Valine overproduction in Methanothermobacter marburgensis with temperature-induced promoter system and allosteric resistant acetolactate synthase variants

In times of a climate crisis caused by extreme emissions of green-house gases on a global scale, mitigation solutions need to be found. One solution is the system of carbon capture and utilization (CCU), where C1 gases, such as carbon monoxide (CO), carbon dioxide (CO 2 ), or methane, are either redirected from industrial off-gas streams or directly air-captured. A biotechnological process for CCU is the use of Methanothermobacter marburgensis for CO 2 fixation and production of value-added compounds. In this study, we focused on valine production, an amino acid important for human or feedstock nutrition. We demonstrated overproduction of valine from CO 2 in M. marburgensis with temperature-induced promoters. Here, we reached a 12.9-fold increase in valine production between the OFF- and ON states of the inducible promoter with a maximal specific production rate of 14.17 mg gCDW −1 h −1 of valine in closed batch experiments. In the second approach for valine production, we overexpressed acetolactate synthase genes with resistance to allosteric valine inhibition from Methanothermobacter thermautotrophicus recombinant in M. marburgensis . We identified a strong reduction in allosteric inhibition towards valine. This resulted in specific valine productivity of up to 40 mg gCDW −1 h −1 and states the highest specific productivity on an individual amino acid in methanogens. With those findings, we expanded the toolbox for genetic modification of M. marburgensis by a thermo-inducible promoter system and applied protein engineering for enhanced production of value-added compounds to M. marburgensis . This proof of concept shows the feasibility of archaeal cell factories generation via genetic engineering for industrial production of value-added compounds with thermophilic methanogens.