Abstract
Genetic circuits and metabolic pathways can be reengineered to allow organisms to process signals and manufacture useful chemicals. However, their functions currently rely on organism-specific regulatory parts, fragmenting synthetic biology and metabolic engineering into host-specific domains. To unify efforts, here we have engineered a cross-species expression resource that enables circuits and pathways to reuse the same genetic parts, while functioning similarly across diverse organisms. Our engineered system combines mixed feedback control loops and cross-species translation signals to autonomously self-regulate expression of an orthogonal polymerase without host-specific promoters, achieving nontoxic and tuneable gene expression in diverse Gram-positive and Gram-negative bacteria. Combining 50 characterized system variants with mechanistic modelling, we show how the cross-species expression resource's dynamics, capacity and toxicity are controlled by the control loops' architecture and feedback strengths. We also demonstrate one application of the resource by reusing the same genetic parts to express a biosynthesis pathway in both model and non-model hosts.
Highlights
Genetic circuits and metabolic pathways can be reengineered to allow organisms to process signals and manufacture useful chemicals
Cross-species portability can be achieved by building genetic circuitry that autonomously regulates the production of an orthogonal polymerase, creating a portable power supply that enables the same genetic system to be expressed in different hosts, while using the same set of host-independent regulatory genetic parts[32,33]
The genetic implementation of the Universal Bacterial Expression Resource (UBER) system combines expression of T7 RNA polymerase (T7 RNAP) and tetracycline repressor (TetR) within a self-regulating genetic circuit that uses a cross-species priming promoter, wild-type T7 promoters, an engineered T7 promoter flanked by TetO operator sites (T7-TetO promoter), cross-species translation signals and efficient transcriptional terminators (Fig. 1a)
Summary
Genetic circuits and metabolic pathways can be reengineered to allow organisms to process signals and manufacture useful chemicals. Such incompatibilities are caused by the differences in gene expression machinery across species[17,18,19,20], which have fragmented the fields of Synthetic Biology and Metabolic Engineering into host-specific domains where specialized genetic parts are tailormade for each organism of interest[21,22] To unify these efforts, a new paradigm is needed that enables one to use the same regulatory genetic parts in different hosts with similar functionality. By developing a system-wide mechanistic model alongside our experiments, we dissect the role of auto-activation, expression level dosage, competitive T7 RNA polymerase binding and feedback loop strengths on controlling the expression resource’s dynamics, capacity and host toxicity During this process, new design rules are formulated for genetic systems that use orthogonal polymerases. We demonstrate how our expression resource enables cross-species metabolic pathway engineering
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