• Archives of Pharmacal Research
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• 제28권12호
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• pp.1341-1344
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• 2005

Bioassay-guided fractionation of $CHCl_{3}$ extract from the fermentation broth of a sponge Mycale plumose-derived actinomycete Saccharopolyspora sp. nov., led to the isolation of two known prodigiosin analogs - metacycloprodigiosin (1) and undecylprodigiosin (2). These compounds exhibited significant cytotoxic activities against five cancer cell lines: P388, HL60, A-549, BEL­7402, and SPCA4. This is the first report on the significant cytotoxicity of metacycloprodigiosin (1) against human cancer cell lines.

• Journal of Microbiology and Biotechnology
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• 제32권9호
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• pp.1134-1145
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• 2022

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.

• Journal of Microbiology
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• 제35권4호
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• pp.271-276
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• 1997

We isolated phenotypic suppressors of an absB (antibiotics synthesis suppression) strain. In the absB colonies, all four antibiotics including two pigmented antibiotics were blocked so that no pigmentation could be found. We assumed that in the colonies with the wuppressive(or reversive) mutation, both pigmentation would be restored so that the strains with suppressive mutation could be cisually detected. Harvested absB spores were treated with chemical mutagen along with electric shock, and were spread on specially fromulated minimal medium plates. The pigmented colonies were isolated from the unpigmented majorities. In one candidate strain, the restoration and significant overproduction of actinorhodin and undecylprodigiosin were recognized. In three other candidate strains, the overproduction of actinorhodin and restoraion of undecylprodigiosin were observed. The production of the two unpigmented antibiotics (CDA and methylenomycin) were visualized in the tested candidate strains. The strains with wuppressive mutations would be very useful in dlucidating the regulation network of antiviotics synthesis and overproduction of the antibiotics.

• Journal of Microbiology and Biotechnology
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• 제27권10호
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• pp.1867-1876
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• 2017

Most of the biosynthetic pathways for secondary metabolites are influenced by carbon metabolism and supply of cytosolic NADPH. We engineered carbon distribution to the pentose phosphate pathway (PPP) and redesigned the host to produce high levels of NADPH and primary intermediates from the PPP. The main enzymes producing NADPH in the PPP, glucose 6-phosphate dehydrogenase (encoded by zwf1 and zwf2) and 6-phosphogluconate dehydrogenase (encoded by zwf3), were overexpressed with opc encoding a positive allosteric effector essential for Zwf activity in various combinations in Streptomyces lividans TK24. Most S. lividans transformants showed better cell growth and higher concentration of cytosolic NADPH than those of the control, and S. lividans TK24/pWHM3-Z23O2 containing zwf2+zwf3+opc2 showed the highest NADPH concentration but poor sporulation in R2YE medium. S. lividans TK24/pWHM3-Z23O2 in minimal medium showed the maximum growth (6.2 mg/ml) at day 4. Thereafter, a gradual decrease of biomass and a sharp increase of cytosolic NADPH and sedoheptulose 7-phosphate between days 2 and 4 and between days 1 and 3, respectively, were observed. Moreover, S. lividans TK24/pWHM3-Z23O2 produced 0.9 times less actinorhodin but 1.8 times more undecylprodigiosin than the control. These results suggested that the increased NADPH concentration and various intermediates from the PPP specifically triggered undecylprodigiosin biosynthesis that required many precursors and NADPH-dependent reduction reaction. This study is the first report on bespoke metabolic engineering of PPP routes especially suitable for producing secondary metabolites that need diverse primary precursors and NADPH, which is useful information for metabolic engineering in Streptomyces.

• Journal of Microbiology and Biotechnology
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• 제9권4호
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• pp.488-497
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• 1999

