Heterologous production of a plant biostimulant in Streptomyces albidoflavus.

Climate change-associated abiotic stresses threaten agricultural productivity, creating a need for sustainable strategies that improve plant resilience. Pteridic acids F and H (PTA-F and PTA-H), originally isolated from Streptomyces iranensis HM 35, are plant growth-promoting polyketides with reported activity under drought and salinity stress. However, reported production was extremely low (~ 0.08 and 0.02 mg/L), limiting further development and application. Here, we established a heterologous production platform for PTA biosynthesis by cloning the 68-kb type I polyketide synthase biosynthetic gene cluster using Cas12a-assisted precise targeted cloning using in vivo Cre-lox recombination (CAPTURE), followed by CRISPR-Cas9-mediated genomic integration and promoter engineering in Streptomyces hosts. Initial heterologous expression resulted in detectable elaiophylin production but not PTA, whereas BGC engineering with the strong constitutive kasOp* promoter enabled PTA production (although below the limit of quantification). Genome-scale metabolic model-guided media optimization further improved production and fed-batch fermentation yielded 1.7 mg/L PTA in J1074-PTA-kasOp* and 2.8 mg/L PTA in NBC1270-PTA-kasOp*. These titers represent a more tha n 20-fold increase compared with the native producer under comparable conditions. This work provides the first functional heterologous platform for PTA biosynthesis and demonstrates how synthetic biology and genome-scale metabolic modeling can be combined to improve production of complex plant-beneficial polyketides. KEY POINTS: • Direct BGC cloning and engineering enabled production of PTA in heterologous host. • Genome-scale metabolic models (GEMs) guided media optimization for PTA production. • Fed-batch fermentation achieved > 20-fold PTA titer improvement over native strain.