• Bulletin of the Korean Chemical Society
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• 제30권8호
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• pp.1839-1844
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• 2009

A 20-residue hybrid peptide CA(1-8)-MA(1-12) (CAMA) incorporating residues 1-8 of cecropin A (CA) and residues 1-12 of magainin 2 (MA) has high antimicrobial activity without toxicity. To investigate the effects of the total positive charges of CAMA on the antibacterial activity and toxicity, a hybrid peptide analogue (CAMA-syn) was designed with substitutions of $Ile^{10}\;and\;Ser^{16}$ with Lys. According to CD spectra, structure of CAMA-syn with increase of cationicity was very similar to that of CAMA in DPC micelle. CAMA-syn showed antimicrobial activity similar with CAMA while CAMA-syn has no hemolytic activity and much lower cytotoxicity against RAW 264.7 macrophage cells than CAMA. Also, CAMA and CAMA-syn significantly inhibited NO production by LPSstimulated RAW264.7 macrophage at 10.0∼20.0 $\mu$M. CAMA-syn displayed salt resistance on antimicrobial activity against Escherichia coli at the physiological concentrations of $CaCl_2\;and\;MgCl_2$. The combination studies of peptides and antibiotics showed that CAMA-syn has synergistic effects with synthetic compound and flavonoid against Enterococcus faecalis and VREF. CAMA-syn can be a good candidate for the development of new antibiotics with potent antibacterial and synergistic activity but without cytotoxicity.

• Tuberculosis and Respiratory Diseases
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• 제83권1호
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• pp.81-88
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• 2020

Background: Use of appropriate antibiotics for the treatment of pneumonia is integral in patients admitted to intensive care units (ICUs). Although it is recommended that empirical treatment regimens should be based on the local distribution of pathogens in patients with suspected hospital-acquired pneumonia, few studies observe patients admitted to ICUs with nursing home-acquired pneumonia (NHAP). We found factors associated with the use of inappropriate antibiotics in patients with pneumonia admitted to the ICU via the emergency room (ER). Methods: We performed a retrospective cohort study of 83 pneumonia patients with confirmed causative bacteria admitted to ICUs via ER March 2015-May 2017. We compared clinical parameters, between patients who received appropriate or inappropriate antibiotics using the Mann-Whitney U, Pearson's chi-square, and Fisher's exact tests. We investigated independent factors associated with inappropriate antibiotic use in patients using multivariate logistic regression. Results: Among 83 patients, 30 patients (36.1%) received inappropriate antibiotics. NHAP patients were more frequently treated with inappropriate antibiotics than with appropriate antibiotics (47.2% vs. 96.7%, p<0.001). Methicillin-resistant Staphylococcus aureus was more frequently isolated from individuals in the inappropriate antibiotics-treated group than in the appropriate antibiotics-treated group (7.5% vs. 70.0%, p<0.001). In multivariate analysis, NHAP was independently associated with the use of inappropriate antibiotics in patients with pneumonia admitted to the ICU via ER. Conclusion: NHAP is a risk factor associated with the use of inappropriate antibiotics in patients with pneumonia admitted to the ICU via the ER.

• Journal of Plant Biotechnology
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• 제7권4호
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• pp.241-246
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• 2005

Factors affecting regeneration of different Rubus varieties (blackberry, raspberry and their hybrid) were examined and a reliable regeneration system was established. Media for stock plant maintenance were tested; different explants and media were investigated to find the best circumstances for the regeneration. The effect of the commonly used antibiotics was studied to determine the most suitable one for selection of the transformants. We found that both MS and LS media supplemented by $20\;gL^{-1}$ sucrose are suitable for the stock plant maintenance. The optimal hormone content for the stock plants is $0.125\;mgL^{-1}$ 6-benzylaminopurine (BAP) with $0.01\;mgL^{-1}$ indole-3- butyric acid (IBA). The highest regeneration rate was observed on medium containing MS salts with B5 vitamins complemented with glucose, sucrose, maltose, $10\;gL^{-1}$ each, supplemented with benzylaminopurine riboside (BAR) ($2\;mgL^{-1}$) and indole-3-acetic acid (IAA) ($0.1\;mgL^{-1}$). The regenerated shoots appeared directly from the cut edges, without callus phase. Hygromycin and geneticin proved to be good selection agents for the Rubus explants, but due to their severe effect on the tissues we propose to use marker-free constructions for the transformation.

