Comparison of Isomerase and Weimberg Pathway for γ-PGA Production From Xylose by Engineered Bacillus subtilis

Abstract

The production of poly-γ-glutamic acid (γ-PGA), a biopolymer consisting of D-and L-glutamic acid monomers, currently relies on L-glutamate, or citrate as carbon substrates. Here we aimed at using plant biomass-derived substrates such as xylose. γ-PGA producing microorganisms including Bacillus subtilis natively metabolize xylose via the isomerase pathway. The Weimberg pathway, a xylose utilization pathway first described for Caulobacter crescentus, offers a carbon-efficient alternative converting xylose to 2-oxoglutarate without carbon loss. We engineered a recombinant B. subtilis strain that was able to grow on xylose with a growth rate of 0.43 h −1 using a recombinant Weimberg pathway. Although ion-pair reversed-phase LC/MS/MS metabolome analysis revealed lower concentrations of γ-PGA precursors such as 2-oxoglutarate, the γ-PGA titer was increased 6-fold compared to the native xylose isomerase strain. Further metabolome analysis indicates a metabolic bottleneck in the phosphoenolpyruvate-pyruvate-oxaloacetate node causing bi-phasic (diauxic) growth of the recombinant Weimberg strain. Flux balance analysis (FBA) of the γ-PGA producing B. subtilis indicated that a maximal theoretical γ-PGA yield is achieved on D-xylose/ D-glucose mixtures. The results of the B. subtilis strain harboring the Weimberg pathway on such D-xylose/ D-glucose mixtures demonstrate indeed resource efficient, high yield γ-PGA production from biomass-derived substrates.

Putative functions of EpsK in teichuronic acid synthesis and phosphate starvation in Bacillus licheniformis

“…Modified Bradford Protein Assay Kit (Sangon Biotech, Shanghai, China) was used to measure the protein content. The γ-PGA content was detected based on CTAB assay with incubation time adjusted to 10 min [ 22 ].…”

Enzymology of Alternative Carbohydrate Catabolic Pathways

“…This observation is claimed to be the first demonstration of a functional Weimberg pathway in fungi. Halmsschlag et al [87] compared the production from xylose of poly-γ-glutamic acid by the isomerase and Weimberg pathways by a genetically engineered strain of B. subtilis, and showed that the limiting diauxic growth in the strain could be overcome by the addition of glucose, resulting in high-yield production of the polymer from biomass substrates. Borgström et al [86] described the channelling of xylose by S. cerevisiae through the pentose phosphate pathway and into glycolysis.…”

Online measurement of the viscosity in shake flasks enables monitoring of γ‐PGA production in depolymerase knockout mutants of Bacillus subtilis with the phosphate‐starvation inducible promoter Ppst

“…One uses the power input measurement during the cultivation, which allows the calculation of the viscosity 33 . The other one is the recently published Viscosity Monitoring Online System (ViMOS), which optically measures the cultivation broth viscosity 34 and has already been used to analyze γ‐PGA production 35 . The power input measurement method is based on the effect of viscosity changes on the power input in unbaffled shake flasks 36,37 .…”

“…33 The other one is the recently published Viscosity Monitoring Online System (ViMOS), which optically measures the cultivation broth viscosity 34 and has already been used to analyze γ-PGA production. 35 The power input measurement method is based on the effect of viscosity changes on the power input in unbaffled shake flasks. 36,37 An increase of fermentation broth viscosity results in an increase of the power input.…”