[P002] Poster

1:00 PM - 1:50 PM JST(4:00 AM - 4:50 AM UTC)

[P01-904] Accelerated Evolution of Metabolic Pathways using Biosensors

〇Ashvinath Narenderan^1,2,3,4, Shujian Ong^1,2,3,4, and Julius Fredens^1,2,3,4 (¹ NUS Synthetic Biology for Clinical and Technological Innovation (SynCTI), National University of Singapore.
² Synthetic Biology Translational Research Programme, Yong Loo Lin School of Medicine, National University of Singapore.
³ Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore.
⁴ National Centre for Engineering Biology (NCEB), Singapore.)

Naringenin is a pharmaceutically important flavonoid whose microbial biosynthesis remains limited by enzyme inefficiencies and metabolic flux imbalances. Here we use LySE, a semi-continuous directed evolution platform combining T7 phage-mediated mutagenesis with intracellular biosensor-driven selection to optimize a five-enzyme naringenin biosynthetic pathway in Escherichia coli. The pathway - comprising RsTAL, At4CL, PhCHS, MatB, and CmCHI - is encoded on a phagemid that undergoes iterative rounds of error-prone replication driven by an anhydrotetracycline-inducible error-prone T7 DNA polymerase (epT7DNAP). Selection is implemented using two intracellular biosensors deployed sequentially: a high-sensitivity TtgR transcription factor-based GFP reporter is used in early selection rounds to enrich low-to-moderate producers, followed by a more stringent naringenin-responsive riboswitch in later rounds to isolate high producers. We present the design and characterization of this integrated platform alongside preliminary naringenin biosynthesis and biosensor validation data. Together, these results establish LySE as a generalizable framework for semi-continuous, selection-guided optimization of polyketide biosynthetic pathways, with broader implications for flavonoid biomanufacturing.