Lactis NZ9000 Research Articles

Disruption of the signal recognition particle pathway leading to impaired growth, sugar metabolism and acid resistance of Lactococcus lactis

Lactococcus lactis is a well-known workhorse for dairy products, whose important industrial traits are tightly associated with numerous cytoplasmic membrane proteins. However, roles of the signal recognition particle (SRP) pathway responsible for membrane protein targeting have not been studied in L. lactis. In this work, the putative genes ffh and ftsY encoding SRP pathway components were identified in the genome of L. lactis NZ9000. Experimental evidence showed that sequence mutation in either the ffh or ftsY was not lethal, but prolonged the lag phase of the resultant mutants Δffh and ΔftsY by 2 h and lowered their biomass to 85.7 % of the wild type under static conditions, as well as deprived the mutants of improved growth capacity under aerobic respiration conditions. Besides, the speeds of glucose consumption and lactate production were significantly decreased in the mutants. Then, the impact of the SPR components on acid resistance was detected, showing that the ffh and ftsY were transcriptionally upregulated by 3.02 ± 1.21 and 8.66 ± 1.01-fold in the wild type during acid challenge at pH 3.0, and cell survival of the Δffh and ΔftsY decreased by10- and 100-fold compared with the wild type. To explore the possible mechanism about the SRP pathway involved in the above physiological traits, proteomics analysis was performed and revealed that disruption of the Ffh or FtsY led to decrease in ribosomal proteins, but increase in DnaK, GroEL and heat shock protein GrpE, indicating that the SRP pathway was closely linked to protein synthesis and folding in L. lactis. Decrease in the fructose-bisphosphate aldolase, respiratory complexes NADH dehydrogenase, as well as glutamate decarboxylase was also detected in the Δffh and ΔftsY, which is consistent with the phenomena of impaired sugar metabolism and acid resistance. Our results demonstrated the dispensable SRP pathway could contribute to the maintenance of metabolism homeostasis and acid resistance of L. lactis.

Construction of Anthocyanin Biosynthesis System Using Chalcone as a Substrate in Lactococcus lactis NZ9000.

Anthocyanins are high-value natural compounds, but to date, their production still mainly relies on extraction from plants. A five-step metabolic pathway was constructed in probiotic Lactococcus lactis NZ9000 for rapid, stable, and glycosylated anthocyanin biosynthesis using chalcone as a substrate. The genes were cloned from anthocyanin-rich blueberry: chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), anthocyanin synthase (ANS), and UDPG-flavonoid 3-O-glycosyltransferase (3GT). Using HR, the polysaccharide pellicle (PSP) segment of the cell wall polysaccharide synthesis (cwps) gene cluster from L. lactis NZ9000 was cloned into vector p15A-Cm-repDE. Then, CHI and F3H were placed sequentially under the control of NZProm 3 of this gene cluster in the vector, which was transformed into L. lactis NZ9000 to obtain Strain A. Furthermore, Strain B was constructed by placing F3H-DFR-ANS and 3GT under NZProm 2 and 3, respectively. Using LC-MS/MS analysis, several types of anthocyanins, including callistephin chloride, oenin chloride, malvidin O-hexoside, malvidin 3,5-diglucoside, and pelargonidin 3-O-malonyl-malonylhexoside, increased in the supernatant of the co-culture of Strains A and B compared to that of L. lactis NZ9000. This is the first time that a five-step metabolic pathway has been developed for anthocyanin biosynthesis in probiotic L. lactis NZ9000. This work lays the groundwork for novel anthocyanin production by a process involving the placement of several biosynthesis genes under the control of a gene cluster.

Co-expression of endoglucanase and cellobiohydrolase from yak rumen in lactic acid bacteria and its preliminary application in whole-plant corn silage fermentation.

