Reprogramming a high robust Geobacillus thermoglucosidasius for efficient synthesis of polymer-grade lactic acid under extremely high temperature (60℃)

Optically pure lactic acid is an important precursor for the synthesis of biodegradable plastic polylactic acid. Geobacillus thermoglucosidasius is a thermophilic bacterial chassis with industrial potential. In this study, we reprogrammed this chassis to efficiently produce polymer-grade lactic acid by combining rational and semi-rational design strategies. We obtained optically pure L-lactic acid- and D-lactic acid-producing strains, GTD17 and GTD7, respectively, via rational strategies: constructing a lactic acid synthesis module, deleting by-product synthesis, and enhancing lactic acid production. At an extremely high temperature (60{degrees}C), the engineered strain GTD17 produced 94.2 g L-1 of L-lactic acid with an overall yield and productivity of 91.5% and 2.0 g L-1 h-1, respectively, and the optical purity of L-lactic acid was 99.5%. We then performed semi-rational adaptive evolution on the engineered strain GTD7 to obtain an optically pure D-lactic acid-producing strain GTD7-144; at the extremely high temperature (60{degrees}C), it produced 153.1 g L-1 of D-lactic acid with an overall yield and productivity of 93.0% and 3.2 g L-1 h-1, respectively. The optical purity of the D-lactic acid was 99.6%. Genome resequencing and analysis of the strain GTD7-144 revealed potential genetic targets that may improve production performance. This study represents an advancement in the high-temperature production of polymer-grade lactic acid by G. thermoglucosidasius.