Abstract
Antimicrobial resistance is an ever-growing threat to modern medicine; with bacteria evolving to have multi-drug resistance, hospital stays are longer, fatality rates are increasing as is the threat of a post-antibiotic era in which minor surgeries and chemotherapy could be compromised. Despite this grave reality, the appetite to research and develop novel antimicrobials has slowed to almost a halt in the pharmaceutical industry as there is a perceived too great of a financial risk compared to the reward.This thesis explores the genome mining and expression of NRPS with similarity to the teixobactin-producing NRPS region found in uncultivatable Eleftheria terrae. Initial results highlighted a similar NRPS region within Lysobacter enzymogenes and so efforts begun to uncover a pipeline to biosynthesise novel natural products with potential to have antimicrobial activity. Lysobacter are Gram-negative soil dwelling bacteria that are well known to produce secondary metabolites with antimicrobial activity such as lysobactin, cephalosporins and lysocins. For this reason, the Lysobacter genus is becoming increasingly popular with researchers.
The identified biosynthetic gene cluster in selected strain L. enzymogenes ATCC 29487, found to contain two sequential NRPS regions but at 68,954 nt in size, the BGC was too large to isolate as a whole and so a cloning vector was selected to assist this process and a cosmid library was constructed. The library was screened using PCR and three cosmids containing overlapping DNA to cover the length of the NRPS regions were identified. Combination of the cosmids, and subsequent reconstruction of the BGC, was unfortunately unsuccessful due to the incompatible orientation of DNA in one of the three cosmids.
Fortunately, in the same region of the genome as the double NRPS gene cluster, there is a predicted class II lanthipeptide. Lanthipeptides often are found to exhibit antimicrobial activity, with those that do being named ‘lantibiotics’. The DNA for this lanthipeptide was isolated within a cosmid and heterologously expressed in E. coli. A novel lanthipeptide, Zhisin, and its truncated species were analysed using LC-MS. Investigations ensued for the optimisation of expression, extraction, and analysis of this lanthipeptide.
The research implications and industrial relevance of the work presented here, after the proposed future work, could assist in the removal of risk from pharmaceutical companies by providing the foundation of a biosynthetic pipeline in the development of novel antimicrobials. In addition, the acquisition of the L. enzymogenes ATCC 29487 cosmid library is invaluable for the complete mapping of the strains genome and to investigate other novel secondary metabolites that are predicted from the strain that are not covered in this body of work.
| Date of Award | 18 Jun 2025 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Linda Popplewell (Supervisor), Claire Jennings (Supervisor) & Hong Gao (Supervisor) |