• Title/Summary/Keyword: Paracoccus haeundaensis

Search Result 10, Processing Time 0.017 seconds

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.

Characterization of Geranylgeranyl Pyrophosphate Synthase from the Marine Bacterium, Paracoccus haeundaensis

  • Seo, Yong-Bae;Lee, Jae-Hyung;Kim, Young-Tae
    • Fisheries and Aquatic Sciences
    • /
    • v.12 no.1
    • /
    • pp.54-59
    • /
    • 2009
  • Carotenoids such as $\beta$-carotene and astaxanthin are used as food colorants, animal feed supplements and for nutritional and cosmetic purposes. In a previous study, an astaxanthin biosynthesis gene cluster was isolated from the marine bacterium, Paracoccus haeundaensis. Geranylgeranyl pyrophosphate (GGPP) synthase (CrtE), encoded by the ortE gene, catalyzes the formation of GGPP from farnesyl pyrophosphate (FPP), which is an essential enzyme for the biosynthesis of carotenoids in early steps. In order to study the biochemical and enzymatic characteristics of this important enzyme, a large quantity of purified GGPP synthase is required. To overproduce GGPP synthase, the crtE gene was subcloned into a pET-44a(+) expression vector and transformed into the Escherichia coli BL21(DE3) codon plus cell. Transformants harboring the crtE gene were cultured and the crtE gene was over-expressed. The expressed protein was purified to homogeneity by affinity chromatography and applied to study its biochemical properties and molecular characteristics.

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.

Overexpression and Characterization of Lycopene Cyclase (CrtY) from Marine Bacterium Paracoccus haeundaensis

  • Jeong, Tae Hyug;Ji, Keunho;Kim, Young Tae
    • Journal of Microbiology and Biotechnology
    • /
    • v.23 no.2
    • /
    • pp.144-148
    • /
    • 2013
  • Lycopene cyclase converts lycopene to ${\beta}$-carotene by catalyzing the formation of two beta-rings at each end of the linear carotene structure. This reaction takes place as a two-step reaction in which both sides of of the lycopene molecule are cyclized into ${\beta}$-carotene rings via the monocyclic ${\gamma}$-carotene as an intermediate. The crtY gene coding for lycopene cyclase from Paracoccus haeundaensis consists of 1,158 base pairs encoding 386 amino acids residues. An expression plasmid containing the crtY gene (pET44a-CrtY) was constructed and expressed in Escherichia coli, and produced a recombinant protein of approximately 43 kDa, corresponding to the molecular mass of lycopene cyclase. The expressed protein was purified to homogeneity by His-tag affinity chromatography and showed enzymatic activity corresponding to lycopene cyclase. We also determined the lycopene substrate specificity and NADPH cofactor requirements of the purified protein. The $K_m$ values for lycopene and NADPH were 3.5 ${\mu}M$ and 2 mM, respectively. The results obtained from this study will provide a wider base of knowledge on the enzyme characterization of lycopene cyclase at the molecular level.

Enhanced Production of Astaxanthin in Paracoccus haeundaensis Strain by Physical and Chemical Mutagenesis (물리·화학적 돌연변이 유도를 통한 Paracoccus haeundaensis의 astaxanthin 생산량 증대)

  • Seo, Yong Bae;Jeong, Tae Hyug;Choi, Seong Seok;Lim, Han Kyu;Kim, Gun-Do
    • Journal of Life Science
    • /
    • v.27 no.3
    • /
    • pp.339-345
    • /
    • 2017
  • Carotenoids are natural lipid-soluble pigments, which are produced primarily by bacteria, algae, and plants. Many studies have focused on the identification, production, and utilization of natural sources of astaxanthin from algae, yeast, and crustacean byproducts as an alternative to the synthetic pigment, which is mostly used today. The aim of the present study was to identify a mutant of Paracoccus haeundaensis by exposure to UV and ethyl methanesulfonate (EMS). The mutant was then exposed to nutrient stress conditions to isolate an astaxanthin-hyperproducing strain, followed by characterization of the mutant. The survival rate decreased in accordance with an increase in the UV exposure time and an increase in the EMS concentration. A mutant of the original P. haeundaensis strain was identified that showed hyperproduction of astaxanthin following exposure to UV irradiation (20 min) and EMS treatment (0.4 M concentration). The optimal culture conditions for the PUE mutant were $25^{\circ}C$, pH 7-8, and 3% NaCl. The effects of various carbon and nitrogen sources on the growth and astaxanthin production of PUE were examined. The addition of 1% raffinose and 3% potassium nitrate influenced cell growth and astaxanthin production. The selected mutant exhibited an increase of 1.58 folds in astaxanthin content compared to initial wild type strain. A genetically stable mutant strain obtained using mutagen (UV irradiation and EMS treatment) may be a suitable candidate for further industrial scale production of astaxanthin.

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.

Enhanced Production of Astaxanthin by Metabolic Engineered Isoprenoid Pathway in Escherichia coli (대장균에서 이소프레노이드 생합성 경로의 대사공학적 개량에 의한 아스타잔틴의 생산성 향상)

  • Lee, Jae-Hyung;Seo, Yong-Bae;Kim, Young-Tae
    • Journal of Life Science
    • /
    • v.18 no.12
    • /
    • pp.1764-1770
    • /
    • 2008
  • The goal of this study is to increase production of astaxanthin in recombinant Escherichia coli by engineered isoprenoid pathway. We have previously reported structural and functional analysis of the astaxanthin biosynthesis genes from a marine bacterium, Paracoccus haeundaensis. The carotenoid biosynthesis gene cluster involved in astaxanthin production contained six carotenogenic genes (crtW, crtZ, crtY, crtI, crtB, and crtE genes) and recombinant E. coli harboring six carotenogenic genes from P. haeundaensis produced 400 ${\mu}g$/g dry cell weight (DCW) of astaxanthin. In order to increase production of astaxanthin in recombinant E. coli, we have cloned 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (lytB), farnesyl diphosphate (FPP) synthase (ispA), and isopentenyl (IPP) diphossphate isomerase (idi) in the isoprenoid pathway from E. coli and coexpressed these genes in recombinant E. coli harboring the astaxanthin biosynthesis genes. This engineered E. coli strain containing both isoprenoid pathway gene and astaxanthin biosynthesis gene cluster produced 1,200 ${\mu}g$/g DCW of astaxanthin, resulting 3-fold increased production of astaxanthin.

Enhanced Production of Astaxanthin by Metabolically Engineered Non-mevalonate Pathway in Escherichia coli

  • Jeong, Tae Hyug;Cho, Youn Su;Choi, Seong-Seok;Kim, Gun-Do;Lim, Han Kyu
    • Microbiology and Biotechnology Letters
    • /
    • v.46 no.2
    • /
    • pp.114-119
    • /
    • 2018
  • Astaxanthin is one of the major carotenoids used in pigment has a great economical value in pharmaceutical markets, feeding, nutraceutical and food industries. This study was to increase the production of astaxanthin by co-expression with transformed Escherichia coli using six genes involved in the non-mevalonate pathway. Involved in the non-mevalonate biosynthetic pathway of the strain Kocuria gwangalliensis were cloned dxs, ispC, ispD, ispE, ispF, ispG, ispH and idi genes in order to increase astaxanthin production from the transformed E. coli. And co-expression with the genes to compared the amount of astaxanthin production. This engineered E. coli, containing both the non-mevalonate pathway gene and the astaxanthin biosynthesis gene cluster, produced astaxanthin at $1,100{\mu}g/g$ DCW (dry cell weight), resulting in approximately three times the production of astaxanthin.

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.