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Enabling the Direct Enzymatic Dehydration of d-Glycerate to Pyruvate as the Key Step in Synthetic Enzyme Cascades Used in the Cell-Free Production of Fine Chemicals

  • Samuel Sutiono
    Samuel Sutiono
    Chair of Chemistry of Biogenic Resources, Campus Straubing for Biotechnology and Sustainability, Technical University of Munich, Schulgasse 16, 94315 Straubing, Germany
  • Mariko Teshima
    Mariko Teshima
    Chair of Chemistry of Biogenic Resources, Campus Straubing for Biotechnology and Sustainability, Technical University of Munich, Schulgasse 16, 94315 Straubing, Germany
  • Barbara Beer
    Barbara Beer
    Chair of Chemistry of Biogenic Resources, Campus Straubing for Biotechnology and Sustainability, Technical University of Munich, Schulgasse 16, 94315 Straubing, Germany
    More by Barbara Beer
  • Gerhard Schenk
    Gerhard Schenk
    School of Chemistry and Molecular Biosciences, The University of Queensland, 68 Copper Road, St. Lucia, Queensland 4072, Australia
    Sustainable Minerals Institute, The University of Queensland, 47 Staff House Road, St. Lucia, Queensland 4072, Australia
  • , and 
  • Volker Sieber*
    Volker Sieber
    Chair of Chemistry of Biogenic Resources, Campus Straubing for Biotechnology and Sustainability, Technical University of Munich, Schulgasse 16, 94315 Straubing, Germany
    Catalytic Research Center, Technical University of Munich, Ernst-Otto-Fischer-Straße 1, 85748 Garching, Germany
    School of Chemistry and Molecular Biosciences, The University of Queensland, 68 Copper Road, St. Lucia, Queensland 4072, Australia
    Straubing Branch BioCat, Fraunhofer IGB, Schulgasse 11a, 94315 Straubing, Germany
    *Email: [email protected]
Cite this: ACS Catal. 2020, 10, 5, 3110–3118
Publication Date (Web):February 3, 2020
https://doi.org/10.1021/acscatal.9b05068
Copyright © 2020 American Chemical Society
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Abstract

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A high degree of dependence on fossil fuels is one of the major problems faced by modern societies. d-Glucose and glycerol have emerged in recent years as prospective replacements for fossil fuels used in the production of high-value chemicals, and cell-free bioproduction routes are expected to play a crucial role in such processes. Recently, several synthetic cascades used for the cell-free biotransformations of d-glucose and glycerol to pyruvate and beyond have been described. However, these were limited by the very slow dehydration step of d-glycerate to pyruvate catalyzed by a dehydratase from Sulfolobus solfataricus (SsDHAD), making this step by far the major bottleneck. By combining the vast number of available genomes with a sequence-based discovery approach, we have identified signature sequences leading to the discovery of two distinct classes of dehydratases which exhibit promising activity and total turnover number (TTN) toward d-glycerate. In particular, the dehydratase from Paralcaligenes ureilyticus (PuDHT) demonstrated >100-fold higher activity and TTN for d-glycerate in comparison to SsDHAD. In addition, PuDHT showed exceptionally high activity and TTN toward d-gluconate. The replacement of SsDHAD by PuDHT in our model cascade from d-glucose to ethanol enhanced the production rate 10-fold, reaching a 92% theoretical yield at 50 °C. PuDHT was also suitable for the conversion of glycerol to pyruvate at ambient temperature, leading to a >5-fold improvement in production rate in comparison to the system utilizing SsDHAD at 50 °C and attaining 97% of the theoretical yield. PuDHT was also compatible when crude glycerol was used as the substrate, and it no longer caused a bottleneck in the enzymatic cascade.

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acscatal.9b05068.

  • SDS-PAGE of all dehydratases purified, multiple sequence alignment of representative dehydratases, and protein annotations, as well as the source of the microorganisms, Michaelis–Menten kinetics of PuDHT, HsDHT, and FtDHAD, preliminary work on an ethanol cascade as well as detailed auxiliary enzymes used in the biotransformations of d-glucose and glycerol, chromatograms of substrates, intermediates, and product separation as well as a comparison of pure and crude glycerol, and a detailed experimental section (PDF)

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Cited By

This article is cited by 15 publications.

