Forward genetics in Thermoanaerobacter kivui: transposases as both problem and solution

• Applicant: Benjamin Zeldes, Ph.D.
• Subject Area: Microbial Ecology and Applied Microbiology
• Term: since 2026
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 577588413

Thermoanaerobacter kivui is one of only a few known thermophilic acetogens, and has gained recent interest for industrial applications due to its robust growth and ease of genetic manipulation. However, as a member of two groups of relatively understudied bacteria (thermophiles and acetogens), much remains unclear about its metabolism and lifestyle. For example, a quarter of genes in the T. kivui genome are “hypothetical” genes of unknown function. Forward genetics techniques are ideal for assigning functions to such a large number of uncharacterized genes, by first identifying strains with new phenotypes, then sequencing to determine what genetic mutations are responsible for the phenotype. Adaptive laboratory evolution (ALE) has been used to generate T. kivui strains with new phenotypes, such as ability to grow on carbon monoxide (CO). Genome sequencing of the resulting evolved strains has identified mutations that likely caused the new phenotypes (Forward genetics). This sequencing also revealed the existence of a highly active Insertion Sequence (IS) element consisting of a single transposase gene, and preliminary experiments indicate that IS element insertion can cause mutations leading to the new phenotypes. Since transposases tend to have a preferred target sequence for insertion, it is likely that results of ALE are biased by oversampling mutations in these genome regions. There have also been instances where IS elements have disrupted an intended genetic manipulation. To determine the extent to which mutation rate and the end results of ALE were influenced by IS elements, the transposases of T. kivui will be deleted using a CRISPR genome editing system. The use of CRISPR will allow for inactivation of all genome copies of a specific transposase simultaneously. Characterization of the resulting transposase-knockouts will provide a better understanding of mutation rates in T. kivui, and how they are influenced by IS elements. The transposase-free strains will also provide an unbiased background for further ALE experiments, and will be advantageous for industrial applications due to their improved genetic stability. An additional aim of the project is to develop a random transposon mutant library in T. kivui, using a synthetic transposase with minimal insertional bias. This random mutant library will serve as a tool for subsequent Forward genetics research, to help identify the function of the many remaining hypothetical genes in T. kivui’s genome.

• DFG Programme: Research Grants