Programming mRNA decay to modulate synthetic circuit resource allocation

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.

A quasi-integral controller for adaptation of genetic modules to variable ribosome demand

“…Yet, unintended interactions arising from synthetic TX devices remains a problem, and there currently does not exist a sufficiently large library of orthogonal ribosomes to assign an independent resource to each TX device. In [40], Venturelli et al engineered a sequencespecific ribonuclease to cleave certain host cell transcripts, thus diverting resources to synthetic proteins to increase productivity of synthetic metabolic pathways. Conversely, Ceroni et al constructed a dCas9-based feedback controller that represses the synthetic circuit when its resource demand is high, thus reallocating resources to maintain constant host cell growth [41].…”

“…Yet, unintended interactions arising from synthetic TX devices remains a problem, and there currently does not exist a sufficiently large library of orthogonal ribosomes to assign an independent resource to each TX device. In [37], Venturelli et al engineered a sequence-specific ribonuclease to cleave certain host cell transcripts, thus diverting resources to synthetic proteins to increase productivity of synthetic metabolic pathways. In [25], Darlington et al partitioned the ribosome pool into synthetic circuit-specific and host-specific ribosomes using orthogonal rRNAs, and implemented a feedback controller to increase the portion of synthetic circuit-specific ribosomes when more are demanded.…”

Quantitative understanding of molecular competition as a hidden layer of gene regulatory network

“…Models incorporating circuit-host competition effects can predict synthetic gene behaviors better (Liao et al, 2017). Reallocating the cellular translational resources by introducing the endoribonuclease MazF circuit can significantly enhance exogenous enzyme expression to promote metabolite production (Venturelli et al, 2017). Utilizing synthetic miRNA and its competitive binding RNA sponges, a RNAbased AND gate circuit was designed for selectively triggering T cell-mediated killing of cancer cells (Nissim et al, 2017).…”

Host-aware synthetic biology

“…Programming mRNA decay to modulate synthetic circuit resource allocation [39] Using the MazF sequence-dependent endonuclease, the authors demonstrated that protecting the mRNA of synthetic constructs against MazF degradation leads to improved productivity and titer of gluconate due to translational resource reallocation.…”