Novel tools and methods supporting high-throughput screening and metabolic engineering in model and non-model microbial host chassis

There is increasing interest in the industrial use of microorganisms as cell factories to enhance bioproduction with reduced environmental impact. To realize this, new tools are needed that facilitate discovery of novel enzymes, pathways, and strains relevant to sustainable biotechnology innovation, and that promote scalable implementation in real-world contexts. In this thesis I describe establishment of various tools and methods supporting high-throughput screening and metabolic engineering in model and non-model microbial host chassis, with emphasis on E. coli and selected cyanobacteria as promising cell factories. In the process, I developed an all-in-one CRISPR-Cas9 toolkit and CRISPR guide-RNA design workflow and evaluated their potential for targeted gene regulation in E. coli. Using this toolkit, I addressed a limitation to functional metagenomic screening in E. coli EPI300 by, for the first time, using CRISPR-interference to identify genes encoding phenotypes of interest encoded on fosmid clones. I went on to insert a landing pad into the E. coli EPI300 chromosome using the chassis-independent recombinase-assisted genome engineering (CRAGE) system, to support targeted insertion of up to 48 kb of exogenous DNA sequence. I used this system to insert a chromosomal RFP into E. coli EPI300 for use in studying co-culture dynamics, and applied the resulting marked strain to evaluate the behaviour of the PemrR:GFP biosensor used for the recovery of lignin transforming phenotypes from fosmid libraries. In addition to tool and methods development, I participated in characterization of a novel cyanobacterial host chassis, Sodalinema yuhuli AB48, with potential biotechnological application. I explored the multi-omic underpinnings of this potential and placed S. yuhuli AB48 within a modern phylogenetic context needed for pangenome analysis. I then designed, built, and tested a consistent lighting system for microplate-based cultivation of photosynthetic microorganisms, creating new opportunities for media optimization and strain selection with direct application to development of photosynthetic microbial cell factories. Collectively, my work effort led to improved methods for recovering biological parts from environmental genomes, discovery and characterization of a novel photosynthetic microorganism with biotechnological potential, and invention of new platform technologies needed for sustainable biotechnology innovation at the interface of microbial ecology, biological engineering and bioinformatics.