Endoglucanase (EG) and cellobiohydrolase (CBH) which produced by microorganisms, have been widely used in industrial applications. In order to construct recombinant bacteria that produce high activity EG and CBH, in this study, eg (endoglucanase) and cbh (cellobiohydrolase) were cloned from the rumen microbial genome of yak and subsequently expressed independently and co-expressed within Lactococcus lactis NZ9000 (L. lactis NZ9000). The recombinant strains L. lactis NZ9000/pMG36e-usp45-cbh (L. lactis-cbh), L. lactis NZ9000/pMG36e-usp45-eg (L. lactis-eg), and L. lactis NZ9000/pMG36e-usp45-eg-usp45-cbh (L. lactis-eg-cbh) were successfully constructed and demonstrated the ability to secrete EG, CBH, and EG-CBH. The sodium carboxymethyl cellulose activity of the recombinant enzyme EG was the highest, and the regenerated amorphous cellulose (RAC) was the specific substrate of the recombinant enzyme CBH, and EG-CBH. The optimum reaction temperature of the recombinant enzyme CBH was 60°C, while the recombinant enzymes EG and EG-CBH were tolerant to higher temperatures (80°C). The optimum reaction pH of EG, CBH, and EG-CBH was 6.0. Mn2+, Fe2+, Cu2+, and Co2+ could promote the activity of CBH. Similarly, Fe2+, Ba2+, and higher concentrations of Ca2+, Cu2+, and Co2+ could promote the activity of EG-CBH. The addition of engineered strains to whole-plant corn silage improved the nutritional quality of the feed, with the lowest pH, acid detergent fiber (ADF), and neutral detergent fiber (NDF) contents observed in silage from the L. lactis-eg group (p < 0.05), and the lowest ammonia nitrogen (NH3-N), and highest lactic acid (LA) and crude protein (CP) contents in silage from the L. lactis-eg + L. lactis-cbh group (p < 0.05), while the silage quality in the L. lactis-cbh group was not satisfactory. Consequently, the recombinant strains L. lactis-cbh, L. lactis-eg, and L. lactis-eg-cbh were successfully constructed, which could successfully expressed EG, CBH, and EG-CBH. L. lactis-eg promoted silage fermentation by degrading cellulose to produce sugar, enabling the secretory expression of EG, CBH, and EG-CBH for potential industrial applications in cellulose degradation.

THE RECOMBINANT BACTERIA LACTOCOCCUS LACTIS EXPRESSING SARS-COV-2 SPIKE PROTEIN OR ITS RECEPTOR-BINDING DOMAIN ELICITS AN IMMUNE RESPONSE FOLLOWING ORAL IMMUNISATION OF RATS

Mucosal delivery of antigens represents an effective strategy for the development of vaccines to control diseases with the upper respiratory and/or gastrointestinal tracts as the main routes of transmission. Lactic acid bacteria (LAB) are food-grade bacteria used for the expression of target antigens in the development of mucosal vaccines. Over the last two decades, research on the use of LAB has focused on the construction of genetically modified strains. Current study aimed to develop and examine the recombinant Lactococcus lactis strains, expressing SARS-CoV-2 spike (S) protein or the receptor-binding domain (RBD) of S protein as immunizing antigens.The spike gene from the component II of Sputnik V vaccine and two synthesized coding sequences of the RBD region of SARS-CoV-2 spike protein (319–529 residues named HA-spike and 318–590 residues named mini-spike) were subcloned into expression vector pNZ8121. The resulting plasmids were verified by sequencing and subsequently transformed into competent L. lactis NZ3900 cells. The expression of recombinant proteins in obtained strains was analyzed by western blot using SARS-COV-2 Spike RBD antibodies. The results showed that the spike gene could be efficiently expressed in the cells of recombinant L. lactis. The major 150 kDa band detected in the cell lysate of L. lactis pNZ::spike represented the full-length S protein. Protein bands of 35 kDa and 23 kDa from the lysates of L. lactis pNZ::mini-spike and L. lactis pNZ::HA-spike respectively were detected indicating that the recombinant RBD protein was also successfully expressed. The mini-spike was expressed more abundantly than the HA-spike variant. Based on the growth kinetics and protein expression profiles of recombinant strains, the highest protein yield was obtained under the following conditions: overnight culture was inoculated (1/25 inoculum) and grown until reaching an optical density of 0,9 at 600 nm, then gene expression was induced by adding nisin (1 ng/ml), cells were harvested after 3 h incubation with the inducer. To examine the immunogenicity of recombinant strains, 1 ml of cell suspension (1,5×109 CFU) of L. lactis pNZ::spike, pNZ::mini-spike or L. lactis pNZ::HA-spike was orally administered to Wistar rats. The same volume of peptone water was administered to the control group. Serum was collected from the animals at 28 days and IgG level was determined by ELISA for the quantitative determination of IgG antibodies to SARS-CoV-2 (COVID-19) Spike RBD protein in rat serum. As expected, the serum from control animals lacked antibodies to the RBD antigen. The serum of rats immunised once with cell suspension of the recombinant strains showed the following levels of specific IgG: 124,24±12,56 ng/ml in group pNZ::mini-spike, 165,91±59,13 ng/ml in group pNZ::spike, and 213,60±54,08 ng/ml in group pNZ::HA-spike. The highest IgG level (545,56±230,24 ng/ml) was detected in the serum of rats immunized with ultrasound-treated cell suspension of L. lactis pNZ::spike. No adverse effects were observed in vaccinated animals at regular check-ups throughout the study period.The current results have demonstrated that the RBD and full spike protein were successfully expressed in L. lactis. Oral administration of the recombinant L. lactis expressing S protein or RBD to rats appeared to generate the anti-SARS-CoV-2 antibodies in serum, positioning this bacteria as promising basis for creating oral vaccine.

