Optimized Expression of <Emphasis Type="Italic">Helicobacter pylori</Emphasis><Emphasis Type="Italic">ureB</Emphasis> Gene in the <Emphasis Type="Italic">Lactococcus lactis</Emphasis> Nisin-Controlled Gene Expression (NICE) System and Experimental Study of Its Immunoreactivity

In the development of an oral vaccine against Helicobacter pylori, H. pylori urease subunit B (UreB) was expressed in a food-grade delivery vehicle, Lactococcus lactis NZ3900. The ureB gene (Genbank accession no. FJ436980) was amplified by polymerase chain reaction (PCR) from MEL-Hp27. The PCR-amplified ureB gene was cloned in the E. coli–L. lactis shuttle vector pNZ8110 and transformed into E. coli MC1061. After the transformant had been identified, the recombinant plasmid was purified and electrotransformed into L. lactis NZ3900. The conditions of UreB expression in the L. lactis transformant were optimized by orthogonal experiment. The maltose binding protein (MBP)-UreB fusion protein expressed by TB₁/pMAL-c2X-ureB was used to cultivate mice polyclonal anti-UreB serum after purification by the amylose prepacked column. The Western blot method was adopted to confirm whether the UreB expressed by L. lactis transformant had immunoreactivity. The optimized conditions for UreB expression were as follows. Nisin 40 ng/ml was added to the medium when the recombinant grew to OD₆₀₀≈0.30–0.40 and the induction time lasted 5 h. As a result, the maximum yield of UreB was 27.26 μg/mL of medium, and the maximum percentage of UreB in cell extracts of the L. lactis transformant reached its peak at 20.19%. Western blot analysis showed that the UreB protein expressed by L. lactis transformant had favorable immunoreactivity. All these results make an appealing case for construction of the food-grade vaccine for H. pylori.     

Improvement in lactic acid production from starch using α-amylase-secreting <Emphasis Type="Italic">Lactococcus lactis</Emphasis> cells adapted to maltose or starch

To achieve direct and efficient lactic acid production from starch, a genetically modified Lactococcus lactis IL 1403 secreting α-amylase, which was obtained from Streptococcus bovis 148, was constructed. Using this strain, the fermentation of soluble starch was achieved, although its rate was far from efficient (0.09 g l⁻¹ h⁻¹ lactate). High-performance liquid chromatography revealed that maltose accumulated during fermentation, and this was thought to lead to inefficient fermentation. To accelerate maltose consumption, starch fermentation was examined using L. lactis cells adapted to maltose instead of glucose. This led to a decrease in the amount of maltose accumulation in the culture, and, as a result, a more rapid fermentation was accomplished (1.31 g l⁻¹ h⁻¹ lactate). Maximum volumetric lactate productivity was further increased (1.57 g l⁻¹ h⁻¹ lactate) using cells adapted to starch, and a high yield of lactate (0.89 g of lactate per gram of consumed sugar) of high optical purity (99.2% of l-lactate) was achieved. In this study, we propose a new approach to lactate production by α-amylase-secreting L. lactis that allows efficient fermentation from starch using cells adapted to maltose or starch before fermentation.     

Diacetyl and acetoin production from whey permeate using engineered <Emphasis Type="Italic">Lactobacillus casei</Emphasis>

The capability of Lactobacillus casei to produce the flavor-related compounds diacetyl and acetoin from whey permeate has been examined by a metabolic engineering approach. An L. casei strain in which the ilvBN genes from Lactococcus lactis, encoding acetohydroxyacid synthase, were expressed from the lactose operon was mutated in the lactate dehydrogenase gene (ldh) and in the pdhC gene, which codes for the E2 subunit of the pyruvate dehydrogenase complex. The introduction of these mutations resulted in an increased capacity to synthesize diacetyl/acetoin from lactose in whey permeate (1,400 mg/l at pH 5.5). The results showed that L. casei can be manipulated to synthesize added-value metabolites from dairy industry by-products.     

