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The chemotaxonomic diversity of 20 Lactiplantibacillus plantarum strains was investigated using non-targeted metabolite profiling under different culture conditions. Multivariate and metabolic pathway analyses based on GC-MS and LC-MS/MS datasets showed that amino acid metabolism, especially 2-hydroxy acids, was enriched under aerobic conditions (AE), whereas fatty acid & sugar metabolism was increased under anaerobic conditions (AN). Based on the metabolite profiles, L. plantarum strains were clustered into three main groups (A, B, and C). Overall, 79 and 83 significantly discriminant metabolites were characterized as chemical markers of AE and AN growth conditions, respectively. Notably, alcohols were more abundant in group A whereas amino acids, peptides, purines, and pyrimidines were significantly higher in group C. 2-hydroxy acids and oxylipins biosynthesized through amino acid and fatty acid metabolism, respectively, were more abundant in groups A and B. Furthermore, we observed a strong correlation between the chemical diversity of L. plantarum groups and their antioxidant activity from metabolite extracts. We propose a non-targeted metabolomic workflow to comprehensively characterize the chemodiversity of L. plantarum strain under different culture conditions, which may help reveal specific biomarkers of individual strains depending on the culture conditions.
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Aminoácidos , Espectrometria de Massas em Tandem , Anaerobiose , Cromatografia Líquida , Hidroxiácidos , Ácidos GraxosRESUMO
BACKGROUND: Probiotic starters can improve the flavor profile, texture, and health-promoting properties of fermented foods. Tetragenococcus halophilus is a halophilic lactic acid bacterium that is a candidate starter for high-salt fermented foods. However, the species is known to produce biogenic amines, which are associated with neurotoxicity. Here, we report a probiotic starter strain of T. halophilus, EFEL7002, that is suitable for use in high-salt fermentation. RESULTS: EFEL7002 was isolated from Korean meju (fermented soybean) and identified as T. halophilus, with 99.85% similarity. The strain is safe for use in food as it is a non-hemolytic and non-biogenic amine producer. EFEL7002 is tolerant to gastrointestinal conditions and can adhere to Caco-2 cells. This strain showed antioxidant, anti-inflammatory, and protective effects against the human gut epithelial barrier. EFEL7002 grew well in media containing 0-18% NaCl showing maximum cell densities in 6% or 12% NaCl. CONCLUSIONS: T. halophilus EFEL7002 can be used as a health-promoting probiotic starter culture for various salty fermented foods.
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Probióticos , Cloreto de Sódio , Aminas Biogênicas/análise , Células CACO-2 , Enterococcaceae , Fermentação , Microbiologia de Alimentos , Humanos , República da Coreia , Glycine maxRESUMO
While zinc oxide (ZnO) with a mesoporous network has long been explored for adsorption of dyes and as an electron-transporting medium in dye-sensitized solar cells (DSSCs), the performance of ZnO-based DSSCs remains unsatisfactory. Despite the importance of understanding the surface characteristics of ZnO in DSSC applications, most of the studies relevant to ZnO-based DSSCs are focused on the synthesis of unique nanostructures of ZnO. In this study, we not only introduce a novel disk-shaped ZnO nanostructure, but also provide insight into the distinctive surface properties of ZnO and its influence on DSSC performance. When utilized in DSSCs, the mesoporous ZnO nanodisk yields 60% better power conversion efficiency (PCE) compared to commercial ZnO nanoparticles, which is attributed to less surface and bulk trap densities as concluded by an in-depth open-circuit voltage decay (OCVD) analysis and electrochemical impedance spectroscopy (EIS). Another aspect that contributes to the higher PCE is the better connectivity of primary particles that join together to form secondary disk-shaped particles. Furthermore, a 40% improvement in the PCE was observed by coating the mesoporous ZnO nanodisk with TiO2, which results from the passivation of the surface defects that aid in suppressing the interfacial charge recombination.
