Lactis NZ9000 Research Articles

Enabling the biosynthesis of malic acid in Lactococcus lactis by establishing the reductive TCA pathway and promoter engineering

Lactococcus lactis, a homofermentative lactic acid bacterium, is widely applied in fermentation industry. In this study, a reductive TCA pathway was constructed in L. lactis for the biosynthesis of malic acid, a C4-dicarboxylic acid platform molecule. First, genes encoding malate dehydrogenase (mdh) from Actinobacillus succinogenes and pyruvate carboxylase (pycAY715T) from L. lactis were expressed in L. lactis NZ9000 to establish the reductive TCA pathway. The gene encoding lactate dehydrogenase (ldh) was simultaneously deleted. Malic acid was accumulated in the resulting strain NZ02-1 with a titer of 1.92 g/L. Subsequently, genes including ldhB, ldhX, als, and pfl, were deleted, increasing the titer to 3.13 g/L in strain NZ03. A promoter library was developed based on the core region of the phosphofructose kinase promoter Ppfk, and a stronger, constitutive promoter was screened for the enhanced expression of pycAY715T, leading to an increased titer of 5.75 g/L in strain NZ20. Through fed-batch fermentation, the titer in strain NZ20 was boosted to 14.1 g/L with a yield of 0.19 g/g glucose. Through a dual-phase fermentation, the titer was improved to 20.52 g/L with a yield of 0.29 g/g. Our study enabled the biosynthesis of malic acid in L. lactis, expanding the application of this strain as a chassis strain in fermentation industry.

The immune response to a recombinant Lactococcus lactis oral vaccine against foot-and-mouth disease virus in mice.

ObjectiveDevelopment of an effective mucosal vaccine to induce specific immune responses against Foot-and-mouth disease virus (FMDV).ResultsFor this purpose, the FMDV VP1 gene (SPVP1) was optimized and synthesized based on the codon bias of Lactococcus lactis (L. lactis), and then incorporated in the plasmid pNZ8148. L. lactis NZ9000 containing the pNZ8148-SPVP1 recombinant plasmid was used as an oral delivery vehicle to induce anti-FMDV mucosal and systemic immune responses in mice. After confirmation that the SPVP1 protein was expressed successfully in the recombinant L. latic, the mice were orally challenged with NZ9000-pNZ8148, NZ9000-pNZ8148-SPVP1, phosphate-buffered saline as a mock infection group, or with inactivated vaccine as a positive group. Mice immunized with NZ9000-pNZ8148-SPVP1 produced high levels of mucosal secretory IgA (sIgA), antigen-specific serum IgG, IgA, and neutralizing antibodies, and developed stronger cell-mediated immune reactions and significant T spleen lymphocyte proliferation. Furthermore, the recombinant group generated much higher levels of IFN-γ, IL-2, IL-4, IL-5, and IL-10 than the other groups.ConclusionsPotent immune responses were successfully elicited in mice with FMDV VP1 delivered through L. lactis.

Nucleotide sequence and analysis of pRC12 and pRC18, two theta-replicating plasmids harbored by Lactobacillus curvatus CRL 705.

The nucleotide sequences of plasmids pRC12 (12,342 bp; GC 43.99%) and pRC18 (18,664 bp; GC 34.33%), harbored by the bacteriocin-producer Lactobacillus curvatus CRL 705, were determined and analyzed. Plasmids pRC12 and pRC18 share a region with high DNA identity (> 83% identity between RepA, a Type II toxin-antitoxin system and a tyrosine integrase genes) and are stably maintained in their natural host L. curvatus CRL 705. Both plasmids are low copy number and belong to the theta-type replicating group. While pRC12 is a pUCL287-like plasmid that possesses iterons and the repA and repB genes for replication, pRC18 harbors a 168 amino acid replication protein affiliated to RepB, which was named RepB’. Plasmid pRC18 also possesses a pUCL287-like repA gene but it was disrupted by an 11 kb insertion element that contains RepB’, several transposases/IS elements, and the lactocin Lac705 operon. An Escherichia coli / Lactobacillus shuttle vector, named plasmid p3B1, carrying the pRC18 replicon (i.e. repB’ and replication origin), a chloramphenicol resistance gene and a pBluescript backbone, was constructed and used to define the host range of RepB’. Chloramphenicol-resistant transformants were obtained after electroporation of Lactobacillus plantarum CRL 691, Lactobacillus sakei 23K and a plasmid-cured derivative of L. curvatus CRL 705, but not of L. curvatus DSM 20019 or Lactococcus lactis NZ9000. Depending on the host, transformation efficiency ranged from 102 to 107 per μg of DNA; in the new hosts, the plasmid was relatively stable as 29–53% of recombinants kept it after cell growth for 100 generations in the absence of selective pressure. Plasmid p3B1 could therefore be used for cloning and functional studies in several Lactobacillus species.

