• Title/Summary/Keyword: crtI

Search Result 61, Processing Time 0.035 seconds

Molecular Cloning and Characterization of the Gene Encoding Phytoene Desaturase from Kocuria gwangalliensis (Kocuria gwangalliensis 유래 phytoene desaturase 유전자의 cloning과 특성 연구)

  • Seo, Yong Bae;Choi, Seong Seok;Nam, Soo-Wan;Kim, Gun-Do
    • Microbiology and Biotechnology Letters
    • /
    • v.45 no.3
    • /
    • pp.226-235
    • /
    • 2017
  • Carotenoids such as phytoene, lycopene, and ${\beta}-carotene$ are used as food colorants, animal feed supplements, and for human nutrition and cosmetic purposes. Previously, we reported the isolation of a novel marine bacterium, Kocuria gwangalliensis, which produces a pink-orange pigment. Phytoene desaturase (CrtI), encoded by the gene crtI, catalyzes lycopene formation from phytoene and is an essential enzyme in the early steps of carotenoid biosynthesis. CrtI is one of the key enzymes regulating carotenoid biosynthesis and has been implicated as a rate-limiting enzyme of the pathway in various carotenoid synthesizing organisms. Here, we report the cloning of the crtI gene responsible for lycopene biosynthesis from K. gwangalliensis. The gene consisted of 1,584 bases encoding 527 amino acid residues. The nucleotide sequence of the crtI gene was compared with that of other species, including Kocuria rhizophila and Myxococcus xanthus, and was found to be well conserved during evolution. An expression plasmid containing the crtI gene was constructed (pCcrt1), and Escherichia coli cells were transformed with this plasmid to produce a recombinant protein of approximately 57 kDa, corresponding to the molecular weight of phytoene desaturase. Lycopene biosynthesis was confirmed when the plasmid pCcrtI was co-transformed into E. coli containing the plasmid pRScrtEB carrying the crtE and crtB genes required for lycopene biosynthesis. The results from this study will provide valuable information on the primary structure of K. gwangalliensis CrtI at the molecular level.

Cloning of Geranylgeranyl Pyrophosphate Synthase (CrtE) Gene from Kocuria gwangalliensis and Its Functional Co-expression in Escherichia coli (코쿠리아 광안리엔시스의 제라닐제라닐 피로인산염 합성 효소의 클로닝과 대장균에서 공발현을 통한 효소 활성에 관한 연구)

  • Seo, Yong-Bae;Kim, Gun-Do;Lee, Jae-Hyung
    • Journal of Life Science
    • /
    • v.22 no.8
    • /
    • pp.1024-1033
    • /
    • 2012
  • A gene encoding a novel geranylgeranyl pyrophosphate (GGPP) synthase from Kocuria gwangalliensis has been cloned and expressed in Escherichia coli. The deduced amino acid sequence showed 59.6% identity with a putative GGPP synthase (CrtE) from K. rhizophila. An expression plasmid containing the crtE gene was constructed, and E. coli cells containing this plasmid produced a recombinant protein with a theoretical molecular mass of 41 kDa, corresponding to the molecular weight of GGPP synthase. Due to the lack of crtE, crtB, and crtI in E. coli, the biosynthesis of lycopene was only obtained when the plasmid pCcrtE was co-transformed into E. coli expressing the pRScrtBI-carrying carotenoid biosynthesis crtB and crtI genes, which were sub-cloned from Paracoccus haeundaensis. The biochemical studies on the expressed proteins were performed via HPLC. The results obtained from this study will provide a wider base of knowledge regarding the primary structure of CrtE cloned from K. gwangalliensis at the molecular level.

Cloning and Characterization of the Zeaxanthin Glucosyltransferase Gene (crtX) from the Astaxanthin-Producing Marine Bacterium, Paracoccus haeundaensis

  • Seo, Yong-Bae;Choi, Seong-Seok;Nam, Soo-Wan;Lee, Jae-Hyung;Kim, Young-Tae
    • Journal of Microbiology and Biotechnology
    • /
    • v.19 no.12
    • /
    • pp.1542-1546
    • /
    • 2009
  • Zeaxanthin glucosyltransferase (CrtX) mediates the formation of zeaxanthin to zeaxanthin diglucoside. Here, we report cloning of the crtX gene responsible for zeaxanthin diglucoside biosynthesis from Paracoccus haeundaensis and the production of the corresponding carotenoids in transformed cells carrying this gene. An expression plasmid containing the crtX gene (pSTCRT-X) was constructed, and Escherichia coli cells containing this plasmid produced the recombinant protein of approximately 46 kDa. Biosynthesis of zeaxanthin diglucoside was obtained when the plasmid pSTCRT-X was co-transformed into E. coli containing the pET-44a(+)-CrtEBIYZ carrying crtE, crtB, crtI, crtY, and crtZ genes required for zeaxanthin $\beta$-D-diglucoside biosynthesis.

