Modular magnetic recyclable dual-Escherichia coli cell factory with in situ UDP-glucose cofactor regeneration for high-efficiency green biosynthesis of isovitexin

Abstract Background Flavone C-glycoside isovitexin exhibits potent antioxidant, anti-inflammatory and neuroprotective biological activities. Its existing biomanufacturing strategies are limited to low-yield plant extraction or purified free-enzyme cascade catalysis, which are plagued by expensive protein purification steps, absent endogenous cofactor circulation, and single-use non-recyclable biocatalysts. Recombinant whole-cell microbial cell factories offer a sustainable substitute, yet magnetically retrievable modular dual-strain chassis integrated with in situ UDP-glucose regeneration for isovitexin C-glycosylation biosynthesis has rarely been reported. Results In this work, we fabricated a magnetically separable modular dual- Escherichia coli cell factory via co-immobilization of two complementary engineered strains in Fe₃O₄-incorporated sodium alginate-polyvinyl alcohol-chitosan (SA-PVA-CS) ternary composite hydrogel beads. One strain expressed MBP-fused C-glycosyltransferase WjGT1 to catalyze apigenin C-glucosylation, while the other produced sucrose synthase SuSyAc to drive closed-loop intracellular UDP-glucose cofactor regeneration. Of three screened CGT enzymes, MBP-WjGT1 delivered the maximum soluble protein yield of 374 µg per mg wet cell and enabled 98.3% conversion of apigenin. Validated by UDP concentration gradient assays, the dual-strain cascade pathway fully sustained cofactor self-supplementation without external UDP addition under optimized parameters (WjGT1:SuSyAc mass ratio = 99.5:0.5, 10 mM sucrose, 5% DMSO, 0.5 mM apigenin). After 16 h bioconversion, 98.5% apigenin conversion and an isovitexin titer of 212.6 mg/L were achieved. The magnetically immobilized cell factory retained 80% catalytic activity compared with free dual whole-cell catalysts, and preserved 64% of its initial conversion capacity over seven successive magnetic recovery batches. Conclusions This modular magnetically recyclable dual- E. coli cell factory bypasses tedious enzyme purification and achieves autonomous cofactor recycling, constructing an economical, eco-friendly and reusable biomanufacturing platform for green isovitexin production. Moreover, this co-immobilization chassis can be generalized for scalable biosynthesis of various flavonoid C-glycosides, offering a robust engineering framework for developing industrial-oriented microbial cell factories targeting natural flavonoid compounds.