Cloning and engineering of novel coleopteran luciferase

Bate, Jack 2022. Cloning and engineering of novel coleopteran luciferase. PhD Thesis, Cardiff University. Item availability restricted.

Preview	PDF (PhD Thesis) - Accepted Post-Print Version Download (14MB) | Preview
	PDF (Cardiff University Electronic Publication Form) - Supplemental Material Restricted to Repository staff only Download (1MB)

Abstract

Firefly luciferases produce visible light by catalysing a reaction utilizing D-luciferin, Mg2+, ATP, and molecular oxygen, in a widely studied bioluminescent system which has been developed into a range of applications relevant to industry and academia. Advancement of firefly luciferase applications depends upon the continued discovery of novel luciferase phenotypes and their respective sequences, regardless of whether these are derived from nature or mutagenic means. The feasibility of bioprospecting novel luciferase gene sequences from dry preserved Coleoptera provided by Amgueddfa Cymru – National Museum Wales was explored. A non-destructive DNA extraction method was followed to enable the preservation of all specimens. A cross-species capture hybridisation method was developed, using biotinylated probes of the luciferase gene from Photinus pyralis, to enrich for luciferase gene fragments prior to Illumina sequencing and the recovery of luciferase gene sequences with a bioinformatics strategy. With this approach, a gene sequence was recovered from an unidentified Costa Rican firefly, which encoded a novel luciferase capable of catalysing a bioluminescence reaction, termed CRLuc. Simultaneously, engineering of the luciferase from Phosphaenus hemipterus (PhemLuc) was attempted to generate two discrete variants which possessed improved compatibility with the synthetic substrate analogue infraluciferin, and increased thermostability. PhemLuc was selected as it presented the opportunity to discover novel mutagenic functionality in a previously uncharacterized enzyme. Homology models of PhemLuc were constructed to identify residues in proximity to the bound substrate for targeted mutagenesis. From 32 targets mutagenized, the mutations H245W and A313G produced considerably increased bioluminescence with infraluciferin, and conferred cumulative effects when combined into a dual mutant termed x2 Infra. Thermostabilisation of PhemLuc was attempted using 15 mutations reported to thermostabilise Photinus pyralis luciferase. Although this mutant was inadequate, DNA shuffling identified an x14 reversion mutant with restored bioluminescence and improved thermostability. This mutant was further improved using epPCR to include mutations I231V and L306H to complete a final x16 thermostable mutant, later demonstrated as capable of functioning in LAMP-BART. The work conducted here demonstrates the potential of targeted bioprospecting for genetic discoveries using museum materials, in addition to the identification of several novel mutations in PhemLuc which warrant further investigation in related luciferase variants.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Biosciences
Subjects:	Q Science > Q Science (General)
Date of First Compliant Deposit:	6 October 2022
Date of Acceptance:	11 September 2022
Last Modified:	05 Jan 2024 08:19
URI:	https://orca.cardiff.ac.uk/id/eprint/153108

Actions (repository staff only)

Edit Item