Oral administration of recombinant Lactococcus lactis expressing largemouth bass (Micropterus salmoides) IFNa3 protein enhances immune response against largemouth bass virus (LMBV) infection

Largemouth bass virus (LMBV) is a highly pathogenic pathogen that often causes high mortality of affected largemouth bass and significant financial losses. Type I interferon as an effective and broad spectrum tool has been successfully used for therapeutic or prophylactic treatment some viral infections. However, the implementation of immunotherapies based on interferon administration to combat LMBV infections has not been reported. And Lactic Acid Bacteria (LAB) are a powerful vehicle for expressing cytokines or immunostimulant peptides at the gastrointestinal level after oral administration. In this study, Lactococcus lactis (L. lactis) expression system with lactose as a screening marker was utilized to express the Micropterus salmoides interferon a3 (IFNa3) protein and orally administered to largemouth bass. The genetically engineered strain pNZ8149-Usp45-IFNa3-6His/L. lactis NZ3900 was successfully constructed, and its potential to elicit immune protection response by oral administration was evaluated. After orally administration, the recombinant L. lactis was detected in guts of experimental fish and remained detectable for 72 h. Additionally, IFNa3 was able to enhance the test fish's immune response, as determined by the relatively increased mRNA relative expression of immune-related genes in the liver, spleen, and kidney tissues, including IFN-γ, TNF-α, IL-1β, IL-8, IgM and IgT. Following LMBV challenge, the experiment group of pNZ8149-Usp45-IFNa3-6His/L. lactis NZ3900 exhibited a 70 % survival rate, while survival rate were 15 % in the PBS control group, 45 % in the pNZ8149/L. lactis NZ3900 group. Furthermore, the viral load in the surviving fish was significantly lower than that of the control groups. These findings suggest that oral administration of recombinant L. lactis producing IFNa3 induces largemouth bass immune responses at a systemic level to effective prevent and combat of LMBV infection.

The MarR family regulator RmaH mediates acid tolerance of Lactococcus lactis through regulating peptidoglycan modification genes

Lactococcus lactis, widely used in the food fermentation industry, has developed various ways to regulate acid adaptation in the process of evolution. The investigation into how peptidoglycan (PG) senses and responds to acid stress is an expanding field. Here, we addressed the regulation of murT-gatD genes which are responsible for the amidation of PG D-Glu. We found that lactic acid stress reduced murT-gatD expression, and overexpressing these genes notably decreased acid tolerance of L. lactis NZ9000, possibly due to a reduction in PG's negative charge, facilitating the influx of extracellular protons into the cell. Subsequently, using a combination of DNA pull-down assay and electrophoretic mobility shift assay (EMSA), we identified a novel MarR family regulator, RmaH, as an activator of murT-gatD transcription. Further MEME motif prediction, EMSA verification and fluorescent protein reporter assay showed that RmaH directly bound to the DNA motif 5'-KGVAWWTTTTGCT-3' located in the upstream region of murT-gatD. Beyond the mechanistic investigation of RmaH activation of murT-gatD, this study provides new insight into how peptidoglycan modification is regulated and responds to lactic acid stress.

