Building the molecular toolkit to exploit Burkholderia bacteria as biotechnological agents

Petrova, Yoana 2022. Building the molecular toolkit to exploit Burkholderia bacteria as biotechnological agents. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

Burkholderia sensu lato is a diverse multi-genus group encompassing environmental, plant-beneficial bacteria and opportunistic pathogens of cystic fibrosis and immunocompromised patients. Members of this complex have large genomes, rich in biosynthetic gene clusters (BGCs) encoding for specialised metabolites, which makes Burkholderia bacteria an untapped resource for discovery of antimicrobial natural products. Many BGCs are silent under standard laboratory conditions and the limited molecular toolkit for efficient genetic manipulations in Burkholderia makes it challenging to engineer and activate those cryptic pathways. A further challenge with using these bacteria for biotechnological applications, in particular as biological control agents in agriculture, are the concerns with their opportunistic pathogenicity. The main aim of this study was to develop further the molecular toolkit available for the manipulations of Burkholderiales and focus it on exploiting the Saccharomyces cerevisiae yeast homologous recombination ability in vivo, a technique not previously applied to Burkholderiales, for the cloning of Burkholderia polyyne cepacin- and caryoynencin-encoding BGCs. The polyyne BGCs were heterologously expressed in B. ambifaria and Paraburkholderia hosts. Paraburkholderia strains were of interest as heterologous hosts for biopesticide development, as this genus constitutes mainly of environmental isolates with lesser potential to cause human disease and certain strains were found to exhibit a stunted growth at 37°C as compared to lower temperatures. One of the key challenges in the use of recombinant Paraburkholderia as biopesticides was the lack of a plasmid vector which was stable in the absence of antibiotic selection. This study attempted to address that by designing two new vectors (chromosomally integrative and a self-replicating plasmid), compatible with the yeast recombination cloning, and evaluating their stability. P. phytofirmans PsJN strain, expressing caryoynencin from the stable integrative vector, conferred significantly higher protection of germinating peas against Gl. ultimum-induced damping-off disease than the wild type strain. This study also used RNA sequencing to understand the differential gene expression in a recombinant caryoynencin producing P. phytofirmans PsJN strain and to identify strong promoters that can be used to increase expression in the heterologous host. In conclusion, this PhD work added to the molecular toolkit available for the genetic manipulation of Burkholderiales and laid the foundation towards designing new Paraburkholderia-based biopesticides for use in agriculture.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Biosciences
Subjects:	Q Science > Q Science (General)
Date of First Compliant Deposit:	23 March 2023
Date of Acceptance:	26 January 2023
Last Modified:	23 Mar 2023 10:51
URI:	https://orca.cardiff.ac.uk/id/eprint/156289

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