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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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

Recently, Microbulbifer thermotolerans JAMB-A94 endo-β-agarase I was expressed as catalytic domain (GH16) without a carbohydrate-binding module (CBM). In this study, we successfully constructed different fusions of GH16 with its original CBM6 and CBM13 derived from Catenovulum agarivorans. The optimum temperature and pH for fusions GH16-CBM6, GH16-CBM13, GH16-CBM6-CBM13 and GH16-CBM13-CBM6 were similar to GH16, at 55°C and pH 7. All the constructed fusions significantly enhanced the GH16 affinity (Km) and the catalytic efficiency (Kcat/Km) toward agar. Among them, GH16-CBM6-CBM13 exhibited the highest agarolytic activity, for which Km decreased from 3.67 to 2.11mg/mL and Kcat/Km increased from 98.6 (mg/mL) -1 sec -1 to 400.6 (mg/mL) -1 sec -1 . Moreover, all fusions selectively increased GH16 binding ability to agar, in which the highest binding ability of 95% was obtained with fusion GH16-CBM6-CBM13. Melted agar was prehydrolyzed with GH16-CBM6-CBM13, resulting in a degree of liquefaction of 45.3% and reducing sugar yield of 14.2%. Further addition of Saccharophagus degradans agarolytic enzymes resulted in mono-sugar yields of 35.4% for galactose and 31.5% for 3,6-anhydro-l-galactose. There was no pH neutralization step required and no 5-hydroxymethylfurfural detected, suggesting the potential of a new enzymatic prehydrolysis process for efficient production of bio-products such as biofuels., (Copyright © 2016 Elsevier Inc. All rights reserved.)

White rose (Rosa hybrida) petals were extracted with ethanol (EtOH) or butanol (BuOH), and tested for their antimicrobial activities against two species of Gram-positive bacteria, six species of Gram-negative bacteria, and two species of fungi. On in vitro antimicrobial assays, Helicobacter pylori and Propionibacterium acnes were highly susceptible to white rose petal extract (WRPE)-EtOH and WRPE-BuOH, leading to minimal inhibitory concentrations of 100 and 10 μg/mL for H. pylori and 400 and 40 μg/mL for P. acnes, respectively. In in vivo experiments, C57BL/6 mice were infected with H. pylori by intragastric inoculation (1 × 10(8) CFU/mouse) 3 times, and orally treated twice a day for 14 days with WRPE-EtOH and WRPE-BuOH. On a CLO kit assay, 200 mg/kg of WRPE-EtOH fully eliminated the bacteria from the gastric mucosa, and the effect of 100 mg/kg of ethanol fraction was similar to pantoprazole (30 mg/kg), displaying 75% elimination. WRPE-BuOH was more effective, exhibiting 75% elimination at 20 mg/kg. The CLO test results were confirmed by bacterial identification. WRPE-EtOH and WRPE-BuOH inhibited the growth of various bacteria and fungi, and in particular, they effectively killed H. pylori and eliminated the bacteria from the mouse stomach. The results indicate that WRPE-EtOH and WRPE-BuOH could be good candidates for the elimination of H. pylori., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293 is a lactic acid bacterium that converts pyruvate mainly to d-(-)-lactic acid by using d-(-)-lactate dehydrogenase (ldhD). The aim of this study was to identify the gene responsible for d-lactic acid formation in this organism and to characterize the enzyme to facilitate the production of optically pure d-lactic acid. A genomic analysis of L. mesenteroides ATCC 8293 revealed that 7 genes encode lactate-related dehydrogenase. According to transcriptomic, proteomic, and phylogenetic analyses, LEUM_1756 was the major gene responsible for the production of d-lactic acid. The LEUM_1756 gene, of 996bp and encoding 332 amino acids (36.5kDa), was cloned and overexpressed in Escherichia coli BL21(DE3) Star from an inducible pET-21a(+) vector. The enzyme was purified by Ni-NTA column chromatography and showed a specific activity of 4450U/mg, significantly higher than those of other previously reported ldhDs. The gel permeation chromatography analysis showed that the purified enzyme exists as tetramers in solution and this was the first report among lactic acid bacteria. The pH and temperature optima were pH 8.0 and 30°C, respectively, for the pyruvate reduction reaction, and pH 11.0 and 20°C, respectively, for the lactate oxidation reaction. The K(m) kinetic parameters for pyruvate and lactate were 0.58mM and 260mM, respectively. In addition, the k(cat) values for pyruvate and lactate were 2900s(-1) and 2280s(-1), respectively. The enzyme was not inhibited by Ca(2+), Co(2+), Cu(2+), Mg(2+), Mn(2+), Na(+), or urea, but was inhibited by 1mM Zn(2+) and 1mM SDS., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Resveratrol, a polyphenol compound found in grape skins, has been proposed to account for the beneficial effects of red wine against heart disease. To produce resveratrol in Saccharomyces cerevisiae, four heterologous genes were introduced: the phenylalanine ammonia lyase gene from Rhodosporidium toruloides, the cinnamic acid 4-hydroxylase and 4-coumarate:coenzyme A ligase genes both from Arabidopsis thaliana, and the stilbene synthase gene from Arachis hypogaea. When this recombinant yeast was cultivated by batch fermentation in YP medium containing 2% galactose, it produced 2.6 mg/L p-coumaric acid and 3.3 mg/L resveratrol. In order to increase the pool of malonyl-CoA, a key precursor in resveratrol biosynthesis, the acetyl-CoA carboxylase (ACC1) gene was additionally overexpressed in the yeast by replacing the native promoter of the ACC1 gene with the stronger GAL1 promoter and this resulted in enhanced production of resveratrol (4.3 mg/L). Furthermore, when tyrosine was supplemented in the medium, the concentration of resveratrol increased up to 5.8 mg/L. This result illustrates a possible strategy for developing metabolically engineered yeast strain for the economical production of resveratrol from cheap amino acids., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Resveratrol is a well-known polyphenol present in red wine and exerts antioxidative and anti-carcinogenic effects on the human body. To produce resveratrol in a food-grade yeast, the 4-coumarate:coenzyme A ligase gene (4CL1) from Arabidopsis thaliana and stilbene synthase gene (STS) from Arachis hypogaea were cloned and transformed into Saccharomyces cerevisiae W303-1A. The resveratrol produced was unglycosylated and secreted into the culture medium. A batch culture with 15.3mg/l p-coumaric acid used as precursor resulted in the production of 3.1mg/l resveratrol with 14.4 mol% yield. Deletion of the putative phenyl acrylic acid decarboxylase gene (PAD1) did not enhance resveratrol production., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Nattokinase was produced by batch and fed-batch culture of Bacillus subtilis in flask and fermentor. Effect of supplementing complex media (peptone, yeast extract, or tryptone) was investigated on the production of nattokinase. In flask culture, the highest cell growth and nattokinase activity were obtained with 50 g/L of peptone supplementation. In this condition, nattokinase activity was 630 unit/ml at 12 h. In batch culture of B. subtilis in fermentor, the highest nattokinase activity of 3400 unit/ml was obtained at 10h with 50 g/L of peptone supplementation. From the batch kinetics data, it was shown that nattokinase production was growth-associated and culture should be harvested before stationary phase for maximum nattokinase production. In fed-batch culture of B. subtilis using pH-stat feeding strategy, cell growth (optical density monitored at 600 nm) increased to ca. 100 at 22 h, which was 2.5 times higher than that in batch culture. The highest nattokinase activity was 7100 unit/ml at 19 h, which was also 2.1 times higher than that in batch culture., (Copyright 2010 Elsevier B.V. All rights reserved.)

