Development of CRISPR Tools for Metabolic Engineering of Specialized Metabolite Production in Streptomyces

Soil microorganisms make up the second largest fraction of biomass on Earth. The soil microcosm is incredibly diverse and encompasses a vast diversity of species, often participating in complex interspecies interactions, even across kingdoms of life. These interactions are often facilitated through specialized metabolites, molecules of fascinating complexity and chemical diversity. Specialized metabolites are of great importance for modern society, for example as antibiotics, antifungals, anticancer compounds, or plant growth-promoting agents. Of all soil bacteria, members of the genus Streptomyces were found to encode the largest biosynthetic potential by far. This biosynthetic potential is encoded in genes clustered on the genomes, and thus referred to as biosynthetic gene clusters (BGCs). However, under laboratory conditions, the majority of BGCs are not expressed, complicating the assignment of specialized metabolites to predicted BGCs. Activating, studying, and modifying BGCs, as well as strain engineering of production strains, relies heavily on our ability to efficiently engineer Streptomyces species. However, introduction of just one chromosomal modification can require months of laboratory work using classical methods. This Ph.D. thesis aimed to improve our metabolic engineering capabilities of Streptomyces species through the development of novel CRISPR-based tools, enabling engineering with unprecedented speed, efficiency, and throughput.

As a contextual starting point, this thesis provides a review of the Design-Build-Test-Learn cycle for metabolic engineering of Streptomyces species. For each cycle stage, important resources and considerations are highlighted. Furthermore, a protocol for all existing CRISPR tools for streptomycetes, including CRISPR-Cas9, CRISPRi, and CRISPR-BEST, is provided. Given the slow throughput of current engineering methods, multiplexed cytosine base editing was studied at various scales, revealing key performance parameters, and highlighting important bottlenecks. The system achieved the highest number of simultaneously edited targets in Streptomyces species using any technology, highlighting its great promise. Given the frequent need for full deletions of genomic regions, a novel CRISPR tool based on CASCADE-Cas3 was developed with superior efficiency to CRISPR-Cas9. This tool, the first developed for Streptomyces based on a type I CRISPR system, was shown to work efficiently in several species and was used for the streamlined construction of a genome-minimized expression host. Finally, the first CRISPR-Prime tool for Escherichia coli was developed, serving as a stepping stone towards the establishment of CRISPR-Prime editing in Streptomyces species. This tool enables the introduction of complicated mutations and represents a platform for further tool development. The developed tools greatly expand the engineering capabilities for Streptomyces species, bringing us one step closer to closing the gap between computationally predicted, and experimentally characterized BGCs. This will soon allow us to greatly expand our access to Nature’s biosynthetic potential, and ultimately urgently needed solutions for medicine, agriculture, and sustainability.