Mechanisms underlying enhanced IgA production in Peyer's patch cells by membrane vesicles derived from Lactobacillus sakei.

We analyzed the mechanisms underlying enhanced IgA production in the cells of Peyer's patch cells via membrane vesicles derived from Lactobacillus sakei subsp. sakei NBRC 15893. Depletion of CD11c+ cells from Peyer's patch cells suppressed the enhanced IgA production mediated by membrane vesicles. Meanwhile, the stimulation of bone-marrow-derived dendritic cells with membrane vesicles increased gene expression of inducible nitric oxide synthase, retinaldehyde dehydrogenase 2, and several inflammatory cytokines. The production of nitric oxide and interleukin (IL)-6 by membrane vesicle stimulation was induced via Toll-like receptor 2 on bone marrow-derived dendritic cells. Inhibition of inducible nitric oxide synthase and retinaldehyde dehydrogenase 2, as well as the neutralization of IL-6 in Peyer's patch cells, suppressed the enhanced IgA production by membrane vesicle stimulation. Hence, nitric oxide, retinoic acid, and IL-6 induced by membrane vesicles play crucial roles in the enhanced IgA production elicited by membrane vesicles in Peyer's patch cells.

RNA-Based Anti-Inflammatory Effects of Membrane Vesicles Derived from Lactiplantibacillus plantarum.

Bacteria generally release extracellular membrane vesicles (MVs), which are nanoparticles that play important roles in bacterial-bacterial and bacterial-host communication. As probiotics, lactic acid bacteria provide diverse health benefits to their hosts. In this study, we found that the Gram-positive lactic acid bacteria Lactiplantibacillus plantarum subsp. plantarum NBRC 15891 produce high amounts of MVs (LpMVs), and that LpMVs inhibit interleukin (IL)-8 production induced by lipopolysaccharide in intestinal epithelial HT29 cells. Heat- or UV-killed bacterial cells did not exhibit anti-inflammatory effects, and there was no uptake of these bacterial cells; contrarily, LpMVs were taken up into the cytoplasm of HT29 cells. Small RNAs extracted from LpMVs also suppressed IL-8 production in HT29 cells, suggesting that RNAs in the cytoplasm of bacterial cells are encapsulated in the MVs and released from the cells, which may be delivered to HT29 cells to exert their anti-inflammatory effects. In addition, administration of LpMVs to mice with dextran sodium sulfate-induced colitis alleviated colitis-induced weight loss and colon length shortening, indicating that LpMV intake is likely to be effective in preventing or ameliorating colitis.

High productivity of immunostimulatory membrane vesicles of Limosilactobacillus antri using glycine.

Nanosized membrane vesicles (MVs) released by bacteria play important roles in both bacteria-bacteria and bacteria-host interactions. Some gram-positive lactic acid bacteria produce MVs exhibiting immunoregulatory activity in the host. We found that both bacterial cells and MVs of Limosilactobacillus antri JCM 15950, isolated from the human stomach mucosa, enhance immunoglobulin A production by murine Peyer's patch cells. However, the thick cell walls of gram-positive bacteria resulted in low MV production, limiting experiments and applications using MVs. In this study, we evaluated the effects of glycine, which inhibits cell wall synthesis, on the immunostimulatory MV productivity of L. antri. Glycine inhibited bacterial growth while increasing MV production, with 20 g/L glycine increasing MV production approximately 12-fold. Glycine was most effective at increasing MV production when added in the early exponential phase, which indicated that cell division in the presence of glycine increased MV production. Finally, glycine increased MV productivity approximately 16-fold. Furthermore, glycine-induced MVs promoted interleukin-6 production by macrophage-like J774.1 cells, and the immunostimulatory activity was comparable to that of spontaneously produced MVs. Our results indicate that glycine is an effective agent for improving the production of MVs with immunostimulatory activity in gram-positive lactic acid bacteria, which can be applied as mucosal adjuvants and functional foods.

Analysis of the effects of specific growth rate of Lactococcus lactis MG1363 on aerobic metabolism and its application to high-density culture.

