Rapid Assembly of Pyrrole-Ligated 1,3,4-Oxadiazoles and Excellent Antibacterial Activity of Iodophenol Substituents.

Pyrrole-ligated 1,3,4-oxadiazole is a very important pharmacophore which exhibits broad therapeutic effects such as anti-tuberculosis, anti-epileptic, anti-HIV, anti-cancer, anti-inflammatory, antioxidant, and antibacterial activities. A one-pot Maillard reaction between D-Ribose and an L-amino methyl ester in DMSO with oxalic acid at 2.5 atm and 80 °C expeditiously produced pyrrole-2-carbaldehyde platform chemicals in reasonable yields, which were utilized for the synthesis of pyrrole-ligated 1,3,4-oxadiazoles. Benzohydrazide reacted with the formyl group of the pyrrole platforms to provide the corresponding imine intermediates, which underwent I2-mediated oxidative cyclization to the pyrrole-ligated 1,3,4-oxadiazole skeleton. The structure and activity relationship (SAR) of the target compounds with varying alkyl or aryl substituents of the amino acids and electron-withdrawing or electron-donating substituents on the phenyl ring of benzohydrazide were evaluated for antibacterial activity against Escherichia coli, Staphylococcus aureus, and Acinetobacter baumannii as representative Gram(-) and Gram(+) bacteria. Branched alkyl groups from the amino acid showed better antibacterial activities. Absolutely superior activities were observed for 5f-1 with an iodophenol substituent against A. baumannii (MIC < 2 µg/mL), a bacterial pathogen that displays a high resistance to commonly used antibiotics.

LuxR-Type SCO6993 Negatively Regulates Antibiotic Production at the Transcriptional Stage by Binding to Promoters of Pathway-Specific Regulatory Genes in Streptomyces coelicolor.

SCO6993 (606 amino acids) in Streptomyces coelicolor belongs to the large ATP-binding regulators of the LuxR family regulators having one DNA-binding motif. Our previous findings predicted that SCO6993 may suppress the production of pigmented antibiotics, actinorhodin, and undecylprodigiosin, in S. coelicolor, resulting in the characterization of its properties at the molecular level. SCO6993-disruptant, S. coelicolor ΔSCO6993 produced excess pigments in R2YE plates as early as the third day of culture and showed 9.0-fold and 1.8-fold increased production of actinorhodin and undecylprodigiosin in R2YE broth, respectively, compared with that by the wild strain and S. coelicolor ΔSCO6993/SCO6993+. Real-time polymerase chain reaction analysis showed that the transcription of actA and actII-ORF4 in the actinorhodin biosynthetic gene cluster and that of redD and redQ in the undecylprodigiosin biosynthetic gene cluster were significantly increased by SCO6993-disruptant. Electrophoretic mobility shift assay and DNase footprinting analysis confirmed that SCO6993 protein could bind only to the promoters of pathway-specific transcriptional activator genes, actII-ORF4 and redD, and a specific palindromic sequence is essential for SCO6993 binding. Moreover, SCO6993 bound to two palindromic sequences on its promoter region. These results indicate that SCO6993 suppresses the expression of other biosynthetic genes in the cluster by repressing the transcription of actII-ORF4 and redD and consequently negatively regulating antibiotic production.

Implications of agar and agarase in industrial applications of sustainable marine biomass.

Agar, a major component of the cell wall of red algae, is an interesting heteropolysaccharide containing an unusual sugar, 3,6-anhydro-L-galactose. It is widely used as a valuable material in various industrial and experimental applications due to its characteristic gelling and stabilizing properties. Agar-derived oligosaccharides or mono-sugars produced by various agarases have become a promising subject for research owing to their unique biological activities, including anti-obesity, anti-diabetic, immunomodulatory, anti-tumor, antioxidant, skin-whitening, skin-moisturizing, anti-fatigue, and anti-cariogenic activities. Agar is also considered as an alternative sustainable source of biomass for chemical feedstock and biofuel production to substitute for the fossil resource. In this review, we summarize various biochemically characterized agarases, which are useful for industrial applications, such as neoagarooligosaccharide or agarooligosaccharide production and saccharification of agar. Additionally, we succinctly discuss various recent studies that have been conducted to investigate the versatile biological activities of agar-derived saccharides and biofuel production from agar biomass. This review provides a basic framework for understanding the importance of agarases and agar-derived saccharides with broad applications in pharmaceutical, cosmetic, food, and bioenergy industries.

Expression and characterization of the processive exo-ß-1,4-cellobiohydrolase SCO6546 from Streptomyces coelicolor A(3).

The sco6546 gene of Streptomyces coelicolor A3(2) was annotated as a putative glycosyl hydrolase belonging to family 48. It is predicted to encode a 973-amino acid polypeptide (103.4 kDa) with a 39-amino acid secretion signal. Here, the SCO6546 protein was overexpressed in Streptomyces lividans TK24, and the purified protein showed the expected molecular weight of the mature secreted form (934 aa, 99.4 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. SCO6546 showed high activity toward Avicel and carboxymethyl cellulose, but low activity toward filter paper and ß-glucan. SCO6546 showed maximum cellulase activity toward Avicel at pH 5.0 and 50 °C, which is similar to the conditions for maximum activity toward cellotetraose and cellopentaose substrates. The kinetic parameters kcat and KM , for cellotetraose at pH 5.0 and 50 °C were 13.3 s-1 and 2.7 mM, respectively. Thin layer chromatography (TLC) of the Avicel hydrolyzed products generated by SCO6546 showed cellobiose only, which was confirmed by mass spectral analysis. TLC analysis of the cello-oligosaccharide and chromogenic substrate hydrolysates generated by SCO6546 revealed that it can hydrolyze cellodextrins mainly from the non-reducing end into cellobiose. These data clearly demonstrated that SCO6546 is an exo-ß-1,4-cellobiohydrolase (EC 3.2.1.91), acting on nonreducing end of cellulose.