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Enterococcus faecium is a prevalent species found in fermented soybean products, known for its contributions to flavor development and inhibition of pathogenic microorganisms during fermentation. This study aims to provide comprehensive phenotypic and genomic evidence supporting the probiotic characteristics of E. faecium EFEL8600, a bacteriocin-producing strain isolated from Korean soy-meju. Phenotypic analysis revealed that EFEL8600 produced a peptide with inhibitory activity against Listeria monocytogenes, estimated to be 4.6 kDa, corresponding to the size of enterocins P or Q. Furthermore, EFEL8600 exhibited probiotic traits, such as resilience in gastrointestinal conditions, antioxidant and anti-inflammatory activities, and protection of the intestinal barrier. Safety assessments demonstrated no hemolytic and bile salt deconjugation activities. Genomic analysis revealed the presence of several genes associated with probiotic characteristics and bacteriocin production, while few deleterious genes with a low likelihood of expression or transferring were detected. Overall, this study highlights E. faecium EFEL8600 as a potent anti-listeria probiotic strain suitable for use as a starter culture in soymilk fermentation, providing potential health benefits to consumers.
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This study investigates the synergistic impact of fermenting kale juice with Limosilactobacillus strains on its antioxidant and anti-inflammatory properties. Kale's rich nutrient profile, especially its flavonoids, offers potential health benefits. Probiotic lactic acid bacteria are employed in kale fermentation to enhance nutrient bioavailability and generate bioactive compounds. Kale juices fermented with L. reuteri EFEL6901 or L. fermentum EFEL6800 exhibited superior microbial growth. Free sugars and amino acids were converted to alcohols and organic acids, affecting the organoleptic and health-related properties of the product. In addition, fermentation increased quercetin and kaempferol content, indicating improved availability. Furthermore, the fermented juice exhibited notable antioxidant activity and suppressed nitric oxide (NO) production, revealing anti-inflammatory potential. Gene expression analysis confirmed reduced pro-inflammatory markers such as iNOS, COX-2, IL-6, and IL-1ß and elevated anti-inflammatory cytokines, including IL-10. This research highlights the promising potential of fermented kale juice, enriched with Limosilactobacillus strains, as a functional food with combined antioxidant and anti-inflammatory benefits.
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The CRISPR-Cas system has emerged as the most efficient genome editing technique for a wide range of cells. Delivery of the Cas9-sgRNA ribonucleoprotein complex (Cas9 RNP) has gained popularity. The objective of this study was to develop a quantitative polymerase chain reaction (qPCR)-based assay to quantify the double-strand break reaction mediated by Cas9 RNP. To accomplish this, the dextransucrase gene (dsr) from Leuconostoc citreum was selected as the target DNA. The Cas9 protein was produced using recombinant Escherichia coli BL21, and two sgRNAs were synthesized through in vitro transcription to facilitate binding with the dsr gene. Under optimized in vitro conditions, the 2.6 kb dsr DNA was specifically cleaved into 1.1 and 1.5 kb fragments by both Cas9-sgRNA365 and Cas9-sgRNA433. By monitoring changes in dsr concentration using qPCR, the endonuclease activities of the two Cas9 RNPs were measured, and their efficiencies were compared. Specifically, the specific activities of dsr365RNP and dsr433RNP were 28.74 and 34.48 (unit/µg RNP), respectively. The versatility of this method was also verified using different target genes, uracil phosphoribosyl transferase (upp) gene, of Bifidobacterium bifidum and specific sgRNAs. The assay method was also utilized to determine the impact of high electrical field on Cas9 RNP activity during an efficient electroporation process. Overall, the results demonstrated that the qPCR-based method is an effective tool for measuring the endonuclease activity of Cas9 RNP.
