The oxidative Weimberg pathway for the five-step pentose degradation to α ketoglutarate from Caulobacter crescentus is a key route for sustainable bioconversion of lignocellulosic biomass to added-value products and biofuels. Here, we developed a novel iterative approach involving initial rate kinetics, progress curves, and enzyme cascades, with high resolution NMR analysis of intermediate dynamics, and multiple cycles of kinetic modelling analyses to construct and validate a quantitative model for the Weimberg pathway.

**DOI:**
10.15490/fairdomhub.1.investigation.284.2

**Zenodo URL:**
None

**Created at:** 30th Jan 2020 at 10:08

#### Initial rate kinetics

Initial rate kinetics for the purified Caulinobacter crescentus Weimberg pathway enzymes, including substrate dependence, and product inhibition.

#### XDH

Kinetic characterisation and mathematical modelling of XDH.

#### XDH-initial rate kinetics data

Initial rate kinetics for xylose dehydrogenase of Caulobacter crescentus, saturation with xylose and NAD, and inhibition by NADH and xylonolactone.

- XDH-initial rate kinetics.xlsx

#### XDH model

Model for the Caulobacter crescentus xylose dehydrogenase, describing the initial rate kinetics including substrate dependence and product inhibition. If the Mathematica notebook is downloaded and the data file for the XDH kinetics is downloaded in the same directory, then the notebook can be evaluated. The model in the notebook will then be parameterised and the figures in the manuscript for XDH will be reproduced.

- XDH-initial rate.nb

#### XLA

Kinetic characterisation and mathematical modelling of XLA.

#### XLA-initial rate kinetics data

Initial rate kinetics for the xylonolactonase reaction of Caulobacter crescentus, xylonolactone saturation for the enzyme catalysed reaction, and for the non-enzymatic reaction.

- XLA-initial rate kinetics.xlsx

#### XLA model

Model for the Caulobacter crescentus xylonolactonase, describing the initial rate kinetics and substrate dependence. If the Mathematica notebook is downloaded and the data file for the XLA kinetics is downloaded in the same directory, then the notebook can be evaluated. The model in the notebook will then be parameterised and the figures in the manuscript for XLA will be reproduced.

- XLA-initial rate.nb

#### XAD

Kinetic characterisation and mathematical modelling of XAD.

#### XAD-initial rate kinetics data

Initial rate kinetics for the xylonate dehydratase of Caulobacter crescentus, xylonate saturation.

- XAD-initial rate kinetics.xlsx

#### XAD model

Model for the Caulobacter crescentus xylonate dehydratase, describing the initial rate kinetics for substrate dependence. If the Mathematica notebook is downloaded and the data file for the XAD kinetics is downloaded in the same directory, then the notebook can be evaluated. The model in the notebook will then be parameterised and the figures in the manuscript for XAD will be reproduced.

- XAD-initial rate.nb

#### KDXD

Kinetic characterisation and mathematical modelling of KDXD.

#### KDXD-initial rate kinetics data

Initial rate kinetics for the 2-keto-3-deoxy-D-xylonate dehydrates of Caulobacter crescentus, 2-keto-3-deoxy-D-xylonate saturation and inhibitor titrations.

- KDXD-initial rate kinetics.xlsx

#### KDXD model

Model for the Caulobacter crescentus 2-keto-3-deoxy-D-xylonate dehydratase, describing the initial rate kinetics for substrate dependence and product inhibition. If the Mathematica notebook is downloaded and the data file for the XAD kinetics is downloaded in the same directory, then the notebook can be evaluated. The model in the notebook will then be parameterised and the figures in the manuscript for KDXD will be reproduced.

- KDXD-initial rate.nb

#### KGSADH

Kinetic characterisation and mathematical modelling of KGSADH.

#### KGSADH-initial rate kinetics data

Initial rate kinetics for the α-ketoglutarate semialdehyde dehydrogenase of Caulobacter crescentus, α-ketoglutarate semialdehyde and NAD saturation, and α-ketoglutarate, NADH and 2-keto-3-deoxy-D-xylonate inhibition.

- KGSADH-initial rate kinetics.xlsx

#### KGSADH model

Model for the Caulobacter crescentus Î±-ketoglutarate semialdehyde dehydrogenase, describing the initial rate kinetics for substrate dependence and product inhibition. If the Mathematica notebook is downloaded and the data file for the XAD kinetics is downloaded in the same directory, then the notebook can be evaluated. The model in the notebook will then be parameterised and the figures in the manuscript for KGSADH will be reproduced.

