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

Poly-[gamma]-glutamic acid ([gamma]-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 [gamma]-PGA production, especially for those glutamate-independent [gamma]-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 [gamma]-PGA producing strain might lead to increased intracellular glutamate availability, and thus ultimate [gamma]-PGA production. In this study, the unique glutamate synthesis features from C. glutamicum was introduced into the glutamate-independent [gamma]-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 [gamma]-PGA production. Moreover, an optimized metabolic toggle switch for controlling the expression of É-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 [gamma]-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 [gamma]-PGA production but rather the resultant [gamma]-PGA production was even lower than that in the NK-1 strain. We proposed new metabolic engineering strategies to improve the [gamma]-PGA production in B. amyloliquefaciens. The NK-1 (pHT315-gdh) strain with the introduction of NADPH-GDH pathway showed 9.1% improvement in [gamma]-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 [gamma]-PGA production than the NK-1 strain. This work proposed a new strategy for improving the target product in microbial cell factories.