Lactic acid bacteria (LAB) are known to produce a large amount of lactate when cultured under non-aerated conditions, which inhibits their growth at high concentrations. Our previous studies have shown that LAB can be cultured without lactate production under aerated conditions at a low specific growth rate. In this study, we investigated the effects of specific growth rate on cell yield and the specific production rates of metabolites in aerated fed-batch cultures of Lactococcus lactis MG1363. The results showed that lactate and acetoin production could be suppressed at specific growth rates below 0.2 h-1, whereas acetate production was the highest at a specific growth rate of 0.2 h-1. When LAB was cultured at a specific growth rate of 0.25 h-1 with the addition of 5 mg/L heme to assist ATP production by respiration, lactate and acetate production was suppressed, and cell concentration reached 19 g-dry-cell/L (5.6 × 10ˆ10 cfu/mL) with a high cell yield of 0.42 ± 0.02 g-dry-cell/g-glucose.

Isolation of a novel promoter for efficient protein expression by Aspergillus oryzae in solid-state culture.

A novel promoter from a hemolysin-like protein encoding the gene, hlyA, was characterized for protein overexpression in Aspergillus oryzae grown in solid-state culture. Using endo-1,4-ß-glucanase from A. oryzae (CelA) as the reporter, promoter activity was found to be higher than that of the α-amylase (amyA) and manganese superoxide dismutase (sodM) genes not only in wheat bran solid-state culture but also in liquid culture. Expression of the A. oryzae endoglucanase CelB and two heterologous endoglucanases (TrEglI and TrEglIII from Trichoderma reesei) under the control of the hlyA promoter were also found to be stronger than under the control of the amyA promoter in A. oryzae grown in wheat bran solid-state culture, suggesting that the hlyA promoter may be useful for the overproduction of other proteins as well. In wheat bran solid-state culture, the productivity of the hlyA promoter in terms of protein produced was high when the cultivation temperature was 30°C or 37°C, when the water content was 0.6 or 0.8 ml/g wheat bran, and from 48 to 72 h after inoculation. Because A. oryzae sporulated actively under these conditions and because hemolysin has been reported to play a role in fungal fruiting body formation, high-level expression of hlyA may be related to sporulation.

Adhesion mechanisms of Bifidobacterium animalis subsp. lactis JCM 10602 to dietary fiber.

Adherence of probiotics to dietary fibers present in the intestinal tract may affect adhesion to intestinal epithelial cells. The properties of the adhesion of bifidobacteria to mucin or epithelial cells have been well studied; however, adhesion of bifidobacteria to dietary fiber has not been investigated. The adhesion ratio of six Bifidobacterium strains to cellulose and chitin was examined; among the strains, Bifidobacterium animalis subsp. lactis JCM 10602 showed high adherence to both cellulose and chitin, and two strains showed high adherence to only chitin. The ratios of adhesion of B. animalis to cellulose and chitin were positively and negatively correlated with ionic strength, respectively. These data suggest that hydrophobic and electrostatic interactions are involved in the adhesion to cellulose and chitin, respectively. The adhesion ratios of the cells in the late logarithmic phase to cellulose and chitin decreased by approximately 40% and 70% of the cells in the early logarithmic phase, respectively. Furthermore, the adhesion ratio to cellulose decreased with increasing bile concentration regardless of the culture phase of the cells. On the other hand, the adhesion ratio to chitin of cells in the early logarithmic phase decreased with increasing bile concentration; however, that of cells in the late logarithmic phase increased slightly, suggesting that adhesins differ depending on the culture phase. Our results indicated the importance of considering adhesion to both dietary fibers and the intestinal mucosa when using bifidobacteria as probiotics.

Lactic acid bacteria display on the cell surface cytosolic proteins that recognize yeast mannan.

Fluorescent-labeled invertase, a hyperglycosylated mannoprotein from Saccharomyces cerevisiae, was found to bind to Lactococcus lactis IL1403 at acidic pH. Proteins on the cell wall of the bacterium affinity-purified using invertase as a ligand were identified to be heat shock proteins such as DnaK and GroEL and glycolytic enzymes such as pyruvate kinase and glyceraldehyde-3-phosphate dehydrogenase. DnaK bound to both the bacterium and yeast at pH 4 and aggregated them at above 0.1 mg/ml, whereas no significant difference between the circular dichroism spectra of DnaK at pH 4 and 7 was observed. These results indicate that the cytosolic proteins, including DnaK displayed on the cell wall, cause the lactic acid bacterium to adhere to the yeast.

Ethanol production from biomass by repetitive solid-state fed-batch fermentation with continuous recovery of ethanol.

