Precise Genomic Riboregulator Control of Metabolic Flux in Microbial Systems

Abstract

Engineered microbes can be used for producing value-added chemicals from renewable feedstocks, relieving the dependency on nonrenewable resources such as petroleum. These microbes often are composed of synthetic metabolic pathways; however, one major problem in establishing a synthetic pathway is the challenge of precisely controlling competing metabolic routes, some of which could be crucial for fitness and survival. While traditional gene deletion and/or coarse overexpression approaches do not provide precise regulation, cis-repressors (CRs) are RNA-based regulatory elements that can control the production levels of a particular protein in a tunable manner. Here, we describe a protocol for a generally applicable fluorescence-activated cell sorting technique used to isolate eight subpopulations of CRs from a semidegenerate library in Escherichia coli, followed by deep sequencing that permitted the identification of 15 individual CRs with a broad range of protein production profiles. Using these new CRs, we demonstrated a change in production levels of a fluorescent reporter by over two orders of magnitude and further showed that these CRs are easily ported from E. coli to Pseudomonas putida. We next used four CRs to tune the production of the enzyme PpsA, involved in pyruvate to phosphoenolpyruvate (PEP) conversion, to alter the pool of PEP that feeds into the shikimate pathway. In an engineered P. putida strain, where carbon flux in the shikimate pathway is diverted to the synthesis of the commodity chemical cis,cis-muconate, we found that tuning PpsA translation levels increased the overall titer of muconate. Therefore, CRs provide an approach to precisely tune protein levels in metabolic pathways and will be an important tool for other metabolic engineering efforts.

Metabolic Engineering of Microorganisms to Produce Pyruvate and Derived Compounds

“…In addition, RNA-based engineering of cis -repressors has been successfully applied in metabolic engineering and synthetic biology to control specific proteins in a modifiable manner and alter metabolic fluxes to produce valuable chemicals [ 124 , 125 ]. The cis -repressors have the advantage of being portable and do not require an inducer due to the built-in set threshold for protein synthesis [ 125 ]. The complexity of regulation and metabolite crosstalk in metabolic pathways is also a major difficulty in the development of metabolic engineering.…”

Targeted mutagenesis and high-throughput screening of diversified gene and promoter libraries for isolating gain-of-function mutations

“…Mutation libraries of the RBS have been used to modulate and optimize translation rates in a variety of applications (Oesterle et al, 2017). Similarly, cis-acting elements on the mRNA can have a profound effect on translation rates (Gebauer et al, 2012;Rhodius et al, 2012) and have also been diversified to generate libraries with a wide range of expression levels of downstream genes (White, 2015;Pandey et al, 2022).…”

A portable regulatory RNA array design enables tunable and complex regulation across diverse bacteria

“…Additionally, most RNA switches use RNA motifs (e.g., RBS, transcriptional terminators) that have relatively conserved sequence-function relations 16 – 18 , and enact regulation through interactions with host-cell machinery that is ubiquitous. While there have been a handful of demonstrations of the portability of synthetic RNA switches 19 – 23 , we currently lack systematic investigation. Additionally, it remains hard to tune input-output relations (i.e., gain).…”