Probing Streptomyces albus ATCC 21838 chromosomal DNA with a proline tRNA sequence resulted in an isolation of a putative integrating element in the 6.4-kb EcoRI fragment. It was found that Streptomyces lividans TK-24 transformed with a cloned DNA fragment on a multicopy plasmid, produced a higher level of spore pigment and mycelial red pigment on a regeneration agar. Furthermore, the transformant S. lividans TK-24 produced a markedly increased level of undecylprodigiosin in a broth culture. A nucleotide sequence analysis of the cloned region revealed several open reading frames homologous to the integrases of integrating plasmids or temperate bacteriophages, signal-transducing regulatory proteins with a conserved ATP-binding domain, oxidoreductases ($\beta$-ketoacyl reductase), and an AraC-like transcriptional regulator. To examine the effect on antibiotic production, each coding region was overexpressed separately from the other genes in the region in S. lividans TK-24 with; pJHS3044 for the expression of the signal-transducing regulatory protein homologue, pJHS3045 for the homologue of oxidoreductase, and pJHS3051 for the homologue of the AraC-like transcriptional regulator. Phenotypic studies of S. lividans TK-24 strains harboring plasmids for the overexpression of individual genes suggested the following effects of the genes on antibiotic production: The oxidoreductase homologue stimulated the production of actinorhodin and undecylprodigiosin, which was influenced by the culture conditions; the homologue of the AraC-like transcriptional regulator was the most effective factor in antibiotic production within all the culture conditions tested; the signal-transducing regulatory protein homologue repressed the effect due to the homologue of the AraC-like transcriptional regulator, however, the antibiotic production was derepressed upon entering the stationary phase.

• Journal of Microbiology and Biotechnology
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• 제31권11호
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• pp.1591-1600
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• 2021

Streptomyces coelicolor is a filamentous soil bacterium producing several kinds of antibiotics. S. coelicolor abs8752 is an abs (antibiotic synthesis deficient)-type mutation at the absR locus; it is characterized by an incapacity to produce any of the four antibiotics synthesized by its parental strain J1501. A chromosomal DNA fragment from S. coelicolor J1501, capable of complementing the abs- phenotype of the abs8752 mutant, was cloned and analyzed. DNA sequencing revealed that two complete ORFs (SCO6992 and SCO6993) were present in opposite directions in the clone. Introduction of SCO6992 in the mutant strain resulted in a remarkable increase in the production of two pigmented antibiotics, actinorhodin and undecylprodigiosin, in S. coelicolor J1501 and abs8752. However, introduction of SCO6993 did not show any significant difference compared to the control, suggesting that SCO6992 is primarily involved in stimulating the biosynthesis of antibiotics in S. coelicolor. In silico analysis of SCO6992 (359 aa, 39.5 kDa) revealed that sequences homologous to SCO6992 were all annotated as hypothetical proteins. Although a metalloprotease domain with a conserved metal-binding motif was found in SCO6992, the recombinant rSCO6992 did not show any protease activity. Instead, it showed very strong β-glucuronidase activity in an API ZYM assay and toward two artificial substrates, p-nitrophenyl-β-D-glucuronide and AS-BI-β-D-glucuronide. The binding between rSCO6992 and Zn²⁺ was confirmed by circular dichroism spectroscopy. We report for the first time that SCO6992 is a novel protein with β-glucuronidase activity, that has a distinct primary structure and physiological role from those of previously reported β-glucuronidases.

• Journal of Microbiology and Biotechnology
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• 제11권6호
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• pp.1077-1086
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• 2001

The sprA and sprB gene encoding chymotrypsin-like proteases Streptomyces griseus protease A (SGPA) and Streptomyces griseus protease B (SGPB) and the sprT gene that encodes Streptomyces griseus trypsin (SGT) were cloned from Streptomyces griseus ATCC10137 and overexpressed in Streptomyces lividans TK24 as a heterologous host. The chymotrypsin activity of tole culture broth measured with the artificial chromogenic substrate , N-succinyl-ala-ala-pro-phe-p-nitroanilide, was 10, 14 and 14 units/mg in the transformants haboring the sprA, sprB and sprD genes, respectively. The growth of S. lividans reached the maximum cell mass after 4 days of culture, yet SGPA and SGPD production started in the stationary phase of cell growth and kept increasing for up to 10 days of culture in an R2YE medium. The trypsin activity of the culture broth measured with the artificial chromogenic substrate , N-${\alpha}$-benzoyl-DL- arginine-p-nitroanilide , was 16 units/mg and SGT production started in the stationary phase of cell growth and kept increasing for up to 10 days of culture in an R2YE medium. The introduction of the sprA gene into S, lividans TK24 triggered the biosynthesis of pigmented antibiotics, actinorhodin and undecylprodigiosin, and induced significant morphological changes in the colonies in Benedict, R2YE, and R1R2 media. In addition, the introduction of the sprT gene also induced morphological changes in the colony shape without affecting the antibiotic production, thereby implying that certain proteases would appear to play very important and specific roles in secondary-metabolites formation and morphological differentiation in Streptomyces.

