UC Riverside

A multigene integration tool takes advantage of type II CRISPR/Cas9 induced breaks on the genome as a selection for rapid one-step integration of up to three gene expression cassettes. Using this tool, a full factorial library of KmARO4, KmARO7, and KmPHA2, each driven by three different promoters that span a wide expression range, was constructed for Shikimate pathway refactoring. With high expressions of the tyrosine-deregulated KmARO4(K221L), native KmPHA2 and KmARO10, and medium expression of feedback insensitive KmARO7(G141S), the best strain achieves 1943 ± 63 mg/L 2-phenyl ethanol. Our highly efficient chemical and heat shock induced method achieving 6 × 10^5 transformants and electroporation for 1.5 × 10^6, facilitate functional genomic studies by enabling adequate representation of a complex screening library with less host cells and transformed DNA. With the CRISPR/Cas9 system and efficient DNA transformation, we characterized a 4-fold single-guide RNA (sgRNA) library targeting both promoters and coding regions of the K. marxianus CBS 6556 genome. With culturing library transformants in three carbon sources, glucose, lactose, and xylose, the differentially induced editing efficiencies of the same sgRNA make the genome-wide CRISPR/Cas9 library a powerful tool to elucidate gene regulations involved in glucose repression. Knocking out ALPHA3 and KAT1 significantly avoids single cell mating type switching because of homothallism, which enables K. marxianus a much more robust platform for high throughput screens heavily dependent on differentially accumulated gene editing fitness from a population growing to cell confluence. An efficient CRISPR RNA (crRNA) library with adequate gene coverage was obtained using six CRISPR guide activity prediction algorithms provided by CHOPCHOP v3. In-house uniqueness assessment criteria were applied to minimize off-target effects and save the highly active to the maximum possible extent. This optimized library enabled us to identify the first set of essential genes for Kluyveromyces genus. Overall, this dissertation developed stable alpha/a haploid strains for K. marxianus CBS 6556, advanced CRISPR-mediated genetic tools, and highly efficient transformation protocols. They all facilitate genetic engineering and system metabolic engineering in K. marxianus, as well as better understanding of its genetics and metabolism.