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 Pveg and (2) Varying induction strength of the xylose inducible promoter Pxyl. 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.
Highlights
Poly-γ-glutamic acid (γ-PGA) is an anionic, biodegradable, non-toxic polymer composed of Dand L-glutamic acid units linked by γ-glutamyl bonds. γ-PGA production with Bacillus subtilis is mainly known from B. subtilis in the production of natto, a traditional Japanese dish
To identify metabolites that are highly correlated with γ-PGA production from glucose in B. subtilis, intracellular metabolite measurements for strains with varying γ-PGA synthesis rates were carried out
Poly-γ-glutamic acid production with glucose as sole carbon source requires strong flux rerouting in the metabolic network of B. subtilis
Summary
Poly-γ-glutamic acid (γ-PGA) is an anionic, biodegradable, non-toxic polymer composed of Dand L-glutamic acid units linked by γ-glutamyl bonds. γ-PGA production with Bacillus subtilis is mainly known from B. subtilis (natto) in the production of natto, a traditional Japanese dish. B. subtilis has been considered as a promising organism for industrial production of γ-PGA. Higher γ-PGA titers have been achieved by process optimization. The use of several substrates for γ-PGA production and different process strategies like fed-batch cultivations have been reported (Ogawa et al, 1997; Yoon et al, 2000). Wild-type γ-PGA producers like B. subtilis (natto) (Ogawa et al, 1997) and Bacillus licheniformis ATCC9945 (Cromwick et al, 1996) have been used for the Metabolic Impact of γ-PGA Production production focusing rather on process optimization than on metabolic engineering strategies to achieve a more efficient production. In order to find metabolic engineering targets to enhance γ-PGA production, a genetically amenable host organism is favorable
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