Flux Connections Between Gluconate Pathway, Glycolysis, and Pentose–Phosphate Pathway During Carbohydrate Metabolism in Bacillus megaterium QM B1551

Bacillus megaterium is a bacterium of great importance as a plant-beneficial bacterium in agricultural applications and in industrial bioproduction of proteins. Understanding intracellular processing of carbohydrates in this species is crucial to predicting natural carbon utilization as well as informing strategies in metabolic engineering. Here, we applied stable isotope-assisted metabolomics profiling and metabolic flux analysis to elucidate, at high resolution, the connections of the different catabolic routes for carbohydrate metabolism immediately following substrate uptake in B. megaterium QM B1551. We performed multiple 13C tracer experiments to obtain both kinetic and long-term 13C profiling of intracellular metabolites. In addition to the direct phosphorylation of glucose to glucose-6-phosphate (G6P) prior to oxidation to 6-phosphogluconate (6P-gluconate), the labeling data also captured glucose catabolism through the gluconate pathway involving glucose oxidation to gluconate followed by phosphorylation to 6P-gluconate. Our data further confirmed the absence of the Entner–Doudoroff pathway in B. megaterium and showed that subsequent catabolism of 6P-gluconate was instead through the oxidative pentose–phosphate (PP) pathway. Quantitative flux analysis of glucose-grown cells showed equal partition of consumed glucose from G6P to the Embden–Meyerhof–Parnas (EMP) pathway and from G6P to the PP pathway through 6P-gluconate. Growth on fructose alone or xylose alone was consistent with the ability of B. megaterium to use each substrate as a sole source of carbon. However, a detailed 13C mapping during simultaneous feeding of B. megaterium on glucose, fructose, and xylose indicated non-uniform intracellular investment of the different carbohydrate substrates. Flux of glucose-derived carbons dominated the gluconate pathway and the PP pathway, whereas carbon flux from both glucose and fructose populated the EMP pathway; there was no assimilatory flux of xylose-derived carbons. Collectively, our findings provide new quantitative insights on the contribution of the different catabolic routes involved in initiating carbohydrate catabolism in B. megaterium and related Bacillus species

Elucidating the central carbon metabolism of Bacillus Megaterium QM B1551

We investigated the central carbon metabolism of Bacillus megaterium QM B1551 cells using various carbon substrates, long-term isotopic enrichment experiments with 13C-labeled carbons, metabolic flux analysis (MFA), and kinetic incorporation of 13C-labeled carbons. We determined that gluconate can be incorporated into the metabolic network structure of B. megaterium QM B1551, particularly when gluconate was present in the environment. It was shown in the same experiment that B. megaterium QM B1551 did not use the Entner-Doudoroff (ED) pathway for glucose (Gluc) metabolism, and instead relied primarily on the Embden-Mayerhof-Parnas (EMP) and oxidative pentose phosphate (PP) pathways. The TCA cycle of this bacterium was incompletely bifurcated, although this bifurcation did not impede the ability of the bacterium to grow. There were bottlenecks in the fluxes from metabolite nodes alpha-ketoglutarate (αKG) to succinate, from succinate to fumarate, and from oxaloacetate (OAA) to fumarate. This bifurcation was overcome with a direct input of substrate into the TCA cycle. The stereochemistry of citrate formation and cleavage was different compared to other bacterial flux studies (Sasnow et al., 2016). It was hypothesized that the carboxylation reaction which combines CO2 and pyruvate (PYR) added CO2 behind the third carbon position of PYR to form OAA. It was further hypothesized that OAA was flipped such that the fourth carbon position of OAA became the fourth carbon position of citrate. Finally, it was observed that B. megaterium QM B1551 had an early preference for glutamate catabolism in the TCA cycle, followed by a preference for Gluc catabolism across the metabolic network. I hope these results clarified contradictions in the literature, and that they will direct research to an optimized industrial use of this bacterium

Validation and Application of a Low-Cost Sorting Device for Fumonisin Reduction in Maize

Fumonisin mycotoxins are a persistent challenge to human and livestock health in tropical and sub-tropical maize cropping systems, and more efficient methods are needed to reduce their presence in food systems. We constructed a novel, low-cost device for sorting grain, the “DropSort”, and tested its effectiveness on both plastic kernel models and fumonisin-contaminated maize. Sorting plastic kernels of known size and shape enabled us to optimize the sorting performance of the DropSort. The device sorted maize into three distinct fractions as measured by bulk density and 100-kernel weight. The level of fumonisin was lower in the heaviest fractions of maize compared to the unsorted samples. Based on correlations among fumonisin and bulk characteristics of each fraction, we found that light fraction 100-kernel weight could be an inexpensive proxy for unsorted fumonisin concentration. Single kernel analysis revealed significant relationships among kernel fumonisin content and physical characteristics that could prove useful for future sorting efforts. The availability of a low-cost device (materials~USD 300) that can be used to reduce fumonisin in maize could improve food safety in resource-limited contexts in which fumonisin contamination remains a pressing challenge