Engineering Non-Growth Metabolism in Escherichia coli for Improved Productivity and Yield of Biochemical Synthesis

Bothfeld, William Henry

doi:https://doi.org/10.21985/n2-anpp-ph91

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Engineering Non-Growth Metabolism in Escherichia coli for Improved Productivity and Yield of Biochemical Synthesis

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The fields of metabolic engineering and synthetic biology have emerged in recent years with the heavily overlapping goals of sustainable bioproduction of chemical goods and predictable and precise engineering of biological function.1 However, efforts to reach commercially relevant titers have stalled.2 Many challenges arise during optimization of each production platform, as rationally engineered biological systems misbehave in unpredictable ways and evolution reverses many strain improvements to ensure robust growth. To date, there has been limited commercial success producing chemicals biologically, so there is a great need to push towards industrial scale biochemical production platforms to address issues of energy, climate change, and human health. In this dissertation, we created tools to enact a non-growth E. coli bioproduction pipeline, which has potential to shift how chemicals are produced biologically. It would enable full utilization of feedstocks for production during the conversion phase and would prevent growth-based mutational escape to break our biological pipeline. Additionally, non-growth products allows toxic chemicals to be produced biologically. First, we developed an autonomously activated glucose-sensing toggle switch to precisely turn on a burdensome heterologous pathway with smart feeding strategies. Second, we generated non-growing E. coli using synthetic auxotrophs for an essential biomolecule and measured the resulting metabolism. Next, we identified knockout targets from stress and metabolite networks in E. coli to find targets to improve non-growth metabolism. These knockouts were constructed and tested for improved limonene biosynthesis. Together, these tools contribute towards realizing an industrial non-growth production pipeline, that should be applicable to a wide portfolio of chemicals.

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