Enhancing Alfalfa Hemicellulose Degradation by Anaerobic Bioprocessing with Engineered Xylanase-Secreting <i>Pediococcus pentosaceus</i>

To enhance hemicellulose utilization in alfalfa silage, <i>Lactococcus lactis</i> (NE) and <i>Pediococcus pentosaceus</i> (PE) expressing xylanase were engineered. The effects of recombinant LAB on hemicellulose utilization, fermentation characteristics, and microbiota diversity during alfalfa ensiling were investigated. The recombinant strains exhibited a 2.35-fold higher xylan degradation efficiency in comparison to the wild-type strains. Recombinant PE treatment significantly improved silage quality, with lactic acid (LA) production surging 149%, pH declining from 5.9 to 4.7 during the early stage (3 days), and hemicellulose degradation increased by 39.5% after 60 days of ensiling. Correlation analysis and KEGG pathway analysis confirmed that the relative abundance of <i>Pediococcus</i> affects the changes in the LA, hemicellulose, and carbohydrate catabolism. Compared to the control group, recombinant PE treatment increased <i>in vitro</i> dry matter digestibility (IVDMD, 20.2%), neutral detergent fiber digestibility (IVNDFD, 12.1%), and acid detergent fiber digestibility (IVADFD, 18.0%). Thus, the engineered strain secretes xylanase to enhance silage fermentation and fiber digestibility.