• Journal of Microbiology and Biotechnology
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• 제18권1호
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• pp.88-94
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• 2008

Two sugar biosynthetic cassette plasm ids were used to direct the biosynthesis of a deoxyaminosugar. The pOTBP1 plasmid containing TDP-glucose synthase (desIII), TDP-glucose-4,6-dehydratase (desIV), and glycosyltransferase (desVII/desVIII) was constructed and transformed into S. venezuelae YJ003, a strain in which the entire gene cluster of desosamine biosynthesis is deleted. The expression plasmid pOTBP3 containing 4-aminotransferase (gerB) and 3,5-epimerase (orf9) was transformed again into S. venezuelae YJ003-OTBP1 to obtain S. venezuelae YJ003-OTBP3 for the production of 4-amino-4,6-dideoxy-L-glucose derivatives. The crude extracts obtained from S. venezuelae ATCC 15439, S. venezuelae YJ003, and S. venezuelae YJ003-OTBP3 were further analyzed by TLC, bioassay, HPLC, ESI/MS, LC/MS, and MS/MS. The results of our study clearly shows that S. venezuelae YJ003-OTBP3 constructs other new hybrid macrolide derivatives including 4-amino-4,6-dideoxy-L-glycosylated YC-17 (3, [M+ $Na^+$] m/z=464.5), methymycin (4, m/z=480.5), novamethymycin (6, m/z=496.5), and pikromycin (5, m/z=536.5) from a 12-membered ring aglycon (10-deoxymethynolide, 1) and a 14-membered ring aglycon (narbonolide, 2). These results suggest a successful engineering of a deoxysugar pathway to generate novel hybrid macrolide derivatives, including deoxyaminosugar.

• Journal of Microbiology and Biotechnology
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• 제17권12호
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• pp.1909-1921
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• 2007

Significant progress has recently been made concerning the engineering of deoxysugar biosynthesis. The biosynthetic gene clusters of several deoxysugars from various polyketides and aminoglycosides-producing microorganisms have been cloned and studied. This review introduces the biosynthetic pathways of several deoxysugars and the generation of novel hybrid macrolide antibiotics via the coexpression of deoxysugar biosynthetic gene cassettes and the substrate-flexible glycosyltransferases in a host organism as well as the production of TDP-deoxysugar derivatives via one-pot enzymatic reactions with the identified enzymes. These recent developments in the engineering of deoxysugars biosynthesis may pave the way to create novel secondary metabolites with potential biological activities.

• 한국미생물·생명공학회지
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• 제22권4호
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• pp.368-372
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• 1994

Three more unusual macrolides in addition to concnamycin B were isolated from the mycelium of Streptomyces sp. strain Bal6. These four compounds showed a potent cytotoxity to hunian cancer cell lines, SNU-1 (stomach cancer cell line), SNU-354 (liver cancer cell line), MCF- 7 (breast cancer cell line) and KB-3-1 (oral epidermoid carcinoma cell line). Interestingly, these compounds confered slight differential cytotoxity on RHEK-1, a human epidermal keratinocyte cell line immotalized by AD12-SV40 hybrid virus and RHEK-1/pSV$_{2}$ ras which was resulted from H-ras transfomation of RHEK-1. These compounds were determined to be concanamycin A, conca- namycin E and 0-methyl concanamycin B by NMR and other spectral analysis.

• 한국미생물생명공학회:학술대회논문집
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• 한국미생물생명공학회 2002년도 학술발표대회
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• pp.18-25
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• 2002