Molecular Cloning and Overexpression of Phytoene Desaturase (CrtI) from Paracoccus haeundaensis

  • Choi, Seong-Seok;Seo, Yong Bae;Lim, Han Kyu;Nam, Soo-Wan;Kim, Gun-Do
    • Microbiology and Biotechnology Letters
    • /
    • v.46 no.2
    • /
    • pp.145-153
    • /
    • 2018
  • Among the carotenoid biosynthesis genes, crtI gene encodes the phytoene desaturase (CrtI) enzyme, and phytoene desaturase convert phytoene to lycopene. Phytoene desaturase is involved in the dehydrogenation reaction, in which four single bonds in the phytoene are introduced into a double bond, eliminating eight hydrogen atoms in the process. Phytoene desaturase is one of the key regulating enzyme in carotenoid biosynthetic pathway of various carotenoid biosynthetic organisms. The crtI gene in genomic DNA of Paracoccus haeundaensis was amplified and cloned into a T-vector to analyze the nucleotide sequence. As a result, the crtI gene coding for phytoene desaturase from P. haeundaensis consists of 1,503 base pairs encoding 501 amino acids residues. An expression plasmid containing the crtI gene was constructed, and Escherichia coli cells containing this plasmid produced the recombinant protein of approximately 55 kDa, equivalent to the molecular weight of phytoene desaturase. The expressed protein in cell lysate showed enzymatic activity similar to phytoene desaturase. Phytoene and lycopene were analyzed by HPLC and measured at wavelength of 280 nm and 470 nm, respectively. The $K_m$ values for phytoene and NADPH were $11.1{\mu}M$ and $129.3{\mu}M$, respectively.

Reduction of thickness of steel strip applied for Inner Magnetic Shields (of CRT) manufacturing from 0,15mm to 0,1mm.

  • Leonov, Evgeny A.;Zubov, Valery I.;Uvarov, Boris N.
    • 한국정보디스플레이학회:학술대회논문집
    • /
    • 2002.08a
    • /
    • pp.1072-1073
    • /
    • 2002
  • We report using thin steel strip (0,1mm) for manufacturing Inner Magnetic Shield of CRT. Here we report that the reducing of steel strip thickness from 0,15mm to 0,1mm is possible as for one-piece IMS and for two-piece IMS without any reflection on quality of CRT. Also we report about advantages, which the producer of CRT could reach using steel with lowed carbon content up to 0,0005% by mass.

  • PDF

Enhancement of Lycopene Production in Escherichia coli by Optimization of the Lycopene Synthetic Pathway

  • KANG MIN-JUNG;YOON SANG-HWAL;LEE YOUNG-MI;LEE SOOK-HEE;KIM JU-EUN;JUNG KYUNG-HWA;SHIN YONG-CHUL;KIM SEON-WON
    • Journal of Microbiology and Biotechnology
    • /
    • v.15 no.4
    • /
    • pp.880-886
    • /
    • 2005
  • Using carotenoid genes of Erwinia herbicola, metabolic engineering was carried out for lycopene production with the pAC-LYCO4 plasmid, which was composed of a chromosomal DNA fragment of E. herbicola containing the crtE, crtB, and crtI genes under the control of the tetracycline promoter and the ipi gene of Haematococcus pluvialis with the trc promoter. Plasmid pAC-LYCm4 was constructed for efficient expression of the four exogenous genes using a strong RBS sequence and the same tetracycline promoter. The optimized expression construct of pAC-LYCm4 increased Iycopene production three times as compared with pAC-LYCO4. pAC-LYCm5 containing ispA behind the four exogenous genes was constructed. There was no significant difference in Iycopene production and cell growth between pAC-LYCm4 and pAC-LYCm5. FPP synthase encoded by ispA was not rate-limiting for Iycopene production. Each gene of crtE, crtB, crtI, and ipi was overexpressed, using pBAD-crtE, pBAD-crtIB, and pBAD-ipiHPI, in addition to their expression from pAC-LYCm4. However, there was no increase oflycopene production with the additional overexpression of each exogenous gene. The four exogenous genes appeared to be not rate-limiting in cells harboring pAC-LYCm4. When pDdxs, pBAD24 containing dxs, was introduced into cells harboring lycopene synthetic plasmids, lycopene production of pAC-LYCO4, pAC-LYCm4, and pAC-LYCm5 was increased by 4.7-, 2.2-, and 2.2-fold, respectively. Lycopene production of pBAD-DXm4 containing crtE, crtB, crtI, ipi, and dxs was 5.2 mg/g dry cell weight with $0.2\%$ arabinose, which was 8.7-fold higher than that of the initial strain with pAC-LYC04. Therefore, the present study showed that proper regulation of a metabolically engineered pathway is important for Iycopene production.

