A sufficient supply of reducing equivalents is essential for obtaining the maximum yield of target products in anaerobic fermentation. The pyruvate dehydrogenase (PDH) complex controls the critical step in pyruvate conversion to acetyl-CoA and NADH. However, in anaerobic Escherichia coil, PDH residing in the dihydrolipoamide dehydrogenase (LPD) component is normally inactive due to inhibition by NADH. In this study, the protein engineering of LPD by structural analysis was explored to eliminate this inhibition. A novel IAA350/351/ 358VVV triple mutant was successfully verified to be more effective than other LPD mutants reported till date. Notably, PDH activity with the triple mutant at an [NADFI]/[NAD(+)] ratio of 0.15 was still higher than that of the wild-type without NADH addition. The altered enzyme of the PDH complex was also active in the presence of such high NADH levels. This is the first study concerning protein engineering of PDH by structure-guided design. The presence and functional activity of such an NADH-insensitive PDH complex provides a useful metabolic element for fermentation products and has potential for biotechnological application.