Expression of the hybrid bacteriocin Ent35-MccV in Lactococcus lactis and its use for controlling Listeria monocytogenes and Escherichia coli in milk

Bacteriocins are antimicrobial peptides synthesised ribosomally. One of the main drawbacks of bacteriocins as food biopreservative agents is their restricted action spectrum. To obtain a broad-spectrum bacteriocin-producing lactic acid bacterium, we engineered a fusion protein between the enterocin P signal peptide and the broad-spectrum chimerical bacteriocin Ent35-MccV. Lactococcus lactis NZ9000 transformed with the recombinant plasmid (pRUK) was tested to identify the bacteriocin production by antimicrobial activity and colony MALDI-TOF mass spectrometry. Additionally, we performed mixed cultures in skim milk to determine the inhibition of Lc. lactis NZ9000 (pRUK) on both Listeria monocytogenes and Escherichia coli strains isolated from food. The results showed that Lc. lactis NZ9000 (pRUK) was able to diminish the growth of both L. monocytogenes and E. coli in skim milk due to Ent35-MccV production. Ent35-MccV is the first linear, genetically engineered bacteriocin produced in situ in food products used effectively to control Gram-negative and Gram-positive foodborne pathogens.

Recombinant Lactococcus lactis co-expressing dendritic cell target peptide and E. tenella 3-1E protein: immune response and efficacy against homologous challenge

ABSTRACT In the present study, recombinant L. lactis NZ9000 displaying anchored dendritic cell-targeting peptide (DCpep) and E. tenella 3-1E protein was constructed. Chickens were orally immunized with the recombinant lactococci for three times at 2-week intervals. After three immunizations, chickens were challenged with E. tenella sporulated oocyst, and the immune response and protective efficacy were evaluated. The finding from animal experiments showed recombinant lactococci expressing anchored DCpep-3-1E protein elicited higher levels of 3-1E specific serum IgG and secretory IgA (sIgA) in caecal lavage, higher proportions of CD4+ and CD8α+ cells in peripheral blood, and higher mRNA expression levels of IL-2 and IFN-γ in the spleen compared to 3-1E-expressing lactococci, and hence offered obvious protective efficacy against homologous challenge. The present work demonstrated that cell surface-displayed target protein and immune enhancers in lactococci may be a promising approach to enhance immunity and immune efficacy against pathogen infection.

Recombinant Lactococcus lactis NZ3900 expressing bioactive human FGF21 reduced body weight of Db/Db mice through the activity of brown adipose tissue.

Fibroblast growth factor 21 (FGF21), a metabolism regulator, has an important effect on metabolic diseases, such as obesity and diabetes. It is also expressed in mice, and the murine source has high homology with human FGF21. Recently, it has been extensively studied and has become a potential drug target for the treatment of metabolic diseases. As it is a protein-based hormone, FGF21 cannot be easily and quickly absorbed into the blood through oral administration. Moreover, it has a 0-2 h half-life in vivo, as shown in a previous study, thus its efficacy lasts for a short period of time when used to treat metabolic diseases, limiting its clinical applications. To avoid these limitations, we used Lactococcus lactis, a food-grade bacterium, as the host to express FGF21. It could be used successfully for the expression and long-term effect of FGF21 in vivo. Instead of antibiotic resistance genes, the LacF gene was used as a selection marker in the NZ3900/PNZ8149 expression system, which is safe and could reduce the antibiotic resistance crisis. In this study, we a constructed human FGF21 expressing L. lactis strain and administered it to Db/Db mice by gavage. Compared with the control group, the body weight of mice in the experimental group was significantly reduced, and the overall homeostasis was improved in mice treated with human FGF21. Moreover, the activity of brown adipose tissue was enhanced. These results revealed that oral administration of FGF21 through heterologous expression in L. lactis appears to be an effective approach for its clinical application.

