Identification of Key Metabolites in Poly-γ-Glutamic Acid Production by Tuning γ-PGA Synthetase Expression

Abstract

Poly-γ-glutamic acid (γ-PGA) production is commonly achieved using glycerol, citrate, and L-glutamic acid as substrates. The constitutive expression of the γ-PGA synthetase enabled γ-PGA production with Bacillus subtilis from glucose only. The precursors for γ-PGA synthesis, D-and L-glutamate, are ubiquitous metabolites. Hence, the metabolic flux toward γ-PGA directly depends on the concentration and activity of the synthetase and thereby on its expression. To identify pathway bottlenecks and important metabolites that are highly correlated with γ-PGA production from glucose, we engineered B. subtilis strains with varying γ-PGA synthesis rates. To alter the rate of γ-PGA synthesis, the expression level was controlled by two approaches: (1) Using promoter variants from the constitutive promoter P veg and (2) Varying induction strength of the xylose inducible promoter P xyl . The variation in the metabolism caused by γ-PGA production was investigated using metabolome analysis. The xylose-induction strategy revealed that the γ-PGA production rate increased the total fluxes through metabolism indicating a driven by demand adaption of the metabolism. Metabolic bottlenecks during γ-PGA from glucose were identified by generation of a model that correlates γ-PGA production rate with intracellular metabolite levels. The generated model indicates the correlation of certain metabolites such as phosphoenolpyruvate with γ-PGA production. The identified metabolites are targets for strain improvement to achieve high level γ-PGA production from glucose.

Quantification of the actual composition of polymeric nanocapsules: a quality control analysis

“…It could be speculated that the polymer molecules that are not part of the NCs’ shell may be solubilized and/or form micelles in the suspending medium. In addition, complexation products between some components, especially polymers with counter-charged surfactants, might be formed, as it is described, for example, for CTAB and PGA [ 36 ]. Finally, a highly dense PEGylated NC surface (as in SE- b- PEG-HA) might hinder the electrostatic interactions between the polymers and the counter-charged surfactants present at the oil/water interphase.…”

Functional Caracterization of CapBCA in Controlling Poly-γ-Glutamic Acid Synthesis in Corynebacterium Glutamicum

“…Besides, γ-PGA yield has been reported to increase with increasing the expression of γ-PGA synthase [32]. Moreover, the strength of pgsB and pgsC expression has been noted to have a greater impact on γ-PGA synthesis [14,20,[33][34][35].…”

Poly-Gamma-Glutamic Acid Secretion Protects Bacillus subtilis from Zinc and Copper Intoxication

“… B. subtilis PGA production was measured using a cetryltrimethylammonium bromide (CTAB) assay as previously described ( 38 , 39 ). Cells were grown in LB broth containing various concentrations of ZnSO 4 or CuSO 4 in LB media overnight at 37°C for 16–18 h. Cells were then pelleted from 15 mL of culture by centrifugation at 10000 × g for 30 min at 4°C and the supernatant was retained.…”