Sucrose contributed to the biofilm formation of Tetragenococcus halophilus and changed the biofilm structure

Based on the previous research results that the addition of sucrose in the medium improved the biofilm formation of Tetragenococcus halophilus, the influence of sucrose on biofilm formation was explored. Moreover, the influence of exogenous expression of related genes sacA and galE from T. halophilus on the biofilm formation of L. lactis NZ9000 was investigated. The results showed that the addition of sucrose in the medium improved the biofilm formation, the resistance of biofilm cells to freeze-drying stress, and the contents of exopolysaccharides (EPS) and eDNA in the T. halophilus biofilms. Meanwhile, the addition of sucrose in the medium changed the monosaccharide composition of EPS and increased the proportion of glucose and galactose in the monosaccharide composition. Under 2.5% (m/v) salt stress condition, the expression of gene sacA promoted the biofilm formation and the EPS production of L. lactis NZ9000 with the sucrose addition in the medium and changed the EPS monosaccharide composition. The expression of gene galE up-regulated the proportion of rhamnose, galactose, and arabinose in the monosaccharide composition of EPS, and down-regulated the proportion of glucose and mannose. This study will provide a theoretical basis for regulating the biofilm formation of T. halophilus, and provide a reference for the subsequent research on lactic acid bacteria biofilms.

Heterologous expression of P9 from Akkermansia muciniphila increases the GLP-1 secretion of intestinal L cells.

Glucagon-like peptide-1(GLP-1) is an incretin hormone secreted primarily from the intestinal L-cells in response to meals. GLP-1 is a key regulator of energy metabolism and food intake. It has been proven that P9 protein from A. muciniphila could increase GLP-1 release and improve glucose homeostasis in HFD-induced mice. To obtain an engineered Lactococcus lactis which produced P9 protein, mature polypeptide chain of P9 was codon-optimized, fused with N-terminal signal peptide Usp45, and expressed in L. lactis NZ9000. Heterologous secretion of P9 by recombinant L. lactis NZP9 were successfully detected by SDS-PAGE and western blotting. Notably, the supernatant of L. lactis NZP9 stimulated GLP-1 production of NCI-H716 cells. The relative expression level of GLP-1 biosynthesis gene GCG and PCSK1 were upregulated by 1.63 and 1.53 folds, respectively. To our knowledge, this is the first report on the secretory expression of carboxyl-terminal processing protease P9 from A. muciniphila in L. lactis. Our results suggest that genetically engineered L. lactis which expressed P9 may have therapeutic potential for the treatment of diabetes, obesity and other metabolic disorders.

Simulation of microbial growth based on Euler’s method

Microorganisms such as bacteria, fungi and yeast produce valuable metabolites when they are grown in suitable culture conditions. The cultivation condition affects the cell growth, metabolism, and product production in a sophisticated and nonlinear way. Therefore, in this research, the growth of Lactococcus lactis NZ9000 in response to the growth conditions was simulated using different growth models. The objective was to simulate the effect of temperature, agitation speed, carbon and nitrogen sources, on the cell growth using the exponential model, logistic and Monod equations. All equations were solved according to the Euler’s method using MATLAB R2021a for simulation. The experimental data used for the simulation were from literature. The accuracy of the model was expressed as percentage relative error between the maximum value of experimental and simulated data. Simulation results shows that the optimum conditions for cell growth was achieved at temperature 27°C, agitation speed of 100 rpm with glucose and peptone as the carbon and nitrogen sources respectively equation. The maximum cell concentration by logistic equation gives the lowest percentage error of 6.40% and 0.33% for the effect of temperature and agitation respectively. While Monod equation give the closest accuracy of 1.84% and 7.11% for carbon and nitrogen sources respectively. Thus, it was shown that the complexity of the microorganism growth was able to be simulated using suitable model such as logistic equation with the lowest relative error.

Co-extrusion microencapsulation of Lactococcus lactis NZ3900 and its survivability in coconut water

Co-extrusion microencapsulation of Lactococcus lactis NZ3900 and its survivability in coconut water

Application of Recombinant Lactic Acid Bacteria (LAB) Live Vector Oral Vaccine in the Prevention of F4+ Enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) causes severe diarrhea in piglets. The current primary approach for ETEC prevention and control relies on antibiotics, as few effective vaccines are available. Consequently, an urgent clinical demand exists for developing an effective vaccine to combat this disease. Here, we utilized food-grade Lactococcus lactis NZ3900 and expression plasmid pNZ8149 as live vectors, together with the secreted expression peptide Usp45 and the cell wall non-covalent linking motif LysM, to effectively present the mutant LTA subunit, the LTB subunit of heat-labile enterotoxin, and the FaeG of F4 pilus on the surface of recombinant lactic acid bacteria (LAB). Combining three recombinant LAB as a live vector oral vaccine, we assessed its efficacy in preventing F4+ ETEC infection. The results demonstrate that oral immunization conferred effective protection against F4+ ETEC infection in mice and piglets lacking maternal antibodies during weaning. Sow immunization during late pregnancy generated significantly elevated antibodies in colostrum, which protected piglets against F4+ ETEC infection during lactation. Moreover, booster immunization on piglets during lactation significantly enhanced their resistance to F4+ ETEC infection during the weaning stage. This study highlights the efficacy of an oral LAB vaccine in preventing F4+ ETEC infection in piglets by combining the sow immunization and booster immunization of piglets, providing a promising vaccination strategy for future prevention and control of ETEC-induced diarrhea in piglets.

