Tingting Guo

This work constructed a recombinant lactic acid bacterium secreting β-galactosidase for GOS formation during milk fermentation. First, Gal_INF, a β-galactosidase derived from infant feces, was characterized to effectively produce GOS in milk, reaching a content of 10.03 g/L. To export Gal_INF in Lactococcus lactis, six signal peptide candidates were employed, resulting in extracellular activities as low as 52.83-85.65 U/L. Then, Gal_INF (114.6 kDa) was split into two complementary modules, M₁-P₇₂₃ and A₇₂₄-I₁₀₂₃, which could be independently secreted and actively reconstituted with the help of the protein scaffold SpyCatcher/SpyTag. The resultant Lc. lactis B1RG exhibited an extracellular β-galactosidase activity of 544.22 U/L. Fermentation of pasteurized milk with Lc. lactis B1RG and the traditional yogurt starters reduced lactose to 19.67 g/L and yielded 7.17 g/L GOS. This work established an effective strategy to export large-sized proteins extracellularly and demonstrated the applicability of LAB secreting β-galactosidase for GOS-enriched fermented dairy products.

Limosilactobacillus reuteri is a well-recognized probiotic that improves gut homeostasis through the tryptophan metabolite indole-3-lactic acid (ILA), while the functional pathway underlying ILA biosynthesis remains unidentified. In this study, we identified two Lb. reuteri gut isolates, SDMCC050455 and SDMCC050493 exhibiting significant and negligible ILA production, respectively. Comparative genomic analysis and transcriptional determination suggested that six aromatic amino acid aminotransferases (AraT1 to AraT6) and two indole lactate dehydrogenases (FldH2 and FldH3) might participate in ILA biosynthesis. To identify the key aminotransferase, all six transcriptionally active AraTs were overexpressed, and AraT1 increased ILA production by 24.8% in SDMCC050455 and restored ILA production in SDMCC050493. Conversely, transcription suppression of AraT1 significantly reduced the ILA yield in SDMCC050455. Biochemical characterization confirmed AraT1 as a real aminotransferase with high affinity for l-tryptophan. Finally, an ILA biosynthesis pathway was reconstructed by coexpressing AraT1 with either FldH2 or FldH3, endowing Lactococcus lactis with ILA-producing ability. Homology searches using AraT1 and FldH2/3 identified sequences with over 90% identity in 720 of 1020 Lb. reuteri genomes, indicating prevalence of the ILA biosynthesis pathway. Our results demonstrated a novel AraT and functional FldHs forming a complete pathway for ILA biosynthesis in Lb. reuteri, providing insights for probiotic screening and customized ILA production.

A novel bivalent oral vaccine candidate against H5N1 and H9N2 avian influenza virus (AIV) was developed using Lactobacillus surface display technology without genetic modification. The hemagglutinin subunit 1 (HA1) antigens from both subtypes were fused to the surface layer-binding domain of Lactobacillus crispatus K313, expressed in Escherichia coli, and purified. Wild-type Lactobacillus johnsonii H31, isolated from chicken intestine, served as a delivery vehicle by adsorbing and stably displaying the HA1 proteins on its surface. This approach eliminates the need for bacterial engineering while utilizing lactobacilli’s natural capacity to protect surface-displayed antigens, as evidenced by HA1’s protease resistance. Mouse immunization studies demonstrated induction of strong systemic IgG and mucosal IgA responses against both H5N1 and H9N2 HA1. The system offers several advantages, including safety through non-GMO probiotics, potential for multivalent vaccine expansion, and intrinsic antigen protection by lactobacilli. These findings suggest this platform could enable development of cost-effective, multivalent AIV vaccines.

2-O-α-D-glucosyl glycerol (2-αGG) is a valuable ingredient in cosmetics, health-care and food fields. Sucrose phosphorylase (SPase) is a favorable choice for biosynthesis of 2-αGG, while its glucosyl-acceptor affinity and thermodynamic feature remain largely unknown, limiting 2-αGG manufacturing. Here, three SPases were obtained from lactobacilli and bifidobacteria, and the one encoded by Lb. reuteri SDMCC050455 (LrSP) had the best transglucosylation ability, with 2-αGG accounting for 86.01% in the total product. However, the LrSP exhibited an initial forward reaction rate of 11.83/s and reached equilibrium of 56.90% at 110 h, indicating low glycerol affinity and conversion rate. To improve catalytic efficiency, the LrSP was overexpressed in Lb. paracasei BL-SP, of which the intracellular SPase activity increased by 6.67-fold compared with Lb. reuteri SDMCC050455. After chemically permeabilized with Triton X-100, the whole-cell biocatalysis of Lb. paracasei BL-SP was prepared and showed the highest activity, with the initial forward reaction rate improved to 50.17/s and conversion rate risen to 80.79% within 17 h. Using the whole-cell biocatalyst, the final yield of 2-αGG was 203.21 g/L from 1 M sucrose and 1 M glycerol. The food grade strain Lb. paracasei was used for the first time as cell factory to recombinantly express the LrSP and construct a whole-cell biocatalyst for the production of 2-αGG. After condition optimization and cell permeabilization, the whole-cell biocatalyst exhibited 23.89% higher equilibrium conversion and 9.10-fold of productivity compared with the pure enzyme catalytic system. This work would provide a reference for large-scale bioprocess of 2-αGG.

