Ad2-TAm also exhibited promising halotolerance, showing no decrease in conversion up to 1.5 M (8.8%) NaCl. The industrial applicability of Ad2-TAm was demonstrated further with acceptance of the versatile substrate, furfural (61.9% conversion to furfurylamine over 16 h), and an ability to function in seawater medium.TAms were also produced using a Haloferax volcanii-based model, capable of expressing soluble protein from the haloarchaea where E. coli was not. The first haloarchaeal TAm to be studied for application in biocatalysis, BC61-TAm, was isolated from the haloarchaeon Halorubrum sp. CSM-61. This enzyme showed a typically halophilic profile, with optimal conversions observed at 1 M NaCl and KCl, with a high degree of relative activity still seen at 4 M for each salt. BC61-TAm also displayed some thermotolerance (optimal conversion at 50 °C), and an (R)-selective enantiopreference, advantageous given the relative scarcity of currently available (R)-selective TAms. Consistent with other halophilic enzymes, BC61-TAm exhibited tolerance to organic solvents, with maximum relative conversion seen in the presence of 30% DMF. Molecular modelling and substrate docking predicted equivalent amino acid residues present in the active site of BC61-TAm to known TAms from bacteria, suggesting haloarchaeal TAms have a similar mechanistic action to their bacterial counterparts. Molecular dynamics predicted the structural basis of BC61-TAm’s organic solvent tolerance, showing an increased flexibility profile across protein residues and an ability to localise available water around the enzyme’s active site, contributing to the maintenance of catalysis.Access to TAms using culture-based approaches was expanded through use of enhanced isolation techniques and cross-domain protein expression. The use of an improvised ‘iChip’ device resulted in the isolation of a number of species and genera which had previously been uncultured within the research group on the same media using conventional isolation techniques alone. Cross-domain expression was used to produce the enzyme Glut18-TAm from the obligately halophilic bacterial isolate KSM18. Glut18-TAm was expressed in the insoluble fraction only, using E. coli, but was expressed in soluble form using the haloarchaeal host Hfx. volcanii.A metagenomic dataset was obtained from a brine sample taken from Kilroot salt mine, allowing for more complete biodiversity profiling to be undertaken on this environment. Taxonomic analysis of the metagenomic dataset showed similar trends to the cultured microbiome of Kilroot salt mine, with Halorubrum and Halomonas the most dominant genera for archaea and bacteria respectively, in both the metagenomic and culture-derived data. In both datasets, biodiversity was greater among the bacteria, with archaea predominating in terms of overall numbers of isolates or sequences, -ivaccounting for 71% of all cellular organisms in the metagenome. As expected, there were a number of species and genera present in the metagenomic dataset which were not present in the cultured isolate library, given the high proportion of uncultivable microbiome from any environment.The acquisition of a metagenomic dataset also provided access to TAms which were previously unattainable using culture-dependent methods alone. Using TAms from Vibrio fluvialis, Arthrobacter sp. KNK168, and Halomonas sp. CSM-2 as query sequences, Kilroot metagenome (KMG) was interrogated for homologous sequences as a means of discovering novel TAms. KMG-TAm4, discovered using this approach, was shown to be an (S)-selective ω-TAm, with a temperature optimum of 30 °C and pH optimum of 7.0. Molecular modelling and substrate docking revealed the presence of conserved active site residues to a number of previously described TAms, including a catalytic lysine residue at position 284. Despite having a similar annotation to Ad2-TAm, KMG-TAm4 showed a much more restricted substrate scope and limited halotolerance, with only 32.1% relative activity seen at 1 M NaCl, compared to 100% observed with Ad2-TAm.Halophilic proteins are known to possess a number of robust and industrially useful characteristics, such as tolerance to salt, organic solvents, and elevated pH and temperatures. To date, the characterisation and application of halophilic TAms has remained relatively understudied. Work presented within this thesis represents an extensive TAm discovery programme from halophilic microorganisms from the ancient hypersaline environment, Kilroot salt mine. TAms have been characterised from both bacteria and archaea, and produced in a number of expression models using both culture-dependent and independent methods. As well as discovering a number of potential candidates for industrial application, this research has helped provide a scaffold for rational design of enzymes with robust properties. Work presented within this thesis has created a platform for future TAm discovery from halophiles, and has highlighted the potential value of halophilic TAms for application in biocatalysis.
Date of AwardSep 2018
Original languageEnglish
Awarding Institution
  • Queen's University Belfast
SupervisorBrendan Gilmore (Supervisor), Christopher C R Allen (Supervisor) & Tom Moody (Supervisor)

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