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Sistemas CRISPR-Cas , RNA Guia de Sistemas CRISPR-Cas , Edição de Genes/métodos , Proteína 9 Associada à CRISPR/genética , DNA , Ribonucleoproteínas/genéticaRESUMO
Lactiplantibacillus plantarum PMO 08 has been used as a probiotic starter culture for plant-based fermented beverages, with various health-promoting effects such as cholesterol-lowering and anti-inflammatory activities. This study aimed to analyze the genome sequence of Lp. plantarum PMO 08 and identify its safety and probiotic characteristics at the genomic level. For this, complete genome sequencing was conducted to investigate the genes associated with risk and probiotic characteristics by using Pacbio combined with Illumina HiSeq. This bacterial strain has one circular chromosome of 3,247,789 bp with 44.5% G + C content and two plasmids of 50,296 bp with 39.0% G + C content and 19,592 bp with 40.5% G + C content. Orthologous average nucleotide identity analysis showed that PMO 08 belongs to the Lp. plantarum group with 99.14% similarity to Lp. plantarum WCFS1. No deleterious genes were determined in the virulence factor analysis, and no hemolysin activity or secondary bile salt synthesis were detected in vitro test. In the case of antibiotic resistance analysis, PMO 08 was resistant to ampicillin in vitro test, but these genes were not transferable. In addition, the strain showed same carbohydrate utilization with Lp. plantarum WCFS1, except for mannopyranoside, which only our strain can metabolize. The strain also harbors a gene for inositol monophosphatase family protein related with phytate hydrolysis and have several genes for metabolizing various carbohydrate which were rich in plant environment. Furthermore, in probiotic characteristics several genes involved in phenotypes such as acid/bile tolerance, adhesion ability, and oxidative stress response were detected in genome analysis. This study demonstrates that Lp. plantarum PMO 08 harbors several probiotic-related genes (with no deleterious genes) and is a suitable probiotic starter for plant-based fermentation.
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Alimentos Fermentados , Lactobacillus plantarum , Probióticos , Ampicilina/metabolismo , Ácidos e Sais Biliares/metabolismo , Colesterol/metabolismo , Lactobacillus plantarum/fisiologia , Manose/metabolismo , Nucleotídeos/metabolismo , Ácido Fítico/metabolismo , Probióticos/metabolismo , Fatores de Virulência/metabolismoRESUMO
Leuconostoc citreum is a heterofermentative lactic acid bacterium frequently found in the various fermented foods. L. citreum EFEL2700 isolated from Korean kimchi has been used as a host strain for biotechnological applications. For the use as a food-grade host to over-produce food ingredients or enzymes, strong endogenous promoters guarantying high expression levels of target genes are necessary. In this study, transcriptomic analysis of L. citreum EFEL2700 was performed using RNA-Seq and three promoters of the most highly expressed genes were selected: glyceraldehyde 3-phosphate dehydrogenase (G3PD), 6-phosphogluconate dehydrogenase (6PGD), and phosphoketolase (PPK). Thereafter, they were used as promoters to express ß-galactosidase gene from Lactobacillus plantarum WCFS1 in L. citreum EFEL2700 and the levels were compared with the control promoter P710 from L. mesenteroides ATCC 8293. As results, the ß-galactosidase activities of the transformants were 2.73, 0.27, 37.43, and 9.25 units/mg under the P710, G3PD, 6PGD, and PPK promoters, respectively. The expression level of endogenous promoter 6PGD was superior to the heterologous P710 promoter previously used in a Leuconostoc-Escherichia coli shuttle vector. The 6PGD developed in this study can be used as the most suitable promoter for ß-galactosidase expression in L. citreum EFEL2700.
Assuntos
Galactosidases , Lactobacillus plantarum , Perfilação da Expressão Gênica , Lactobacillus plantarum/genética , Leuconostoc/genéticaRESUMO
The goal of this study was to develop a starter strain of Limosilactobacillus fermentum which is beneficial for human health and suitable for rice fermentation. To achieve the goal, the characteristics of 25 strains of L. fermentum were compared in terms of health promoting potentials and rice fermenting abilities. L. fermentum MG7011 was selected as a superior strain to meet the required properties. First, as probiotic traits, the strain had tolerance to gastrointestinal conditions and ability to adhere to Caco-2 and HT-29 cells. The strain showed the antioxidative activity, anti-inflammatory activity, and a protective effect on the epithelial barrier. Next, as starter traits for rice fermentation, MG7011 exhibited proper fermentation profiles in rice solution, such as fast growth rate, pH and metabolite changes, amylase and phytase activities, and optimal viscosity changes for beverage. In conclusion, L. fermentum MG7011 has excellent probiotic activities and proper starter traits in rice, thereby it can be used as a suitable probiotic starter for rice fermentation.