Fluorescent detection of nisin by genetically modified Lactococcus lactis strains in milk and a colonic model: Application of whole-cell nisin biosensors

Detection of bioactive peptides in complex ecosystems like intestinal environment is a difficult task. In this study, we developed two new bioreporters for nisin based on Lactococcus lactis NZ9000 transformed with the vector pNZ:Nis-aFP or pNZ:Nis-mCherry, that encoded for the anaerobic fluorescent protein evoglow-Pp1 (aFP) or the fluorescent protein mCherry, respectively. The biosensors were used to study nisin A production by L.lactis INIA 650 in milk and in a colonic model. The use of L.lactis NZ9000 pNZ:Nis-aFP as a biosensor allowed the detection of nisin produced by L.lactis INIA 650 in milk, but not in the invitro colonic model. In milk, this reporter was induced by direct addition of 10ng/ml nisin while, in the colonic model, nisin concentrations of 50ng/ml were necessary. However, the reporter system based on pNZ:Nis-mCherry showed a higher sensibility, detecting nisin concentrations of 1ng/ml produced by L.lactis INIA 650 in colonic media using agar diffusion or cross streak bioassays.

Short communication: An inducible CRISPR/dCas9 gene repression system in Lactococcus lactis

Lactococcus lactis, one of the most important probiotic lactic acid bacteria (LAB), is widely used in the dairy industry as a cell factory for recombinant protein production. Currently, a nisin-controlled inducible expression system is used for this purpose and represents the only commercial expression system in LAB. However, the available genetic modification methods are rather limited for modulating gene expression in L. lactis. Here, we developed a 2-plasmid system for gene transcription repression in L. lactis NZ9000 that uses inducible clustered regularly interspaced short palindromic repeats (CRISPR)-dCas9. An inducible promoter Pnisin was used to drive the expression of dCas9 from Streptococcus pyogenes, whereas a strong constitutive promoter P44 drove single guide RNA expression for single or multiple target genes. dCas9 enabled CRISPR interference-mediated silencing of single or multiple target genes with significant reduction of gene expression, up to 99%. In addition, LLNZ_07335, a putative penicillin acylase, was identified as bile salt hydrolase for bile salt resistance in NZ9000 using this system. To our knowledge, this report is the first for a functional gene for bile salt tolerance in L. lactis. Overall, our work introduces a new gene repression tool for various applications in L. lactis or other LAB.

Restructured Lactococcus lactis strains with emergent properties constructed by a novel highly efficient screening system

BackgroundAfter 2.83% genome reduction in Lactococcus lactis NZ9000, a good candidate host for proteins production was obtained in our previous work. However, the gene deletion process was time consuming and laborious. Here, we proposed a convenient gene deletion method suitable for large-scale genome reduction in L. lactis NZ9000.ResultsPlasmid pNZ5417 containing a visually selectable marker PnisZ-lacZ was constructed, which allowed more efficient and convenient screening of gene deletion mutants. Using this plasmid, two large nonessential DNA regions, L-4A and L-5A, accounting for 1.25% of the chromosome were deleted stepwise in L. lactis 9k-3. When compared with the parent strain, the mutant L. lactis 9k-5A showed better growth characteristics, transformability, carbon metabolic capacity, and amino acids biosynthesis.ConclusionsThus, this study provides a convenient and efficient system for large-scale genome deletion in L. lactis through application of visually selectable marker, which could be helpful for rapid genome streamlining and generation of restructured L. lactis strains that can be used as cell factories.

