Biphasic Metabolic Control of Shikimic Acid Production in E. coli via an Antisense RNA

Escherichia coli is an ideal microbial chassis for producing high-value compounds due to its well-characterized genome and extensive genetic toolbox. Shikimic acid, an intermediate in the aromatic amino acid pathway and the main precursor for oseltamivir, a drug used to treat influenza, is of considerable industrial importance. Here, we present a proof-of-concept metabolic control strategy that combines a biphasic control module with antisense RNA (asRNA) to redirect carbon flux toward shikimic acid accumulation. An asRNA targeting the aroL gene was designed to include the MicF M7.4 Hfq-binding motif and a complementary region targeting the translation initiation sequence of aroL. The construct was expressed from the PBAD promoter in E. coli JW5947-1 (ΔaroK) using the plasmid pBR322. The biphasic system enables staged control of carbon partitioning. In the absence of the PBAD inducer, antisense RNA expression is repressed, allowing shikimic acid utilization for aromatic amino acid synthesis. Upon induction, antisense RNA expression represses shikimate kinase II and promotes shikimic acid accumulation. Shake-flask fermentations showed that extracellular shikimic acid increased to 12.86 ± 1.70 mg L^-1 compared to 6.20 ± 0.68 mg L^-1 in the control. Yield increased (∼2.6-fold) to 157.1 μg/g (±19 μg/g) after induction compared to 82.9 μg/g (±17 μg/g) prior to induction. This work introduces a portable, nongenome-editing, trans-acting regulatory module that enables biphasic flux control and can be integrated with chromosomal or transporter engineering for scalable production.