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The use of probiotic starters can improve the sensory and health-promoting properties of fermented foods. This study aimed to evaluate the suitability of probiotic lactic acid bacteria (LAB) as a starter for kimchi fermentation. Seventeen probiotic type strains were tested for their growth rates, volatile aroma compounds, metabolites, and sensory characteristics of kimchi, and their characteristics were compared to those of Leuconostoc (Le.) mesenteroides DRC 1506, a commercial kimchi starter. Among the tested strains, Limosilactobacillus fermentum, Limosilactobacillus reuteri, Lacticaseibacillus rhamnosus, Lacticaseibacillus paracasei, and Ligilactobacillus salivarius exhibited high or moderate growth rates in simulated kimchi juice (SKJ) at 37 °C and 15 °C. When these five strains were inoculated in kimchi and metabolite profiles were analyzed during fermentation using GC/MS and 1H-NMR, data from the principal component analysis (PCA) showed that L. fermentum and L. reuteri were highly correlated with Le. mesenteroides in concentrations of sugar, mannitol, lactate, acetate, and total volatile compounds. Sensory test results also indicated that these three strains showed similar sensory preferences. In conclusion, L. fermentum and L. reuteri can be considered potential candidates as probiotic starters or cocultures to develop health-promoting kimchi products.
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Laphet is a traditional fermented food in Myanmar, made from tea leaves (Camellia sinensis) by fermentation with limited air passage. We performed microbial diversity analyses on 14 Laphet products collected from different locations in Myanmar. Amplicon-based sequencing results revealed Lactobacillus and Acetobacter were abundant bacteria and Candida, Pichia, Cyberlindnera, and Debaryomyces were abundant yeast. Using selective media, eight species of lactic acid bacteria and nine species of yeast were isolated; Lactobacillus plantarum and L. collinoides were dominant bacteria and Pichia manshurica, Candida boidinii, and Cyberlindnera jadinii were major yeasts. PCR-DGGE analysis confirmed that most of the dominant bacterial and yeast species found in culture dependent analysis were present in Laphet samples. Microbial diversity and pH of Laphet were different between samples from tea plantation area and local markets due to possible differences in incubation time periods. When tannase activity was tested, 23 among 29 bacterial isolates and two among 36 yeast isolates showed positive activities. These findings provide new insights into microbial diversity of Laphet and increased our understanding of the core bacterial and yeast species involved in the manufacture of Laphet.
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Bactérias/isolamento & purificação , Camellia sinensis/microbiologia , Alimentos Fermentados/microbiologia , Microbiologia de Alimentos , Fungos/isolamento & purificação , Bactérias/classificação , Bactérias/genética , Bactérias/metabolismo , Biodiversidade , Hidrolases de Éster Carboxílico/metabolismo , Fermentação , Fungos/classificação , Fungos/genética , Fungos/metabolismo , Mianmar , Folhas de Planta/microbiologiaRESUMO
We report the whole-genome sequence of Lactobacillus plantarum SPC-SNU 72-2, a probiotic starter for sourdough. Genome sequencing was completed using the Pacific Biosciences RS II and Illumina platforms. This study will facilitate the understanding of microbial characteristics of L. plantarum SPC-SNU 72-2 and its roles during sourdough fermentation.
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Fermenting garlic and onion provides the advantages of storage life extension, anti-oxidative and anti-diabetic activities, and their metabolite, allyl mercaptan, offers a strong aroma and various health benefits. Here, we report the probiotic properties of Lactobacillus pentosus SMB718 isolated from Korean traditional paste and its high allyl mercaptan productivity in garlic and onion fermentation. This strain was safe for use in food fermentation, as it was a non-biogenic amine producer and non-hemolytic. It showed high stability under simulated human gastrointestinal conditions and good adhesion ability to intestinal epithelial cells, including both Caco-2 and HT-29. This strain had antioxidant and anti-microbial activities. In addition, the heat-killed cells and lysate exerted anti-inflammatory effects on both LPS-stimulated RAW 264.7 cells and mouse macrophages by inhibition of pro-inflammatory cytokines and induction of anti-inflammatory cytokines. Furthermore, this strain possessed good fermentation properties in garlic and onion-enriched radish juice (GORJ); it grew well decreasing the pH and provided a rich aroma compound during fermentation. When L. pentosus SMB718 was used as a starter in garlic and onion fermentation, a significantly higher amount of allyl mercaptan (344 ppb) was produced compared with that produced by the type strain (82 ppb). In conclusion, L. pentosus SMB718 can be used as a beneficial probiotic starter for better flavor production in the fermentation of allium species plants.
