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Tsevelkhoroloo, Maral,Li, Xiaoqiang,Jin, Xue-Mei,Shin, Jung-Ho,Lee, Chang-Ro,Kang, Yup,Hong, Soon-Kwang The Korean Society for Microbiology and Biotechnol 2022 Journal of microbiology and biotechnology Vol.32 No.9
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<sup>+</sup>. 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.
Yu Jin Jung,Maral Tsevelkhoroloo,Hyun Ju Lee,Yeo Jin Jung,Hyo Ju Lee,Yong Gu Cho,Kwon Kyoo Kang 한국육종학회 2015 한국육종학회 심포지엄 Vol.2015 No.07
Zinc finger nucleases (ZFNs) have been used for targeted mutagenesis in eukaryotic cells. Custom-designed ZFNs can induce double-strand breaks (DSBs) at a specific locus. Our custom ZFN dimer was designed 3-finger of left and 4-finger of right with 2 kb size using 2A. A Ti-plasmid vector, pTA7002 containing the target site of SSS4A gene for a ZFN pair, that was shown to be active in yeast, was integrated in the rice genome. This promising technique for genome engineering was induced into 4 exon region of SSS4A gene in rice genome using Agrobacterium-mediated transformation. The SSS4A full-length cDNA was 5,070 bp consisting of a 318 bp 5′-untranslated region (UTR), a complete ORF of 2,928 bp encoding a polypeptide of 975 amino acids and a 3′-UTR of 1,824 bp. The vector is based on glucocorticoid receptor inducible gene expression system. Thus, SSS4A::ZFN expression was tightly controlled and the phenotype in low concentrations 10uM of the glucocorticoid hormone dexamethasone (DEX). In plant cells, transient ZFN expression is achieved by direct gene transfer into the target cells. For an alternative, ZFN delivery and production of mutant plants using a tobacco transient expression system for indirect transient delivery of ZFNs into a variety of tissues and cells of plants. ZFN activity was determined by PCR and sequence analysis of the target site. ZFN induced plants were obtained in up to 2% of the PCR products, consisting of deletions ranging between 1and 100 bp and insertions ranging between 1 and 10 bp. Our results describe an alternative to direct gene transfer for ZFN delivery and for the production of mutated rice.
상추에서 LED광질에 따른 플라보노이드 생합성 관련 유전자들의 발현 및 이차대사산물의 성분 분석
정유진,강대현,Maral Tsevelkhoroloo,문준관,강권규 한국식물생명공학회 2015 식물생명공학회지 Vol.42 No.2
We analyzed the effects of various LED light treatments (red 655 nm, blue 456 nm, white and mixed light) on growth pattern, gene expression and flavonoid contents in lettuce leaf. Plants treated with mixed light (red+blue+ white) showed better growth performance than those treated with single LED and fluorescent lamp (FL). Expression analysis of the eight genes involved in flavonoid biosynthesis in plants treated with LED light was examined. Results showed that red lettuce grown under mixed light showed high expression levels of LsC4H, LsF3H and LsDRF genes. Morever, the same treatment plants possessed higher content of gallic acid, chlorogenic acid and quercetin contents than those in plants exposed to single light. However, the highest total anthocyanin content was identified in plants treated with red+blue light and the lowest content was identified in plants exposed to white fluorescent lamp and single LED light condition. Thus, this study indicates that the ratio of blue to red LEDs is important for the morphology, growth, and phenolic compounds with anthocyanin properties in the two lettuce cultivars tested.
( Xue-mei Jin ),( Mu-yong Choi ),( Maral Tsevelkhoroloo ),( Uhnmee Park ),( Joo-won Suh ),( Soon-kwang Hong ) 한국미생물 · 생명공학회 2021 Journal of microbiology and biotechnology Vol.31 No.11
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 Zn<sup>2+</sup> 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.