Evolution, environmental distribution, and engineering of the abyssomicin biosynthetic gene cluster

Ph. D. Thesis.Evolution, environmental distribution, and engineering of the abyssomicin biosynthetic gene cluster. Microbial secondary metabolites constitute a great source of pharmaceutically interesting biomolecules. In particular, the tetronate family of natural products is a structurally and functionally diverse group of secondary metabolites whose potent bioactivities make them attractive targets for clinical and industrial exploitation. The abyssomicins are an actively growing family of small spirotetronate natural products that has been widely studied due to the unique structural features and bioactivities that some of its members exhibit, including antimicrobial activity against Gram-positive bacteria such as methicillin- and vancomycinresistant Staphylococcus aureus and different Mycobacteria strains, HIV inhibitory and reactivator properties and anti-influenza A virus activity. Abyssomicin C and its atrop- isomer, produced by the slow growing marine Actinobacteria Micromonospora maris AB-18-032 T , are type I polyketide antibiotics that inhibit the formation of p-aminobenzoic acid, a constituent of the folate pathway. Abyssomicin biosynthesis is highly amenable to reengineering, as the enzymes involved in the synthesis of the tetronate (AbyA1) and the spiro-tetronate-forming Diels-Alderase (AbyU) are both capable of accepting structurally diverse substrates. The aim of this project was to set up the grounds for the discovery and production of novel abyssomicins with applications in the biopharmaceutical industry. First, in order to understand the environmental distribution and evolution of the abyssomicin biosynthetic gene clusters (BGCs) present in nature, an analysis of publicly available genomic and metagenomic data was carried out. The strategy of selecting a pathwayspecific enzyme to direct the mining proved to be an excellent strategy; 74 new Diels–Alderase homologs were identified and a surprising prevalence of the abyssomicin BGC within terrestrial habitats, mainly soil and plant-associated, was unveiled. Five complete and 12 partial new abyssomicin BGCs and 23 new potential abyssomicin BGCs were also identified, suggesting that a plethora of abyssomicins remain to be discovered. A preliminary study on the abyssomicin production potential of five of the strains containing potential abyssomicin BGCs was also carried out although no abyssomicins were found. After that, with the final goal of producing abyssomicins of various lengths and different saturation/oxidation patterns, it was necessary to express the aby BGC of M. maris AB-18-032 in a well-established heterologous host. This cluster was successfully moved into E. coli and various Streptomyces species, the abyssomicin production potential of these strains was evaluated in various conditions and some of the hosts were promoter engineered to force the expression of the aby BGC. Active gene expression was demonstrated, but despite the efforts, none of the heterologous hosts produced abyssomicins. Later analysis unveiled the presence of several mutations within abyB1, the first polyketide synthase gene in the aby BGC, suggesting this could be the reason for the lack of production. Since the approach to heterologously produce abyssomicins was not fruitful, this work then focused on increasing abyssomicin production in M. maris AB-18-032 and developing genetic tools for this system. First, through ribosome engineering, a library of M. maris drug-resistant mutants capable of producing up to 3.4-fold abyssomicin C in comparison to the wild-type strain was generated. Then, using statistical Design of Experiments (DOE), an efficient electroporation protocol that could accelerate targeted genetic manipulations in M. maris was developed. Together, increased abyssomicin production and a quick and easy electroporation protocol for M. maris, will facilitate future engineering of the aby BGC directly in M. maris to produce diverse non-natural abyssomicin