• Journal of Microbiology and Biotechnology
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• v.14 no.2
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• pp.356-365
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• 2004

In analyzing the region of the Amycolatopsis mediterranei S699 chromosome responsible for the biosynthesis of the ansamycin antibiotic rifamycin, we identified a gene, designated orj0, which is located immediately upstream of the rifamycin polyketide synthase (PKS). Orj0 encodes a protein, on the basis of sequence-comparative analysis, that is similar to several cytochrome P450 monooxygenases from different sources. The rifamycin producer, A. mediterranei, predominantly produces rifamycin B from its macrocyclic intermediate, proansamycin X, through dehydrogenation and hydroxylation steps. However, an A. mediterranei strain, deleted in orj0 by gene replacement, no longer produced rifamycin B. Furthermore, a versatile replicative vector in A. mediterranei was constructed and rifamycin B production was restored in a complementation experiment of orj0 using this novel vector. These consecutive results verified that the arf0 protein, which is a P450 hydroxylase, is required for the production of rifamycin B in A. mediterranei.

• Journal of Microbiology and Biotechnology
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• v.13 no.4
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• pp.613-619
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• 2003

The polyketide backbone of rifamycin B is assembled by the type I rifamycin polyketide synthase (PKS) encoded by the rifA-rifE genes. In order to produce novel analogs of rifamycin via engineering of the PKS genes, inactivation of the ${\beta}-ketoacyl:acyl$ carrier protein reductase (KR) domain in module 8 of rifD, by site-specific mutagenesis of the NADPH binding site, was attempted. Module 8 contains a nonfunctional dehydratase (DH) domain and a functional KR domain that is involved in the reduction of the ${\beta}-carbonyl$ group, resulting in the C-21 hydroxyl of rifamycin B. This mutant strain produced linear polyketides, from tetraketide to octaketide, which were also produced by a rifD-disruption mutant as a consequence of premature termination of the polyketide assembly. Another attempt to replace the DH domain of module 7, which has been considered nonfunctional, with a functional homolog derived from module 7 of rapamycin-producing PKS also resulted in the production of linear polyketides, including the heptaketide intermediate and its precursors. Premature release of the carbon chain assembly intermediates is an unusual property of the rifamycin PKS. that is not seen in other PKSs such as the erythromycin PKS.

• Journal of Microbiology and Biotechnology
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• v.1 no.1
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• pp.70-74
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• 1991

The rheological properties of rifamycin B fermentation broth using Nocardia mediterranei were characterized in concomitance with the chemical changes of the fermentation broth. The data were interpreted with various rheological models. As results, it was found that the rheological behaviour at the early growth phases of the culture was the Newtonian fluid, but it was changed to the non-Newtonian fluid (Casson plastic behaviour) at the later phases of the culture. Rheological parameters viz., apparent viscosity ($\mu_a$), yield shear stress ($\tau_o$), and Casson constant ($K_c$) were changed through the fermentation where cell concentrations were changed and mycelia were greatly branched. Those results indicated that cell concentration and cell morphology played important roles to determine the rheological characters.

• Microbiology and Biotechnology Letters
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• v.9 no.4
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• pp.225-230
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• 1981

The kinetic study of rifamycin B production in batch culture of Nocardia mediterranei was undertaken in part of our endeavor to optimize the fermentation condition. The growth parameters such as $\mu$$_{m}$ and Ks values for nitrogen source were evaluated by employing Monod equation. From the experiments, $\mu$$_{m}$ and Ks were 0.15hr$^{-1}$ and 8.35g/1, respectively. The growth kinetics in batch culture was found successfully interpreted by logistic law, i.e., the initial specific growth rate and the maximum cell mass concentration were determined as function of pH and both found to have maxima. For the production of rifamycin B, a non-growth associated production kinetics was employed and the specific productivity as a function of pH was found to have two maximum points. The yield coefficient and the specific productivity were calculated as mean values in production phase. Utilizing these experimental data as a function of pH, the optimal condition for the rifamycin B production was discussed with regad to the pH effect on the cell growth and production of the antibiotic. As a result, growth phase at pH 6.5 and production phase at pH 7.0 were found to be recommended.ded.

