Engineering of secondary metabolite production in streptomycetes

Abstract

Streptomycetes are known for their ability to produce a range of different secondary metabolites, including antibiotics, immunosuppressive, anti-fungals, and anti-cancer compounds. Of these compounds, antibiotics play an important role in the clinics for treatment of both mild and severe bacterial infections. However, with the rise of multi-resistant pathogens, the demand for new antibiotics or derivatives of old ones, with improved properties, is now higher than ever. Recent efforts in genome sequencing and mining have revealed a so far untapped potential of streptomycetes and related actinomycetes as evident from so-called “silent” biosynthetic gene clusters, whose products remain undetectable under standard laboratory conditions. These clusters harbour all information necessary for production of potentially novel bioactive compounds, and hence provide high priority candidates for engineering to activate their production. With this knowledge, the need for better molecular tools to harness the potential of the gifted microorganisms is now greater than ever. One such molecular tool, which has truly revolutionised the field of genome engineering, is the CRISPR-Cas9 genome engineering system. In this thesis, the CRISPR-Cas9 system for genome engineering of actinomycetes was expanded for future applications in a high-throughput semi-automatic setting. First, a toolbox and workflow for construction of CRISPR plasmids, for a range of different engineering purposes was developed, including the computational prediction of suitable 20 bp protospacers for the single guide RNAs and a USER-cloning method for construction of the CRISPR plasmids. Additional improvement to the system was achieved through the development of an optimised USER assembly workflow for cheaper and faster plasmid construction. The workflow was verified by manual knock-down of two biosynthetic gene clusters in model organism Streptomyces coelicolor A3(2), which confirmed the applicability of the system. A second part of the thesis was devoted to engineering of Streptomyces collinus Tü 365, which is a known producer of the narrow-spectrum antibiotic kirromycin. While there exists several studies addressing the PKS scaffold biosynthesis of kirromycin, knowledge about the supply of the precursor ethylmalonyl-CoA and most of the tailoring reactions remained scarce. In this thesis, the role of the gene kirN, believed to be involved in precursor supply, and the six genes kirM, kirHIV, kirHV, kirHVI, kirOI and kirOII, all predicted to be involved in tailoring reactions, were investigated by gene inactivations, complementations, and characterisation of the biosynthetic products of the generated mutants. Within our studies, four novel kirromycin derivatives were generated and characterised. Our investigations allowed for closing some of the missing gaps in the biosynthesis of kirromycin, along with providing us with a toolbox of new mutants, which produce derivatives of the original compound. These derivatives could serve as scaffolds for future bioderivatization efforts. This thesis lays the groundwork for future engineering of streptomycetes to improve secondary metabolite production. For the USER-CRISPR-Cas9 platform, the next logical step will be to implement the workflow in a robotic setting. Furthermore, the mutants of S. collinus Tü 365 will be included in a derivatization platform to produce new kirromycin analogues with improved pharmacokinetic properties.