Effects of Disruption of Genes With Unknown Function in the C1-Fixing Gene Cluster on a Gas-Fermenting Acetogen

Abstract

Gas fermentation is a promising technology for producing chemicals, fuels, and feed proteins from greenhouse gases and solid waste using gas-fermenting microbes. Acetogens are the most attractive biocatalysts for gas fermentation as they can use C1 gases carbon monoxide (CO) and carbon dioxide (CO2) as their sole carbon source using the Wood-Ljungdahl pathway (WLP). Genes encoding WLP enzymes are located in a C1-fixing gene cluster that also contains several genes with unclear function. In this work, the role of two such genes were investigated through gene disruption in the model-acetogen Clostridium autoethanogenum. For this, CRISPR/Cas9n plasmids were constructed and used for knock-out (KO) of the glycine cleavage system H protein (gcvH) and double knock-out (dKO) of gcvH and the first 80 bp of the adjacent acsV gene. Phenotypic characterisation of ∆gcvH in autotrophic batch cultures revealed significant growth defects and altered yields of acetate and 2,3-butanediol (2,3-BDO) compared to the parental LAbrini strain. Additionally, ∆gcvH showed a very long lag phase during growth without yeast extract. Supplementing cultures with glycine shortened the lag phase but did not restore normal growth, suggesting gcvH involvement in glycine synthesis. Notably, the dKO ∆gcvH∆acsV strain failed to grow autotrophically and phenotypic characterisation of heterotrophic batch cultures revealed significant growth defects and higher 2,3-BDO and ethanol product yields compared to LAbrini. Our findings shed light on the potential links between the glycine cleavage system and WLP and contribute towards a better understanding of carbon fixation in acetogens.