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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.

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