Pathway swapping: Toward modular engineering of essential cellular processes

Significance Replacement of petrochemistry by bio-based processes requires microbes equipped with novel-to-nature capabilities. The efficiency of such engineered microbes strongly depends on their native metabolic networks, which, forged by eons of evolution, are complex and encoded by mosaic microbial genomes. Absence of a modular organization of genomes tremendously restricts genetic accessibility and presents a major hurdle for fundamental understanding and rational engineering of central metabolism. Using as a paradigm the nearly ubiquitous glycolytic pathway, we introduce a radical approach, enabling the “transplantation” of essential metabolic routes in the model and industrial yeast Saccharomyces cerevisiae. This achievement demonstrates that a modular design of synthetic genomes offers unprecedented possibilities for fast, combinatorial exploration, and optimization of the biological function of essential cellular processes. Recent developments in synthetic biology enable one-step implementation of entire metabolic pathways in industrial microorganisms. A similarly radical remodelling of central metabolism could greatly accelerate fundamental and applied research, but is impeded by the mosaic organization of microbial genomes. To eliminate this limitation, we propose and explore the concept of “pathway swapping,” using yeast glycolysis as the experimental model. Construction of a “single-locus glycolysis” Saccharomyces cerevisiae platform enabled quick and easy replacement of this yeast’s entire complement of 26 glycolytic isoenzymes by any alternative, functional glycolytic pathway configuration. The potential of this approach was demonstrated by the construction and characterization of S. cerevisiae strains whose growth depended on two nonnative glycolytic pathways: a complete glycolysis from the related yeast Saccharomyces kudriavzevii and a mosaic glycolysis consisting of yeast and human enzymes. This work demonstrates the feasibility and potential of modular, combinatorial approaches to engineering and analysis of core cellular processes.

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