4-Hydroxyphenylpyruvate dioxygenase (HPPD) is a Fe(II)-dependent, non-heme oxygenase that converts 4-hydroxyphenylpyruvate to homogentisate. Essential cofactors, such as plastoquinone and tocopherol, are produced by HPPD-dependent anabolic pathways in plants. To isolate a novel hppd using culture-independent method, a cosmid metagenomic library was constructed from soil in Korea. Screening of Escherichia coli metagenomic libraries led to the identification of a positive clone, YS103B, producing dark brown pigment in Luria-Bertani medium supplemented with l-tyrosine. In vitro transposon mutagenesis of YS103B showed that the 1.3kb insert was sufficient to produce the hemolytic brown pigment. Sequence analysis of YS103B disclosed one open reading frame encoding a 41.4kDa protein with the well-conserved prokaryotic oxygenase motif of the HPPD family of enzymes. The HPPD-specific beta-triketone herbicide, sulcotrione, inhibited YS103B pigmentation. The recombinant protein expressed in E. coli generated homogentisic acid. Thus, we present the successful heterologous expression of a previously uncharacterized hppd gene from an uncultured soil bacterium.

It was observed that Bacillus stearothermophilus maltogenic amylase cleaved the first glycosidic bond of acarbose to produce glucose and a pseudotrisaccharide (PTS) that was transferred to C-6 of the glucose to give an alpha-(1-->6) glycosidic linkage and the formation of isoacarbose. The addition of a number of different carbohydrates to the digest gave transfer products in which PTS was primarily attached alpha-(1-->6) to D-glucose, D-mannose, D-galactose, and methyl alpha-D-glucopyranoside. With D-fructopyranose and D-xylopyranose, PTS was linked alpha-(1-->5) and alpha-(1-->4), respectively. PTS was primarily transferred to C-6 of the nonreducing residue of maltose, cellobiose, lactose, and gentiobiose. Lesser amounts of alpha-(1-->3) and/or alpha-(1-->4) transfer products were also observed for these carbohydrate acceptors. The major transfer product to sucrose gave PTS linked alpha-(1-->4) to the glucose residue. alpha,alpha-Trehalose gave two major products with PTS linked alpha-(1-->6) and alpha-(1-->4). Maltitol gave two major products with PTS linked alpha-(1-->6) and alpha-(1-->4) to the glucopyranose residue. Raffinose gave two major products with PTS linked alpha-(1-->6) and alpha-(1-->4) to the D-galactopyranose residue. Maltotriose gave two major products with PTS linked alpha-(1-->6) and alpha-(1-->4) to the nonreducing end glucopyranose residue. Xylitol gave PTS linked alpha-(1-->5) as the major product and D-glucitol gave PTS linked alpha-(1-->6) as the only product. The structures of the transfer products were determined using thin-layer chromatography, high-performance ion chromatography, enzyme hydrolysis, methylation analysis and 13C NMR spectroscopy. The best acceptor was gentiobiose, followed closely by maltose and cellobiose, and the weakest acceptor was D-glucitol.