Sugar-inducible promoters for "knock-down" of essential genes in a thermophilic acetogen

Thermoanaerobacter kivui is an anaerobic thermophilic bacterium (T_opt 66°C) growing on sugars or H₂+CO₂. It produces acetate as a main product using the Wood-Ljungdahl Pathway (WLP), and it is promising for biotechnological applications to produce valuable organic molecules from syngas (mixture of H₂, CO₂, and CO). However, for its application, a thorough understanding of the metabolism, as well as methods of manipulating it, are required. As some genes are essential for the cell"s survival, e.g. these encoding some enzymes of the WLP, it is difficult to study the metabolism of T. kivui solely by gene knockouts. For that reason, "knock-downs" of putative essential genes were performed using newly identified sugar-inducible promoters. The aim of this work was to characterise the resulting strains to better understand the redox and energy metabolism of T. kivui.

To generate "knock-down" strains of T. kivui, a ∆pyrE acceptor strain was transformed using PCR products containing a pyrE gene¹, the new sugar-inducible promoter, and flanking regions for homologous recombination to replace the native promoter. An inducible promoter was inserted in front of a gene of interest, so that it could be regulated by the presence or absence of the inducing sugar. Growth and metabolite analysis were used to compare the phenotype of cells in different conditions, and qPCR was performed to assess the expression levels of regulated genes.

Growth of T. kivui strains modified with inducible promoters was found to be inhibited when cells were cultured on non-inducing sugars. A "knock-down" strain with a mannitol inducible promoter (P_Man) in front of the major WLP gene operon accumulated formate when grown on glucose due to a metabolic bottleneck, but growth on mannitol eliminated formate accumulation. This demonstrates that sugar-inducible promoters can be used to control essential genes in T. kivui. Further characterization of additional strains will help to fill gaps in the current understanding of T. kivui"s metabolism. The new promoters also show promise for future development of metabolically engineered strains for industrial applications.

¹ Basen et al. 2018. AEM, 84, e02210-17