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Title: Towards development of a platform process for novel lantiobic production
Author: Khan, Haroon Dawood Sadullah
ISNI:       0000 0004 7231 1094
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2018
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The worldwide increase in antibiotic resistant bacteria has been identified as a major concern to the continued provision of healthcare. The lack of new antibiotic discoveries and the diminishing number effective against multi-drug resistant bacteria has increased efforts to identify new natural antimicrobials and to use synthetic biology to engineer artificial ones. Lantibiotics, a class of ribosomally synthesized antimicrobial peptides, with a broad spectrum of activity, have been identified as potential candidates to combat such bacteria. However no clinical lantibiotic products currently exist, owing to low product titres and lack of a suitable manufacturing process. Initial studies on the lantibiotic gallidermin have managed to increase product titre by modifying the producer strain, Stapylococcus gallinarum Tϋ3928, to synthesize the biologically inactive precursor, pregallidermin. This helps overcome product auto-toxicity and may be a generic route to enhancing the production of novel engineered lantibiotics. This thesis investigates the scalability of a pregallidermin production process to determine whether it could serve as a production platform for novel lantibiotics. Initially, fed-batch cultures were performed in 7.5 L stirred tank bioreactors to characterise the parameters necessary for achieving high cell density. These identified aeration, agitation and nutrient feed rates as important parameters to optimal product formation. The fed-batch culture was repeated at pilot scale (30 L) under the same process conditions, yielding 0.7 g.L-1 of pregallidermin. A scalable downstream process was also developed and evaluated. The overall process flowsheet comprised of pregallidermin capture on the hydrophobic resin Amberlite XAD-7, followed by cation exchange chromatography to purify the pregallidermin. This was then trypsinised to release the required gallidermin. The final purity of gallidermin was 70 % (w/w) with an overall process yield of 13.5 %. Using this downstream process sequence 1.9 g (0.09 g.L-1) of pregallidermin was isolated from the 30 L fermentation. Confirmation of the identity of the purified pregallidermin and gallidermin was obtained by HPLC and mass spectrometry. Microscale methods were developed to optimise the downstream process and determine product physico-chemical properties with a view to informing process design. These determined that higher purity gallidermin recovery was possible with the optimisation of the XAD-7 adsorption/desorption. Optimal conditions for pregallidermin adsorption, resin wash and pregallidermin elution from resin were determined using the microscale methods. A redesign of the downstream process based on optimised pregallidermin capture increased overall recovery of mature gallidermin 3-fold to 0.3 g.L-1. In summary, this work has demonstrated a novel process for lantibiotic production and purification at pilot scale. The process is sufficiently generic that it could serve as a manufacturing platform for production of next generation engineered lantibiotics. It has also demonstrated the utility of microscale methods in optimising the production process.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available