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bioRxiv · 10.1101/2023.07.10.548313

Engineering and Evolution of the Complete Reductive Glycine Pathway in Saccharomyces cerevisiae for Formate and CO2 Assimilation

Bysani, V. R.·Alam, A. S. M.·Bar-Even, A.·Machens, F.
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Abstract

Using captured CO2 and C1-feedstocks like formate and methanol derived from electrochemical activation of CO2 are key solutions for transforming industrial processes towards a circular carbon economy. Engineering formate and CO2-based growth in the biotechnologically relevant yeast Saccharomyces cerevisiae could boost the emergence of a formate-mediated circular bio-economy. This study adopts a growth-coupled selection scheme for modular implementation of the Reductive Glycine Pathway (RGP) and subsequent Adaptive Laboratory Evolution (ALE) to enable formate and CO2 assimilation for biomass formation in yeast. We first constructed a serine biosensor strain and then implemented the serine synthesis module of the RGP into yeast, establishing glycine and serine synthesis from formate and CO2. ALE improved the RGP-dependent growth by 8-fold. 13C-labeling experiments reveal glycine, serine, and pyruvate synthesis via the RGP, demonstrating the complete pathway activity. Further, we reestablished formate and CO2-dependent growth in non-evolved biosensor strains via reverse-engineering a mutation in GDH1 identified from ALE. This mutation led to significantly more 13C-formate assimilation than in WT without any selection or overexpression of the RGP. Overall, we demonstrated the activity of the complete RGP, showing evidence for carbon transfer from formate to pyruvate coupled with CO2 assimilation.

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Bysani, V. R., Alam, A. S. M., Bar-Even, A., Machens, F.. 2023-07-10. Engineering and Evolution of the Complete Reductive Glycine Pathway in Saccharomyces cerevisiae for Formate and CO2 Assimilation. https://doi.org/10.1101/2023.07.10.548313

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