Purification and immobilization of the soluble and insoluble portions of recombinant lipase by gram-positive enhancer matrix (GEM) particles

Lipases are important enzyme for industries. In this work, the recombinant lipase with an AcmA tag working as purification and immobilization tag was expressed in Escherichia coli. Gram-positive enhancer matrix (GEM) particles work to purify and immobilize the recombinant lipase. GEM particles are produced by boiling the cells of Lactococcus lactis NZ9000 to remove the DNA and most proteins. GEM particles specifically bind protein with the AcmA tag in the C-terminal. The recombinant lipase was in two forms, the soluble part and the inclusion body. GEM particles could purify and immobilize the lipase from the soluble part in one step. After the inclusion body being dissolved by 8 M urea, the enzyme activity was recycled by the GEM particles. The GEM particles could immobilize over 75% of the enzyme activity. The lipase immobilized was a basophilla enzyme with the optimal temperature was 30 °C. The activity of the lipase immobilized was 47.1U/OD600 GEM particles at optimal conditions. The enzyme catalysis did not need the ions added to improve the activity. The GEM particles had excellent enzyme activity reusability.

Preventative delivery of IL-35 by Lactococcus lactis ameliorates DSS-induced colitis in mice.

Ulcerative colitis (UC) is one of the two major forms of inflammatory bowel disease (IBD) characterized by superficial mucosal inflammation, rectal bleeding, diarrhea, and abdominal pain. Anti-inflammatory and immunosuppressive drugs have been used in the therapy of human UC. Interleukin (IL)-35, which functions as an anti-inflammatory cytokine, has been shown to play a potential therapeutic role in a UC-like mouse colitis induced by dextran sodium sulfate (DSS). However, the contribution of IL-35 via oral administration to colitis prevention has not been determined. In order to explore its preventative potentiality, a dairy Lactococcus lactis NZ9000 strain was engineered to express murine IL-35 (NZ9000/IL-35), and this recombinant bacteria was applied to prevent and limit the development of DSS-induced mouse colitis. We found that oral administration of NZ9000/IL-35 induced the accumulation of IL-35 in the gut lumen of normal mice. When administrated preventatively, NZ9000/IL-35-gavaged mice exhibited decreased weight loss, DAI score, colon shortening as well as colitis-associated histopathological changes in colon, indicating that the oral administration of NZ9000/35 contributed to the suppression of DSS-induced colitis progression. Moreover, much less Th17 cells and higher level of Treg cells in lamina propria, as well as increased colon and serum levels of IL-10 with a concomitant reduced pro-inflammatory cytokines, IL-6, IL-17A, IFN-γ, and TNF-α were apparently regulated by NZ9000/IL-35 in colitis mice. Together, we put forward direct evidence pinpointing the effectiveness of NZ9000/IL-35 in preventing UC-like mouse colitis, implying a potential candidate of this recombinant Lactococcus lactis that prevent the progression of IBD.

Surface display of hirame novirhabdovirus (HIRRV) G protein in Lactococcus lactis and its immune protection in flounder (Paralichthys olivaceus)

BackgroundHirame novirhabdovirus (HIRRV) can infect a wide range of marine and freshwater fish, causing huge economic losses to aquaculture industry. Vaccine development, especially oral vaccine, has become an effective and convenient way to control aquatic infectious diseases. HIRRV glycoprotein (G), an immunogenic viral protein is a potential vaccine candidate for prevention of the disease. Here, we aimed to construct a recombinant Lactococcus lactis strain expressing HIRRV-G on the cell surface as an oral vaccine to prevent HIRRV.ResultsGlycoprotein gene of HIRRV was successfully cloned and expressed in L. lactis NZ9000 in a surface-displayed form, yielding Ll:pSLC-G. An approximately 81 kDa recombinant G protein (containing LysM anchoring motif) was confirmed by SDS-PAGE, western blotting and mass spectrometry analysis. The surface-displayed G protein was also verified by immunofluorescence and flow cytometry assays. Furthermore, to evaluate the potential of Ll:pSLC-G as oral vaccine candidate, flounders were continuously fed with commercial diet pellets coated with 1.0 × 109 cfu/g of induced Ll:pSLC-G for 1 week. Four weeks later, booster vaccination was performed with the same procedure. Compared with the controls, Ll:pSLC-G elicited significantly higher levels of specific IgM against HIRRV in flounder gut mucus at the second week and in serum at the fourth week (p < 0.05). Meanwhile, oral immunization with Ll:pSLC-G could provide 60.7% protection against HIRRV infection and a significantly lower virus load was detected than the controls on the third day post-challenge (p < 0.01). Moreover, on the first day post 1-week feeding, approximately 104–105 recombinant L. lactis cells were detected in every gram of foregut, midgut and hindgut of flounder, which were mainly localized at the bottom of gut mucus layer; and on day 21, 102–103L. lactis cells could still be recovered.ConclusionsHIRRV-G protein was successfully expressed on the surface of L. lactis cells, which could trigger mucosal and humoral immune response of flounder and provide considerable immune protection against HIRRV. It suggests that genetically engineered L. lactis expressing G protein can be employed as a promising oral vaccine against HIRRV infection.