The pikromycin biosynthetic system in Streptomyces venezuleae is unique for its ability to produce two groups of antibiotics that include the 12-membered ring macrolides methymycin and neomethymycin, and the 14-membered ring macrolides narbomycin and pikromycin. The metabolic pathway also contains two post polyketide-modification enzymes, a glycosyltransferase and P450 hydroxylase that have unusually broad substrate specificities. In order to explore further the substrate flexibility of these enzymes a series of hybrid polyketide synthases were constructed and their metabolic products characterized. The plasmid-based replacement of the multifunctional protein subunits of the pikromycin PKS in S. venezuelae by the corresponding subunits from heterologous modular PKSs resulted in recombinant strains that produce both 12- and 14-membered ring macrolactones with predicted structural alterations. In all cases, novel macrolactones were produced and further modified by the DesVII glycosyltransferase and PikC hydroxylase leading to biologically active macrolide structures. These results demonstrate that hybrid PKSs in S. venezuelae can produce a multiplicity of new macrolactones that are modified further by the highly flexible DesVII glycosyltransferase and PikC hydroxylase tailoring enzymes. This work demonstrates the unique capacity of the S. venezuelae pikromycin pathway to expand the toolbox of combinatorial biosynthesis and to accelerate the creation of novel biologically active natural products. The polyketide backbone of rifamycin B is assembled through successive condensation and ${\beta}$-carbonyl processing of the extender units by the modular rifamycin PKS. The eighth module, in the RifD protein, contains nonfunctional DH domain and functional KR domain, which specify the reduction of the ${\beta}$-carbonyl group resulting in the C-21 bydroxyl of rifamycin B. A four amino acid substitution and one amino acid deletion were introduced in the putative NADPH binding motif in the proposed KR domain encoded by rifD. This strategy of mutation was based on the amino acid sequences of the corresponding motif of the KR domain of module 3 in the RifA protein, which is believed dysfunctional, so as to introduce a minimum alteration and retain the reading frame intact, yet ensure loss of function. The resulting strain produces linear polyketides, from tetraketide to octaketide, which are also produced by a rifD disrupted mutant as a consequence of premature termination of polyketide assembly. Much of the structural diversity within the polyketide superfamily of natural products is due to the ability of PKSs to vary the reduction level of every other alternate carbon atom in the backbone. Thus, the ability to introduce heterologous reductive segments such as ketoreductase (KR), dehydratase (DH), and enoylreductase (ER) into modules that naturally lack these activities would increase the power of the combinatorial biosynthetic toolbox. The dehydratase domain of module 7 of the rifamycin PKS, which is predicted to be nonfunctional in view of the sequence of the apparent active site, was replaced with its functional homolog from module 7 of rapamycin-producing polyketide synthase. The resulting mutant strain behaved like a rifC disrupted mutant, i.e., it accumulated the heptaketide intermediate and its precursors. This result points out a major difficulty we have encountered with all the Amycolatopsis mediterranei strain containing hybrid polyketide synthases: all the engineered strains prepared so far accumulate a plethora of products derived from the polyketide chain assembly intermediates as major products instead of just analogs of rifamycin B or its ansamycin precursors.

• Journal of Microbiology and Biotechnology
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• 제15권2호
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• pp.346-353
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• 2005

A 50-kb chromosome DNA region was isolated from Streptomyces kanamyceticus by screening the fosmid genomic library, using the 16S rRNA methylase gene (kmr) as a probe. Sequence analysis of this region revealed 42 putative open reading frames (ORFs), which included biosynthetic genes such as genes responsible for 2-deoxystreptamine (2­DOS) biosynthesis as well as genes for resistance and regulatory function. Also, the kanamycin acetyltransferase gene (kac) was characterized by in vitro enzyme assay, which conferred E. coli BL21 (DE3) with 10, 50, and 80-times higher resistance to kanamycin A, tobramycin, and amikacin, respectively, than the control strain had, thus strongly indicating that the isolated gene cluster is very likely involved in kanamycin biosynthesis. This work provides a solid basis for further elucidation of the kanamycin biosynthesis pathway as well as the productivity improvement and construction of new hybrid antibiotics.

• Journal of Microbiology and Biotechnology
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• 제15권3호
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• pp.640-645
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• 2005

The function of the desVIII gene in the pikromycin producer Streptomyces venezuelae was characterized by gene deletion and complementation analysis. In addition to the DesVII glycosyltransferase, the desVIII gene that has previously been suggested to be required for the incorporation of endogenous deoxysugar, TDP-D-desosamine, into the aglycone of pikromycin is also required for the transfer of exogenous deoxysugars, TDP-D-quinovose and TDP-D-olivose.

• Journal of Microbiology and Biotechnology
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• 제18권5호
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• pp.866-873
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• 2008

2-Deoxystreptamine is a core aglycon that is vital to backbone formation in various aminoglycosides. This core structure can be modified to develop hybrid types of aminoglycoside antibiotics. We obtained three genes responsible for 2-deoxystreptamine production, neo7, neo6, and neo5, which encode 2-deoxy-scyllo-inosose synthase, L-glutamine: 2-deoxy-scyllo-inosose aminotransferase, and dehydrogenase, respectively, from the neomycin gene cluster. These genes were cloned into pIBR25, a Streptomyces expression vector, resulting in pNDOS. The recombinant pNDOS was transformed into a non-aminoglycoside-producing host, Streptomyces venezuelae YJ003, for heterologous expression. Based on comparisons of the retention time on LC-ESI/MS and ESI-MS data with those of the 2-deoxystreptamine standard, a compound produced by S. venezuelae YJ003/pNDOS was found to be 2-deoxystreptamine.