# Identification and functional analysis of two oligopeptide transporters supporting the growth of Lacticaseibacillus paracasei strain Shirota in milk

## TL;DR

This study identifies two oligopeptide transporters in Lacticaseibacillus paracasei Shirota that are essential for its growth in milk by transporting nitrogen sources.

## Contribution

The first experimental evidence of oligopeptide transporters in Lactobacilli capable of transporting peptides up to eight residues long.

## Key findings

- OppLc and DppLc are the primary oligopeptide transporters in LcS, essential for growth in milk.
- OppLc transports peptides of 4–8 residues, while DppLc transports peptides of 3–7 residues.
- OppA1 is the only oligopeptide-binding protein in OppLc, while DppA2 and DppA3 may function as subunits in DppLc.

## Abstract

Lacticaseibacillus paracasei strain Shirota (LcS) employs two distinct oligopeptide transporters, OppLc and DppLc, to facilitate nitrogen acquisition during growth in milk. These transporters exhibit differential substrate specificity, enabling the uptake of oligopeptides of varying lengths derived from milk proteins.

Oligopeptide transporters are important proteins in several lactic acid bacteria (LAB) that facilitate the transport of oligopeptides, the primary nitrogen source for growth in milk. Although the proteolytic mechanisms are well understood in some LAB species, limited research has been conducted on the peptide transport systems of Lacticaseibacillus paracasei (formerly Lactobacillus casei) strain Shirota (LcS), particularly its oligopeptide transporters. This study investigated the nitrogen uptake mechanism of LcS, a probiotic lactic acid bacterium, by generating gene knockout (KO) strains of two oligopeptide transporters, OppLc and DppLc. Consequently, the disruption of these genes eliminated the ability of the bacterium to grow in milk, identifying OppLc and DppLc as the primary oligopeptide transporters in LcS. Growth in a leucine-free chemically defined medium with a Leu-containing peptide as the sole nitrogen source indicated that OppLc and DppLc transport peptides of 4–8 and 3–7 residues, respectively. To our knowledge, this study provides the first experimental evidence of oligopeptide transporters in Lactobacilli capable of transporting peptides up to eight residues long. Analysis of KO strains targeting OppA1 or DppA1 to identify other oligopeptide-binding proteins (OBPs) within each oligopeptide transporter operon that may influence substrate specificity revealed that OppA1 is the only OBPs in OppLc. However, DppA2 and DppA3, encoded at chromosomal locations distant from the DppLc operon, may function as subunits constituting DppLc and DppA1. These findings enhance our understanding of nitrogen source utilization in lactobacilli and might inform future strategies to optimize nitrogen sources for LcS and improve culture technology for LcS-based products.