Tunable Gene Expression System Independent of Downstream Coding Sequence

Fine control of the expression levels
of proteins constitutes a
major challenge in synthetic biology and metabolic engineering. However,
the dependence of translation initiation on the downstream coding
sequence (CDS) obscures accurate prediction of the protein expression
levels from mRNA sequences. Here, we present a tunable gene-expression
system comprising 24 expression cassettes that produce predefined
relative expression levels of proteins ranging from 0.001 to 1 without
being influenced by the downstream CDS. To validate the practical
utility of the tunable expression system, it was applied to a synthetic
circuit displaying three states of fluorescence depending on the difference
in protein expression levels. To demonstrate the suitability of application
to metabolic engineering, this system was used to diversify the levels
of key metabolic enzymes. As a result, expression-optimized strains
were capable of producing 2.25 g/L of cadaverine, 2.59 g/L of L-proline,
and 95.7 mg/L of 1-propanol. Collectively, the tunable expression
system could be utilized to optimize genetic circuits for desired
operation and to optimize metabolic fluxes through biosynthetic pathways
for enhancing production yields of bioproducts. This tunable system
will be useful for studying basic and applied biological sciences
in addition to applications in synthetic biology and metabolic engineering.