Investigation by NMR of metabolic routes to bacterial γ-poly(glutamic acid) using13C-labeled citrate and glutamate as media carbon sources

Abstract

Bacillus licheniformis ATCC 9945A produces γ-poly(glutamic acid) (γ-PGA) when using glutamate, citrate, and glycerol in combination as media carbon sources. Also, various aspects of B. licheniformis cellular physiology are affected by the concentration of Mn(II) salts in culture media. Thus, the metabolism of carbon sources into γ-PGA as a function of MnSO4 concentration was studied by enriching the media with either L-[1,2-¹³C]glutamic acid (NH₂—²CH(¹COOH)—(CH₂)₂-COOH) or [1,5-¹³C]citric acid (HOO¹C—CH₂—C(OH)(COOH)—CH₂—⁵COOH) at two media MnSO₄concentrations (615 and 0 μM). Enrichment factors (EF) from¹³C-NMR spectra were calculated from the ratio of peak intensities from¹³C-enriched γ-PGA divided by the ratio of peak intensities for nonenriched γ-PGA. EF values were than used to determine the percentage of repeat units that were formed from glutamate and citrate. The percentage of repeat units formed from provided glutamate at the 0 and 615 μM Mn(II) media concentrations was 89 ± 14 and 67 ± 11%, respectively. These respective products have 51 ± 9 and 39 ± 11% of their repeat units formed with apparent retention of the glutamate carbon skeleton. Also, enrichment of γ-PGA repeat units at C-1 was found to be lower than C-2 at both Mn(II) levels. Thus, provided glutamate was used to a large extent for polymer formation with both retention of the carbon skeleton as well as decarboxylation at C-1. Provided citrate was also used as a source of carbon to form repeat units. At the 0 and 615 μM media Mn(II) levels, 9 ± 4 and 19 ± 5% of repeat units were formed from citrate. It is believed that citrate is metabolized to γ-PGA by entry into the citric acid cycle and formation of α-ketoglutarate. Analysis of products from cultivations where both glutamate and citrate were¹³C enriched indicated that citrate and glutamate carbon source metabolism to γ-PGA occurs via independent pathways to common monomer precursors without multiple recycling of these carbon sources through catabolic and anabolic pathways.Key words: γ-poly(glutamic acid), metabolism, manganese, Bacillus licheniformis, nuclear magnetic resonance.