Effect of treatment with Lactococcus lactis NZ9000 on intestinal microbiota and mucosal immune responses against Clostridium perfringens in broiler chickens.

Alterations in intestinal microbiota can modulate the developing avian intestinal immune system and, subsequently, may impact on resistance to enteric pathogens. The aim was to demonstrate that early life exposure to Lactococcus lactis, could affect either susceptibility or resistance of broilers to necrotic enteritis (NE). L. lactis NZ9000 (rL. lactis) pre-treatment at 1, 7, 14 and 21 days of age (DOA) led to a significant decrease in NE lesion scores in Clostridium perfringens infected chickens. C. perfringens Infection was associated with spatial and temporal decreases in mononuclear phagocytes and CD4+ αβ T cells. However, rL. Lactis pre-treatment and subsequent C. perfringens infection led to a significant increase in mononuclear phagocytes, CD8α + γδ T, αβ T cells (CD4+ and CD8α+) and B cells (IgM+, IgA+ and IgY+), as well as IL-12p40, IFN-γ and CD40. Differential expression of interleukin (IL)-6, IL-8, IL-10, IL-13, IL-18, IL-22, and transforming growth factor (TGF)-β were observed in L. lactis treated chickens when compared to C. perfringens infected chickens. Microbiota analysis in C. perfringens infected chickens demonstrated an increase in abundance of Bacillota, Bacteroidota, Pseudomonadota and Actinomycetota. These findings suggests that modulation of the chicken intestinal immune system by L. lactis confers partial protection 30 against NE.

Software-based screening for efficient sgRNAs in Lactococcus lactis.

The two essential editing elements in the clustered regularly interspaced short palindromic repeats (CRISPR) editing system are promoter and single-guide RNA (sgRNA), the latter of which determines whether Cas protein can precisely target a specific location to edit the targeted gene. Therefore, the selection of sgRNA is crucial to the efficiency of the CRISPR editing system. Various online prediction tools for sgRNA are currently available. These tools can predict all possible sgRNAs of the targeted gene and rank sgRNAs according to certain scoring criteria in obedience to the demands of the user. We designed sgRNAs for Lactococcus lactis NZ9000 LLNZ_RS02020 (ldh) and LLNZ_RS10925 (upp) individually using online prediction software - CRISPOR and successfully constructed a series of knockout strains to allow comparison of the knockout efficiency of each sgRNA and analyze the differences between software predictions and actual experimental results. Our experimental results showed that the actual editing efficiency of the screened sgRNAs did not match the predicted results, a phenomenon that suggests that established findings from eukaryotic studies are not universally applicable to prokaryotes. software prediction can still be used as a tool for the initial screening of sgRNAs before further selection of suitable sgRNAs through experimental experience. This article is protected by copyright. All rights reserved.

Freeze drying of food-grade recombinant Lactococcus lactis NZ3900-fermented milk with different protecting agents

Lactococcus lactis NZ3900 expressing K-ras mimotope as a live vaccine is a promising non-invasive form of targeted immunotherapy for K-ras-mutated colorectal cancers. However, the stability and delivery of live oral vaccines remain a challenge. Different protectants (maltodextrin, trehalose, whey protein isolate, sodium caseinate) and different ratios of maltodextrin to trehalose were investigated for the freeze-drying of L. lactis NZ3900-fermented milk. Maltodextrin and trehalose gave the highest survivability after freeze-drying (>89%) and high survivability of L. lactis in simulated gastrointestinal conditions (>82%). The combination of maltodextrin and trehalose was shown to be an effective protectant and additive for the L. lactis-freeze-dried fermented milk powder expressing mutated K-ras mimotopes. A 1:1 ratio of maltodextrin and trehalose was found to be the most suitable, giving the lowest moisture content, water activity, and moisture absorption, and the highest survivability after freeze-drying. K-ras peptide expression was confirmed.