  1. Shiming Tang, Daocheng Liao, Xuewen Li, Ying Lin, Shuangyan Han, Suiping Zheng. Cell-Free Biosynthesis System: Methodology and Perspective of in Vitro Efficient Platform for Pyruvate Biosynthesis and Transformation. ACS Synthetic Biology 2021, 10 (10) , 2417-2433. https://doi.org/10.1021/acssynbio.1c00252
  2. Tao Li, Xuexian Cui, Yinglu Cui, Jinyuan Sun, Yanchun Chen, Tong Zhu, Chuijian Li, Ruifeng Li, Bian Wu. Exploration of Transaminase Diversity for the Oxidative Conversion of Natural Amino Acids into 2-Ketoacids and High-Value Chemicals. ACS Catalysis 2020, 10 (14) , 7950-7957. https://doi.org/10.1021/acscatal.0c01895
  3. Yipeng Zhang, Liting Ma, Weikang Sun, Dan Xiao, Chuanjuan Lü, Qian Wang, Ping Xu, Cuiqing Ma, Chao Gao. An in vitro cascade with four enzymes for the production of d -3,4-dihydroxybutyric acid from d -xylose. Green Chemistry 2022, 24 (19) , 7602-7611. https://doi.org/10.1039/D2GC02177C
  4. You Lv, Shan Zheng, Adi Goldenzweig, Fengjiang Liu, Yan Gao, Xiuna Yang, Ajit Kandale, Ross P. McGeary, Simon Williams, Bostjan Kobe, Mark A. Schembri, Michael J. Landsberg, Bin Wu, Thomas B. Brück, Volker Sieber, Mikael Boden, Zihe Rao, Sarel J. Fleishman, Gerhard Schenk, Luke W. Guddat. Enhancing the Thermal and Kinetic Stability of Ketol-Acid Reductoisomerase, a Central Catalyst of a Cell-Free Enzyme Cascade for the Manufacture of Platform Chemicals. Applied Biosciences 2022, 1 (2) , 163-178. https://doi.org/10.3390/applbiosci1020011
  5. Tenuun Bayaraa, Jose Gaete, Samuel Sutiono, Julia Kurz, Thierry Lonhienne, Jeffrey R. Harmer, Paul V. Bernhardt, Volker Sieber, Luke Guddat, Gerhard Schenk. Dihydroxy‐Acid Dehydratases From Pathogenic Bacteria: Emerging Drug Targets to Combat Antibiotic Resistance. Chemistry – A European Journal 2022, 28 (44) https://doi.org/10.1002/chem.202200927
  6. Okke Melse, Samuel Sutiono, Magdalena Haslbeck, Gerhard Schenk, Iris Antes, Volker Sieber. Structure‐Guided Modulation of the Catalytic Properties of [2Fe−2S]‐Dependent Dehydratases. ChemBioChem 2022, 23 (10) https://doi.org/10.1002/cbic.202200088
  7. Roland Wohlgemuth. Selective Biocatalytic Defunctionalization of Raw Materials. ChemSusChem 2022, 15 (9) https://doi.org/10.1002/cssc.202200402
  8. Juan Wang, Ge Qu, Leipeng Xie, Chao Gao, Yingying Jiang, Yi-Heng P. Job Zhang, Zhoutong Sun, Chun You. Engineering of a thermophilic dihydroxy-acid dehydratase toward glycerate dehydration for in vitro biosystems. Applied Microbiology and Biotechnology 2022, 106 (9-10) , 3625-3637. https://doi.org/10.1007/s00253-022-11936-9
  9. Wensi Meng, Cuiqing Ma, Ping Xu, Chao Gao. Biotechnological production of chiral acetoin. Trends in Biotechnology 2022, 32 https://doi.org/10.1016/j.tibtech.2022.01.008
  10. Shiting Guo, Xiaoxu Tan, Yuxian Wang, Kai Li, Chuanjuan Lü, Cuiqing Ma, Chao Gao. Enhanced In Vitro Cascade Catalysis of Glycerol into Pyruvate and Acetoin by Integration with Dihydroxy Acid Dehydratase from Paralcaligenes ureilyticus. Catalysts 2021, 11 (11) , 1282. https://doi.org/10.3390/catal11111282
  11. Yi Zhou, Shuke Wu, Uwe T. Bornscheuer. Recent advances in (chemo)enzymatic cascades for upgrading bio-based resources. Chemical Communications 2021, 57 (82) , 10661-10674. https://doi.org/10.1039/D1CC04243B
  12. Samuel Sutiono, André Pick, Volker Sieber. Converging conversion – using promiscuous biocatalysts for the cell-free synthesis of chemicals from heterogeneous biomass. Green Chemistry 2021, 23 (10) , 3656-3663. https://doi.org/10.1039/D0GC04288A
  13. Benjamin Begander, Anna Huber, Josef Sperl, Volker Sieber. Development of a Cofactor Balanced, Multi Enzymatic Cascade Reaction for the Simultaneous Production of L-Alanine and L-Serine from 2-Keto-3-deoxy-gluconate. Catalysts 2021, 11 (1) , 31. https://doi.org/10.3390/catal11010031
  14. Peter L. Bergquist, Sana Siddiqui, Anwar Sunna. Cell-Free Biocatalysis for the Production of Platform Chemicals. Frontiers in Energy Research 2020, 8 https://doi.org/10.3389/fenrg.2020.00193
  15. Samuel Sutiono, Bettina Siebers, Volker Sieber. Characterization of highly active 2-keto-3-deoxy-L-arabinonate and 2-keto-3-deoxy-D-xylonate dehydratases in terms of the biotransformation of hemicellulose sugars to chemicals. Applied Microbiology and Biotechnology 2020, 104 (16) , 7023-7035. https://doi.org/10.1007/s00253-020-10742-5

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