Dynamic allocation of orthogonal ribosomes facilitates uncoupling of co-expressed genes

Abstract

Introduction of synthetic circuits into microbes creates competition between circuit and host genes for shared cellular resources, such as ribosomes. This can lead to the emergence of unwanted coupling between the expression of different circuit genes, complicating the design process and potentially leading to circuit failure. By expressing a synthetic 16S rRNA with altered specificity, we can partition the ribosome pool into host-specific and circuit-specific activities. We show mathematically and experimentally that the effects of resource competition can be alleviated by targeting genes to different ribosomal pools. This division of labour can be used to increase flux through a metabolic pathway. We develop a model of cell physiology which is able to capture these observations and use it to design a dynamic resource allocation controller. When implemented, this controller acts to decouple genes by increasing orthogonal ribosome production as the demand for translational resources by a synthetic circuit increases.

Exploiting the Feedstock Flexibility of the Emergent Synthetic Biology Chassis Vibrio natriegens for Engineered Natural Product Production

“…natriegens intrinsically has many desirable characteristics for a bioproduction chassis: Fast growth rate, genetic tractability, biological safety, and growth on a wide variety of carbon sources [4][5][6][7][8][9][10][11][12][13]46]. In regards to growth rate in particular, it has been shown that expression of some heterologous genes can decrease expression of others, due to competition for finite resources in the cell such as RNA polymerase and ribosomes [47,48]. One could expect that fast growth would require more demand on these finite resources, which would then be less available for heterologous expression and a liability for bioproduction.…”

Growth Defects and Loss-of-Function in Synthetic Gene Circuits

“…Gorochowski et al 15 showed that transcriptional profiles can be used to debug failure modes of various gene circuits. Most recently, Rugbjerg and colleagues 16 coupled metabolite production to the expression of essential endogenous genes, which allowed to exploit evolutionary forces and increase production, while Darlington et al engineered a system that alleviates resource bottlenecks via an orthogonal ribosome 17 . Recent work further showed that promoters linked to the heat shock response of E. coli respond to heterologous expression, and then employed them to build a CRISPR/dCas9-based feedback system to control expression 18 .…”

Winner-Takes-All Resource Competition Redirects Cascading Cell Fate Transitions

“…Several approaches have been proposed to counteract the effects of resource competition, either by finetuning the parameters in the gene circuit 5,7 or manipulating the size of the orthogonal resource pools [31][32][33][34] . Additionally, a burden-driven negative feedback loop was implemented to control gene expression by monitoring the cellular burden [35][36][37] .…”