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

ABSTRACT: The production of poly-?-glutamic acid (?-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. ?-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 ?-PGA precursors such as 2-oxoglutarate, the ?-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 ?-PGA producing B. subtilis indicated that a maximal theoretical ?-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 ?-PGA production from biomass-derived substrates.

SUBMITTER: Halmschlag B

PROVIDER: S-EPMC6985040 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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

Halmschlag Birthe B Hoffmann Kyra K Hanke René R Putri Sastia P SP Fukusaki Eiichiro E Büchs Jochen J Blank Lars M LM

Frontiers in bioengineering and biotechnology 20200121

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 altern ...[more]

PMID: 32039180

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Project description:Glucose, xylose and arabinose are the three most abundant monosaccharide found in lignocellulosic biomass. Effectively and simultaneously utilization of these sugars by microorganisms for production of the biofuels and bio-chemicals is essential toward directly fermentation of the lignocellulosic biomass. In our previous study, the recombinant Bacillus subtilis 168ARSRCPΔacoAΔbdhA strain was already shown to efficiently utilize xylose for production of acetoin, with a yield of 0.36 g/g xylose. In the current study, the Bacillus subtilis168ARSRCPΔacoAΔbdhA strain was further engineered to produce acetoin from a glucose, xylose, and arabinose mixtures. To accomplish this, the endogenous xylose transport protein AraE, the exogenous xylose isomerase gene xylA and the xylulokinase gene xylB from E. coli were co-overexpressed in the Bacillus subtilis 168ARSRCPΔacoAΔbdhA strain, which enabled the resulting strain, denoted ZB02, to simultaneously utilize glucose and xylose. Unexpectedly, the ZB02 strain could simultaneously utilize glucose and arabinose also. Further results indicated that the transcriptional inhibition of the arabinose transport protein gene araE was the main limiting factor for arabinose utilization in the presence of glucose. Additionally, the arabinose operon in B. subtilis could be activated by the addition of arabinose, even in the presence of glucose. Through fed-batch fermentation, strain ZB02 could simultaneously utilize glucose, xylose, and arabinose, with an average sugar consumption rate of 3.00 g/l/h and an average production of 62.2 g/l acetoin at a rate of 0.864 g/l/h. Finally, the strain produced 11.2 g/l acetoin from lignocellulosic hydrolysate (containing 20.6g/l glucose, 12.1 g/l xylose and 0.45 g/l arabinose) in flask cultivation, with an acetoin yield of 0.34 g/g total sugar. The result demonstrates that this strain has good potential for the utilization of lignocellulosic hydrolysate for production of acetoin.

Project description:BackgroundWith D-xylose being the second most abundant sugar in nature, its conversion into products could significantly improve biomass-based process economy. There are two well-studied phosphorylative pathways for D-xylose metabolism. One is isomerase pathway mainly found in bacteria, and the other one is oxo-reductive pathway that always exists in fungi. Except for these two pathways, there are also non-phosphorylative pathways named xylose oxidative pathways and they have several advantages over traditional phosphorylative pathways. In Myceliophthora thermophila, D-xylose can be metabolized through oxo-reductive pathway after plant biomass degradation. The survey of non-phosphorylative pathways in this filamentous fungus will offer a potential way for carbon-efficient production of fuels and chemicals using D-xylose.ResultsIn this study, an alternative for utilization of D-xylose, the non-phosphorylative Weimberg pathway was established in M. thermophila. Growth on D-xylose of strains whose D-xylose reductase gene was disrupted, was restored after overexpression of the entire Weimberg pathway. During the construction, a native D-xylose dehydrogenase with highest activity in M. thermophila was discovered. Here, M. thermophila was also engineered to produce 1,2,4-butanetriol using D-xylose through non-phosphorylative pathway. Afterwards, transcriptome analysis revealed that the D-xylose dehydrogenase gene was obviously upregulated after deletion of D-xylose reductase gene when cultured in a D-xylose medium. Besides, genes involved in growth were enriched in strains containing the Weimberg pathway.ConclusionsThe Weimberg pathway was established in M. thermophila to support its growth with D-xylose being the sole carbon source. Besides, M. thermophila was engineered to produce 1,2,4-butanetriol using D-xylose through non-phosphorylative pathway. To our knowledge, this is the first report of non-phosphorylative pathway recombinant in filamentous fungi, which shows great potential to convert D-xylose to valuable chemicals.

Dataset Information

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

Publications

Comparison of Isomerase and Weimberg Pathway for γ-PGA Production From Xylose by Engineered <i>Bacillus subtilis</i>.

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