- KGSADH-initial rate-SEEK.nb

#### Progress curves

Progress curves for the purified Caulinobacter crescentus Weimberg pathway enzymes. Each reaction is followed up to completion and then the next enzyme in the pathway is added, i.e. XDH-XLA-XAD-KDXD and finally KGSADH

#### Progress curve XDH

Conversion of Xyl to XLAC by Caulobacter crescentus XDH, measured in NMR.

#### Progress curve analysis data

Data files for the conversion of XYL to KG, via the sequential addition of the Caulobacter crescentus Weimberg pathway enzymes, XDH, XLA, XAD, KDXD, KGSADH.

- progress curve analysis data.xlsx

#### Progress curve analysis XDH

Model for the Caulobacter crescentus Weimberg pathway, describing the conversion of Xyl to KG upon sequential adition of purified enzymes. If the Mathematica notebook is downloaded and the data file is downloaded in the same directory, then the notebook can be evaluated, and the figure in the manuscript for the progress curves will be reproduced.

- progressCurves-XDH-SEEK.nb

#### Progress curve analysis (shen1)

Model for the Caulobacter crescentus Weimberg pathway, describing the conversion of Xyl to KG, with sequential addition of purified enzymes.

- shen1.xml

#### Progress curve XLA

Conversion of XLAC to XA by Caulobacter crescentus XLA, measured in NMR.

#### Progress curve analysis data

Data files for the conversion of XYL to KG, via the sequential addition of the Caulobacter crescentus Weimberg pathway enzymes, XDH, XLA, XAD, KDXD, KGSADH.

- progress curve analysis data.xlsx

#### Progress curve analysis XLA

Model for the Caulobacter crescentus Weimberg pathway, describing the conversion of Xyl to KG upon sequential adition of purified enzymes. If the Mathematica notebook is downloaded and the data file is downloaded in the same directory, then the notebook can be evaluated, and the figure in the manuscript for the progress curves will be reproduced.

- progressCurves-XLA-SEEK.nb

#### Progress curve analysis (shen1)

Model for the Caulobacter crescentus Weimberg pathway, describing the conversion of Xyl to KG, with sequential addition of purified enzymes.

- shen1.xml

#### Progress curve XAD

Conversion of XA to KDX by Caulobacter crescentus XAD, measured in NMR.

#### Progress curve analysis data

Data files for the conversion of XYL to KG, via the sequential addition of the Caulobacter crescentus Weimberg pathway enzymes, XDH, XLA, XAD, KDXD, KGSADH.

- progress curve analysis data.xlsx

#### Progress curve analysis XAD

Model for the Caulobacter crescentus Weimberg pathway, describing the conversion of Xyl to KG upon sequential adition of purified enzymes. If the Mathematica notebook is downloaded and the data file is downloaded in the same directory, then the notebook can be evaluated, and the figure in the manuscript for the progress curves will be reproduced.

- progressCurves-XAD-SEEK.nb

#### Progress curve analysis (shen1)

Model for the Caulobacter crescentus Weimberg pathway, describing the conversion of Xyl to KG, with sequential addition of purified enzymes.

- shen1.xml

#### Progress curve KDXD

Conversion of KDX to KGSA by Caulobacter crescentus KDXD, measured in NMR.

#### Progress curve analysis data

- progress curve analysis data.xlsx

#### Progress curve analysis KDXD

- progressCurves-KDXD-SEEK.nb

#### Progress curve analysis (shen1)

- shen1.xml

#### Progress curve KGSADH

Conversion of KGSA to KG by Caulobacter crescentus KGSADH, measured in NMR.

#### Progress curve analysis data

- progress curve analysis data.xlsx

#### Progress curve analysis KGSADH

- progressCurves-KGSADH-SEEK.nb

#### Progress curve analysis (shen1)

- shen1.xml

#### Progress curves combined

Conversion of XYL to KG by sequential addition of Weimberg pathway enzymes of Caulobacter crescentus, measured in NMR.

https://jjj.bio.vu.nl/models/experiments/shen2020_fig2c/simulate

#### Progress curve analysis data

- progress curve analysis data.xlsx

#### Progress curve analysis combined

- progressCurves-combined-SEEK.nb

#### Progress curve analysis (shen1)

- shen1.xml

#### One pot cascade

One pot cascade - pathway analysis for the purified Caulinobacter crescentus Weimberg pathway enzymes. Effect of co-factor recycling, removal of XLA, and optimisation on Xylose to aKG is studied.

