Engineering mitochondrial lipoate metabolism through lplA expression improves alpha-lipoic acid production in Pichia...

α-Lipoic acid (ALA) is an essential metabolic cofactor involved in oxidative metabolism and redox regulation, with increasing interest for its biotechnological production due to the limitations of chemical synthesis. In this study, we evaluated a heterologous approach to modulate lipoate metabolism in Pichia pastoris, a characterized by high-cell-density, strictly aerobic production host, by expressing a mitochondrially targeted bacterial lipoate-protein ligase (LplA) under the control of the constitutive P_GAP promoter. His design aimed to couple an exogenous activation pathway with endogenous lipoylation. Expression of LplA did not result in lipoylated proteins levels under flask conditions. Notably, when cultivated in a stirred-tank bioreactor under control conditions (40% dissolved oxygen, 400 rpm, 28 °C), strain Y2 showed up to 1.5-fold higher biomass and a 42% increase in ALA accumulation at the end of the exponential phase compared to the wild-type strain. This increase was associated with higher volumetric productivity (2.41 vs 2.07 µg/mL·h), despite a reduction in specific productivity. Then, expression of LplA can influence intracellular lipoate metabolism and promote ALA accumulation under specific physiological conditions without precursor supplementation. This study highlights the potential of P. pastoris as a platform for developing alternative strategies for ALA production.