Expression of Helicobacter pylori cag12 gene in Lactococcus lactis MG1363 and its oral administration to induce systemic anti-Cag12 immune response in mice

To develop an oral vaccine against Helicobacter pylori infection, we have expressed the H. pylori cag12 (HP0532) gene, encoding the outer membrane protein Cag12 (31 kDa), in a live delivery vehicle Lactococcus lactis. The cag12 gene was amplified by polymerase chain reaction (PCR) using the genomic DNA of H. pylori K51 isolated from Korean patients. DNA sequence analysis revealed that the cag12 gene of H. pylori K51 has 98.1 and 97.4% identity with individual cag12 genes of the H. pylori 26695 and J99, respectively. The GST–Cag12 fusion protein, produced using the Escherichia coli expression system, was used to raise a rat polyclonal anti-Cag12 antibody. The PCR-amplified cag12 gene of H. pylori K51 was cloned in the E. coli–L. lactis shuttle vector (pMG36e) and transformed into L. lactis. Western blot analysis demonstrated that the Cag12 protein was expressed in the L. lactis transformant, with a maximum level at the log phase without extracelluar secretion. The oral administration of the transformant into mice resulted in the generation of the anti-Cag12 antibody in serum in two out of five cases. These results suggest that the recombinant L. lactis, which expresses Cag12, may be applicable as an oral vaccine to induce protective immunity against H. pylori.     

Overproduction of Heterologous Mannitol 1-Phosphatase: a Key Factor for Engineering Mannitol Production by Lactococcus lactis

To achieve high mannitol production by Lactococcus lactis, the mannitol 1-phosphatase gene of Eimeria tenella and the mannitol 1-phosphate dehydrogenase gene mtlD of Lactobacillus plantarum were cloned in the nisin-dependent L. lactis NICE overexpression system. As predicted by a kinetic L. lactis glycolysis model, increase in mannitol 1-phosphate dehydrogenase and mannitol 1-phosphatase activities resulted in increased mannitol production. Overexpression of both genes in growing cells resulted in glucose-mannitol conversions of 11, 21, and 27% by the L. lactis parental strain, a strain with reduced phosphofructokinase activity, and a lactate dehydrogenase-deficient strain, respectively. Improved induction conditions and increased substrate concentrations resulted in an even higher glucose-to-mannitol conversion of 50% by the lactate dehydrogenase-deficient L. lactis strain, close to the theoretical mannitol yield of 67%. Moreover, a clear correlation between mannitol 1-phosphatase activity and mannitol production was shown, demonstrating the usefulness of this metabolic engineering approach.     

Oral immunization of mice with <Emphasis Type="Italic">Lactococcus lactis</Emphasis> expressing the rotavirus VP8* protein

The efficacy of recombinant Lactococcus lactis as a delivery vehicle for a rotavirus antigen was evaluated in a mouse model. The rotavirus VP8* protein was expressed intracellularly and extracellularly in L. lactis wild type and in an alr mutant deficient in alanine racemase activity, necessary for the synthesis of the cell-wall component d-alanine. When the mucosal immune response was evaluated by measuring VP8*-specific IgA antibody in faeces, wild-type L. lactis triggered a low IgA synthesis only when the secreting strain was used. In contrast, VP8*-specific IgA was detected in faeces of both groups of mice orally given the alr mutant expressing extracellular VP8* and intracellular VP8*, which reached levels similar to that obtained with the wild type secreting strain. However, oral administration of the recombinant strains did not induce serum IgG or IgA responses. L. lactis cell-wall mutants may therefore provide certain advantages when low-antigenic proteins are expressed intracellularly. However, the low immune response obtained by using this antigen-bacterial host combination prompts to the use of new strains and vaccination protocols in order to develop acceptable rotavirus immunization levels.     

Characterization of a plasmid involved with cointegrate formation and lactose metabolism in Lactococcus lactis subsp. lactis OZS1

A 55 kilobase (kb) plasmid (pOZS550) in the non-clumping Lactococcus lactis subsp. lactis strain OZS1 carrying genes for lactose metabolism was characterised. A mobilizable cointegrate plasmid which is formed between pOZS550 and pOZS448 carries the necessary information for conjugation and transfer. Cointegrate formation was found to involve an insertional element located on pOZS550. The insertion sequence was found to be identical to ISS1 located on pSK08 in the clumping L. lactis subsp. lactis strain ML3. Restriction maps of pOZS550 and pSK08 were similar suggesting a close ancestral relationship, although pSK08, in addition to the lactose metabolism genes, expressed genes for proteinase activity and cell clumping, which were not expressed by pOZS550, and carried two copies of ISS1 compared to one on pOZS550. Furthermore, hybridization of the 18 base pair inverted repeat, of the insertion sequence, with various L. lactis subsp. lactis strains and two L. lactis subsp. cremoris strains showed moderate to strong hybridization to one plasmid in each organism.     

