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Mixing and matching methylotrophic enzymes to design a novel methanol utilization pathway in E. coli

Abstract : One-carbon (C1) compounds, such as methanol, have recently gained attention as alternative low-cost and non-food feedstocks for microbial bioprocesses. Considerable research efforts are thus currently focused on the generation of synthetic methylotrophs by transferring methanol assimilation pathways into established bacterial production hosts. In this study, we used an iterative combination of dry and wet approaches to design, implement and optimize this metabolic trait in the most common chassis, E. coli. Through in silico modelling, we designed a new route that “mixed and matched” two methylotrophic enzymes: a bacterial methanol dehydrogenase (Mdh) and a dihydroxyacetone synthase (Das) from yeast. To identify the best combination of enzymes to introduce into E. coli, we built a library of 266 pathway variants containing different combinations of Mdh and Das homologues and screened it using high-throughput 13C-labeling experiments. The highest level of incorporation of methanol into central metabolism intermediates (e.g. 22% into the PEP), was obtained using a variant composed of a Mdh from A. gerneri and a codon-optimized version of P. angusta Das. Finally, the activity of this new synthetic pathway was further improved by engineering strategic metabolic targets identified using omics and modelling approaches. The final synthetic strain had 1.5 to 5.9 times higher methanol assimilation in intracellular metabolites and proteinogenic amino acids than the starting strain did. Broadening the repertoire of methanol assimilation pathways is one step further toward synthetic methylotrophy in E. coli.
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https://hal.inrae.fr/hal-02933567
Déposant : Stephanie Heux <>
Soumis le : mardi 8 septembre 2020 - 15:30:17
Dernière modification le : mercredi 9 septembre 2020 - 09:14:43

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A. de Simone, C.M. Vicente, C. Peiro, L. Gales, F. Bellvert, et al.. Mixing and matching methylotrophic enzymes to design a novel methanol utilization pathway in E. coli. Metabolic Engineering, Elsevier, 2020, 61, pp.315-325. ⟨10.1016/j.ymben.2020.07.005⟩. ⟨hal-02933567⟩

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