Optimization of plasmid curing from genetically engineered Clostridium autoethanogenum

Abstract

The accumulation of greenhouse gases (GHGs) released by harmful human activities involving the combustion of fossil-fuels is a driver of climate change that threatens biosustainability on Earth. Microbial gas fermentation provides an attractive option to capture CO2 while also enabling biomanufacturing of chemicals, fuels, and proteins. Acetogens are the preferred biocatalysts for gas fermentation as they can use CO2 as their sole carbon source (with H2 as energy source). However, genetic engineering of acetogens to better understand their metabolism and develop cell factories is challenged by slow growth, very low transformation efficiencies, and inefficient plasmid curing. In this thesis, we developed a CRISPR/Cas9-based curing plasmid (C-plasmid) tool for optimized plasmid curing from the model-acetogen Clostridium autoethanogenum. Firstly, the C-plasmid was constructed to express Cas9 and a gRNA targeting the ColE1 origin of replication in both the C-plasmid and an editing plasmid (E-plasmid). Next, the C-plasmid and the non-template gRNA plasmid (N-plasmid) were electroporated into C. autoethanogenum harboring an E-plasmid used for gene deletion and culture were plated on agar. Plate counting and PCR screening showed no presence of plasmids in colonies transformed with either C-plasmid or N-plasmid. This implies that cells were cured of plasmids by the act of electroporation and transformation of a C-plasmid might not be needed. In any case, this thesis seems to have identified a significantly more efficient plasmid curing method for C. autoethanogenum. Further tests are needed to confirm these observations and its applicability to other genetically-engineered C. autoethanogenum strains. The methodology has potential to contribute towards improving genetic engineering workflows for acetogens.