Enhanced acid-stress tolerance in Lactococcus lactis NZ9000 by overexpression of ABC transporters

BackgroundMicrobial cell factories are widely used in the production of acidic products such as organic acids and amino acids. However, the metabolic activity of microbial cells and their production efficiency are severely inhibited with the accumulation of intracellular acidic metabolites. Therefore, it remains a key issue to enhance the acid tolerance of microbial cells. In this study, we investigated the effects of four ATP-binding cassette (ABC) transporters on acid stress tolerance in Lactococcus lactis.ResultsOverexpressing the rbsA, rbsB, msmK, and dppA genes exhibited 5.8-, 12.2-, 213.7-, and 5.2-fold higher survival rates than the control strain, respectively, after acid shock for 3 h at pH 4.0. Subsequently, transcriptional profile alterations in recombinant strains were analyzed during acid stress. The differentially expressed genes associated with cold-shock proteins (csp), fatty acid biosynthesis (fabH), and coenzyme A biosynthesis (coaD) were up-regulated in the four recombinant strains during acid stress. Additionally, some genes were differentially expressed in specific recombinant strains. For example, in L. lactis (RbsB), genes involved in the pyrimidine biosynthetic pathway (pyrCBDEK) and glycine or betaine transport process (busAA and busAB) were up-regulated during acid stress, and the argG genes showed up-regulations in L. lactis (MsmK). Finally, we found that overexpression of the ABC transporters RbsB and MsmK increased intracellular ATP concentrations to protect cells against acidic damage in the initial stage of acid stress. Furthermore, L. lactis (MsmK) consistently maintained elevated ATP concentrations under acid stress.ConclusionsThis study elucidates the common and specific mechanisms underlying improved acid tolerance by manipulating ABC transporters and provides a further understanding of the role of ABC transporters in acid-stress tolerance.

Biosynthesis of Polysaccharides-Capped Selenium Nanoparticles Using Lactococcus lactis NZ9000 and Their Antioxidant and Anti-inflammatory Activities.

Lactococcus lactis (L. lactis) NZ9000, which has been genetically modified, is the most commonly used host strain for nisin regulated gene expression. Selenium (Se) is an essential trace element in the diet of humans and animals important for the maintenance of health and growth. Biosynthesized Se nanoparticles (SeNPs) that use microorganisms as a vehicle are uniquely advantages in terms of low costs, low toxicity and high bioavailability. This study was aimed at preparing novel functionalized SeNPs by L. lactis NZ9000 through eco-friendly and economic biotechnology methods. Moreover, its physicochemical characteristics, antioxidant and anti-inflammatory activities were investigated. L. lactis NZ9000 synthesized elemental red SeNPs when co-cultivated with sodium selenite under anaerobic conditions. Biosynthesized SeNPs by L. lactis NZ9000 were mainly capped with polysaccharides and significantly alleviated the increase of malondialdehyde (MDA) concentration, the decrease of glutathione peroxidase (GPx) and total superoxide dismutase (T-SOD) activity in porcine intestinal epithelial cells (IPEC-J2) challenged by hydrogen peroxide (H2O2). SeNPs also prevented the H2O2-caused reduction of transepithelial electrical resistance (TEER) and the increase of FITC-Dextran fluxes across IPEC-J2. Moreover, SeNPs attenuated the increase of reactive oxygen species (ROS), the reduction of adenosine triphosphate (ATP) and the mitochondrial membrane potential (MMP) and maintained intestinal epithelial permeability in IPEC-J2 cells exposed to H2O2. In addition, SeNPs pretreatment alleviated the cytotoxicity of Enterotoxigenic Escherichia coli (ETEC) K88 on IPEC-J2 cells and maintained the intestinal epithelial barrier integrity by up-regulating the expression of Occludin and Claudin-1 and modulating inflammatory cytokines. Biosynthesized SeNPs by L. lactis NZ9000 are a promising selenium supplement with antioxidant and anti-inflammatory activities.

