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.