• Journal of Microbiology and Biotechnology
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• 제24권8호
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• pp.1065-1072
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• 2014

Doxorubicin, produced by Streptomyces peucetius ATCC 27952, is tightly regulated by dnrO, dnrN, and dnrI regulators. Genome mining of S. peucetius revealed the presence of the IclR (doxR) type family of transcription regulator mediating the signal-dependent expression of operons at the nonribosomal peptide synthetase gene cluster. Overexpression of doxR in native strain strongly repressed the drug production. Furthermore, it also had a negative effect on the regulatory system of doxorubicin, wherein the transcript of dnrI was reduced to the maximum level in comparision with the other two. Interestingly, the overexpression of the same gene also had strong inhibitory effects on the production of actinorhodin (blue pigment) and undecylprodigiosin (red pigment) in Streptomyces coelicolor M145, herboxidiene production in Streptomyces chromofuscus ATCC 49982, and spinosyn production in Saccharopolyspora spinosa NRRL 18395, respectively. Moreover, DoxR exhibited pleiotropic effects on the production of blue and red pigments in S. coelicolor when grown in different agar media, wherein the production of blue pigment was inhibited in R2YE medium and the red pigment was inhibited in YEME medium. However, the production of both blue and red pigments from S. coelicolor harboring doxR was halted in ISP2 medium, whereas S. coelicolor produced both pigmented antibiotics in the same plate. These consequences demonstrate that the on and off production of these antibiotics was not due to salt stress or media compositions, but was selectively controlled in actinomycetes.

• Journal of Microbiology and Biotechnology
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• 제8권4호
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• pp.325-332
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• 1998

In vitro phosphorylation experiments with a cell extract of Streptomyces coelicolor A3(2) M130 in the presence of ${\gamma}-[^32P]$]ATP revealed the presence of multiple phosphorylated proteins, including the AfsR/AfsK kinases which control the biosynthesis of A-factor, actinorhodin, and undecylprodigiosin. Phosphorylation of AfsR by a cell extract as an AfsK source was significantly inhibited by Ser/Thr protein kinase inhibitors, staurosporine and K-252a, at concentrations giving 50% inhibition ($IC_50$) of $1{\mu}M\;and\;0.1{\mu}M$, respectively. Further in vitro experiments with the cell extracts showed that phosphorylation of multiple proteins was inhibited by various protein kinase inhibitors with different inhibitory profiles. Manganese and calcium ions in the reaction mixture also modulate phosphorylation of multiple proteins. Manganese at 10 mM greatly enhanced the phosphorylation and partially circumvented the inhibition caused by staurosporine and K-252a. A calcium-activated protein kinase(s) was little affected by these inhibitors. Herbimycin and radicicol, which are known as tyrosine kinase inhibitors, did not show any significant inhibition of AfsR phosphorylation. Consistent with the in vitro effect of the kinase inhibitors, they inhibited aerial mycelium formation and pigmented antibiotic production on solid media. On the contrary, when assayed in liquid culture, the amount of actinorhodin produced was increased by staurosporine and K-252a and greatly decreased by manganese. All of these data clearly show that the genus Streptomyces possesses several protein kinases of eukaryotic types which are involved in the regulatory network for morphogenesis and secondary metabolism.

• Journal of Microbiology and Biotechnology
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• 제17권11호
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• pp.1818-1825
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• 2007

S-Adenosylmethionine (SAM) was previously documented to activate secondary metabolism in a variety of Streptomyces spp. and to promote actinorhodin (ACT) and undecylprodigiosin (RED) in Streptomyces coelicolor. The SAM-induced proteins in S. coelicolor include several ABC transporter components (SCO5260 and SCO5477) including BldKB, the component of a well-known regulatory factor for differentiations. In order to assess the role of these ABC transporter complexes in differentiation of Streptomyces, SCO5260 and SCO5476, the first genes from the cognate complex clusters, were individually inactivated by gene replacement. Inactivation of either SCO5260 or SCO5476 led to impaired sporulation on agar medium, with the more drastic defect in the SCO5260 null mutant (${\Delta}SCO5260$). ${\Delta}SCO5260$ displayed growth retardation and reduced yields of ACT and RED in liquid cultures. In addition, SAM supplementation failed in promoting the production of ACT and RED in ${\Delta}SCO5260$. Inactivation of SCO5476 gave no significant change in growth and production of ACT and RED, but impaired the promoting effect of SAM on ACT production without interfering with the effect on RED production. The present study suggests that SAM induces several ABC transporters to modulate secondary metabolism and morphological development in S. coelicolor.