Engineering Lactococcus lactis for D-Lactic Acid Production from Starch.

Bioprocess development is a current requirement to enhance the global production of D-lactic acid. Herein, we report a new bioprocess for D-lactic acid production directly from starch using engineered Lactococcus lactis NZ9000. To modify L. lactis as a D-lactic acid producer, its major endogenous L-lactate dehydrogenase (L-Ldh) gene was replaced with a heterologous D-Ldh gene from Lactobacillusdelbrueckiisubsp.lactis JCM 1107. The resulting strain AH1 showed a somewhat slower growth rate but similar lactic acid production compared to those of the intact strain when cultivated with glucose as a carbon source. The chemical purity of D-lactic acid produced by L. lactis AH1 was 93.8%, and the enzymatic activities of D- and L-Ldh in AH1 were 1.54 U/mL and 0.05 U/mL, respectively. Next, a heterologous α-amylase gene from Streptococcus bovis NRIC1535 cloned into an expression vector pNZ8048 was introduced into AH1. The resulting strain AH2 showed an amylolytic activity of 0.26 U/mL in the culture supernatant. Direct production of D-lactic acid from starch as the carbon source was demonstrated using L. lactis AH2, resulting in D-lactic acid production at a concentration of 15.0g/L after 24h cultivation. To our knowledge, this is the first report on D-lactic acid production in engineered L. lactis.

Bypassing lantibiotic resistance by an effective nisin derivative

The need for new antibiotic compounds is rising and antimicrobial peptides are excellent candidates to fulfill this object. The bacteriocin subgroup lantibiotics, for example, are active in the nanomolar range and target the membranes of mainly Gram-positive bacteria. They bind to lipid II, inhibit cell growth and in some cases form pores within the bacterial membrane, inducing rapid cell death. Pharmaceutical usage of lantibiotics is however hampered by the presence of gene clusters in human pathogenic strains which, when expressed, confer resistance. The human pathogen Streptococcus agalactiae COH1, expresses several lantibiotic resistance proteins resulting in resistance against for example nisin.This study presents a highly potent, pore forming nisin variant as an alternative lantibiotic which bypasses the SaNSR protein. It is shown that this nisin derivate nisinC28P keeps its nanomolar antibacterial activity against L. lactis NZ9000 cells but is not recognized by the nisin resistance protein SaNSR.NisinC28P is cleaved by SaNSR in vitro with a highly decreased efficiency, as shown by an cleavage assay. Furthermore, we show that nisinC28P is still able to form pores in the membranes of L. lactis and is three times more efficient against SaNSR-expressing L. lactis cells than wildtype nisin.

Heterologous expression of azurin from Pseudomonas aeruginosa in food-grade Lactococcus lactis

In this study, azurin, a bacteriocin with anticancer property, was produced by food-grade Lactococcus lactis using the Nisin Controlled Gene Expression (NICE) System. In addition, the antibacterial and cytotoxic properties of recombinant azurin in the culture supernatant were also investigated. Azurin gene from Pseudomonas aeruginosa was cloned into the pNZ8149 vector and the resulting recombinant DNA was transformed into food grade L. lactis NZ3900. The expression of azurin protein was induced by the optimum concentration of nisin for 3 h. Inhibition zones for Escherichia coli and Bacillus cereus were observed at 5.0 and 10 mg/mL concentrations of lyophilized supernatants containing azurin, but no inhibition zone at azurin-free lyophilized supernatants. When HUVEC, HT29, HCT116, and MCF7 cell lines were treated with lyophilized culture supernatants with azurin or without azurin, cell viability decreased with increasing concentrations of the supernatant. Furthermore, the supernatants containing azurin showed more anti-proliferative effect than the azurin-free supernatants. This work provides a practicable method to produce recombinant azurin in the food grade L. lactis strain. As a result, the recombinant L. lactis strain, producing azurin, can be used in the investigation of food biopreservatives and in the development of a therapeutic probiotic.

