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

ABSTRACT: Poly-?-glutamic acid (?-PGA) production is commonly achieved using glycerol, citrate, and L-glutamic acid as substrates. The constitutive expression of the ?-PGA synthetase enabled ?-PGA production with Bacillus subtilis from glucose only. The precursors for ?-PGA synthesis, D- and L-glutamate, are ubiquitous metabolites. Hence, the metabolic flux toward ?-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 ?-PGA production from glucose, we engineered B. subtilis strains with varying ?-PGA synthesis rates. To alter the rate of ?-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 ?-PGA production was investigated using metabolome analysis. The xylose-induction strategy revealed that the ?-PGA production rate increased the total fluxes through metabolism indicating a driven by demand adaption of the metabolism. Metabolic bottlenecks during ?-PGA from glucose were identified by generation of a model that correlates ?-PGA production rate with intracellular metabolite levels. The generated model indicates the correlation of certain metabolites such as phosphoenolpyruvate with ?-PGA production. The identified metabolites are targets for strain improvement to achieve high level ?-PGA production from glucose.

SUBMITTER: Halmschlag B

PROVIDER: S-EPMC7002566 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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

Halmschlag Birthe B Putri Sastia P SP Fukusaki Eiichiro E Blank Lars M LM

Frontiers in bioengineering and biotechnology 20200130

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 <i>Bacillus subtilis</i> 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 bottleneck ...[more]

PMID: 32083073

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Project description:BackgroundPoly-γ-glutamic acid (γ-PGA) is a valuable polymer with glutamate as its sole precursor. Enhancement of the intracellular glutamate synthesis is a very important strategy for the improvement of γ-PGA production, especially for those glutamate-independent γ-PGA producing strains. Corynebacterium glutamicum has long been used for industrial glutamate production and it exhibits some unique features for glutamate synthesis; therefore introduction of these metabolic characters into the γ-PGA producing strain might lead to increased intracellular glutamate availability, and thus ultimate γ-PGA production.ResultsIn this study, the unique glutamate synthesis features from C. glutamicum was introduced into the glutamate-independent γ-PGA producing Bacillus amyloliquefaciens NK-1 strain. After introducing the energy-saving NADPH-dependent glutamate dehydrogenase (NADPH-GDH) pathway, the NK-1 (pHT315-gdh) strain showed slightly increase (by 9.1%) in γ-PGA production. Moreover, an optimized metabolic toggle switch for controlling the expression of ɑ-oxoglutarate dehydrogenase complex (ODHC) was introduced into the NK-1 strain, because it was previously shown that the ODHC in C. glutamicum was completely inhibited when glutamate was actively produced. The obtained NK-PO1 (pHT01-xylR) strain showed 66.2% higher γ-PGA production than the NK-1 strain. However, the further combination of these two strategies (introducing both NADPH-GDH pathway and the metabolic toggle switch) did not lead to further increase of γ-PGA production but rather the resultant γ-PGA production was even lower than that in the NK-1 strain.ConclusionsWe proposed new metabolic engineering strategies to improve the γ-PGA production in B. amyloliquefaciens. The NK-1 (pHT315-gdh) strain with the introduction of NADPH-GDH pathway showed 9.1% improvement in γ-PGA production. The NK-PO1 (pHT01-xylR) strain with the introduction of a metabolic toggle switch for controlling the expression of ODHC showed 66.2% higher γ-PGA production than the NK-1 strain. This work proposed a new strategy for improving the target product in microbial cell factories.

Dataset Information

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

Publications

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

Similar Datasets

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