Lactococcus lactis is one of the most extensively characterized lactic acid bacteria, from physiological traits to industrial exploitation. Since last decade, L. lactis has been developed into cell factories for the production of bioactive compounds such as enzymes, vaccine antigens and natural products. However, its precise and efficient genome editing tools is still required to make L. lactis more suitable candidate for engineered functionality. A high active recombinase, RecT of Enterococcus faecalis ATCC14506, was selected from six candidates and mediated homologous recombination between single-stranded DNA (ssDNA) and the L. lactis chromosomal rpoB locus with an efficiency of 100% after rifampin selection. To screen mutants without an externally selectable phenotype, the CRISPR/Cas9 system was used for counterselection, yielding an upp mutant with an efficiency of 46%. By optimization of the copy number of plasmid carrying the CRISPR/Cas9 system and the length of spacer sequence, the off-target efficiency of the recA, galK, hemN and noxD genes were eliminated. The ability of this optimized tool to perform sequential point mutation was demonstrated using the upp and galK gene loci as targets with improved efficiencies > 75%. Moreover, seamless genomic DNA deletions (50/100 bp) or insertion (a loxP site, 34 bp) was efficiently accomplished within 72 h. The work provided a rapid, versatile and precise tool for L. lactis genomic engineering by combination of ssDNA recombineering with improved CRISPR/Cas9 counterselection. This tool will simplify the production of isogenic strains for assessment of gene function or construction of biosynthetic host.

Lactic acid bacteria (LAB) play important roles in silage fermentation, which depends on the production of sufficient organic acids to inhibit the growth of undesirable microorganisms. However, LAB are not able to degrade cellulose and hemicellulose. Bacteria and fibrolytic enzymes are usually used as inoculants to improve the silage quality and digestibility. In the present study, we isolated four Lactobacillus strains (L. amylovorus CGMCC 11056, L. acidophilus CCTCC AB2010208, L. farciminis CCTCC AB2016237 and L. fermentum CCTCC AB2010204) with feruloyl esterase (FAE) activities from ensiled corn stover (CS) by a plate screening assay. The genes encoding FAEs were cloned and hetero-expressed in Escherichia coli. The optimal temperature and pH of these purified enzymes ranged from 45 to 50ºC and from 7.0 to 8.0, respectively. They could hydrolyze hydroxycinnamoyl esters in a substrate-specific manner when methyl ferulate, methyl caffeate, methyl ρ-coumarate and methyl sinapinate were used as substrates. Moreover, these four FAEs were able to hydrolyze CS to release hydroxycinnamic acids. Furthermore, these strains could degrade hydroxycinnamic esters, and L. amylovorus CGMCC 11056 was the most efficient strain among these four isolates. These results provided a new target for the development of inoculants to improve silage quality and digestibility.

To improve silage quality of crop forages, bacterial inoculants are often employed. In this study, Lactobacillus brevis SDMCC050297 and Lactobacillus parafarraginis SDMCC050300 were used as inoculants to corn stover in lab silos for ensiling. At the initial stage of ensiling, the pH value of the inoculated silages reduced more drastically, and the inoculated silages had higher lactic acid and acetic acid contents. After 20 days of ensiling, a reduction in lactic acid content coupled with an increase in acetic acid and 1,2-propanediol contents was observed in inoculated silages. Furthermore, both the amount of lactic acid bacteria and the abundance of order Lactobacillales in inoculated silages were higher than those of controls in the whole process. Meanwhile, Lb. brevis predominated before day 20 and then the dominance was shifted to Lb. parafarraginis until the late stage of ensiling. In contrast, the epiphytic Lactococcus lactic and Lb. plantarum played major roles at the beginning of naturally fermented silages and then Lb. plantarum and Lb. brevis were the most abundant at the later stage. In conclusion, these two selected strains had capability of improving the silage quality and providing the reproducible ensiling process, thus having the potential as silage inoculants.

Lactic acid bacteria (LAB) are a phylogenetically diverse group with the ability to convert soluble carbohydrates into lactic acid. Many LAB have a long history of safe use in fermented foods and are recognized as food-grade microorganisms. LAB are also natural inhabitants of the human intestinal tract and have beneficial effects on health. Considering these properties, LAB have potential applications as biotherapeutic vehicles to delivery cytokines, antigens and other medicinal molecules. In this review, we summarize the development of, and advances in, genome manipulation techniques for engineering LAB and the expected future development of such genetic tools. These methods are crucial for us to maximize the value of LAB. We also discuss applications of the genome-editing tools in enhancing probiotic characteristics and therapeutic functionalities of LAB.

The endolysin Lyb5, from Lactobacillus fermentum temperate bacteriophage φPYB5, showed a broad lytic spectrum against Gram-positive as well as Gram-negative bacteria. Sequence analysis revealed that the C terminus of the endolysin Lyb5 (Ly5C) contained three putative lysin motif (LysM) repeat regions, implying that Ly5C was involved in bacterial cell wall binding. To investigate the potential of Ly5C for surface display, green fluorescent protein (GFP) was fused to Ly5C at its N or C terminus and the resulting fusion proteins were expressed in Escherichia coli . After being mixed with various cells in vitro , GFP was successfully displayed on the surfaces of Lactococcus lactis , Lactobacillus casei , Lb. brevis , Lb. plantarum , Lb. fermentum , Lb. delbrueckii , Lb. helveticus , and Streptococcus thermophilus cells. Increases in the fluorescence intensities of chemically pretreated L. lactis and Lb. casei cells compared to those of nonpretreated cells suggested that the peptidoglycan was the binding ligand for Ly5C. Moreover, the pH and concentration of sodium chloride were optimized to enhance the binding capacity of GFP-Ly5C, and high-intensity fluorescence of cells was observed under optimal conditions. All results suggested that Ly5C was a novel anchor for constructing a surface display system for lactic acid bacteria (LAB). To demonstrate the applicability of the Ly5C-mediated surface display system, β-galactosidase (β-Gal) from Paenibacillus sp. strain K1, replacing GFP, was functionally displayed on the surfaces of LAB cells via Ly5C. The success in surface display of GFP and β-Gal opened up the feasibility of employing the cell wall anchor of bacteriophage endolysin for surface display in LAB.

Yogurt making traditionally relies on the simultaneous utilization of 2 starters: Streptococcus thermophilus and Lactobacillus bulgaricus, but these 2 strains normally metabolize the glucose portion of lactose and release galactose into extracellular medium, resulting in high levels of residual galactose and unfermented lactose in yogurt, which leads to several industrial and health concerns. In this study, we found that Lactobacillus plantarum could effectively metabolize both lactose and galactose. Comparative genomic analysis demonstrated the constant presence of a chromosome-encoded Leloir pathway for galactose metabolism in Lb. plantarum species, and the gal operon was driven by a strong constitutive promoter in Lb. plantarum WCFS1, displaying great potential in low-sugar yogurt making. To test this hypothesis, Lb. plantarum WCFS1 was co-cultured with S. thermophilus or Lb. bulgaricus in lactose-based medium. Results showed that lactose was consumed completely and galactose was metabolized efficiently. For yogurt making, co-cultivation of Lb. plantarum WCFS1 with yogurt starter cultures produced a higher reduction of total sugar content compared with the traditional fermentation processes. In addition, the sensory analysis indicated that the yogurt fermented with yogurt starter cultures and Lb. plantarum WCFS1 was acceptable to consumers in appearance, texture, and flavor. Therefore, this study emphasized the potential to manufacture low-sugar yogurt by the co-cultivation of Lb. plantarum with yogurt starter cultures.