• Microbiology and Biotechnology Letters
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• v.13 no.2
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• pp.115-118
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• 1985

Rifamycin B oxidase converts rifamycin B to rifamycin S using oxygen as cosubstrate. Humnicola spp. (ATCC 20620) was treated with acetone and the cell powder was immobilized with cellulose acetate. The properties of the immobilized enzyme was examined. The optimum pHs of the immobilized and the free enzymes were 7.2. The optimum temperature of the immobilized enzyme was at 50-55$^{\circ}C$, which was 5$^{\circ}C$ higher than that of the free enzyme. The activities of the immobilized enzyme appeared less sensistive with respect to the changes of temperature and pH as compared to those of the free enzyme. Twenty percent of the enzyme activity was recovered when the enzyme was immobilized in 3mm beads. The storage stability was good below 4$0^{\circ}C$, but the activity decreased very rapidly above 5$0^{\circ}C$. The physical strength of the beads was good and was suitable as packing material in a three-phase enzyme reactor.

• Microbiology and Biotechnology Letters
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• v.20 no.4
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• pp.470-476
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• 1992

Upon lyophilization of Nocardia mediterranei, the effects of cryoprotectants, cell concentration and drying time on viability were examined, The data were treated by computer according to response surface analysis. As a result, the maximum value of presumed viability was 39.3% under the optimal conditions of 1l.6%(v/v) sucrose, $1.16{\times}10^{11}$(CFU/ml) cell concentration, and drying time for 6.18 hrs. We also used the starter cell of rehydrated solution after lyophilization in industrial production, obtained the fermentation pattern and the purity of rifamycin B which were the same with control (FVM) and it is possible for us to use N mediterranei as a starter cell after the storage of lyophilization for 18 months.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2002.10a
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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.

• Archives of Pharmacal Research
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• v.24 no.2
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• pp.100-104
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• 2001

Differential pulse polarographic(DPP) analytical procedure for the rifampicin antibiotic, which can be applied to monitor its synthetic process from the starting antibiotic of rifamycin B or rifamycin SV has been developed based on the electrochemical reduction of an azomethine group. Rifampicin exhibited a cathodic peak due to the azomethine group in the side chain of 3-[(4-methyl-1-piperazinyl)imino]methyl moiety and another cathodic peak due to the carbonyl group in rifamycin SV by DPP. The experimental peak potential shift of an azomethine reduction was -73 mV/pH in the pH range between 3.0 and 7.5, agreeing with involvement of 4 e-and 5 $H^5$ in its reduction. By the cyclic voltammetric(CV) studies, the azomethine and the carbonyl reductions in rifampicin were processed irreversibly on the mercury electrode. The plot of peak currents vs. concentrations of rifampicin ranging $1.0{\times}10^{-7} M~$1.0{\times}10^{-5} M yielded a straight line with a correlation coefficient of 0.9996. The detection limit was $1.0{\times}10^{-8} M with a modulation amplitude of 50 mV DPP has been successfully applied for the determination of rifampicin in the pharmaceutical preparations.

• 한국생물공학회:학술대회논문집
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• 2005.10a
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• pp.623-624
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• 2005

• Korean Journal of Food Science and Technology
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• v.34 no.1
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• pp.73-78
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• 2002

Bacillus polyfermenticus SCD, which is commonly called as Bisroot strain, is being used for functional foods through the treatment of long-term intestinal disorders, since the live strains in the form of active endospores can successfully reach the target intestine in both humans and animals. The cells of B. polyfermenticus SCD were treated for 24 h in artifical bile after incubation for 2 h in artificial gastric juice and final number of the strain was reached to around $3.3{times}10^7\;CFU/mL$. In test of API ZYM kit, ${\beta}-glucuronidase$ or ${\beta}-glucosidase$ was not produced by B. polyfermenticus SCD. B. polyfermenticus SCD was resistant to antibiotics, such as nisin, streptomycin, tetracycline, and rifamycin. B. polyfermenticus SCD was also affected by alcohol concentration up to 4%, but more than 8%, their growth was not affected significantly. Finally, B. polyfermenticus SCD was shown to inhibit the growth of Listeria monocytogenes ATCC 19111 completely within 24 h of incubation, which indicated its bactericidal nature.