Lactate removal by anionic-exchange resin improves nisin production by Lactococcus lactis

When lactate was removed from sucrose fermentation in situ, using the anionic-exchange resin Amberlite IRA-67, by Lactococcus lactis growing in batch culture, nisin production increased by two-fold when compared to the alkali pH-controlled fermentation. In comparison to sucrose, lactate removal increased nisin production 1.5-fold and 0.3-fold when galactose and glucose were used as carbon sources respectively.     

Metabolic engineering of a Lactobacillus plantarum double ldh knockout strain for enhanced ethanol production

Lactobacillus plantarum ferments glucose through the Embden–Meyerhof–Parnas pathway: the central metabolite pyruvate is converted into lactate via lactate dehydrogenase (LDH). By substituting LDH with pyruvate decarboxylase (PDC) activity, pyruvate may be redirected toward ethanol production instead of lactic acid fermentation. A PDC gene from the Gram-positive bacterium Sarcina ventriculi (Spdc) was introduced into an LDH-deficient strain, L. plantarum TF103, in which both the ldhL and ldhD genes were inactivated. Four different fusion genes between Spdc and either the S. ventriculi promoter or three Lactococcus lactis promoters in pTRKH2 were introduced into TF103. PDC activity was detected in all four recombinant strains. The engineered strains were examined for production of ethanol and other metabolites in flask fermentations. The recombinant strains grew slightly faster than the parent TF103 and produced 90–130 mM ethanol. Although slightly more ethanol was observed, carbon flow was not significantly improved toward ethanol, suggesting that a further understanding of this organism’s metabolism is necessary.     

Inducible gene expression systems inLactococcus lactis

Lactococcus lactis is industrially important microorganism used in many dairy fermentations. Numerous genes and gene expression signals from this organism have now been identified and characterized. Recently, several naturally occurring, inducible gene-expression systems have also been described inL. lactis. The main features of these systems can be exploited to design genetically engineered expression cassettes for controlled production of various proteins and enzymes. Novel gene-expression systems inLactococcus have great potential for development of industrial cultures with desirable metabolic traits for a variety of bioprocessing applications.     

Alanine production in an H+-ATPase- and lactate dehydrogenase-defective mutant of Escherichia coli expressing alanine dehydrogenase

Previously, we reported that pyruvate production was markedly improved in TBLA-1, an H⁺-ATPase-defective Escherichia coli mutant derived from W1485lip2, a pyruvate-producing E. coli K-12 strain. TBLA-1 produced more than 30 g/l pyruvate from 50 g/l glucose by jar fermentation, while W1485lip2 produced only 25 g/l pyruvate (Yokota et al. in Biosci Biotechnol Biochem 58:2164–2167, 1994b). In this study, we tested the ability of TBLA-1 to produce alanine by fermentation. The alanine dehydrogenase (ADH) gene from Bacillus stearothermophilus was introduced into TBLA-1, and direct fermentation of alanine from glucose was carried out. However, a considerable amount of lactate was also produced. To reduce lactate accumulation, we knocked out the lactate dehydrogenase gene (ldhA) in TBLA-1. This alanine dehydrogenase-expressing and lactate dehydrogenase-defective mutant of TBLA-1 produced 20 g/l alanine from 50 g/l glucose after 24 h of fermentation. The molar conversion ratio of glucose to alanine was 41%, which is the highest level of alanine production reported to date. This is the first report to show that an H⁺-ATPase-defective mutant of E. coli can be used for amino acid production. Our results further indicate that H⁺-ATPase-defective mutants may be used for fermentative production of various compounds, including alanine.     

