Genomic analysis of regulatory mechanisms governing EPS66A biosynthesis in Streptomyces changanensis HL-66

Streptomyces changanensis HL-66 produces the α-(1,4)/(1,6)-glucan exopolysaccharide EPS66A, a potent plant immune elicitor with promising applications in plant protection. However, its low native fermentation yield limits large-scale application. To investigate the biosynthetic potential and regulatory mechanisms underlying EPS66A production, the whole genome of HL-66 was sequenced and analyzed. The HL-66 genome is 6.82 Mb in size, with a GC content of 74%, and encodes 6081 predicted functional genes. Among these, 1390 genes were annotated to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, 4187 were assigned to Gene Ontology (GO) terms, and 143 were classified into Clusters of Orthologous Groups (COG) categories. antiSMASH analysis identified 22 secondary metabolite biosynthetic gene clusters, including multiple polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) clusters. Functional analyses revealed that the glycosyltransferase gene (GTy) and the global regulatory gene (bldD) are involved in EPS66A biosynthesis. bldD is involved in morphological development and EPS66A production, whereas GTy specifically regulates EPS66A production without affecting growth or development. In both in vivo and potted-plant experiments, EPS66A (200 μg/mL) significantly reduced the severity of tobacco mosaic virus, apple anthracnose leaf spot, walnut bacterial leaf spot, and jujube anthracnose, achieving control efficacies of 90.21%, 87.95%, 77.41%, and 68.55%, respectively, and outperforming a commercial chitosan oligosaccharide control. These findings provide new insights into the genetic architecture and regulatory mechanisms of EPS66A biosynthesis and support its development as a polysaccharide-based green pesticide.