Abstract
Synthetic circuits embedded in host cells compete with cellular processes for limited intracellular resources. Here we show how funnelling of cellular resources, after global transcriptome degradation by the sequence-dependent endoribonuclease MazF, to a synthetic circuit can increase production. Target genes are protected from MazF activity by recoding the gene sequence to eliminate recognition sites, while preserving the amino acid sequence. The expression of a protected fluorescent reporter and flux of a high-value metabolite are significantly enhanced using this genome-scale control strategy. Proteomics measurements discover a host factor in need of protection to improve resource redistribution activity. A computational model demonstrates that the MazF mRNA-decay feedback loop enables proportional control of MazF in an optimal operating regime. Transcriptional profiling of MazF-induced cells elucidates the dynamic shifts in transcript abundance and discovers regulatory design elements. Altogether, our results suggest that manipulation of cellular resource allocation is a key control parameter for synthetic circuit design.
Highlights
Synthetic circuits embedded in host cells compete with cellular processes for limited intracellular resources
Novel cellular behaviours can be programmed by interacting networks of biomolecules to process information from the environment and execute target functions. These synthetic biomolecular circuits interact with endogenous cellular processes through competition over shared resources that include ribosomes, transfer RNAs, RNA polymerases, amino acids and nucleotides[2,3]
Cells operate with a limited resource quota, which manifests as a trade-off in the partitioning of energy between cellular processes and synthetic circuit functions[1,3,5,6]
Summary
Synthetic circuits embedded in host cells compete with cellular processes for limited intracellular resources. We show that MazF activity induces a global cellular physiological shift that can be exploited to enhance synthetic circuit expression These results suggest that the MazF resource allocator controllably redistributed core cellular subsystems to support a synthetic circuit and an engineered metabolic pathway. The former is further enhanced by protection of specific host-cell factors and use of the orthogonal RNA polymerase from T7 bacteriophage (T7 RNA polymerase) to transcribe genes in the synthetic circuit. To pinpoint major parameters that influence MazF-induced decay rates, we examine the number and positioning of MazF recognition sites on the expression of a fluorescent reporter gene These results suggest a platform for global manipulation of resource pools as a key parameter for modulating synthetic circuit behaviour
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