To save cost and input energy for bioethanol production, a consolidated continuous solid-state fermentation system composed of a rotating drum reactor, a humidifier, and a condenser was developed. Biomass, saccharifying enzymes, yeast, and a minimum amount of water are introduced into the system. Ethanol produced by simultaneous saccharification and fermentation is continuously recovered as vapor from the headspace of the reactor, while the humidifier compensates for the water loss. From raw corn starch as a biomass model, 95 +/- 3, 226 +/- 9, 458 +/- 26, and 509 +/- 64 g l(-1) of ethanol solutions were recovered continuously when the ethanol content in reactor was controlled at 10-20, 30-50, 50-70 and 75-85 g kg-mixture(-1), respectively. The residue showed a lesser volume and higher solid content than that obtained by conventional liquid fermentation. The cost and energy for intensive waste water treatment are decreased, and the continuous fermentation enabled the sustainability of enzyme activity and yeast in the system.

Enhanced antibody production following intermediate addition based on flux analysis in mammalian cell continuous culture.

Generally, mammalian cells utilize glucose and glutamine as primary energy sources. To investigate the effect of energy sources on metabolic fluxes and antibody production, glucose- or glutamine-limited serum-free continuous culture of hybridoma 3A21 cells, which produce anti-ribonuclease A antibody, was carried out. The cell volume and dry cell weight were evaluated under various steady-state conditions. The specific consumption and production rates were evaluated on the basis of dry cell weight. On the basis of these results, the fluxes of the metabolic pathway were calculated. It was found that increasing the specific growth rate causes the specific ATP and antibody production rates to decrease. The fluxes between malate and pyruvate also decreased with the increase in specific growth rate. To increase the ATP production rate under steady-state conditions by the enhancement of fluxes between malate and pyruvate, the reduced metabolic fluxes were increased by an intermediate (pyruvate, malate, and citrate) addition. As a result, higher specific ATP and antibody production rates were achieved following the intermediate addition at a constant dilution rate.

Suppression of lactate production of Lactobacillus reuteri JCM1112 by co-feeding glycerol with glucose.

Lactate produced by lactic acid bacteria inhibits their growth. To suppress lactate production, it is necessary to regenerate NAD+ consumed by glycolysis with alternative pathways other than lactate dehydrogenase. In a heterofermentative lactic acid bacterium, Lactobacillus reuteri JCM1112, suppression of lactate production by regenerating NAD+ when producing 1,3-propanediol from glycerol was investigated. The bacterium produced lactate with a yield of 4.7 ± 0.8 g·g-cell-1 in a batch culture using glucose as the sole carbon source. When glycerol was added to glucose at a molar ratio (rGly/Glc) of three in the batch culture, the bacterium produced 1,3-propanediol at 1.6 ± 0.7 g·g-cell-1·h-1 and the lactate yield decreased to 3.6 ± 0.5 g·g-cell-1. When glycerol was co-fed with glucose exponentially to give a target specific growth rate of 0.1 h-1 (rGly/Glc = 3), the lactate yield decreased to 1.5 ± 0.2 g·g-cell-1. The lactate production when glycerol was added together with glucose was reduced to one-third of that observed in the batch culture using glucose as a carbon source.

Suppression of lactate production by aerobic fed-batch cultures of Lactococcus lactis.

Aerobic fed-batch cultures were studied as a means of suppressing the production of lactate, which inhibits the growth of lactic acid bacteria (LAB). LAB produce lactate via lactate dehydrogenase (LDH), regenerating nicotinamide adenine dinucleotide (NAD+) consumed during glycolysis. Therefore, we focused on NADH oxidase (NOX), employing oxygen as an electron acceptor, as an alternative pathway to LDH for NAD+ regeneration. To avoid glucose repression of NOX and NAD+ consumption by glycolysis exceeding NAD+ regeneration by NOX, glucose was fed gradually. When Lactococcus lactis MG 1363 was aerobically fed at a specific growth rate of 0.2 h-1, the amount of lactate produced per amount of grown cell was reduced to 12% of that in anaerobic batch cultures. Metabolic flux analysis revealed that in addition to NAD+ regeneration by NOX, ATP acquisition by production of acetate and NAD+ regeneration by production of acetoin and 2,3-butanediol contributed to suppression of lactate production.

