The industrially relevant biopolymer poly-g-glutamic acid (gamma-PGA) is commonly synthesized using glycerol, citrate, and glutamic acid as carbon sources. In this study, two strains capable of utilizing glucose as sole carbon source for gamma-PGA synthesis were constructed. Efficient gamma-PGA production was achieved with derivatives of the well-investigated laboratory strain Bacillus subtilis 168, by replacing the native promoter of the PGA synthetase operon with the strong constitutive promoter P-veg or with the xylose-inducible promoter P-xyl. The carbon yield for gamma-PGA increased by 129% to 0.131 C-mol C-mol(-1) when using glucose as the sole substrate compared to the conventional carbon source mixture glycerol, citrate, and glutamic acid. The characterization of the produced gamma-PGA demonstrated a time-dependent molecular weight of 1180-1850 kDa and a D-glutamic acid monomer content of 49-62%. To elucidate the consequences of gamma-PGA production, we characterized the engineered strain by metabolomics. While the metabolite concentrations in the TCA cycle leading up to 2-oxoglutarate decreased in gamma-PGA producer strains, the glutamic acid concentration was constant, despite the drastic increase in glutamic acid demand. The results are discussed in the context of metabolic regulation and future metabolic engineering strategies to enhance precursor supply for gamma-PGA synthesis from glucose. (C) 2020, The Society for Biotechnology, Japan. All rights reserved.