| Summary: | <i>Pseudoclostridium thermosuccinogenes</i> is a thermophilic bacterium capable of producing succinate from lignocellulosic-derived sugars and has the potential to be exploited as a platform organism. However, exploitation of <i>P. thermosuccinogenes</i> has been limited partly due to the genetic inaccessibility and lack of genome engineering tools. In this study, we established the genetic accessibility for <i>P. thermosuccinogenes</i> DSM 5809. By overcoming restriction barriers, transformation efficiencies of 10<sup>2</sup> CFU/µg plasmid DNA were achieved. To this end, the plasmid DNA was methylated in vivo when transformed into an engineered <i>E. coli</i> HST04 strain expressing three native methylation systems of the thermophile. This protocol was used to introduce a ThermodCas9-based CRISPRi tool targeting the gene encoding malic enzyme in <i>P. thermosuccinogenes</i>, demonstrating the principle of gene silencing. This resulted in 75% downregulation of its expression and had an impact on the strain’s fermentation profile. Although the details of the functioning of the restriction modification systems require further study, in vivo methylation can already be applied to improve transformation efficiency of <i>P. thermosuccinogenes.</i> Making use of the ThermodCas9-based CRISPRi, this is the first example demonstrating that genetic engineering in <i>P. thermosuccinogenes</i> is feasible and establishing the way for metabolic engineering of this bacterium.
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