Suppression of lactate production by using sucrose as a carbon source in lactic acid bacteria.

Lactic acid bacteria (LAB) grow by producing lactate from sugar. However, the accumulation of lactate inhibits their growth. Here, the lactate productivity per cell in a semi-solid medium prepared with a chlorella powder in several LAB strains was much lower than that in the conventional MRS medium. Furthermore, the lactate production was suppressed not only in semi-solid medium, but also in chlorella liquid medium. The lactate productivity by Lactococcus lactis subsp. lactis NBRC 12007 in the chlorella liquid medium and MRS medium was 3.0 and 6.9 g-lactate·g-cell-1, respectively. The productivity of lactate in the chlorella liquid medium decreased to 44% of that in MRS medium. Gas chromatography/mass spectrometry (GC/MS) analysis of the culture supernatants revealed that the utilization of sucrose in the chlorella powder led to the suppression of lactate production. Comparison of the metabolites extracted from the cells indicated that the two ATP generating pathways, the arginine deiminase pathway and the decarboxylation reaction of glutamate and GABA, which are usually repressed by glucose, are activated in chlorella medium. It was considered that these pathways which do not require NAD+ for generation of ATP are not repressed when sucrose is used as a carbon source. Thus, the utilization of these pathways results in the suppression of the lactate production.

Suppression of lactate production in fed-batch culture of some lactic acid bacteria with sucrose as the carbon source.

We report a method for suppression of lactate production by lactic acid bacteria (LAB) in culture. LAB produce lactate to regenerate NAD+ that is consumed during glycolysis. Glucose suppresses NAD+ regeneration pathways other than lactate dehydrogenase and non-glycolytic ATP production pathways. Therefore, the carbon source was changed to sucrose, and fed-batch culture was performed to limit the glycolytic flux and thus suppress lactate production. As a result, lactate productivity (i.e., the amount of lactate produced per amount of grown cell) in the sucrose/fed-batch culture was decreased compared to that in glucose/batch culture, in all five LAB strains examined. The productivity level decreased to 24% and 46% in Lactobacillus reuteri JCM 1112 and Lactococcus lactis JCM 7638, respectively. Metabolic flux analysis of Lactobacillus reuteri JCM 1112 revealed increased contributions of the mannitol production pathway to NAD+ regeneration and the arginine deiminase pathway to ATP production in the sucrose/fed-batch culture.

Direct immobilization of functional single-chain variable fragment antibodies (scFvs) onto a polystyrene plate by genetic fusion of a polystyrene-binding peptide (PS-tag).

Single-chain Fv antibodies (scFv) genetically fused with polystyrene-binding peptides (PS-tags, (PS19-1; RAFIASRRIRRP, PS19-6; RIIIRRIRR)) were generated by recombinant Escherichia coli for direct and site-specific immobilization of scFv on polystyrene supports with high antigen-binding activity. PS-tag-fused scFvs (scFv-PS-tags) specific for human C-reactive protein (CRP) were successfully over-expressed as an inclusion body and were refolded using the batch-dilution method. When scFv-PS-tags were immobilized on a hydrophilic PS (phi-PS) plate in the presence of Tween 20, they showed high antigen-binding activity comparable to, or greater than, that of a whole monoclonal antibody (mAb) on a hydrophobic PS (pho-PS) plate, which has been the exclusive method for enzyme-linked immunosorbent assay (ELISA). Furthermore, when a scFv-PS-tag was used as a ligand antibody in one- and two-step ELISA, the assay time was reduced without loss of sensitivity. These results indicate that strong and specific attachment of PS-tags onto the phi-PS surface prevented scFv conformational changes and consequently, the high antigen-binding activities of scFvs were preserved. Nearly identical results were obtained by use of PS-tag-fused scFvs with different VH/VL pairs. Therefore, a variety of scFvs could be functionalized onto phi-PS plates by genetic fusion of PS-tags. ScFv-PS-tags, which possess high antigen-binding activity on the phi-PS plate, are more useful ligand antibodies than whole mAbs. Thus, scFv-PS-tags are applicable in both clinical diagnosis and proteomic research.

IgA-enhancing effects of membrane vesicles derived from Lactobacillus sakei subsp. sakei NBRC15893.

