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Whole genome sequencing analysis and functional characterization of Lacticaseibacillus rhamnosus HP-B1083.

Lacticaseibacillus rhamnosus is an important strain for the biotransformation of natural products, and its crude extract exhibits biotransformation effect on glycosidic compounds such as baicalin. To further explore the potential of this strain, particularly given its previously demonstrated high-efficiency β-glucuronidase activity for baicalin conversion, whole-genome sequencing and functional annotation of Lacticaseibacillus rhamnosus HP-B1083 were performed in this study, and its acid tolerance, bile salt tolerance, short-term heat resistance and antibacterial activity were evaluated. The results showed that the strain possessed a circular chromosome with a full length of 3,090,505 bp and a GC content of 46.69%. Gene annotation revealed that the genome contained 2941 coding sequences (CDS) and 112 non-coding RNA genes, including 60 tRNA genes, 1 tmRNA gene, 36 misc_RNA genes and 15 rRNA genes. The functional annotations further reveal that this genome is rich in genes related to carbohydrate metabolism, hydrolases, and transferases, which is highly consistent with its phenotypic characteristics in glycoside transformation and the synthesis of antibacterial substances. In addition, acid tolerance, bile salt tolerance and short-term heat resistance experiments verified that HP-B1083 had acid resistance, bile salt resistance and short-term heat resistance. Antibacterial activity tests confirmed that HP-B1083 produced inhibition zone diameters over 10 mm against common foodborne pathogenic bacteria such as Escherichia coli and Bacillus cereus. Therefore, Lacticaseibacillus rhamnosus HP-B1083 has important application prospects in the development of functional foods, preparation of enzyme preparations and pharmaceutical industry.

Whole Genome Sequencing

Hybrid genome assembly and phenotypic assays reveal carbohydrate metabolism diversity in Lacticaseibacillus strains.

Investigation of carbohydrate metabolism in lactic acid bacteria is essential for the rational selection of strains for fermentation processes, particularly in emerging applications involving non-conventional substrates or building of synthetic microbial consortia. However, establishing robust genotype-phenotype relationships remains challenging, as gene presence alone often fails to explain observed metabolic traits without considering the genomic context and regulatory architecture. In the present study, we combined hybrid genome assembly (Illumina and Oxford Nanopore) with high-throughput phenotype profiling (Biolog GENIII and PM2A) to investigate carbohydrate utilization in five Lacticaseibacillus strains. Phenotypic assays revealed clear intra- and inter-specific variability in substrate utilization. We therefore investigated whether such differences could be attributed to the organization and regulatory context of carbohydrate-associated loci, rather than to gene presence alone. Functional annotation based on COG and CAZyme databases revealed candidate genomic regions potentially involved in carbohydrate metabolism. Comparative analysis between predicted and experimentally observed substrate usage highlighted specific loci associated with carbohydrate utilization profile. The trehalose (tre) operon was conserved across all strains, while at least two distinct cellobiose-associated loci were detected in each genome. Despite the presence of these loci, L. paracasei strains were unable to metabolize cellobiose, a phenotype likely linked to the presence of a downstream TetR-type transcriptional repressor within the cellobiose (cel) operon. Additionally, a genomic region uniquely found in L. rhamnosus strains was associated with gentiobiose utilization, consistent with phenotypic observations. Overall, these findings highlight the importance of integrating phenotypic validation with complete genome context to support the identification of candidate structural and regulatory determinants of carbohydrate utilization in lactic acid bacteria. KEY POINTS: • Phenotype microarrays reveal metabolic traits of interest in isolated strains. • Regulatory context is key to understanding carbohydrate metabolism differences. • Basis of subspecies-dependent cellobiose metabolism in L. paracasei is provided.

Carbohydrate Metabolism