https://jjj.bio.vu.nl/models/experiments/shen2020_fig3a/simulate

https://jjj.bio.vu.nl/models/experiments/shen2020_fig3b/simulate

https://jjj.bio.vu.nl/models/experiments/shen2020_fig3c/simulate

https://jjj.bio.vu.nl/models/experiments/shen2020_fig3d/simulate

#### One pot cascade 10

Conversion of XYL to KG in one pot cascade of Weimberg pathway enzymes of Caulobacter crescentus, measured in NMR.

https://jjj.bio.vu.nl/models/experiments/shen2020_fig3a/simulate

#### One-Pot-Cascade 10 data

Data files for the conversion of XYL to KG, via the Caulobacter crescentus Weimberg pathway enzymes, XDH, XLA, XAD, KDXD, KGSADH.

- OnePotCascade-10.xlsx

#### One-Pot-Cascade 10

Model for the Caulobacter crescentus Weimberg pathway, describing the conversion of Xyl to KG. If the Mathematica notebook is downloaded and the data file is downloaded in the same directory, then the notebook can be evaluated, and the figure in the manuscript for cascade 10 will be reproduced.

- onePotCasc10-SEEK.nb

#### Cascade analysis (shen2)

Model for the Caulobacter crescentus Weimberg pathway, describing the conversion of Xyl to KG.

- shen2.xml

#### One pot cascade 12

Conversion of XYL to KG in one pot cascade of Weimberg pathway enzymes of Caulobacter crescentus, omitting XLA, measured in NMR.

https://jjj.bio.vu.nl/models/experiments/shen2020_fig3c/simulate

#### One-Pot-Cascade 12 data

Data files for the conversion of XYL to KG, via the Caulobacter crescentus Weimberg pathway enzymes, omitting XLA: XDH, XAD, KDXD, KGSADH.

- OnePotCascade-12.xlsx

#### One pot cascade 12

Model for the Caulobacter crescentus Weimberg pathway, describing the conversion of Xyl to KG. If the Mathematica notebook is downloaded and the data file is downloaded in the same directory, then the notebook can be evaluated, and the figure in the manuscript for cascade 12 will be reproduced.

- onePotCasc12-SEEK.nb

#### Cascade analysis (shen2)

Model for the Caulobacter crescentus Weimberg pathway, describing the conversion of Xyl to KG.

- shen2.xml

#### One pot cascade 13

Conversion of XYL to KG in one pot cascade of Weimberg pathway enzymes of Caulobacter crescentus, with NAD recycling, measured in NMR.

https://jjj.bio.vu.nl/models/experiments/shen2020_fig3b/simulate

#### One-Pot-Cascade 13 data

Data files for the conversion of XYL to KG, via the Caulobacter crescentus Weimberg pathway enzymes: XDH, XLA, XAD, KDXD, KGSADH, with NAD recycling.

- OnePotCascade-13.xlsx

#### One-Pot-Cascade 13

Model for the Caulobacter crescentus Weimberg pathway, describing the conversion of Xyl to KG, with NAD recycling. If the Mathematica notebook is downloaded and the data file is downloaded in the same directory, then the notebook can be evaluated, and the figure in the manuscript for cascade 13 will be reproduced.

- onePotCasc13-SEEK.nb

#### Cascade analysis (shen2)

Model for the Caulobacter crescentus Weimberg pathway, describing the conversion of Xyl to KG.

- shen2.xml

#### One pot cascade 16

Conversion of XYL to KG in one pot cascade of Weimberg pathway enzymes of Caulobacter crescentus, using old enzymes with optimal protein distribution, with NAD recycling, measured in NMR.

https://jjj.bio.vu.nl/models/experiments/shen2020_fig3d/simulate

#### One-Pot-Cascade 16 data

Data files for the conversion of XYL to KG, via the Caulobacter crescentus Weimberg pathway, using old enzymes: XDH, XLA, XAD, KDXD, KGSADH, with NAD recycling, and optimal protein distribution.

- OnePotCascade-16.xlsx

#### One pot cascade 16

Model for the Caulobacter crescentus Weimberg pathway, describing the conversion of Xyl to KG, using old enzymes, with optimal protein distribution. If the Mathematica notebook is downloaded and the data file is downloaded in the same directory, then the notebook can be evaluated, and the figure in the manuscript for cascade 16 will be reproduced.