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Fermentação , Alimentos Fermentados , Alho/química , Lactobacillus pentosus/fisiologia , Cebolas/química , Probióticos , Compostos de Sulfidrila/metabolismo , Animais , Anti-Infecciosos/farmacologia , Anti-Inflamatórios , Antioxidantes/farmacologia , Células CACO-2 , Citocinas , Células Epiteliais , Alho/microbiologia , Células HT29 , Humanos , Camundongos , Cebolas/microbiologia , Células RAW 264.7RESUMO
Phosphomannomutase (ManB) converts mannose-6-phosphate (M-6-P) to mannose-1-phosphate (M-1-P), which is a key metabolic precursor for the production of GDP-D-mannose used for production of glycoconjugates and post-translational modification of proteins. The aim of this study was to express the manB gene from Escherichia coli in Lactococcus lactis subsp. cremoris NZ9000 and to characterize the encoded enzyme. The manB gene from E. coli K12, of 1,371 bp and encoding 457 amino acids (52 kDa), was cloned and overexpressed in L. lactis NZ9000 using the nisin-controlled expression system. The enzyme was purified by Ni-NTA column chromatography and exhibited a specific activity of 5.34 units/mg, significantly higher than that of other previously reported ManB enzymes. The pH and temperature optima were 8.0 and 50°C, respectively. Interestingly, the ManB used in this study had two substrate specificity for both mannose-1-phosphate and glucose-1-phosphate, and the specific activity for glucose-1-phosphate was 3.76 units/mg showing 70% relative activity to that of mannose-1-phosphate. This is the first study on heterologous expression and characterization of ManB in lactic acid bacteria. The ManB expression system constructed in this study canbe used to synthesize rare sugars or glycoconjugates.
Assuntos
Escherichia coli/genética , Expressão Gênica , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Fosfotransferases (Fosfomutases)/genética , Fosfotransferases (Fosfomutases)/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Glucofosfatos/metabolismo , Concentração de Íons de Hidrogênio , Manosefosfatos/metabolismo , Fosfotransferases (Fosfomutases)/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , TemperaturaRESUMO
The industrial application of microorganisms as starters or probiotics requires their preservation to assure viability and metabolic activity. Freezing is routinely used for this purpose, but the cold damage caused by ice crystal formation may result in severe decrease in microbial activity. In this study, adaptive laboratory evolution (ALE) technique was applied to a lactic acid bacterium to select tolerant strains against freezing and thawing stresses. Lactobacillus rhamnosus GG was subjected to freeze-thaw-growth (FTG) for 150 cycles with four replicates. After 150 cycles, FTG-evolved mutants showed improved fitness (survival rates), faster growth rate, and shortened lag phase than those of the ancestor. Genome sequencing analysis of two evolved mutants showed genetic variants at distant loci in six genes and one intergenic space. Loss-of-function mutations were thought to alter the structure of the microbial cell membrane (one insertion in cls), peptidoglycan (two missense mutations in dacA and murQ), and capsular polysaccharides (one missense mutation in wze), resulting in an increase in cellular fluidity. Consequently, L. rhamnosus GG was successfully evolved into stress-tolerant mutants using FTG-ALE in a concerted mode at distal loci of DNA. This study reports for the first time the functioning of dacA and murQ in freeze-thaw sensitivity of cells and demonstrates that simple treatment of ALE designed appropriately can lead to an intelligent genetic changes at multiple target genes in the host microbial cell.