Immunoglobulin (Ig) A in the mucus of the intestinal tract plays an important role in preventing the invasion of pathogenic microorganisms and regulating the composition of the gut microbiota. Several strains of probiotic lactic acid bacteria (LAB) are known to promote intestinal IgA production. Bacteria are also known to naturally release spherical membrane vesicles (MVs) that are involved in various biological functions such as quorum sensing, pathogenesis, and host immunomodulation. However, the production of MVs by LAB and their effects on host immunity remain poorly understood. In this study, we investigated the MV production by Lactobacillus sakei subsp. sakei NBRC15893 isolated from kimoto, the traditional seed mash used for brewing sake. MVs were separated from the culture broth of L. sakei NBRC15893 through filtration and density gradient ultracentrifugation and were observed by transmission electron microscopy. The MVs showed a spherical morphology, with a diameter of 30-400 nm, and contained proteins and nucleic acids. In addition, both the LAB cells and purified MVs promoted IgA production by murine Peyer's patch cells. This MV- and cell-induced IgA production was suppressed by neutralization of Toll-like receptor (TLR) 2, which recognizes cell wall components of gram-positive bacteria, using an anti-TLR2 antibody. Collectively, our results indicate that MVs released from L. sakei NBRC15893 enhance IgA production by activating host TLR2 signaling through its cell wall components. Thus, it is important to consider novel interactions between gut microbiota and hosts via MVs, and MVs derived from probiotic bacteria could have promising applications as safe adjuvants.

Expression of C-terminal repeat region of peptidoglycan hydrolase of Lactococcus lactis IL1403 in methylotrophic yeast Pichia pastoris.

The C-terminal region of the peptidoglycan hydrolase (CPH) of Lactococcus lactis IL1403 produced intracellularly in Escherichia coli was able to attach to the surface of cells of Lactobacillus casei NRRL B-441, Bacillus subtilis 168, E. coli XL1-blue and Saccharomyces cerevisiae IFO0216. Therefore, this domain is a suitable fusion partner for the adhesion of proteins to cell surfaces. The production of cell-surface adhesive proteins using this domain in Pichia pastoris is particularly attractive, because this organism has better capability to allow the correct folding of the recombinant proteins than prokaryotic hosts. However, when this domain is produced in this yeast, its cell-surface binding activity may be limited by glycosylation. In this study, therefore, we constructed a CPH mutant (CPHM) devoid of the potential N-glycosylation sites by site-directed mutagenesis. CPHM was successfully expressed extracellularly in P. pastoris (GS115) using the methanol inducible AOX1 promoter with an alpha-mating factor signal sequence, whereas the native CPH was not produced in this host. Western blot analysis revealed that the apparent molecular size of CPHM was 18 kDa greater than that of CPH produced in E. coli (32 kDa), which is attributed to O-glycosylation. However, CPHM produced in P. pastoris was capable of binding to the cell surfaces despite its modification by the yeast, and its dissociation rate constant from the surface of L. casei NRRL B-441 cells was 3.5-fold lower than that of CPH produced in E. coli. These results demonstrate the applicability of the constructed domain (CPHM) for the production of cell-surface adhesive proteins in P. pastoris.

Rational screening of antibodies and design of sandwich enzyme linked immunosorbant assay on the basis of a kinetic model.

A rational strategy for the rapid establishment of a sensitive sandwich enzyme linked immunosorbant assay was developed. The kinetic properties required for the solid-phase and enzyme-conjugated antibodies of sandwich ELISA were determined rationally on the basis of a kinetic model describing antibody-antigen interaction. Some antibodies possessing the required kinetic properties against a model antigen, C-reactive protein (CRP), were successfully isolated from a phage antibody library under the screening conditions that were designed on the basis of simulation results. The best combination of solid-phase and enzyme-conjugated antibodies that gives the most sensitive sandwich ELISA was determined by simulation on the basis of the apparent association and dissociation rate constants of the isolated antibodies. It was confirmed by experiment that the sandwich ELISA using the best combination of antibodies was actually the most sensitive one. Our strategy would be useful for the rapid establishment of sensitive sandwich ELISAs compared with the traditional hybridoma method in which the best condition is determined by trial and error.

Strategies for reducing supplemental medium cost in bioethanol production from waste house wood hydrolysate by ethanologenic Escherichia coli: inoculum size increase and coculture with Saccharomyces cerevisiae.

