High-Titer and High-Purity Production of GameXPeptide A Through Systematic Metabolic Engineering and Rational Precursor Feeding

Abstract

Agricultural pests pose a severe threat to global food security, but the long-term use of chemical pesticides raises health and environmental concerns. Although entomopathogenic fungi offer an environmentally friendly alternative, their efficacy is limited by the strong innate immune system of insect pests. Due to its immunosuppressive effect, GameXPeptide A (GXP-A) represents a promising lead compound for the development of an insecticidal enhancer, but its industrial application has been hindered by low production titers and co-generation of structural analogs during fermentation. To resolve these bottlenecks, we systematically engineered the native producer Xenorhabdus budapestensis XBD8 through promoter and RBS engineering to enhance expression of the GXP-A synthase gene gxpS, followed by genome simplification via knockout of competing pathways. Finally, precursor engineering was applied to enhance the phenylalanine supply, which was combined with efflux engineering targeting MFS genes. This integrated strategy increased the shake-flask titer of GXP-A from 0.92 to 587.5 mg/L. After further optimization of precursor feeding, the titer in a 5-L bioreactor reached 2598.2 mg/L, with the purity of GXP-A reaching 94.5%. Moreover, we demonstrated that GXP-A enhanced the killing efficacy of the entomopathogenic fungus Metarhizium anisopliae against the Spodoptera litura by 20.6%, which was attributed to suppression of cellular immunity and downregulation of antifungal peptide expression. This study not only offers the highest-titer microbial cell factory and precursor feeding strategy for the production of GXP-A developed to date, but also demonstrates its potential as a synergist for biological insecticides.