This study systematically compared the effects of dietary supplementation with glutamine (Gln) and its precursors, including glutamic acid (GA) and α-ketoglutarate (AKG), on growth performance, serum antioxidant and immune parameters, and multi-region gastrointestinal microbiota in suckling lambs. Forty healthy suckling Hu lambs with similar body weight (7.37 ± 1.18 kg) and age (7 ± 0.8 d) were selected and randomly allocated into four groups (n = 10 per group): a control group (CON, without additive), and three treatment groups (GA, AKG, and Gln), each receiving 2 g per animal per day of the corresponding additive. The experimental period lasted for 42 d. All three additives showed a tendency to increase the final body weight (p = 0.056) and significantly increased the average daily gain (ADG) of lambs (p < 0.05). GA supplementation increased the dry matter intake throughout the entire trial (p < 0.05), whereas the addition of AKG and Gln increased the dry matter intake only during the later period (d 21–42) (p < 0.05). The feed-to-gain ratios did not differ among all groups (p > 0.05). Compared with the CON group, all three treatment groups showed elevated serum activities of catalase, glutathione peroxidase, and total antioxidant capacity, as well as increased IgA and IgG contents (p < 0.05). In addition, malondialdehyde concentration was decreased in all three treatment groups (p < 0.05). Moreover, GA supplementation reduced the ruminal alpha diversity while increasing the abundance of butyrate-producing bacteria (Ruminococcaceae UCG-014) (p < 0.05). All three interventions consistently decreased the abundance of the intestinal pathogen Escherichia-Shigella in the ileum (p < 0.05). Correlation analyses showed that ruminal Treponema 2 abundance was negatively correlated with ADG, whereas jejunal Methylobacterium and ileal [Eubacterium] coprostanoligenes group were positively correlated with final body weight or ADG. In conclusion, glutamine and its precursors play an important role in modulating gastrointestinal bacterial diversity and composition, enhancing antioxidant and immune functions, and improving the growth performance of suckling lambs.

Skatole is a harmful, odorous pollutant in livestock manure. Skatole-degrading strains are mostly harmful Gram-negative bacteria, whereas safe Gram-positive strains demonstrate poor degradation performance, limiting bioremediation applications. To solve this problem, this study was conducted to enhance the skatole degradation capacity of the food-grade strain Lactococcus lactis NZ9000 via heterologous expression of the skatole-degrading Ska enzyme from Acinetobacter piscicola p38. Three gene sequences, designated Ska-Y (original sequence from A. piscicola p38), Ska-D (E. coli codon-optimized), and Ska-R (L. lactis codon-optimized), were separately expressed using constitutive pMG36e and nisin-inducible pNZ8148 plasmids. The constitutive system only transcribed mRNA but produced misfolded, nonfunctional inclusion bodies. By contrast, the codon-optimized pNZ8148-Ska-R strain exhibited prominent skatole degradation ability, even under non-inductive conditions. Under optimal conditions (30 ℃, ultra-low nisin induction), the engineered strain increased the 24 h degradation rate of 50 mg/L skatole from 20% to 88% and completely degraded 25 mg/L skatole. Field tests verified that the strain effectively reduced skatole accumulation in manure compost and lagoon manure. This study achieved efficient functional heterologous expression in Gram-positive bacteria, provides new insights into ultra-low-dose induction and codon optimization, and offers a safe and efficient microbial agent for livestock manure odor remediation.

Catechol 2,3-dioxygenase (C23O) is the rate-limiting enzyme in the meta-cleavage pathway of aromatic compound degradation, yet its functional annotation within the structurally conserved VOC superfamily remains challenging due to high sequence homology. Here, we isolated a catechol-degrading strain from bovine feces and identified it as Bacillus thuringiensis HHY919 via whole-genome sequencing. Genome annotation revealed four VOC genes sharing the same COG annotation (“catechol 2,3-dioxygenase”) but divergent KO annotations (two as glyoxalases and two as C23O), suggesting functional divergence. Using homology modeling and molecular docking, we compared their binding affinities toward catechol and 11 derivatives. All four proteins showed typical meta-cleavage binding energies (−4.9 to −5.4 kcal/mol), with slightly more favorable binding than an ortho-cleavage control. Notably, gene4224 exhibited the broadest and strongest predicted affinities in virtual screening against 212 compounds, particularly for trichlorophenol (−6.0 kcal/mol) and complex natural products (qvina_score ≤ −7.6 kcal/mol). Phylogenetic analysis and docking results jointly identified gene3355 and gene4224 as computationally prioritized C23O candidates, with gene4224 recommended as the top candidate for future enzyme engineering and bioremediation studies. This study provides a computational workflow for resolving functional ambiguity in VOC family enzymes and generating testable hypotheses for experimental validation.

Skatole (3-methylindole) is a primary malodorous compound emitted from livestock manure, posing significant challenges for odor control in intensive animal production. In this study, a skatole-degrading bacterial strain, designated hhy901, was isolated from fresh cattle manure and identified as Bacillus pumilus through polyphasic taxonomy, including 16S rRNA and gyrB gene phylogeny, morphological characterization, biochemical profiling, and whole-cell fatty acid analysis. Under an initial skatole concentration of 100 mg/L in Bacillus liquid medium, strain hhy901 removed 87.2% of skatole within 96 h, as determined by high-performance liquid chromatography (HPLC). Whole-genome sequencing revealed a genome of approximately 3.60 Mb with a GC content of 41.91%, encoding 3564 predicted protein-coding genes. Functional genomic annotation identified 39 genes associated with xenobiotic biodegradation and metabolism, including putative catechol dioxygenase, aldehyde dehydrogenase, and cytochrome P450 genes, which together suggest a potential skatole catabolic pathway. Furthermore, the genome harbored a broad repertoire of genes associated with resistance to heavy metals, disinfectants, and multiple antibiotic classes, indicating a potential for environmental stress adaptation. Virulence factor profiling predominantly identified genes related to nutrient acquisition and microbial competition, with only a single invasion-associated factor detected, suggesting a relatively low invasive pathogenic potential. However, the presence of multiple antimicrobial resistance determinants warrants cautious interpretation, and phenotypic validation of both degradation function and biosafety is required before environmental application. These results establish B. pumilus hhy901 as a promising microbial candidate for further development of bio-deodorization strategies in livestock production, pending functional and safety validation.