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Abstract
Genetically encoded gene editing and regulation systems offer versatile toolkits for constructing microbial cell factories. My doctoral research focused on exploring and optimizing the CRISPR-Cas9 system and a transcription factor-based biosensor system for their applications in metabolic engineering. While CRISPR-Cas9 stands as a revolutionary technology, its targeting efficacy is consistently hindered by the PAM restriction. To address this challenge, my study endeavored to engineer the prototype SpCas9 from Streptococcus pyogenes, originally recognizing a 5’-NGG-3’ PAM, to accommodate a 5’-CAT-3’ PAM. This modification could transform Cas9 into a universal gene repressor capable of targeting the most common ATG start codon. Structure-guided rational engineering of SpCas9 resulted in a PAM-flexible variant that preferentially accommodates 5′-NRN-3′ PAMs. Tunable CRISPRi on competitive genes using the engineered variant yielded a 40.1% increase in mevalonate production. Meanwhile, my work also explored the Cas9 orthologs across Streptococcus, uncovering the occurrence of diverse PAM-binding motifs affording distinct PAM preferences. Utilizing AlphaFold and site-directed mutagenesis, the PAM recognition mechanisms of novel Cas9 orthologs were dissected, guiding further rational engineering and leading to the generation of multiple PAMless or even PAM-free Cas9 variants. One such PAMless variant enabled the implementation of CRISPRi/a for metabolic flux rewiring, resulting in a substantial 2.6-fold enhancement of 4-hydroxycoumarin titer. In addition to CRISPR-Cas9, transcription factor-based biosensors also offer powerful tools for strain development, yet suffer from limited variety. To broaden the repertoire of available biosensors, my research investigated and engineered a diol-responsive transcription factor-based biosensor. Through promoter engineering and protein mutagenesis, a diol-responsive bifunctional gene controller with a 5-fold higher dynamic range was obtained. Overall, my dissertation provided new Cas9 scaffolds with minimal or no PAM limitation, alongside an engineered diol-responsive bifunctional biosensor, enriching the toolkit for versatile metabolic engineering applications.