Preclinical Safety Evaluation of Engineered Lactococcus lactis NZ3900 Biotherapeutics Using a 13-Week Repeated Dose Oral Toxicity Study in Rabbits

ABSTRACT Lactococcus lactis is one of the most important and safe microorganisms used in the dairy industry. Previously, we orally fed cholesterol-fed rabbits with recombinant Ling Zhi 8 (LZ8) protein from L. lactis NZ3900/pNZ8149 and showed anti-inflammatory effects during the development of fatty liver and atheroma. In order to generate a clinically effective oral immunotherapy for the treatment of allergy, L. lactis clones for Der p 2, Per a 2, and Cla c 14 were introduced into L. lactis NZ3900/pNZ8149 to obtain recombinant allergen-based vaccines. In this study, we developed a triple-antigen vaccine formulation, Lactis- IL-1Ra, Lactis-LZ8, Lactis-Triple, and evaluated their preclinical safety in rabbits by oral administration with repeated doses for 90 days. All of the rabbits survived the challenge without any adverse clinical signs or abnormal changes. A notable advantage of this vaccine is that it can be given orally, and thus self-administration at home would be feasible.

Construction of a food-grade gene editing system based on CRISPR-Cas9 and its application in Lactococcus lactis NZ9000.

Clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) system has been widely used in gene editing of various organisms. However, food-grade gene editing systems in lactic acid bacteria are still preliminary. Red/ET-dependent homologous recombination or CRISPR-based systems have been developed to gene editing in Lactococcus lactis, but these methods are overall inefficient. In the present study, a recombinant system based on CRISPR/Cas9 technology combined with Red/ET was developed using the plasmid pMG36e derived from Lactococcus lactis. Then, the developed recombinant system was applied to Lactococcus lactis. Knockout efficiency was significantly higher using the developed system (91%). In addition, this system showed the potential to be used as a high-throughput method for hierarchical screening. Finally, a gene-edited strain was obtained, and no antibiotics or exogenous genes were introduced using the developed gene editing system. Thus, the efficient system in lactic acid bacteria was constructed and optimized.

Expression of β-Glucosidases from the Yak Rumen in Lactic Acid Bacteria: A Genetic Engineering Approach.

β-glucosidase derived from microorganisms has wide industrial applications. In order to generate genetically engineered bacteria with high-efficiency β-glucosidase, in this study two subunits (bglA and bglB) of β-glucosidase obtained from the yak rumen were expressed as independent proteins and fused proteins in lactic acid bacteria (Lactobacillus lactis NZ9000). The engineered strains L. lactis NZ9000/pMG36e-usp45-bglA, L. lactis NZ9000/pMG36e-usp45-bglB, and L. lactis NZ9000/pMG36e-usp45-bglA-usp45-bglB were successfully constructed. These bacteria showed the secretory expression of BglA, BglB, and Bgl, respectively. The molecular weights of BglA, BglB, and Bgl were about 55 kDa, 55 kDa, and 75 kDa, respectively. The enzyme activity of Bgl was significantly higher (p < 0.05) than that of BglA and BglB for substrates such as regenerated amorphous cellulose (RAC), sodium carboxymethyl cellulose (CMC-Na), desiccated cotton, microcrystalline cellulose, filter paper, and 1% salicin. Moreover, 1% salicin appeared to be the most suitable substrate for these three recombinant proteins. The optimum reaction temperatures and pH values for these three recombinant enzymes were 50 °C and 7.0, respectively. In subsequent studies using 1% salicin as the substrate, the enzymatic activities of BglA, BglB, and Bgl were found to be 2.09 U/mL, 2.36 U/mL, and 9.4 U/mL, respectively. The enzyme kinetic parameters (Vmax, Km, Kcat, and Kcat/Km) of the three recombinant strains were analyzed using 1% salicin as the substrate at 50 °C and pH 7.0, respectively. Under conditions of increased K+ and Fe2+ concentrations, the Bgl enzyme activity was significantly higher (p < 0.05) than the BglA and BglB enzyme activity. However, under conditions of increased Zn2+, Hg2+, and Tween20 concentrations, the Bgl enzyme activity was significantly lower (p < 0.05) than the BglA and BglB enzyme activity. Overall, the engineered lactic acid bacteria strains generated in this study could efficiently hydrolyze cellulose, laying the foundation for the industrial application of β-glucosidase.

