Increasing Production of the Antibiotic Abyssomicin C from Micromonospora maris AB-18-032

Abstract

Antibiotic-resistant pathogenic infections pose a significant threat for global human health, to combat which there is a pressing need to discover new antibiotics with novel modes of action. Microbial secondary metabolites have been shown to be valuable sources of inspiration in the development of new antibiotics, in particular those metabolites from actinobacteria. Abyssomicin C, a secondary metabolite from Micromonospora maris AB-18- 032, has shown potent antibacterial activity against pathogenic drug-resistant bacteria methicillin-resistant Staphylococcus aureus(MRSA) and vancomycin-resistant Staphylococcus aureus (VRSA). The mode of action (MOA) of abyssomicin C involves inhibition of the folate biosynthesis pathway, through interfering with the formation a key folate precursor, paraaminobenzoic acid (pABA). However, it is currently difficult to access sufficient quantities of abyssomicin C for further biological study, via either total synthesis or biosynthesis. In this thesis, we aimed to create a library of M. maris Ochi mutants capable of high abyssomicin C production by fermentation, from which large quantities of abyssomicin C could be easily purified. To achieve this, we have applied ribosome engineering methods to M. maris AB-18-032, to generate a library of M. maris Ochi mutants with the potential for increased abyssomicin C production. These M. maris Ochi mutants were rapidly screened via antimicrobial disk diffusion assays, designed to be sensitive to pABA inhibitors. Validation of abyssomicin C production and subsequent quantification via chemical analysis (HPLC, LC-MS and NMR) allowed the identification of best abyssomicin C producers. Whole genome sequencing was used to identify point mutations in selected M. maris Ochi mutants, to better understand the observed increased abyssomicin C production. Additionally, statistical Design of Experiments (DOE) was successful applied to optimise the culture media for high abyssomicin C production from M. maris Ochi mutants, as quantified by HPLC. This work also allowed the serendipitous identification of an Ochi mutant, producing the secondary metabolite ortho-aminobenzoic acid, which was identified via chemical analysis including single crystal X-ray diffraction. We have successfully demonstrated the upregulation of abyssomicin C production by M. maris, through ribosome engineering. We anticipate that improved access to abyssomicin C will facilitate its future study, and developments towards use in the clinic. This work is also the first report of Micromonospora Ochi mutants, which we envisage will enable future work on the development of new and existing antibiotics from Micromonospora species