Enhanced Production of Astaxanthin by Archaea Chaperonin in Escherichia coli (대장균에서 고세균 샤페론을 이용한 아스타잔틴 생산능 향상을 위한 연구)

  • Seo, Yong Bae;Lee, Jong Kyu;Jeong, Tae Hyug;Nam, Soo-Wan;Kim, Gun-Do
    • Journal of Life Science
    • /
    • v.25 no.12
    • /
    • pp.1339-1346
    • /
    • 2015
  • The aim of this study is to increase production of carotenoids in recombinant Escherichia coli by Archaea chaperonin. The carotenoids are a widely distributed class of structurally and functionally diverse yellow, orange, and red natural pigments. These pigments are synthesized in bacteria, algae, fungi, and plants, and have been widely used as a feed supplement from poultry rearing to aquaculture. Carotenoids also exhibit diverse biological properties, such as strong antioxidant and antitumor activities, and enhancement of immune responses. In the microbial world, carotenoids are present in both anoxygenic and oxygenic photosynthetic bacteria and algae and in many fungi. We have previously reported cloning and functional analysis of the carotenoid biosynthesis genes from Paracoccus haeundaensis. The carotenogenic gene cluster involved in astaxanthin production contained seven carotenogenic genes (crtE, crtB, crtI, crtY, crtZ, crtW and crtX genes) and recombinant Escherichia coli harboring seven carotenogenic genes from Paracoccus haeundaensis produced 400 μg/g dry cell weight (DCW) of astaxanthin. In order to increase production of astaxanthin, we have co-expressed chaperone genes (ApCpnA and ApCpnB) in recombinant Escherichia coli harboring the astaxanthin biosynthesis genes. This engineered Escherichia coli strain containing both chaperone gene and astaxanthin biosynthesis gene cluster produced 890 μg/g DCW of astaxanthin, resulting 2-fold increased production of astaxanthin.

In vitro immunoregulatory role of recombinant Ancylostoma ceylanicum calreticulin

  • Tingting Zhuang;Asmaa M. I. Abuzeid;Xiaoyu Chen;Shilan Zhu;Guoqing Li
    • Parasites, Hosts and Diseases
    • /
    • v.62 no.1
    • /
    • pp.75-84
    • /
    • 2024
  • Ancylostoma ceylanicum is a zoonotic soil-derived nematode that parasitizes the intestines of humans and animals (dogs and cats), leading to malnutrition and iron-deficiency anemia. Helminth parasites secrete calreticulin (CRT), which regulates or blocks the host's immune response. However, no data on A. ceylanicum calreticulin (Ace-CRT) are available. We investigated the biological function of recombinant Ace-CRT (rAce-CRT). rAce-CRT showed reliable antigenicity and stimulated the proliferation of mouse splenocytes and canine peripheral blood mononuclear cells. Quantitative reverse-transcription PCR assays revealed that rAce-CRT primarily promoted the expression of T helper 2 cytokines, particularly IL-13, in canine peripheral blood lymphocytes. rAce-CRT inhibited complement-mediated sheep erythrocyte hemolysis in vitro. Our findings indicate that Ace-CRT plays an immunomodulatory role and may be a promising candidate molecule for a hookworm vaccine.

Genome sequence of carotenoid producing Sphingobacteriaceae bacterium SH-48 isolated from freshwater in Korea (카로티노이드 생산 Sphingobacteriaceae SH-48 균주의 유전체 염기서열 분석)

  • Choi, Ahyoung;Chung, Eu Jin;Nam, Young Ho;Choi, Gang-Guk
    • Korean Journal of Microbiology
    • /
    • v.53 no.4
    • /
    • pp.347-350
    • /
    • 2017
  • We sequenced the genome of the Sphingobacteriaceae bacterium SH-48 isolated from the Sohan stream in Republic of Korea by using a dilution-to-extinction culturing method. The sequences were assembled into a draft genome containing 5,650,162 bp with a G + C content of 35.4% and 4,856 protein-coding genes in 2 contigs. This strain contains the carotenoid biosynthesis genes crtY, crtZ, crtD, crtI, crtB, and crtH as gene clusters. This genomic information provides new insights into the carotenoid biosynthesis pathway.

Optimal Mobility Management of PCNs Using Two Types of Cell Residence Time (이동 통신망에 있어서 새로운 셀 체류시간 모형화에 따른 최적 이동성 관리)

  • 홍정식;장인갑;이창훈
    • Journal of the Korean Operations Research and Management Science Society
    • /
    • v.27 no.3
    • /
    • pp.59-74
    • /
    • 2002
  • This study investigates two basic operations of mobility management of PCNs (Personal Communication Networks), i.e., the location update and the paging of the mobile terminal. From the realistic consideration that a user either moves through several cells consecutively or stays in a cell with long time, we model the mobility pattern by introducing two types of CRT (Cell Residence Time). Mobility patterns of the mobile terminal are classified Into various ways by using the ratios of two types of CRT. Cost analysis is performed for distance-based and movement-based location update schemes combined with blanket polling paging and selective paging scheme. It is demonstrated that in a certain condition of mobility pattern and call arrival pattern, 2-state CRT model produces different optimal threshold and so, is more effective than IID ( Independently-Identically-Distributed) CRT model. An analytical model for the new CRT model is compact and easily extendable to the other location update schemes.