Enhancing poly-γ-glutamic acid production in Bacillus amyloliquefaciens by introducing the glutamate synthesis features from Corynebacterium glutamicum

Abstract

Background: Poly-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.

Results: In 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.

Conclusions: We 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.

Keywords: Metabolic toggle switch; NADPH-dependent glutamate dehydrogenase; Poly-γ-glutamic acid.

Fig. 1

Comparison of the two glutamate biosynthetic pathways existing in nature. a GS–GOGAT pathway; b NADPH-dependent glutamate dehydrogenase (GDH) pathway

Fig. 2

γ-PGA synthesis pathway in Bacillus amyloliquefaciens NK-1 and the schematic of the metabolic engineering work carried out in this study

Fig. 3

Comparison of the NADPH-GDH activity (a) and the γ-PGA production (b) among NK-1, NK-1 (pHT315-gdh) and NK-1 (pHT315-cgdh) strains. The reported values represent mean ± SD of triplicates

Fig. 4

Schematic of the metabolic toggle switch construction. a the design of the metabolic toggle switch for controlling odhA expression; b the schematic design for the verification of the metabolic toggle switch using a bgaB reporter gene

Fig. 5

The modified metabolic toggle switch. a the design of the XylR regulated PO1 promoter; b the schematic design of the modified metabolic toggle switch

Fig. 6

Comparison of γ-PGA production through fermentation with NK-1, NK-PO1 and NK-PO1 (pHT01-xylR) strains. 1 mM IPTG was added into each fermentation at different time point (different by 3 h from 0–24 h of the fermentation). Values represent mean ± SD of triplicates