In this paper, we report a simultaneous realization of both efficient ethanol production and saving medium nutrient (corn steep liquor [CSL]) during bioethanol fermentation of overliming-treated hydrolysate of waste house wood (WHW) using ethanologenic Escherichia coli KO11. In cultivation using WHW hydrolysate supplemented with 4% (v/v) CSL and 0.2 g-dry cell weight (DCW)/l E. coli KO11 cells, the overall ethanol yield reached 84% of the theoretical value at 61 h. When we conducted the cultivation with 1% CSL to reduce the supplemental medium cost, the overall ethanol yield remained in the range of 66-72% even at 90 h. We proposed two alternative methods for increasing the overall yield even with 1% CSL. The first method involved increasing the inoculum size of E. coli KO11 up to 0.8 g-DCW/l, where 83% of the overall yield was attained at 60 h of cultivation. The second method involved the coculture of 0.2 g-DCW/l E. coli KO11 together with 0.02 g-DCW/l of Saccharomyces cerevisiae TJ1, and the overall yield reached 81% at 47 h of cultivation.

Bidirectional cell-surface anchoring function of C-terminal repeat region of peptidoglycan hydrolase of Lactococcus lactis IL1403.

With the aim of constructing an efficient protein display system for lactic acid bacteria (LABs), the effect of fusion direction on the cell-surface binding activity of the C-terminal region of the peptidoglycan hydrolase (CPH) of Lactococcus lactis IL1403 was studied. CPH fused to the alpha-amylase (AMY) of Streptococcus bovis 148 either at its C-terminus (CPH-AMY) or at its N-terminus (AMY-CPH) was expressed intracellularly in Escherichia coli. This domain was able to direct binding of AMY to the surface of L. lactis ATCC 19435 in both constructs. However, the number of bound molecules per cell and the specific activity for starch digestion in the case of CPH-AMY were 3 and 14 times greater than those in the case of AMY-CPH, respectively. Of the LABs tested, L. lactis ATCC 19435 showed the highest binding capability for CPH-AMY, up to 6 x 10(4) molecules per cell, with a dissociation rate constant of 5.00 x 10(-5) s(-1). The binding of CPH-AMY to the surface of Lactobacillus delbrueckii ATCC 9649 cells was very stable with a dissociation rate constant of 6.96 x 10(-6) s(-1). The production of CPH-AMY in the soluble form increased 3-fold as a result of coexpression with a molecular chaperone, trigger factor. The results of this study suggest the usefulness of CPH as a bidirectional anchor protein for the production of cell-surface adhesive enzymes in E. coli. Furthermore, the importance of the fusion direction of CPH in determining cell-surface binding and enzymatic activities was shown.

Identification of target genes conferring ethanol stress tolerance to Saccharomyces cerevisiae based on DNA microarray data analysis.

During industrial production process using yeast, cells are exposed to the stress due to the accumulation of ethanol, which affects the cell growth activity and productivity of target products, thus, the ethanol stress-tolerant yeast strains are highly desired. To identify the target gene(s) for constructing ethanol stress tolerant yeast strains, we obtained the gene expression profiles of two strains of Saccharomyces cerevisiae, namely, a laboratory strain and a strain used for brewing Japanese rice wine (sake), in the presence of 5% (v/v) ethanol, using DNA microarray. For the selection of target genes for breeding ethanol stress tolerant strains, clustering of DNA microarray data was performed. For further selection, the ethanol sensitivity of the knockout mutants in each of which the gene selected by DNA microarray analysis is deleted, was also investigated. The integration of the DNA microarray data and the ethanol sensitivity data of knockout strains suggests that the enhancement of expression of genes related to tryptophan biosynthesis might confer the ethanol stress tolerance to yeast cells. Indeed, the strains overexpressing tryptophan biosynthesis genes showed a stress tolerance to 5% ethanol. Moreover, the addition of tryptophan to the culture medium and overexpression of tryptophan permease gene conferred ethanol stress tolerance to yeast cells. These results indicate that overexpression of the genes for trypophan biosynthesis increases the ethanol stress tolerance. Tryptophan supplementation to culture and overexpression of the tryptophan permease gene are also effective for the increase in ethanol stress tolerance. Our methodology for the selection of target genes for constructing ethanol stress tolerant strains, based on the data of DNA microarray analysis and phenotypes of knockout mutants, was validated.