Genetic basis of exopolysaccharide production in Limosilactobacillus fermentum: insights from comparative genomics and GWAS

Exopolysaccharides (EPS) from lactic acid bacteria are critical for the production of fermented foods and probiotics. EPS production varies among Limosilactobacillus fermentum ( L. fermentum ) strains. Conventional phenotypic screening identifies high-yield strains but does not reveal genetic determinants. In this study, we sequenced 40 L. fermentum strains from diverse sources and used comparative genomics, single nucleotide polymorphism (SNP) analysis, and genome-wide association studies (GWAS) to uncover the genetic basis for differential EPS production. EPS production ranged from 79.68 to 228.19 mg/L. Pan-genome analysis revealed an open pan-genome of 6365 genes with strain-specific variations. Core SNP analysis confirmed high genetic diversity, revealing 39,087 SNPs ( π = 0.004279). GWAS identified 24 SNP sites significantly linked to EPS production, nine of which were missense mutations. The most critical variant was Arg375Gln (c.G1124A) in the accessory Sec system glycosyltransferase Asp1 ( LAF_RS00270 ). This mutation, in a wall teichoic acid (WTA) biosynthesis gene, may reduce EPS yield by altering the partitioning of the precursor UDP-glucose between the WTA and EPS synthesis pathways. This study provides genomic insights and candidate targets for rational selection and metabolic engineering of high-EPS-yield L. fermentum for enhanced applications in the food and health industry.