Heterologous expression of the gcc gene cluster and subsequent characterisation of the glycocin F biosynthetic pathway : a thesis presented in partial fulfillment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Manawatu, New Zealand
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2020
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Massey University
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Abstract
The rise of antibiotic-resistant pathogens has motivated research into new treatments to prevent the resurgence of infectious and deadly diseases. GccF is a diglycosylated, 43-amino acid, bacteriostatic peptide produced by the generally regarded as safe bacteria Lactobacillus plantarum KW30. It is active against many Lactobacillus plantarum strains as well as the pathogens Enterococcus faecium and vancomycin resistant Enterococcus faecalis, stopping the growth of susceptible species at low (1-20 nM) concentrations within 2 minutes. Understanding this novel bacteriostatic mechanism could provide a blueprint for the design of a new family of antibiotics. Reported here is the development of an easily modifiable 11.2-kbp plasmid-based heterologous expression system of the gcc cluster that is capable of producing active GccF in L. plantarum NC8 and Lactobacillus sakei 790. Expression of the gccF gene relies on the promotors found naturally within the cluster and results in the production of active GccF matching the concentration produced by the native host. Additionally, the activity of the GccF produced by this system is identical to that of the native producer with 2 nM being sufficient to inhibit the growth of L. plantarum ATCC 8014 by 50 %. Mutations introduced within the coding sequence of five of the cluster genes (gccA and gccC-F), confirmed their roles in the production and maturation of GccF that had previously been predicted using bioinformatic analyses.