Lactococcus lactis and Streptococcus thermophilus are considered as ideal chassis of engineered probiotics, while food-grade genetic tools are limited in those strains. Here, a Zn²⁺-controlled gene expression (ZICE) system was identified in the genome of S. thermophilus CGMCC7.179, including a transcriptional regulator sczA_st and a promoter region of cation transporter czcD (P_czcDst). Specific binding of the SczA_st to the palindromic sequences in P_czcDst was demonstrated by EMSA analysis, suggesting the regulation role of SczA_st on P_czcDst. To evaluate their possibility to control gene expression in vivo, the sczA_st-P_czcDst was employed to drive the expression of green fluorescence protein (GFP) gene in L. lactis NZ9000 and S. thermophilus CGMCC7.179, respectively. Both of the transformants could express GFP under Zn²⁺ induction, while no fluorescence without Zn²⁺ addition. For optimal conditions, Zn²⁺ was used at a final concentration of 0.8 mM in L. lactis and 0.16 mM in S. thermophilus at OD₆₀₀ close to 0.4, and omitting yeast extract powder in the medium unexpectedly improved GFP expression level by 2.2-fold. With the help of the ZICE system, engineered L. lactis and S. thermophilus strains were constructed to secret cytokine interleukin-10 (IL-10) with immunogenicity, and the IL-10 content in the supernatant of the engineered L. lactis was 59.37 % of that under the nisin controlled expression system. This study provided a tightly controlled expression system by the food-grade inducer Zn²⁺, having potential in development of engineered probiotics.

Lactobacillus plantarum is a catalase-negative species and distributes in human intestinal tracts. However, the cytoprotective effects of the catalase-activated L. plantarum strain have yet to be exploited against reactive oxygen species (ROS). Here, a catalase-activated L. plantarum CGMCC 6888 (CatA⁺) was obtained using exogenous added heme. The scavenging free radical abilities of this strain were obviously increased. Moreover, the activated catalase A in L. plantarum CGMCC 6888 endowed the intestinal epithelium NCM460 with lower ROS content after degrading H₂O₂. In addition, the transcription levels of Nrf2 and Nrf2-related antioxidant enzyme genes (HO-1, GCLC, NQO-1 and TXNRD1) and tight junction protein genes (ZO-1, OCLN, and JAM-1) were upregulated significantly when co-incubated with CGMCC 6888/CatA⁺. This work confirmed that the catalase A conferred L. plantarum with the strong protection effects in the intestinal epithelial cells against ROS.

Aim: Developing efficient methods for monitoring the complex microbial community to rapidly assess the health status. Materials & methods: The qPCR-based method was developed, verified and in situ ...

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.

It was reported that oral administration of Bacillus favored the growth of Lactobacillus in the intestinal tract. Here, this phenomenon was confirmed by co-cultivation of Bacillus subtilis 168 and Lactobacillus plantarum SDMCC050204-pL157 in vitro. To explain the possible molecular mechanisms, B. subtilis 168 cells were incubated in simulated intestinal fluid at 37 °C for 24 h, and up to 90% of cells autolysed in the presence of bile salts. Addition of the autolysate to medium inoculated with Lb. plantarum SDMCC050204 decreased the concentration of H₂O₂ in the culture, alleviated DNA damage and increased the survival of Lb. plantarum, as like the results of exogenous heme addition. These results suggested that the autolysate provided heme, which activated the heme-dependent catalase KatA in Lb. plantarum SDMCC050204. HPLC confirmed the presence of heme in the autolysate. Disruption of the Lb. plantarum SDMCC050204 katA gene abolished the protective effect of the B. subtilis 168 autolysate against H₂O₂ stress. We thus hypothesized that the beneficial effect of Bacillus toward Lactobacillus was established through activation of the heme-dependent catalase and remission of the damage of reactive oxygen species against Lactobacillus. This study raised new crosstalk between the two frequently-used probiotics, highlighting heme-dependent catalase as the key mediator.