# A thermostable Cas9-based genome editing system for thermophilic acetogenic bacterium Thermoanaerobacter kivui

## TL;DR

A new genome editing system using thermostable Cas9 was developed for the thermophilic bacterium Thermoanaerobacter kivui, enabling efficient gene knockout and integration.

## Contribution

The development of a thermostable Cas9-based system for genome editing in T. kivui expands genetic tools for thermophilic acetogenic bacteria.

## Key findings

- A thermostable Cas9 system enabled efficient gene knockout and integration in T. kivui.
- Successful integration of the adhE gene from T. ethanolicus allowed ethanol production in engineered T. kivui.
- Gene editing efficiency reached 90% after culturing with kanamycin sulfate.

## Abstract

Thermoanaerobacter kivui is a thermophilic acetogenic bacterium capable of thriving at elevated temperatures up to 66°C. It metabolizes carbohydrates such as glucose, mannose, and fructose and can also grow lithotrophically utilizing hydrogen (H2) and carbon dioxide (CO2) or carbon monoxide (CO), with acetate serving as its main product. A simple and efficient genome editing system for T. kivui would not only facilitate the understanding of the physiological function of enzymes involved in energy and carbon metabolism but also enable metabolic engineering. To address this issue, we developed a thermostable Cas9-based genome editing system for targeted gene knockout and gene integration into the T. kivui genome. Gene knockout assays were conducted on the adh gene, responsible for encoding alcohol dehydrogenase, and the ldh gene, encoding lactate dehydrogenase. Furthermore, the adhE gene from Thermoanaerobacter ethanolicus, which encodes a bifunctional aldehyde/alcohol dehydrogenase enzyme, was successfully integrated into the T. kivui genome. As a result, the engineered strain was able to produce ethanol. Following a liquid culturing period with kanamycin sulfate for about 72 hours, the efficiency of gene editing was enhanced, resulting in a ratio of mutants out of all colonies obtained of 90%. The results confirm the validity and efficiency of the thermostable Cas9-based genome editing system in T. kivui for gene editing.

Thermophilic acetogenic microorganisms represent an emerging metabolic engineering platform for the production of various biochemicals from hydrogen and carbon dioxide, or synthesis gas, under conditions of high-temperature fermentation. Gas fermentation has gained significant research interest due to its excellent thermodynamics, economic feasibility, and multisubstrate utilization. However, a major obstacle to the use of thermophilic acetogenic microorganisms as metabolic engineering platforms is the scarcity of genetic tools. This study demonstrates a proof of concept for a thermostable Cas9-based genome editing of the thermophilic acetogenic bacterium T. kivui. The system is an important expansion to the genetic toolbox of T. kivui, enabling a better understanding of key enzyme functions and the construction of cell factories for the biotechnological conversion of carbon dioxide and organic substrates into value-added products.