(119d) Development and Application of a Crispri System for the Syngas-Fermenting Microbe Clostridium Ljungdahlii

Clostridium ljungdahlii has recently emerged as an attractive candidate for the bioconversion of synthesis gas (CO, CO₂, H₂) to a variety of fuels and chemicals through the Wood-Ljungdahl (WL) pathway. However, the lack of robust tools for generating gene deletions and inducible knockdowns in this organism limits the abiltity to both divert carbon flux away from acetate toward higher-value products, and to assign gene function in the poorly annotated genome. To address this need, here we develop a CRISPRi system for C. ljungdahlii that allows for the controlled knockdown of specific genes using the deactivated Cas9 nuclease (dCas9) and a single-guide RNA (sgRNA). qRT-PCR and enzymatic assays confirmed the ability of the system to regulate key genes with proposed roles in energy conservation and carbon flux. Screening of tightly controlled promoters for dCas9 expression reduced background downregulation, and multiplexing of sgRNAs through the use of a BioBricks assembly method allowed the simultaneous regulation of multiple gene targets in a hight-throughput manner. As a proof of concept to test the ability of the CRISPRi system to divert flux to heterologous product synthesis in an engineered strain, plasmids were constructed that enabled 3-hydroxybutyrate (3HB) synthesis, and production was assessed in strains co-transformed with various CRISPRi plasmids. The results provide interesting insights into the hitherto opaque metabolic and regulatory networks of C. ljungdahlii, and serve as a useful tool to identify gene targets for improving heterologous product yield.