Abstract
Writing artificial logic and dynamic function into complex cellular background to achieve desired phenotypes or improved outputs calls for the development of new genetic tools as well as their innovative use. In this study, we present a sensor-regulator and RNAi-based bifunctional dynamic control network that can provide simultaneous upregulation and downregulation of cellular metabolism for engineered biosynthesis. The promoter-regulator-mediated upregulation function and its transduced downregulation function through RNAi are systematically verified and characterized. We apply this dynamic control network to regulate the phosphoenolpyruvate metabolic node in Escherichia coli and achieve autonomous distribution of carbon flux between its native metabolism and the engineered muconic acid biosynthetic pathway. This allows muconic acid biosynthesis to reach 1.8 g L−1. This study also suggests the circumstances where dynamic control approaches are likely to take effects.
Highlights
Writing artificial logic and dynamic function into complex cellular background to achieve desired phenotypes or improved outputs calls for the development of new genetic tools as well as their innovative use
The dynamic control in the demonstrated applications was largely constrained to mono-function with only upregulation or downregulation being achieved separately due to limited tools and techniques being developed. Addressing these limitations and advancing nature-inspired dynamic regulation in biological systems call for novel control strategies and understanding how to implement them in complex cellular settings. When this manuscript was under review, a study reported the regulation of cellular metabolism using optogenetic tools to improve chemical production[22]
Further disrupting pykA and pykF in the Δppc strain slightly restored the cell growth, which was still significantly retarded compared with the wild-type strain. These results suggested that ppc is responsible for a major PEP competing pathway, its disruption would introduce undesired burden or stress to cellular metabolism by arbitrarily limiting the carbon sources for cell growth
Summary
Writing artificial logic and dynamic function into complex cellular background to achieve desired phenotypes or improved outputs calls for the development of new genetic tools as well as their innovative use. We present a sensor-regulator and RNAi-based bifunctional dynamic control network that can provide simultaneous upregulation and downregulation of cellular metabolism for engineered biosynthesis. The promoter-regulatormediated upregulation function and its transduced downregulation function through RNAi are systematically verified and characterized We apply this dynamic control network to regulate the phosphoenolpyruvate metabolic node in Escherichia coli and achieve autonomous distribution of carbon flux between its native metabolism and the engineered muconic acid biosynthetic pathway. The dynamic control in the demonstrated applications was largely constrained to mono-function with only upregulation or downregulation being achieved separately due to limited tools and techniques being developed Addressing these limitations and advancing nature-inspired dynamic regulation in biological systems call for novel control strategies and understanding how to implement them in complex cellular settings. The results of this study shed light on the questions of whether or when dynamic control can be appropriately used to achieve desired phenotypes or improved outputs
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