Expression in Lactococcus lactis of a β-1,3-1,4-glucanase gene from Bacillus sp. SJ-10 isolated from fermented fish

Bacterial β-1,3-1,4-glucanase (BG) is an endoglucanase that hydrolyzes linear β-glucans containing β-1,3 and β-1,4 linkages, such as barley β-glucans. In this study, a BG gene was transformed into the food-grade plasmid pNZ8149 and successfully expressed in Lactococcus lactis NZ3900 using the nisin-controlled gene expression system. To facilitate extracellular secretion, the signal peptide Usp45 was added during vector construction. A histidine tag was also added for affinity purification. BG was extracellularly secreted and was also present in the cells in soluble form. N-terminal amino acid residue analysis of secreted BG revealed that the Usp45 peptide was removed. The optimum temperature and pH for both intracellular and extracellular BG were 40 °C and 6, respectively. The enzyme kinetic parameters, Vmax, Km, kcat, and kcat/Km, of extracellular BG were 1317.51 μmol min−1, 1.97 mg ml−1, 588.54 s−1, and 298.26 ml s−1∙mg−1, respectively. There was no significant difference in the enzyme kinetic parameters of intracellular and extracellular BG. The growth pattern of transformed L. lactis NZ3900 in β-glucan-containing liquid medium confirmed β-glucan degradation by BG. The transformed strain degraded β-glucans, produced gluco-oligosaccharide, and produced lactic acid. The strain and expression system constructed in this study could be applied to industrial fields requiring BG produced in food-grade lactococcal secretory expression system.

Expression of milk-derived angiotensin I-converting enzyme-inhibitory peptides in Lactococcus lactis

ABSTRACTAngiotensin I-converting enzyme (ACE) plays a key role in the regulation of blood pressure. Currently, most single or tandem repeats of ACE-inhibitory (ACE-I) peptides have been expressed in Escherichia coli. However, in this study, a food-grade system was constructed using Lactococcus lactis (L. lactis) to simultaneously express four different milk-derived ACE-I peptides with antihypertensive activity. Mixed peptides (MPs) fused with green fluorescent protein (GFP) and eight histidines were synthesized. To ensure potent ACE inhibition by the MPs in human digestive juice, pepsin and trypsin cleavage sites were introduced among the four ACE-I peptides. The MP fusion gene was inserted into expression vector pSEC-E7 with nisin induction and expressed in L. lactis NZ9000, then purified by affinity chromatography. The transformants containing pSEC-MP:GFP were identified based on green fluorescence using Leica laser scanning confocal microscopy. The target proteins were detected by SDS-PAGE analysis and displayed obvious immunogenicity by western blot. After hydrolysis with digestive enzymes, the IC50 of the MPs was 118.63 μM. These results suggested that multiple milk-derived ACE-I peptides with antihypertensive properties could be produced using a food-grade lactococcal expression system.

Heterologous expression of Class IIb bacteriocin Plantaricin JK in Lactococcus Lactis

Plantaricin JK (PlnJK) is a Class IIb LAB bacteriocin that includes two peptides; i.e., PlnJ and PlnK, which can synergistically halt many types of gram-positive bacteria, including food spoilage organisms. Purification of these peptides from natural lactic acid bacteria is difficult therefore, their application remains limited. To overproduce this two-peptide bacteriocin, the food-grade nisin-controlled expression (NICE) system was firstly used to heterologous expression of PlnJK. We constructed recombinant plasmids pNZ8124-plnJ and pNZ8124-plnK, and expression of PlnJ and PlnK was achieved in Lactococcus lactis NZ9000. A combination technique of XAD-2 macroporous resin, strong cation column, and reversed-phase high performance liquid chromatography were used to obtain recombinant proteins. Their molecular mass was quantified by ESI-MS and the results were 2929.32 Da and 3502.89 Da, respectively. An antimicrobial activity assay indicated that PlnJK had significant antimicrobial activities toward strains of Staphylococcus and the two peptides acted synergistically. Fluorescence leakage analysis indicated that PlnJK induced the increase of membrane permeabilization, which resulted in intracellular ion leakage, electrolytes efflux and ultimately cell death.