Secretory expression of K88 (F4) fimbrial adhesin FaeG by recombinant <Emphasis Type="Italic">Lactococcus lactis</Emphasis> for oral vaccination and its protective immune response in mice

K88 (F4) fimbrial adhesin, FaeG, was expressed extracellularly in Lactococcus lactis using a nisin-controlled gene expression system. The antibody response and protective efficacy of the recombinant bacteria (L. lactis [spNZ8048-faeG]) against live enterotoxigenic E. coli (ETEC) C₈₃₅₄₉ challenge were evaluated in ICR mice. Mice vaccinated with L. lactis [spNZ8048-faeG] had a significantly increased antigen-specific IgG level in the serum and decreased mortality rate (P < 0.05) compared with the control. This indicates that oral immunization of L. lactis [spNZ8048-faeG] can induce an immune-response protection upon challenge with live ETEC in ICR mice. An erratum to this article can be found at     

Experimental determination of control of glycolysis in Lactococcus lactis

The understanding of control of metabolic processes requires quantitative studies of the importance of the different enzymatic steps for the magnitude of metabolic fluxes and metabolite concentrations. An important element in such studies is the modulation of enzyme activities in small steps above and below the wild-type level. We review a genetic approach that is well suited for both Metabolic Optimization and Metabolic Control Analysis and studies on the importance of a number of glycolytic enzymes for metabolic fluxes in Lactococcus lactis. The glycolytic enzymes phosphofructokinase (PFK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), pyruvate kinase (PYK) and lactate dehydrogenase (LDH) are shown to have no significant control on the glycolytic flux in exponentially growing cells of L. lactis MG1363. Introduction of an uncoupled ATPase activity results in uncoupling of glycolysis from biomass production. With MG1363 growing in defined medium supplemented with glucose, the ATP demanding processes do not have a significant control on the glycolytic flux; it appears that glycolysis is running at maximal rate. It is likely that the flux control is distributed over many enzymes in L. lactis, but it cannot yet be excluded that one of the remaining glycolytic steps is a rate-limiting step for the glycolytic flux.     

Characterization of the impact of acetate and lactate on ethanolic fermentation by Thermoanaerobacter ethanolicus

Ethanolic fermentation of simple sugars is an important step in the production of bioethanol as a renewable fuel. Significant levels of organic acids, which are generally considered inhibitory to microbial metabolism, could be accumulated during ethanolic fermentation, either as a fermentation product or as a by-product generated from pre-treatment steps. To study the impact of elevated concentrations of organic acids on ethanol production, varying levels of exogenous acetate or lactate were added into cultures of Thermoanaerobacter ethanolicus strain 39E with glucose, xylose or cellobiose as the sole fermentation substrate. Our results found that lactate was in general inhibitory to ethanolic fermentation by strain 39E. However, the addition of acetate showed an unexpected stimulatory effect on ethanolic fermentation of sugars by strain 39E, enhancing ethanol production by up to 394%. Similar stimulatory effects of acetate were also evident in two other ethanologens tested, T. ethanolicus X514, and Clostridium thermocellum ATCC 27405, suggesting the potentially broad occurrence of acetate stimulation of ethanolic fermentation. Analysis of fermentation end product profiles further indicated that the uptake of exogenous acetate as a carbon source might contribute to the improved ethanol yield when 0.1% (w/v) yeast extract was added as a nutrient supplement. In contrast, when yeast extract was omitted, increases in sugar utilization appeared to be the likely cause of higher ethanol yields, suggesting that the characteristics of acetate stimulation were growth condition-dependent. Further understanding of the physiological and metabolic basis of the acetate stimulation effect is warranted for its potential application in improving bioethanol fermentation processes.     

Production of nisin Z using <Emphasis Type="Italic">Lactococcus lactis</Emphasis> IO-1 from hydrolyzed sago starch

A membrane bioreactor for production of nisin Z was constructed using Lactococcus lactis IO-1 in continuous culture using hydrolyzed sago starch as carbon source. A strategy used to enhance the productivity of nisin Z was to maintain the cells in a continuous growth at high cell concentration. This resulted in a volumetric productivity of nisin Z, as 50,000 IU l⁻¹ h⁻¹ using a cell concentration of 15 g l⁻¹, 30^°C, pH 5.5 and a dilution rate of 1.24 h⁻¹. Adding 10 g l⁻¹ YE and 2 g l⁻¹ polypeptone, other inducers were unnecessary to maintain production of nisin. The operating conditions of the reactor removed nisin and lactate, thus minimizing their effects which allowed the maintenance of cells in continuous exponential growth phase mode with high metabolic activity.     

