Development of a Highly Selective NAD+-Dependent Glyceraldehyde Dehydrogenase and its Application in Minimal Cell-Free Enzyme Cascades

Mariko Teshima, Samuel Sutiono, Manuel Döring, Barbara Beer, Mikael Boden, Gerhard Schenk, Volker Sieber

Research output: Contribution to journalArticlepeer-review

Abstract

Anthropogenic climate change has been caused by over-exploitation of fossil fuels and CO2 emissions. To counteract this, the chemical industry has shifted its focus to sustainable chemical production and the valorization of renewable resources. However, the biggest challenges in biomanufacturing are technical efficiency and profitability. In our minimal cell-free enzyme cascade generating pyruvate as the central intermediate, the NAD+-dependent, selective oxidation of D-glyceraldehyde was identified as a key reaction step to improve the overall cascade flux. Successive genome mining identified one candidate enzyme with 24-fold enhanced activity and another whose stability is unaffected in 10 % (v/v) ethanol, the final product of our model cascade. Semi-rational engineering improved the substrate selectivity of the enzyme up to 21-fold, thus minimizing side reactions in the one-pot enzyme cascade. The final biotransformation of D-glucose showed a continuous linear production of ethanol (via pyruvate) to a final titer of 4.9 % (v/v) with a molar product yield of 98.7 %. Due to the central role of pyruvate in diverse biotransformations, the optimized production module has great potential for broad biomanufacturing applications.

Original languageEnglish
Article numbere202301132
JournalChemSusChem
Volume17
Issue number4
DOIs
StatePublished - 22 Feb 2024

Keywords

  • Biofuels
  • Cell-Free Enzyme Cascad
  • Enzyme Engineering
  • Glyceraldehyde Dehydrogenase
  • Substrate Selectivity

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