Metabolic Engineering of Bacillus licheniformis for High-Yield L-Lactic Acid and Galactooligosaccharide Retention in Complementary Synbiotics Production

Abstract

Using Bacillus licheniformis H107 as the initial strain, a novel complementary synbiotics production method was developed through comprehensive metabolic engineering strategies. Key modifications included the systematic analysis and reconstruction of the central carbon metabolism pathway through precise gene editing, targeting the deletion of pflB, alsS, ydaP, and pycA genes while disrupting ganA1 and ganA2 to block galactooligosaccharide (GOS) metabolism. Additionally, heterologous expression of the L-lactate dehydrogenase gene (BcoaLDH) was implemented, resulting in the engineered strain H107-06A. Shake-flask fermentation under anaerobic conditions with 20 g/L glucose yielded L-lactic acid production of 4.45 g/L, representing a 15.3-fold increase compared to the wild type. In a 5 L fermenter using GOS syrup as the carbon source, the engineered system synergistically produced complementary synbiotics, comprising L-lactic acid (42.56 g/L), GOS (141.89 g/L, accounting for 75.09% of total sugars), and viable cells (3.82 g/L). These findings provide a foundation for developing innovative and functional fermentation products.