Engineering of Clostridium thermocellum lactate dehydrogenase: Effects on enzyme catalytic properties and bacterial...

Clostridium thermocellum is a promising microorganism for direct fermentation of cheap cellulosic biomass to lactic acid (LA), a chemical with several applications in food, cosmetics, pharmaceuticals and in the synthesis of biodegradable plastic polymers (e.g., polylactide). Here, we investigated the effect of different lactate dehydrogenases (LDH) on C. thermocellum LA production. A mutant LDH (LDH^S161R) showing reduced allosteric activation by fructose 1,6-bisphosphate (FBP), and a different mutant featuring a mutation in the nicotinamide cofactor binding site (LDH^I40R) increased LA yield (32 % and 53 %, respectively) with respect to C. thermocellum expressing wild type (WT) LDH. The LDH^I40R enzyme could recognize both NADH and NADPH as cofactor. Apart from FBP, other metabolic intermediates (namely NAD⁺, GDP, ATP, PP_i, and GTP) act as activity modulators of the LDH enzyme, with NAD⁺ and GDP showing the strongest inhibitory effect. Interestingly, the LDH^S161R and LDH^I40R mutant enzymes showed lower sensitivity to NAD⁺ inhibition compared to the WT enzyme. The LDH^I40R mutant was also less inhibited by ATP. These characteristics likely contributed to their ability to enhance LA yield in C. thermocellum.