Designed peptide surfactants have commercial potential due to their advanced properties. Recombinant production in E. coli from renewable carbon with conventional fed-batch processes has the potential to manufacture designed peptide surfactants at competitive prices with the existing petrochemical route. Sucrose from sugarcane is the ideal feedstock for microbial fermentation due to its superior environmental performances and its lower price than glucose in Australia. Designed peptide surfactants however, are currently produced with an inefficient process from complex medium; furthermore industrial E. coli strains do not metabolize sucrose natively.
In this thesis, a model designed surfactant was efficiently produced from sucrose. Sucrose metabolism was engineered on the host strain and production of the model peptide was investigated; hence limitations in product accumulation were overcome and product titer maximized. A strategy for sucrose metabolism in E. coli was developed to demonstrate the potential of sucrose as a feedstock for microbial fermentation. The proposed approach ensured compatibility with pre-existing strain modifications while not altering performances. The host strain grew on sucrose as it does on glucose, the natural substrate.
The existing production process was tested for product accumulation to create a base for process development and it was implemented: firstly, complex medium was replaced with chemically defined medium to meet industrial conditions; subsequently cell density was increased to maximize product titer. Product titer was about 20-fold higher for the engineered process compared to the reference demonstrating efficient production from minimal medium with high product titer. Sucrose or glucose feedstocks performed similarly, indicating that sucrose can replace glucose for industrial fermentations.
In-silico analysis of intracellular flux distributions unravelled the limiting factors for cell growth and product formation. Limitations were overcome by medium supplementation and specific productivity rose; to avoid medium supplementation, a genetic strategy was adopted, which yielded similar increase in specific productivity as medium supplementation did. Overall, final product titer from sucrose reached more than 6 g/L and maximum accumulation of the recombinant peptide reached 20% of the biomass weight. To the best of our knowledge this is the first time that peptides are manufactured from an E. coli strain engineered for sucrose metabolism and this is the highest product titer reported to date for designed peptides. It is hoped that this thesis will encourage the retrofitting of existing E. coli-based productions to sucrose feedstock and that renewable designed peptide surfactants will replace traditional petrochemical surfactants.