- onePotCasc16-SEEK.nb

#### Cascade analysis (shen2)

Model for the Caulobacter crescentus Weimberg pathway, describing the conversion of Xyl to KG.

- shen2.xml

#### Cell free extract

Cell free extract - pathway analysis for Caulinobacter crescentus Weimberg pathway enzymes. Effect of co-factor recycling, and Mn2+ on Xylose to aKG conversion is studied.

#### Cell free extract, with Mn and NAD recycling

Conversion of XYL to KG in a cell free extract of Caulobacter crescentus, with 0.15 mM Mn2+ added, and with NAD recycling, metabolites measured enzymatically.

https://jjj.bio.vu.nl/models/experiments/shen2020_fig4b/simulate

#### CFE Mn NADrec data

Data files for the conversion of XYL to KG, in Caulobacter crescentus cell free extract, with NAD recycling, and additional Mn2+ (0.15 mM) added.

- cfe-Mn-rec.xlsx

#### CFE Mn NADrec

Model for the Caulobacter crescentus Weimberg pathway, describing the conversion of Xyl to KG in cell free extract. If the Mathematica notebook is downloaded and the data file is downloaded in the same directory, then the notebook can be evaluated, and the figure in the manuscript for the cell free extract with added Mn and NAD rec will be reproduced.

- cfe-Mn-rec-SEEK.nb

#### CFE analysis (shen2)

Model for the Caulobacter crescentus Weimberg pathway, describing the conversion of Xyl to KG. Protein levels need to be adapted to CFE levels, see SED-ML scripts

- shen2.xml

#### Cell free extract, with Mn, no NAD recycling

Conversion of XYL to KG in a cell free extract of Caulobacter crescentus, with 0.15 mM Mn2+ added, but no NAD recycling, metabolites measured enzymatically.

https://jjj.bio.vu.nl/models/experiments/shen2020_fig4c/simulate

#### CFE Mn no-NADrec data

Data files for the conversion of XYL to KG, in Caulobacter crescentus cell free extract, without NAD recycling, but with additional Mn2+ (0.15 mM) added.

- cfe-Mn-noRec.xlsx

#### CFE Mn noNADrec

Model for the Caulobacter crescentus Weimberg pathway, describing the conversion of Xyl to KG in cell free extract. If the Mathematica notebook is downloaded and the data file is downloaded in the same directory, then the notebook can be evaluated, and the figure in the manuscript for the cell free extract with added Mn, but no NAD rec, will be reproduced.

- cfe-Mn-noRec-SEEK.nb

#### CFE analysis (shen2)

Model for the Caulobacter crescentus Weimberg pathway, describing the conversion of Xyl to KG. Protein levels need to be adapted to CFE levels, see SED-ML scripts

- shen2.xml

#### Cell free extract, without added Mn, with NAD recycling

Conversion of XYL to KG in a cell free extract of Caulobacter crescentus, without Mn2+ added, but with NAD recycling, metabolites measured enzymatically.

https://jjj.bio.vu.nl/models/experiments/shen2020_fig4d/simulate

#### CFE no-Mn but with NADrec data

Data files for the conversion of XYL to KG, in Caulobacter crescentus cell free extract, with NAD recycling, but without additional Mn2+ added.

- cfe-noMn-Rec.xlsx

#### CFE no Mn, with NADrec

Model for the Caulobacter crescentus Weimberg pathway, describing the conversion of Xyl to KG in cell free extract. If the Mathematica notebook is downloaded and the data file is downloaded in the same directory, then the notebook can be evaluated, and the figure in the manuscript for the cell free extract with no added Mn, but with NAD rec, will be reproduced.

- cfe-noMn-rec-SEEK.nb

#### CFE analysis (shen2)

Model for the Caulobacter crescentus Weimberg pathway, describing the conversion of Xyl to KG. Protein levels need to be adapted to CFE levels, see SED-ML scripts

- shen2.xml

#### Steady state cell free extract, with Mn and NAD recycling

No description specified

#### CFE steady state analysis (shen3)

Steady state model for the Caulobacter crescentus Weimberg pathway, describing the conversion of Xyl to KG. Protein levels need to be adapted to CFE levels, see SED-ML scripts.

- shen3.xml

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*Caulobacter crescentus Weimberg pathway*. FAIRDOMHub. https://doi.org/10.15490/FAIRDOMHUB.1.INVESTIGATION.284.2

**Snapshot 2**(30th Jan 2020)

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**Created**: 30th Jan 2020 at 10:08

**Last updated**: 30th Jan 2020 at 10:08