Knock-down of genes essential for homoacetogenic growth using sugar inducible promoters in the thermophile Thermoanaerobacter kivui

Abstract

Given recent interest in hydrogen (H2) as a medium for storing and transporting renewable energy, the ability of acetogenic bacteria to use H2 to directly fix carbon dioxide (CO2) shows great promise. Thermophilic bacteria have long been of interest as industrial strains due to their resistance to contamination. Thermoanaerobacter kivui is among the most thermophilic acetogens. It can be genetically manipulated, but as yet only in an all-or-nothing manner, either complete gene knockouts or very strong constitutive promoters leading to protein overexpression. The sugar inducible promoters characterized here provide the finer control necessary to close remaining gaps in understanding of T. kivuis energy metabolism and for controlled expression of recombinant genes of industrial relevance. Acetogenic bacteria are capable of growing on mixtures of the gasses H2, CO2, and CO using the Wood-Ljungdahl pathway, the most energy efficient CO2 fixation pathway. T. kivui is a thermophilic (Topt = 66{degrees}C) acetogen also capable of growth on sugars including glucose, mannitol, and fructose. RNA-sequencing indicated an operon of phosphotransferase import genes up-regulated on mannitol, and growth on a mixture of glucose and mannitol was diauxic. Given this evidence for sugar responsive transcriptional regulation, the promoter region from the mannitol operon, and a similar operon involved in fructose uptake were used to control expression of a thermostable {beta}-galactosidase reporter gene. Both promoters resulted in higher reporter gene activity when grown on their inducing sugar relative to glucose. The inducible promoters were used to create "knock-down" mutants of the proton-pumping Ech1 hydrogenase. Ech1 is believed to be essential because repeated attempts to knock-out the gene operon have failed. The resulting sugar-inducible Ech1 expression strains grew better on their inducing sugar than on glucose. The mannitol inducible promoter was also used to control expression of an essential gene of the Wood-Ljungdahl pathway, formyl-THF synthetase (fhs), involved in consuming the metabolic intermediate formate. When fhs repressed by growth on glucose significantly more formate accumulated in the culture medium than during growth on mannitol, clear evidence of a metabolic bottleneck under the knock-down condition. The promoters characterized here provide finer control of recombinant or native gene expression in T. kivui, while the thermostable reporter gene allows for rapid characterization of new promoters.