Central Metabolism Of Bacillus Thuringiensis During Growth And Sporulation

Several major features of central metabolism during the Bacillus thuringiensis fermentation for bioinsecticide production remain uncertain. Thus it was desired to identify and quantitate the central metabolic pathways of Bt growth and sporulation.;To achieve these objectives it was necessary to identify and measure all potentially significant cellular inputs and outputs. A set of pathways possibly comprising central metabolism was assumed, based on literature and experimental findings, including detection of several intermediates of the three branched-chain amino acid (viz. valine, leucine and isoleucine) pathways. Selected samples were incubated anaerobically to measure inhibition of catabolic flux through branched-chain 2-oxoacid dehydrogenase. Chloramphenicol was present to block enzyme induction and biosynthetic flows. Dehydrogenation rates of two branched-chain 2-oxoacids were calculated from the flux of the third one, by employing rates of branched-chain acyl-CoA use for fatty acid biosynthesis to determine relative pathway flow rates. This permitted model solution through molar balances around acertyl-CoA and pyruvate.;The following liquid-phase analytes were measured: glucose, ammonia and each of 16 amino acids after hydrolysis; volatile organic products: acetate, acetoin, isobutyrate, 2-methylbutyrate and isovalerate; and non-volatile organic products: 2,3-butanediol, lactate, pyruvate, combined 2-keto-isovalerate and 2-hydroxy-isovalerate, 2-keto-3-methylvalerate, 2-keto-isocaproate and succinate. Biomass content of poly-{dollar}\beta{dollar}-hydroxybutyric acid (PHB), poly-{dollar}\beta{dollar}-hydroxyvaleric acid (PHV), and 15 fatty acids was also determined.;Anaerobic incubation generally led to accumulation of isoleucine pathway intermediates, which was used to calculate valine and leucine pathway fluxes. Increased 2-oxoisovalerate biosynthesis accompanied granulation as vegetative growth ended. Greater catabolic flux in valine and leucine pathways led to induction of the glyoxylate cycle and malic enzyme as sporulation began. After forespore membrane completion, relatively increased leucine pathway flux accompanied protein turnover. Subsequently leucine and valine catabolic pathways operated jointly, apparently to complete turnover of mother-cell into spore constituents and metabolic energy.;In conclusion, the three branched-chain amino acid pathways operate as complete amphibolic units, functionally integrated with the known pathways of central metabolism in B. thuringiensis (viz. glycolysis, tricarboxylic acid and glyoxylate cycles). These novel pathways complete potential cycles for interconversion and/or terminal oxidation of metabolic intermediates, especially during cellular differentiation