Food-Grade Expression of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> UreB Subunit in <Emphasis Type="Italic">Lactococcus lactis</Emphasis> and its Immunoreactivity

Helicobacter pylori is the principal cause of chronic active gastritis, peptic ulcer, and gastric cancer. To develop an oral vaccine against H. pylori infection, we had expressed the H. pylori ureB gene (Genbank accession no. FJ436980) in nisin-controlled expression vectors using Lactococcus lactis NZ3900 as host. The ureB gene was amplified by PCR from a H.pylori strain MEL-Hp27. Then the ureB gene was fused translationally downstream of the nisin-inducible promoter nisA in a L. lactis plasmid pNZ8149. Lactose utilization based on the complementation of the lacF gene was used as a dominant selection marker for the food-grade expression system employing L. lactis NZ3900. The conditions of UreB expression in this system were optimized by orthogonal experiment. The optimized conditions have been determined as follows: induction of expression was carried out at the cells density of OD₆₀₀ ≈ 0.4 with 25 ng/ml nisin, and harvest after 5 h. The maximum percentage of recombinant UreB was estimated to be 7% of total soluble cellular proteins and the yield was 12.9 μg/ml. Western blot demonstrated that the UreB protein was expressed in the L. lactis transformant and had favorable immunoreactivity. These results indicated that the lactococci-derived vaccines could be promising candidates as alternative vaccine strategies for preventing H. pylori infection.     

Metabolic Engineering of Mannitol Production in Lactococcus lactis: Influence of Overexpression of Mannitol 1-Phosphate Dehydrogenase in Different Genetic Backgrounds

To obtain a mannitol-producing Lactococcus lactis strain, the mannitol 1-phosphate dehydrogenase gene (mtlD) from Lactobacillus plantarum was overexpressed in a wild-type strain, a lactate dehydrogenase(LDH)-deficient strain, and a strain with reduced phosphofructokinase activity. High-performance liquid chromatography and ¹³C nuclear magnetic resonance analysis revealed that small amounts (<1%) of mannitol were formed by growing cells of mtlD-overexpressing LDH-deficient and phosphofructokinase-reduced strains, whereas resting cells of the LDH-deficient transformant converted 25% of glucose into mannitol. Moreover, the formed mannitol was not reutilized upon glucose depletion. Of the metabolic-engineering strategies investigated in this work, mtlD-overexpressing LDH-deficient L. lactis seemed to be the most promising strain for mannitol production.     

Engineering of Carbon Distribution between Glycolysis and Sugar Nucleotide Biosynthesis in Lactococcus lactis

We describe the effects of modulating the activities of glucokinase, phosphofructokinase, and phosphoglucomutase on the branching point between sugar degradation and the biosynthesis of sugar nucleotides involved in the production of exopolysaccharide biosynthesis by Lactococcus lactis. This was realized by using a described isogenic L. lactis mutant with reduced enzyme activities or by controlled expression of the well-characterized genes for phosphoglucomutase or glucokinase from Escherichia coli or Bacillus subtilis, respectively. The role of decreased metabolic flux was studied in L. lactis strains with decreased phosphofructokinase activities. The concomitant reduction of the activities of phosphofructokinase and other enzymes encoded by the las operon (lactate dehydrogenase and pyruvate kinase) resulted in significant changes in the concentrations of sugar-phosphates. In contrast, a >25-fold overproduction of glucokinase resulted in 7-fold-increased fructose-6-phosphate levels and 2-fold-reduced glucose-1-phosphate and glucose-6-phosphate levels. However, these increased sugar-phosphate concentrations did not affect the levels of sugar nucleotides. Finally, an ~100-fold overproduction of phosphoglucomutase resulted in 5-fold-increased levels of both UDP-glucose and UDP-galactose. While the increased concentrations of sugar-phosphates or sugar nucleotides did not significantly affect the production of exopolysaccharides, they demonstrate the metabolic flexibility of L. lactis.