Lactobacillus plantarum is a food-grade microorganism with industrial and medical relevance belonging to the group of lactic acid bacteria (LAB). Traditional strategies for obtaining gene deletion variants in this organism are mainly vector-based double-crossover methods, which are inefficient and laborious. A feasible possibility to solve this problem is the recombineering, which greatly expands the possibilities for engineering DNA molecules in vivo in various organisms. In this work, a double-stranded DNA (dsDNA) recombineering system was established in L. plantarum. An exonuclease encoded by lp_0642 and a potential host-nuclease inhibitor encoded by lp_0640 involved in dsDNA recombination were identified from a prophage P1 locus in L. plantarum WCFS1. These two proteins, combined with the previously characterized single strand annealing protein encoded by lp_0641, can perform homologous recombination between a heterologous dsDNA substrate and host genomic DNA. Based on this, we developed a method for marker-free genetic manipulation of the chromosome in L. plantarum. This Lp_0640-41-42-mediated recombination allowed easy screening of mutants and could serve as an alternative to other genetic manipulation methods. We expect that this method can help for understanding the probiotic functionality and physiology of LAB.

This study explored the enhancement of exopolysaccharide (EPS) production by Ganoderma lucidum through bidirectional liquid fermentation, employing Clinacanthus nutans (Burm. f.) Lindau leaves as a medicinal substrate. The optimal concentration of C. nutans leaf powder was determined to be 6 g/L, resulting in a significant increase in both mycelial biomass (61.78%) and EPS yield (116.6%). Structural analyses indicated that the EPS supplemented with C. nutans underwent notable modifications. Fourier-transform infrared spectroscopy suggested the introduction of potential carbonyl groups and a shift in glycosidic linkage configuration. Monosaccharide composition analysis revealed a significant transition from a glucose-dominated profile in the control to a galactose-rich, more diverse profile, including uronic acids and amino sugars, in the experimental group. High-performance gel permeation chromatography demonstrated a transformation from a low, homogeneous molecular weight (4.7 kDa) to a heterogeneous, bimodal distribution featuring a prominent high-molecular-weight fraction (38.5 kDa). Consequently, the modified EPS exhibited significantly enhanced antioxidant activities, with scavenging rates for DPPH, hydroxyl, and ABTS radicals increasing to 55.5%, 35.1%, and 88.0%, respectively, at a concentration of 2 mg/mL. These findings demonstrate that C. nutans is an effective supplement for modulating the fermentation process of G. lucidum, not only boosting EPS production but also tailoring its structural characteristics to obtain polysaccharides with superior bioactivities, highlighting its potential in functional food and nutraceutical applications.