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Leuconostoc citreum is an important lactic acid bacterium used as a starter culture for producing kimchi, the traditional Korean fermented vegetables. An efficient host strain for plasmid transformation, L. citreum EFEL2700, was isolated from kimchi, and it has been frequently used for genetic engineering of L. citreum. In this study, we report the whole genome sequence of the strain and its genetic characteristics. Genome assembly yielded 5 contigs (1 chromosome and 4 plasmids), and the complete genome contained 1,923,830 base pairs (bp) with a G + C content of 39.0%. Average nucleotide identity analysis showed high homology (≥ 99%) to the reference strain L. citreum KM 20. The smallest plasmid (4.3 kbp) was used as an Escherichia coli shuttle vector (pCB) for heterologous gene expression, and L. citreum EFEL2700 showed the highest transformation efficiency, 6.7 × 104 CFU µg-1 DNA. Genetic analysis of the genome enabled the construction of primary metabolic pathway showing a typical hetero-type lactic acid fermentation. Notably, no core genes for primary metabolism were observed in plasmid 4 and it could be eliminated to create an efficient host for gene transformation. This report will facilitate the understanding and application of L. citreum EFEL2700 as a food-grade microbial cell factory.
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Engenharia Genética/métodos , Vetores Genéticos/genética , Genoma Bacteriano/genética , Leuconostoc/genética , Sequência de Bases/genética , DNA Bacteriano/genética , Alimentos Fermentados/microbiologia , Leuconostoc/classificação , Leuconostoc/isolamento & purificação , Análise de Sequência de DNARESUMO
Sourdough is made by fermentation of dough by lactic acid bacteria (LAB) and yeast to improve bread properties like volume, flavor, and texture. A Korean traditional sourdough was made by fermenting rice flour with rice wine (makgeolli) and used to make sponge-like bread (jeung-pyun). The aim of this study was to investigate the microbial diversity of makgeolli products and their influence on the organoleptic quality of jeung-pyun. Three commercial makgeolli were tested for jeung-pyun production, with each product exhibiting varied dough swelling rates and organoleptic qualities, and among them, J-product was ranked highest in texture and taste. Microbial analysis of the three makgeolli also showed a big difference in their population and diversity. J-product had the highest LAB and yeast counts, and the predominant species were Lactobacillus casei, Lactobacillus brevis, Leuconostoc pseudomenteroides, and Saccharomyces cerevisiae. Using J-product, sourdough was fermented at 25°C, 30°C, and 35°C, and the microbial growth in and textural properties of jeung-pyun were examined by instrumental and sensory tests. At high temperature (35°C), the rates of dough swelling and acidification were fast due to rapid microbial growth mainly caused by LAB, resulting in a short leavening time and soft and sour jeung-pyun. Sensory tests showed consumer preference for the soft and mild-sour jeung-pyun. This study shows that LAB in makgeolli play key roles in production of jeung-pyun, influencing the textural and sensory properties. For the production of high-quality jeung-pyun, development of LAB starters with high gas productivity and low acidity and establishment of an optimal fermentation procedure for rice dough are necessary.
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Bebidas Alcoólicas/microbiologia , Biodiversidade , Microbiologia de Alimentos , Lactobacillales/classificação , Lactobacillales/isolamento & purificação , Oryza/microbiologia , Sensação , Bebidas Alcoólicas/análise , Pão , DNA Bacteriano , Fermentação , Farinha/análise , Farinha/microbiologia , Concentração de Íons de Hidrogênio , Lactobacillales/crescimento & desenvolvimento , Lactobacillales/metabolismo , Filogenia , RNA Ribossômico 16S , Temperatura , Vinho/microbiologiaRESUMO
GDP-l-fucose is an l-fucose donor to synthesize fucosylated compounds such as human milk oligosaccharides or Lewis antigen. In this study, we used Lactococcus lactis subsp. cremoris NZ9000 to express 4 enzymes, ManB, ManC, Gmd, and WcaG and produced GDP-l-fucose by using one-pot synthesis method with mannose-6-phosphate as substrate and the enzymes as biocatalyst. For preparation of enzyme mixture, 4 genes (manB, manC, gmd, and wcaG) cloned from Escherichia coli were transformed into L. lactis strains using pNZ8008 and the recombinant cell lysates were obtained after cultivation. When mannose-6-phosphate was used as the substrate, the consecutive reactions with ManB, ManC, Gmd, and WcaG resulted in the successful production of GDP-l-fucose (0.13mM). When GDP-d-mannose was used as the substrate, it was entirely converted to GDP-l-fucose (0.2mM; 0.12g/L) via 2 enzymatic reactions mediated by Gmd and WcaG. This is the first report of GDP-l-fucose production by using multiple enzymes expressed in lactic acid bacteria.
