CRISPRi knockdown of acetate and 2,3-butanediol production-related genes in a gas-fermenting acetogen

Abstract

Gas fermentation allows to convert carbon oxide emissions into value-added products using acetogen bacteria. This thesis aimed at creating a CRISPR/Cas9-based gene knockdown (KD) system to reduce expression of two genes in the model-acetogen C. autoethanogenum: 1) alpha-acetolactate decarboxylase (budA) in the 2,3-butanediol (2,3-BDO) and 2) phosphotransacetylase (pta) in the acetate synthesis pathway. Six KD plasmids were designed where single guide RNAs (sgRNAs) targeted three unique positions per gene, plus one plasmid as a non-targeting control. Notably, KD of budA expression abolished 2,3-BDO production but showed varied effects on autotrophic growth. pta KD strains showed strong repression of pta expression, slower growth for two sgRNAs, and, surprisingly, higher acetate-to-ethanol ratios than the control strain. This work contributes towards engineering and understanding of acetogen metabolism by establishing a gene KD system for a model-acetogen.