Enhanced yield of ethylene glycol production from d-xylose by pathway optimization in Escherichia coli.

The microbial production of renewable ethylene glycol (EG) has been gaining attention recently due to its growing importance in chemical and polymer industries. EG has been successfully produced biosynthetically from d-xylose through several novel pathways. The first report on EG biosynthesis employed the Dahms pathway in Escherichia coli wherein 71% of the theoretical yield was achieved. This report further improved the EG yield by implementing metabolic engineering strategies. First, d-xylonic acid accumulation was reduced by employing a weak promoter which provided a tighter control over Xdh expression. Second, EG yield was further improved by expressing the YjgB, which was identified as the most suitable aldehyde reductase endogenous to E. coli. Finally, cellular growth, d-xylose consumption, and EG yield were further increased by blocking a competing reaction. The final strain (WTXB) was able to reach up to 98% of the theoretical yield (25% higher as compared to the first study), the highest reported value for EG production from d-xylose.

Molecular characterization of SCO0765 as a cellotriose releasing endo-ß-1,4-cellulase from Streptomyces coelicolor A(3).

The sco0765 gene was annotated as a glycosyl hydrolase family 5 endoglucanase from the genomic sequence of Streptomyces coelicolor A3(2) and consisted of 2,241 bp encoding a polypeptide of 747 amino acids (molecular weight of 80.5 kDa) with a 29-amino acid signal peptide for secretion. The SCO0765 recombinant protein was heterogeneously over-expressed in Streptomyces lividans TK24 under the control of a strong ermE* promoter. The purified SCO0765 protein showed the expected molecular weight of the mature form (718 aa, 77.6 kDa) on sodium dodecyl sulfate-polyacryl amide gel electrophoresis. SCO0765 showed high activity toward ß-glucan and carboxymethyl cellulose (CMC) and negligible activity to Avicel, xylan, and xyloglucan. The SCO0765 cellulase had a maximum activity at pH 6.0 and 40°C toward CMC and at pH 9.0 and 50-60°C toward ß-glucan. Thin layer chromatography of the hydrolyzed products of CMC and ß-glucan by SCO0765 gave cellotriose as the major product and cellotetraose, cellopentaose, and longer oligosaccharides as the minor products. These results clearly demonstrate that SCO0765 is an endo-ß-1,4-cellulase, hydrolyzing the ß-1,4 glycosidic bond of cellulose into cellotriose.

Biochemical characterization of a novel iron-dependent GH16 ß-agarase, AgaH92, from an agarolytic bacterium Pseudoalteromonas sp. H9.

A putative agarase gene (agaH92) encoding a primary translation product (50.1 kDa) of 445 amino acids with a 19-amino-acid signal peptide and glycoside hydrolase 16 and RICIN superfamily domains was identified in an agarolytic marine bacterium, Pseudoalteromonas sp. H9 ( = KCTC23887). The heterologously expressed protein rAgaH92 in Escherichia coli had an apparent molecular weight of 51 kDa on SDS-PAGE, consistent with the calculated molecular weight. Agarase activity of rAgaH92 was confirmed by a zymogram assay. rAgaH92 hydrolyzed p-nitrophenyl-ß-D-galactopyranoside, but not p-nitrophenyl-α-D-galactopyranoside. The optimum pH and temperature for rAgaH92 were 6.0 and 45°C, respectively. It was thermostable and retained more than 85% of its initial activity after heat treatment at 50°C for 1 h. rAgaH92 required Fe(2+) for agarase activity and inhibition by EDTA was compensated by Fe(2+). TLC analysis, mass spectrometry and NMR spectrometry of the GST-AgaH71 hydrolysis products revealed that rAgaH92 is an endo-type ß-agarase, hydrolyzing agarose into neoagarotetraose and neoagarohexaose.

Escherichia coli enzyme IIANtr regulates the K+ transporter TrkA.

The maintenance of ionic homeostasis in response to changes in the environment is essential for all living cells. Although there are still many important questions concerning the role of the major monovalent cation K(+), cytoplasmic K(+) in bacteria is required for diverse processes. Here, we show that enzyme IIA(Ntr) (EIIA(Ntr)) of the nitrogen-metabolic phosphotransferase system interacts with and regulates the Escherichia coli K(+) transporter TrkA. Previously we reported that an E. coli K-12 mutant in the ptsN gene encoding EIIA(Ntr) was extremely sensitive to growth inhibition by leucine or leucine-containing peptides (LCPs). This sensitivity was due to the requirement of the dephosphorylated form of EIIA(Ntr) for the derepression of ilvBN expression. Whereas the ptsN mutant is extremely sensitive to LCPs, a ptsN trkA double mutant is as resistant as WT. Furthermore, the sensitivity of the ptsN mutant to LCPs decreases as the K(+) level in culture media is lowered. We demonstrate that dephosphorylated EIIA(Ntr), but not its phosphorylated form, forms a tight complex with TrkA that inhibits the accumulation of high intracellular concentrations of K(+). High cellular K(+) levels in a ptsN mutant promote the sensitivity of E. coli K-12 to leucine or LCPs by inhibiting both the expression of ilvBN and the activity of its gene products. Here, we delineate the similarity of regulatory mechanisms for the paralogous carbon and nitrogen phosphotransferase systems. Dephosphorylated EIIA(Glc) regulates a variety of transport systems for carbon sources, whereas dephosphorylated EIIA(Ntr) regulates the transport system for K(+), which has global effects related to nitrogen metabolism.