Engineering and evolution of the complete Reductive Glycine Pathway in Saccharomyces cerevisiae for formate and CO2 assimilation.

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 re-established 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.

Engineering the Reductive Glycine Pathway: A Promising Synthetic Metabolism Approach for C1-Assimilation.

In recent years the reductive glycine pathway (rGlyP) has emerged as a promising pathway for the assimilation of formate and other sustainable C1-feedstocks for future biotechnology. It was originally proposed as an attractive "synthetic pathway" to support formatotrophic growth due to its high ATP efficiency, linear structure, and limited overlap with native pathways in most microbial hosts. Here, we present the current state of research on this pathway including breakthroughs on its engineering. Different variants of the rGlyP are discussed, including its core module for formate to glycine conversion, as well as varying modules for substrate conversion to formate, and glycine assimilation routes. Very recently, the rGlyP has been successfully implemented for synthetic formatotrophic growth, as well as for growth on methanol, in some bacterial hosts. We discuss the engineering strategies employed in these studies, including growth-coupled selection of functional pathway modules. We also compare the rGlyP to other natural and synthetic C1-assimilation pathways. Finally, we provide an outlook on open challenges and opportunities for the rGlyP, including its engineering into more biotechnological hosts, as well as the still-to-be realized production of value-added chemicals via this pathway. We expect that further research on the rGlyP will support the efficient use of sustainable C1-substrates in bioproduction.