In vitro reconstitution and characterisation of the oxidative d-xylose pathway for production of organic acids and alcohols

Harry Boer (Corresponding Author), Martina Andberg, Robert Pylkkänen, Hannu Maaheimo, Anu Koivula

Research output: Contribution to journalArticleScientificpeer-review

Abstract

The oxidative d-xylose pathway, i.e. Dahms pathway, can be utilised to produce from cheap biomass raw material useful chemical intermediates. In vitro metabolic pathways offer a fast way to study the rate-limiting steps and find the most suitable enzymes for each reaction. We have constructed here in vitro multi-enzyme cascades leading from d-xylose or d-xylonolactone to ethylene glycol, glycolic acid and lactic acid, and use simple spectrophotometric assays for the read-out of the efficiency of these pathways. Based on our earlier results, we focussed particularly on the less studied xylonolactone ring opening (hydrolysis) reaction. The bacterial Caulobacter crescentus lactonase (Cc XylC), was shown to be a metal-dependent enzyme clearly improving the formation of d-xylonic acid at pH range from 6 to 8. The following dehydration reaction by the ILVD/EDD family d-xylonate dehydratase is a rate-limiting step in the pathway, and an effort was made to screen for novel enolase family d-xylonate dehydratases, however, no suitable replacing enzymes were found for this reaction. Concerning the oxidation of glycolaldehyde to glycolic acid, several enzyme candidates were also tested. Both Escherichia coli aldehyde dehydrogenase (Ec AldA) and Azospirillum brasilense α-ketoglutarate semialdehyde dehydrogenase (Ab AraE) proved to be suitable enzymes for this reaction.

Original languageEnglish
Article number48
JournalAMB Express
Volume9
Issue number1
DOIs
Publication statusPublished - 2019
MoE publication typeA1 Journal article-refereed

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Xylose
glycolic acid
Alcohols
Acids
Enzymes
Hydro-Lyases
Azospirillum brasilense
Caulobacter crescentus
Aldehyde Dehydrogenase
Ethylene Glycol
Phosphopyruvate Hydratase
Metabolic Networks and Pathways
Dehydration
Biomass
In Vitro Techniques
Lactic Acid
Oxidoreductases
Hydrolysis
Metals
Escherichia coli

Keywords

  • Dahms pathway
  • Ethylene glycol
  • Glycolate
  • In vitro enzyme pathway
  • Lactate
  • Lactonase

Cite this

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abstract = "The oxidative d-xylose pathway, i.e. Dahms pathway, can be utilised to produce from cheap biomass raw material useful chemical intermediates. In vitro metabolic pathways offer a fast way to study the rate-limiting steps and find the most suitable enzymes for each reaction. We have constructed here in vitro multi-enzyme cascades leading from d-xylose or d-xylonolactone to ethylene glycol, glycolic acid and lactic acid, and use simple spectrophotometric assays for the read-out of the efficiency of these pathways. Based on our earlier results, we focussed particularly on the less studied xylonolactone ring opening (hydrolysis) reaction. The bacterial Caulobacter crescentus lactonase (Cc XylC), was shown to be a metal-dependent enzyme clearly improving the formation of d-xylonic acid at pH range from 6 to 8. The following dehydration reaction by the ILVD/EDD family d-xylonate dehydratase is a rate-limiting step in the pathway, and an effort was made to screen for novel enolase family d-xylonate dehydratases, however, no suitable replacing enzymes were found for this reaction. Concerning the oxidation of glycolaldehyde to glycolic acid, several enzyme candidates were also tested. Both Escherichia coli aldehyde dehydrogenase (Ec AldA) and Azospirillum brasilense α-ketoglutarate semialdehyde dehydrogenase (Ab AraE) proved to be suitable enzymes for this reaction.",
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AU - Koivula, Anu

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