Metabolic engineering of streptomyces albulus for polylysine production

Abstract

During the last few decades, Streptomycetes have shown to be an important and adaptable group of bacteria for the production of various beneficial secondary metabolites. One such secondary metabolite, epsilon polylysine (ε-PL), produced by Streptomyces albulus is of particular interest due to its antimicrobial activity. This work aimed to study different facets surrounding ε-PL and its production. Firstly, to grow S. albulus CCRC 11814, using economically viable crude glycerol as a carbon source and subsequently measure ε-PL production using an anionic dye, trypan blue. Secondly, to evaluate the antimicrobial activity of ε-PL against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Aspergillus niger and Penicillium simplissium. Thirdly, to determine whether there is economic feasibility of ε-PL as a food preservative in South Africa. Lastly, to develop and optimise tools for metabolic engineering such as recombineering and group II introns to improve ε-PL production. The results obtained in this study fall into three different areas: In terms of growth studies, S. albulus grew in the presence of crude glycerol, although the growth was suboptimal, 0.48 g/L as compared to 1.04 g/L produced using pure glycerol or glucose. This is due to the pressures on the bacteria from the impurities of crude glycerol such as methanol and salts. ε-PL antimicrobial activity was effective at a concentration of 100 μg/ml against S. aureus, E. coli and A. niger. It was however, ineffective against P. aeruginosa owing to the low outer membrane permeability of the bacteria. Due to the ability of S. albulus to grow in crude glycerol, it could be used as a financially viable option to produce ε-PL as a natural food preservative in South Africa. The economic feasibility of ε-PL as a food preservative in South Africa showed potential in terms of market research as well as the financial evaluations. However; the production volumes are low due to the use of the crude glycerol and may not cater for the large food industry in the country. For these reasons, metabolic engineering could be employed to improve these production volumes. The first step to metabolic engineering was to develop novel tools which can be used for genetic modifications in S. albulus. The group II intron tools for gene knockouts were developed by the construction of a vector which subsequently requires sequencing and testing to perform gene knockouts. Based on current knowledge, this is the first experiment of its kind. In terms of introduction of genetic material post gene knockouts, iii transformation was shown to be a more effective gene transfer technique as opposed to electroporation, producing 7.75 transformants/μg and 0.038 transformants/μg of DNA, respectively. Future work would involve the use of biocatalysis for metabolic engineering of S. albulus by either removing genes inhibiting ε-PL or overexpressing the enzyme responsible for its production. This research has developed the groundwork for future ε-PL production improvement using biocatalysis and economically viable crude glycerol as a carbon source for applications of the secondary metabolite as a food preservative.