Effect of Secretion Efficiency of Mutant KRAS Neoantigen by Lactococcus lactis on the Immune Response of a Mucosal Vaccine Delivery Vehicle Targeting Colorectal Cancer.

Colorectal cancer (CRC) is often caused by mutations in the KRAS oncogene, making KRAS neoantigens a promising vaccine candidate for immunotherapy. Secreting KRAS antigens using live Generally Recognized as Safe (GRAS) vaccine delivery hosts such as Lactococcus lactis is deemed to be an effective strategy in inducing specific desired responses. Recently, through the engineering of a novel signal peptide SPK1 from Pediococcus pentosaceus, an optimized secretion system was developed in the L. lactis NZ9000 host. In this study, the potential of the L. lactis NZ9000 as a vaccine delivery host for the production of two KRAS oncopeptides (mutant 68V-DT and wild-type KRAS) through the use of the signal peptide SPK1 and its mutated derivative (SPKM19) was investigated. The expression and secretion efficiency analyses of KRAS peptides from L. lactis were performed in vitro and in vivo in BALB/c mice. Contradictory to our previous study using the reporter staphylococcal nuclease (NUC), the yield of secreted KRAS antigens mediated by the target mutant signal peptide SPKM19 was significantly lower (by ~1.3-folds) compared to the wild-type SPK1. Consistently, a superior elevation of IgA response against KRAS aided by SPK1 rather than mutant SPKM19 was observed. Despite the lower specific IgA response for SPKM19, a positive IgA immune response from mice intestinal washes was successfully triggered following immunization. Size and secondary conformation of the mature proteins are suggested to be the contributing factors for these discrepancies. This study proves the potential of L. lactis NZ9000 as a host for oral vaccine delivery due to its ability to evoke the desired mucosal immune response in the gastrointestinal tract of mice.

Arginine deiminase pathway of Tetragenococcus halophilus contributes to improve the acid tolerance of lactic acid bacteria

Arginine deiminase pathway, controlled by arginine deiminase, ornithine carbamoyltransferase and carbamate kinase, could affect and modulate the intracellular pH homeostasis of lactic acid bacteria under acid stress. Herein, strategy based on exogenous addition of arginine had been proposed to improve the robustness of Tetragenococcus halophilus during acid stressed condition. Results indicated cells cultured in the presence of arginine acquired high tolerance to acid stress mainly through maintaining the homeostasis of intracellular microenvironment. Additionally, metabolomic analysis and q-PCR showed the content of intracellular metabolites and expression levels of genes involved in ADI pathway significantly increased when cells encountered acid stress with the presence of exogenous arginine. Furthermore, Lactococcus lactis NZ9000 with heterologous overexpression of arcA and arcC from T. halophilus exhibited high stress tolerance to acidic condition. This study may provide an insight into the systematical understanding about the mechanism underlying acid tolerance and improve the fermentation performance of LAB during harsh condition.

Oral Delivery of Mouse β-Defensin 14 (mBD14)-Producing Lactococcus lactis NZ9000 Attenuates Experimental Colitis in Mice

Antimicrobial peptides (AMPs) play essential roles in maintaining intestinal health and have been suggested as possible therapeutic strategies against inflammatory bowel disease (IBD). However, the instability of AMPs in the process of transmission in vivo limits their application in the treatment of IBD. In this study, we constructed the mBD14-producing Lactococcus lactis NZ9000 (L. lactis/mBD14) to achieve enteric delivery of mBD14 and evaluated its protective effect on dextran sodium sulfate (DSS)-induced colitis. Mice treated with L. lactis/mBD14 exhibited milder symptoms of colitis (P < 0.01). Additionally, L. lactis/mBD14 treatment reversed DSS-induced epithelial dysfunction and reduced the production of pro-inflammatory cytokines in colon (P < 0.01). Mechanistically, L. lactis/mBD14 significantly inhibited NOD-like receptor pyrin domain containing three inflammasome-mediated pro-inflammatory response (P < 0.05) and regulated microbiota homeostasis by promoting the abundance of probiotic bacteria Akkermansia muciniphila and Faecalibacterium prausnitzii and decreasing the pathogenic Escherichia coli (P < 0.01). Taken together, this study demonstrates the protective effect of L. lactis/mBD14 in DSS-induced colitis, and suggests that oral administration of L. lactis/mBD14 may represent a potential therapeutic strategy for IBD.