The Absence of Pyruvate Kinase Affects Glucose-Dependent Carbon Catabolite Repression in Bacillus subtilis

Abstract

Pyruvate is a key intermediate of diverse metabolic pathways of central carbon metabolism. In addition to being the end product of glycolysis, pyruvate is an essential carbon distribution point to oxidative metabolism, amino acid and fatty acid syntheses, and overflow metabolite production. Hence, a tight regulation of pyruvate kinase (Pyk) activity is of great importance. This study aimed to analyze targeted metabolites from several pathways and possible changes in Bacillus subtilis lacking Pyk. Wild type and Δpyk cells were cultivated in chemically defined medium with glucose and pyruvate as carbon sources, and the extracted metabolites were analyzed by 1H-NMR, GC-MS, HPLC-MS, and LC-MS/MS. The results showed that the perturbation created in the pyruvate node drove an adaptation to new conditions by altering the nutritional compounds’ consumption. In Δpyk, pyruvate, which is subject to glucose-dependent carbon catabolite repression, did not comply with the hierarchy in carbon source utilization. Other metabolic alterations were observed such as the higher secretion of the overflow metabolites acetoin and 2,3-butanediol by Δpyk. Our results help to elucidate the regulatory transport of glucose and pyruvate in B. subtilis and possible metabolic reroute to alternative pathways in the absence of Pyk.

Production of acetoin and its derivative tetramethylpyrazine from okara hydrolysate with Bacillus subtilis

“…4 c). CcpA, the global transcription factor of Bacillus subtilis , binds to the phosphorylated cofactor HPr to form a ternary complex that represses other carbon-derived catabolite related gene expression during mixed sugar fermentation (Sousa et al 2019 ). It was suggested by Lu Lin et al ( 2020 ) that CCR could be relieved by inhibiting the activities of two enzymes, CcpA (the global transcription factor of Bacillus subtilis ) and HPrK.…”

Unraveling the potential and constraints associated with corn steep liquor as a nutrient source for industrial fermentations

“…B. subtilis reached its first OTR peak of 25 mmol/L/h after 3 h, when grown on glucose‐enriched LB medium. Following the first OTR peak, growth on the LB‐glucose medium displayed three other peaks after 5, 9, and 16 h. As Bacillus subtilis is known to produce acetate, acetoin, and 2,3‐butanediol at excess glucose concentrations, 55–57 different respiratory activities may indicate a change in substrate and overflow metabolite consumption. Growth on the complex components of the LB‐glucose medium led to ammonium ions being release, 6 causing the pH to be increased to 7.5 (Figure 4b).…”

“…This OTR peak was followed by a second OTR peak of 14.5 mmol/L/h. At this highest CSL concentration, B. subtilis is likely to have produced its known overflow metabolites acetate, acetoin, and 2,3-butanediol, [55][56][57] which are then consumed as indicated by the second peak (Figure 5a).…”

Wobble Editing of Cre-box by Unspecific CRISPR/Cas9 Causes CCR Release and Phenotypic Changes in Bacillus pumilus

“…Since previous studies on CCR in Bacillus showed that cre negatively regulates genes related to carbohydrate catabolism ( Sousa et al, 2019 ), mutation of cre could potentially disrupt secondary pathways, and lead to a significant increase in extracellular excretion. When we incubated the mutant LG3145 strain on LB agar, colonies were more adhesive and pigmented under the naked eye, with fuzzy edges, whereas WT colonies appeared smooth, white, and with distinct edges ( Supplementary Figures S6A, 6B ).…”