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Proteínas de Bactérias/metabolismo , Guanosina Difosfato Fucose/metabolismo , Lactococcus lactis/metabolismo , Manosiltransferases/metabolismo , Engenharia Metabólica/métodos , Proteínas de Bactérias/genética , Escherichia coli/genética , Lactococcus lactis/genética , Manose-6-Fosfato Isomerase/genética , Manose-6-Fosfato Isomerase/metabolismo , Manosiltransferases/genética , Redes e Vias Metabólicas/genética , Oxirredutases/genética , Oxirredutases/metabolismo , Plasmídeos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Leuconostoc spp. are important lactic acid bacteria for the fermentation of foods. In particular, L. citreum strains isolated from various foods have been used as host strains for genetic and metabolic engineering studies. In order to develop a food-grade genetic engineering system, L. citreum CB2567 was isolated from Kimchi. However, the isolated bacterium contained a cryptic plasmid which was difficult to eliminate. As the existence of the plasmid might hinder strain engineering, we eliminated the plasmid using an RNA-guided DNA endonuclease CRISPR/Cas9 system. We demonstrated that a plasmid-free L. citreum CB2567 host strain could be efficiently constructed through a two-step procedure: 1) transformation of the "killer" plasmid expressing Cas9 endonuclease and a guide RNA (gRNA) targeting for a specific sequence in the cryptic plasmid, and 2) serial subculture without antibiotics for curing the killer plasmid. When the crude extract of L. citreum expressing Cas9 and the guide RNA was incubated with a PCR fragment containing the specific sequence recognized by the guide RNA, the PCR fragment was cleaved. Also, the cryptic plasmid pCB42 was successfully eliminated from the host strain after transforming the plasmid harboring Cas9 and the guide RNA. The Cas9 and gRNA expression plasmid used in this study can be applied for genome engineering purposes by additionally introducing an editing DNA template to repair the double strand DNA breakage caused by Cas9 in the genome of L. citreum. This study demonstrates the feasibility of developing CRISPR/Cas9-based genetic engineering tools to develop a safe host strain and construct food-grade lactic acid bacteria without residual antibiotic markers.
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Leuconostoc/genética , Plasmídeos , Sistemas CRISPR-Cas , Engenharia GenéticaRESUMO
After recombinant human growth hormone (rhGH) was introduced in the treatment of patients with growth hormone deficiency (GHD) and idiopathic short stature (ISS), many studies have addressed the effect of GH treatment and changes in the height standard deviation score (SDS) after GH treatment. However, few studies comparing the effect of GH in Korean patients with idiopathic GHD and ISS have been designed. Therefore, this study focused on the difference in effect of GH treatment between the two groups. We retrospectively reviewed the height SDS of 34 patients with idiopathic GHD and 12 patients with ISS. The mean ages of the patients with idiopathic GHD and ISS were 9.84±2.09 and 10.72±1.48 years, respectively. All patients were treated with GH for 1 year and body parameters were recorded before and after the GH treatment. Change in height SDS in patients with idiopathic GHD was significantly higher than that in patients with ISS (0.62±0.33 vs. 0.40±0.27, p=0.03). However, body mass index, insulin-like growth factor-1, and insulin-like growth factor binding protein-3 were not significantly different between the two groups after GH treatment. These results suggest that GH treatment has a more powerful effect on increasing height SDS in patients with idiopathic GHD than in patients with ISS.