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Realization of a New-to-Nature Carboxylation Pathway
Most inorganic carbon enters the biosphere via the Calvin-Benson-Bassham (CBB) cycle by its key enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). An unproductive side reaction of RuBisCO with oxygen leads to the formation of 2-phosphoglycolate (2-PG), which is recycled via complex pa...
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| フォーマット: | Dissertation |
| 言語: | 英語 |
| 出版事項: |
Philipps-Universität Marburg
2020
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| オンライン･アクセス: | PDFフルテキスト |
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| 要約: | Most inorganic carbon enters the biosphere via the Calvin-Benson-Bassham (CBB) cycle by its key enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). An unproductive side reaction of RuBisCO with oxygen leads to the formation of 2-phosphoglycolate (2-PG), which is recycled via complex pathways into 3-phosphoglycerate (3-PGA), releasing carbon dioxide in the process. The tartronyl-CoA pathway represents a synthetic pathway that was designed to recycle 2-PG more efficiently, avoiding the release of carbon dioxide, and fixing carbon dioxide instead. It consists of four main reactions steps, which are not known to take part in any natural metabolic pathway. These steps are the activation of glycolate to glycolyl-CoA, the carboxylation of glycolyl-CoA to tartronyl-CoA as its key reaction, and the subsequent two reductions giving rise to glycerate.
In this work, all required enzymes were identified or established by engineering and the tartronyl-CoA pathway was realized in vitro. Promiscuous enzyme candidates performing analogous reactions with similar substrates were screened and further improved to perform their desired functions. These include engineered glycolyl-CoA synthetase and glycolyl-CoA carboxylase (GCC), as well as a tartronyl-CoA reductase. For the engineering of GCC, rational design as well as high-throughput directed evolution was applied resulting in a new-to-nature carboxylase that matches the kinetic properties of natural carboxylases. Moreover, a 1.96 Å resolution cryogenic electron microscopy (cryo-EM) structure of GCC was obtained, highlighting and corroborating the effects of the introduced mutations. The concerted function of all tartronyl-CoA pathway enzymes was confirmed in the context of photorespiration in vitro. The in vitro reconstitution also included the optimization of reaction parameters as well as efficient cofactor recycling. Besides its function as photorespiratory bypass, the tartronyl-CoA pathway was shown to be functional as an additional carbon fixing module, able to connect a synthetic carbon dioxide fixation cycle to central carbon metabolism. Furthermore, the tartronyl-CoA pathway was successfully employed for the in vitro conversion of the plastic waste component ethylene glycol into the central carbon metabolite glycerate. In an initial attempt of an in vivo implementation of the tartronyl-CoA pathway for ethylene glycol assimilation, it was shown that GCC, the key enzyme of the tartronyl-CoA pathway, can be functionally produced in Pseudomonas putida. |
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| 物理的記述: | 181 Seiten |
| DOI: | 10.17192/z2020.0245 |