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

Birthe Halmschlag; Kyra Hoffmann; René Hanke; Sastia P Putri; Eiichiro Fukusaki; Jochen Büchs; Lars M Blank

doi:10.3389/fbioe.2019.00476

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

Front Bioeng Biotechnol. 2020 Jan 21:7:476. doi: 10.3389/fbioe.2019.00476. eCollection 2019.

Authors

Birthe Halmschlag¹, Kyra Hoffmann², René Hanke², Sastia P Putri³, Eiichiro Fukusaki³, Jochen Büchs², Lars M Blank¹

Affiliations

¹ Institute of Applied Microbiology, Aachen Biology and Biotechnology, RWTH Aachen University, Aachen, Germany.
² AVT-Biochemical Engineering, RWTH Aachen University, Aachen, Germany.
³ Department of Biotechnology, Graduate School of Engineering, Osaka University, Osaka, Japan.

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.

Keywords: Bacillus subtilis; metabolic engineering; metabolome analysis; online viscosity measurement; weimberg pathway; xylose; γ-PGA.