Konstruktion eines synthetischen Stoffwechselwegs für die kohlenstoffkonservierende Wertstoffsynthese aus Ethylenglykol und Glykolaldehyd
Zusammenfassung der Projektergebnisse
Development of the bio-based economy requires the exploitation of new carbon sources to satisfy the increasing demand for organic substrates that can be used in microbial product syntheses. The most sustainable strategy to meet this demand is to use carbon dioxide (CO2) as renewable carbon source for production of chemical compounds. To make CO2 accessible as a substrate for heterotrophic microorganisms, it is first chemically converted to methanol or synthesis gas. Both compounds are attractive carbon sources from which the whole panel of industrially relevant chemicals can be produced by microorganisms. However, the performance of these microbial product syntheses is limited by the extremely high oxygen demand of methanol-based biosyntheses and comparatively slow phase transition of the gaseous substrates synthesis gas and oxygen into the liquid culture medium. To solve these technological problems, the Institute of Natural Materials Technology of the Technische Universität Dresden proposes to chemically convert synthesis gas and methanol (MeOH) into the liquid and non-volatile C2-compound ethylene glycol (EG). Using this liquid compound as carbon source solves the phase transition problem. In addition, conversion of EG into value-added products (by the herein proposed synthetic metabolic pathway) has a significantly lower oxygen demand than when directly using MeOH as the substrate. The advantages and disadvantages of EG and MeOH-based product syntheses were systematically analyzed for a representative panel of chemical products and summarized in a review paper. It was shown that EG-based product syntheses have distinct advantages over MeOH, mainly consisting in identical product yields at systematically lower oxygen demand, lower toxicity of the characteristic aldehyde intermediate (comparing IC50, formaldehyde is 40 times more toxic than glycolaldehyde), lower investment cost of fermentation plants since EG is non-volatile and non-toxic as opposed to the toxic and volatile MeOH. These advantages must be weighed against a higher price of EG. Various bacteria are capable of using EG as a carbon source. However, natural EG- assimilating pathways contain several CO2-releasing reactions, which makes them particularly unsuitable for acetyl-CoA dependent product syntheses (maximum carbon yield of acetyl-CoA on EG is 50 %). In this research project, a synthetic metabolic pathway was implemented enabling carbon-conserving biosynthesis of acetyl-CoA from EG. The pathway relies on the initial oxidation of EG to GA and the aldol addition of GA to glyceraldehyde 3-phosphate (GAP). The reaction product arabinose 5-phosphate is further processed by several reaction steps to yield acetyl-CoA and to regenerate the GA-acceptor GAP. The function of the synthetic pathway was demonstrated in a cell-free reaction system by using isolated enzymes. This work was followed by implementation of the pathway in engineered Escherichia coli and Pseudomonas putida strains. In vivo function of the pathway was demonstrated by showing biosynthesis of mevalonate which is derived from acetyl-CoA. When the pathway was expressed in E. coli, EG was assimilated only at very residual rates due to the very unfavorable thermodynamics of the initial NAD-dependent EG oxidation step. Implementation of the pathway in P. putida provided a strong advantage when assimilating EG, since the initial EG- oxidation step in this organism is PQQ dependent and therefore thermodynamically favorable. Even though pathway function could be shown, carbon flux through the pathway and, therefore, productivity were very low despite extensive engineering efforts. The reason is most likely the circular design of the pathway, which requires regeneration of the glycolaldehydeaccepting metabolite glyceraldehyde-3 phosphate. Thus, a linear pathway design for converting EG into acetyl-CoA is preferable. Initial ideas could be developed which will be tested in follow-up research projects. Our study makes an important contribution to the exploitation of alternative carbon sources for microbial product syntheses.
Projektbezogene Publikationen (Auswahl)
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Engineering E. coli for the carbon-conserving conversion of ethylene glycol to acetyl-CoA and derived products. (Bio)Process Engineering - a Key to Sustainable Development, Dechema, Aachen
N. Wagner; F. Bade; K. Rabe & T. Walther
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Engineering E. coli for the carbon-conserving conversion of ethylene glycol to acetyl-CoA and derived products. Dechema Himmelfahrtstagung, Future Bioprocesses for a Sustainable Industry, Mainz
N. Wagner; F. Bade; K. Rabe & T. Walther
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Ethylene glycol is an interesting platform molecule for microbial CO2-based product syntheses. (Bio)Process Engineering - a Key to Sustainable Development, Dechema, Aachen
N. Wagner; C. Frazão & T. Walther
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Construction of a synthetic metabolic pathway for biosynthesis of 2,4-dihydroxybutyric acid from ethylene glycol. Nature Communications, 14(1).
Frazão, Cláudio J. R.; Wagner, Nils; Rabe, Kenny & Walther, Thomas
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Ethylene glycol is an interesting platform molecule for microbial CO2-based product syntheses. Himmelfahrtstagung on Bioprocess Engineering 2023, Weimar
N. Wagner; F. Kraußer; C. Frazão & T. Walther
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In vivo implementation of a synthetic metabolic pathway for the carbon-conserving conversion of glycolaldehyde to acetyl-CoA. Frontiers in Bioengineering and Biotechnology, 11.
Wagner, Nils; Bade, Frederik; Straube, Elly; Rabe, Kenny; Frazão, Cláudio J. R. & Walther, Thomas
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Next-generation feedstocks methanol and ethylene glycol and their potential in industrial biotechnology. Biotechnology Advances, 69, 108276.
Wagner, Nils; Wen, Linxuan; Frazão, Cláudio J.R. & Walther, Thomas
