Glucokinase contributes to glucose phosphorylation in d -lactic acid production by Sporolactobacillus inulinus Y2-8

Sporolactobacillus inulinus , a homofermentative lactic acid bacterium, is a species capable of efficient industrial d -lactic acid production from glucose. Glucose phosphorylation is the key step of glucose metabolism, and fine-tuned expression of which can improve d -lactic acid production. During growth on high-concentration glucose, a fast induction of high glucokinase (GLK) activity was observed, and paralleled the patterns of glucose consumption and d -lactic acid accumulation, while phosphoenolpyruvate phosphotransferase system (PTS) activity was completely repressed. The transmembrane proton gradient of 1.3–1.5 units was expected to generate a large proton motive force to the uptake of glucose. This suggests that the GLK pathway is the major route for glucose utilization, with the uptake of glucose through PTS-independent transport systems and phosphorylation of glucose by GLK in S. inulinus d -lactic acid production. The gene encoding GLK was cloned from S. inulinus and expressed in Escherichia coli . The amino acid sequence revealed significant similarity to GLK sequences from Bacillaceae. The recombinant GLK was purified and shown to be a homodimer with a subunit molecular mass of 34.5 kDa. Strikingly, it demonstrated an unusual broad substrate specificity, catalyzing phosphorylation of 2-deoxyglucose, mannitol, maltose, galactose and glucosamine, in addition to glucose. This report documented the key step concerning glucose phosphorylation of S. inulinus , which will help to understand the regulation of glucose metabolism and d -lactic acid production.