# Chb and nag genes drive N,N′-diacetylchitobiose metabolism in probiotic Lacticaseibacillus paracasei

## TL;DR

This study identifies genes in Lacticaseibacillus paracasei that help it metabolize a sugar found in human and dietary sources, aiding its survival in the gut.

## Contribution

The paper is the first to characterize the chb and nag gene clusters responsible for N,N’-diacetylchitobiose metabolism in Lactobacillales.

## Key findings

- The chb gene cluster is essential for transporting and metabolizing N,N’-diacetylchitobiose in L. paracasei.
- The nagA gene is required to convert N-acetylglucosamine-6P into glucosamine-6P for ChbNAc metabolism.
- ChbR and NagR regulate the chb and nag gene clusters through transcriptional repression.

## Abstract

The persistence of commensal bacteria and administered probiotics in the human gut depends to some extent on their capacity to metabolize diet and host-derived glycans. N,N′-Diacetylchitobiose (N-acetylglucosamine-β-1,4-N-acetylglucosamine; ChbNAc) is a component of N-glycosylated proteins and also the major degradation product of chitin. We have identified in Lacticaseibacillus paracasei BL23 a gene cluster, named chb, involved in the catabolism of ChbNAc. The cluster encodes a transcriptional regulator (ChbR), a cellobiose-type phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) IIC (ChbC), IIA (ChbA) and IIB (ChbB) components, a DUF3284-containing protein (ChbD), and a glycoside hydrolase of the newly identified GH170 family (ChbE). Inactivation of chbC or chbE prevents the growth of L. paracasei in ChbNAc, suggesting that the PTS is involved in its transport and phosphorylation, and that the putative hydrolase ChbE may be acting on the resulting phosphorylated ChbNAc. An L. paracasei mutant with inactivated nagA, encoding an N-acetylglucosamine-6P deacetylase, was also defective in ChbNAc utilization, indicating that the transformation of N-acetylglucosamine-6P into glucosamine-6P by NagA is necessary for ChbNAc metabolism. Transcriptional analysis showed that the chb genes and the nagA gene are regulated by substrate-specific induction mediated by the transcriptional repressors ChbR and NagR, respectively. In addition, both transcriptional regulators repressed the nagB gene, which encodes a glucosamine-6P deaminase that catalyzes the conversion of glucosamine-6P into the glycolytic intermediate fructose-6P. We characterized for the first time the genes responsible for ChbNAc metabolism in a member of the Lactobacillales. The chb and nag clusters may constitute a strategy that allows L. paracasei to adapt to the gastrointestinal environment.

• Lacticaseibacillus paracasei BL23 metabolizes N,N’-diacetylchitobiose

• The chb and nag gene clusters are involved in N,N’-diacetylchitobiose metabolism

• ChbR and NagR transcriptionally repressed the chb and nagAR clusters, respectively

The online version contains supplementary material available at 10.1007/s00253-025-13656-2.