The ability of living systems to carry out the tasks needed to sustain life relies on the existence of a dynamic and complex network of chemical reactions within each cell. Indeed, it is the cell’s capacity for chemistry that allows it to intake simple carbon sources and transform them into the thousands of molecules needed to drive and coordinate the fundamental processes that are the hallmarks of life. Thus, cells possess an enormous synthetic potential that can be engineered for targeted chemical synthesis. By mixing-and-matching enzymes to construct synthetic metabolic pathways, the potential of natural metabolism can be harnessed to achieve multi-step synthetic routes in a single fermentation step in green conditions. As such, these approaches have expanded contributions of biological systems in new areas of the chemical, beauty, fashion, and food sectors as well as providing innovative solutions for sustainability. A major challenge in the development of cell-based chemical synthesis is the re-routing of carbon through a metabolic network that has evolved robust mechanisms to ensure coordination at the local- and system-level for the native function of cell growth and maintenance. In particular, central carbon pathways, such as glycolysis and the tricarboxylic acid cycle (TCA), form many connections with the rest of the network and are difficult to manipulate as their behavior is affected by multiple inputs and outputs and subject to strong homeostatic control.

In this work, we combine rational design and adaptive evolution to achieve a high carbon flux to synthetic pathways by coupling cell growth with product titers. We demonstrated a hybrid approach via the design of synthetic pathways in Escherichia coli to selectively produce three industrially-relevant C4 monomers, 2-hydroxybutanone, 1,3-butanediol, and n-butanol, as bioproduct precursors to methyl vinyl ketone, 1,3-butadiene, and 1-butene. Using a genetic selection, these pathways could be evolved from theoretical yields of 7-20% to near quantitative yield. Genome sequencing of the evolved strains showed that global RNA processors, rpoB/rpoC, pcnB, and rne, were found mutated in the most successful daughter cells, giving rise to the hypothesis that changes in metabolism were related to transcriptional remodeling. Subsequent characterization of these mutations demonstrates that they are sufficient to capture the majority of the evolved phenotype. Further cell profiling experiments show that large-scale shifts do indeed occur in both the transcriptome and metabolome between the parent strains and evolved strains. Notably, we observed that a 25-fold increase in the central building block, acetyl coenzyme A (CoA), could be attained through adaptive evolution. Taken together, these results highlight the possibility of synthetic pathways to be used not only for scalable chemical production but also as a platform for discovery and study of cellular function.

A similar strategy was developed for the eukaryotic host, Saccharomyces cerevisiae, with the goal of exploring metabolic compartmentalization and eukaryotic regulatory mechanisms. Towards this goal, a synthetic n-butanol pathway in yeast was constructed and optimize by a combination of promoter and terminator engineering, enzyme screening, and gene knockout to alter redox balance and cellular regulation of transcription and translation. These efforts yielded a 5-fold increase from ~120 mg L-1 to ~550 mg L-1. In conjunction of the pyruvate dehydrogenase bypass pathway for production of cytosolic acetyl-CoA, we explored the effect of the deletion of GCN5, which consumes acetyl-CoA through its histone acetylase activity. Combining these approaches, we also achieved a 5-fold increase in n-butanol production titer (from ~100 mg L-1 to ~500 mg L-1). Through the knockout of 7 redundant alcohol dehydrogenases for ethanol production, we have initiated the preliminary implementation of adaptive evolution in this system.