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ABSTRACT: Production of Z-type farnesyl diphosphate (FPP) has not been reported in E. coli. Here we present the fusion enzyme (ILRv) of E. coli E,E-FPP synthase (IspA) and Mycobacterium tuberculosis Z,E-FPP synthase (Rv1086), which can produce primarily Z,E-FPP rather than E,E-FPP, the predominant stereoisomer found in most organisms. Z,E-farnesol (FOH) was produced from E. coli harboring the bottom portion of the MVA pathway and the fusion FPP synthase (ILRv) at a titer of 115.6mg/L in 2YT medium containing 1% (v/v) glycerol as a carbon source and 5mM mevalonate. The Z,E-FOH production was improved by 15-fold, compared with 7.7mg/L obtained from the co-overexpression of separate IspA and Rv1086. The Z,E-FPP was not metabolized in native metabolic pathways of E. coli. It would be of interest to produce Z,E-FPP derived sesquiterpenes from recombinant E. coli due to no loss of Z,E-FPP substrate in endogenous metabolism of the host strain.
Metabolic Engineering 04/2013; · 5.61 Impact Factor
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ABSTRACT: Improvement of a microorganism's tolerance against organic solvents is required for a microbial factory producing terpenoid based biofuels. The bacterial genes, marA, imp, cls and cti have been found to increase organic solvent tolerance. Thus, the tolerance against the following terpenoids (isopentenol, geraniol, myrcene, and farnesol) was studied with overexpression of marA, imp, cls and cti genes in Escherichia coli. The marA overexpression significantly enhanced the tolerance of E. coli against geraniol, whereas there was no tolerance improvement against the terpenoids by overexpression of cls and cti genes. The imp overexpression even yielded sensitive phenotype to the tested solvents. The colony forming efficiency of the marA overexpressing E. coli was increased by 10(4)-fold in plate overlay of geraniol compared to that of wild type E. coli and a two-fold decrease of intracellular geraniol accumulation was also observed in liquid culture of geraniol. Single knock-out mutations of marA, or one of the following genes (acrA, acrB and tolC) encoding AcrAB-TolC efflux pump made E. coli hypersensitive to geraniol. The geraniol tolerance conferred by marA overexpression was attributed to the AcrAB-TolC efflux pump that is activated by MarA.
Journal of Bioscience and Bioengineering 11/2012; · 1.79 Impact Factor
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ABSTRACT: Escherichia coli DH5α strain was selected as the recombinant host, and a chemically defined medium supplemented with amino acids was used instead of a complex medium for the efficient production of β-carotene. In a fed-batch culture using glycerol with a chemically defined medium supplemented with amino acids, the concentration, specific content, and productivity of β-carotene were 2,470 mg/l, 72 mg/g cells, and 77 mg/l h after 32 h, respectively. These values were, respectively, 43, 33, and 26 % higher than those obtained using the complex medium. This is the highest β-carotene production that has been reported among the recombinant cells to date.
Biotechnology Letters 10/2012; · 1.68 Impact Factor
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Tae Kyung Hyun,
Yeonggil Rim,
Hui-Jeong Jang,
Cheol Hong Kim,
Jongsun Park,
Ritesh Kumar,
Sunghoon Lee,
Byung Chul Kim,
Jong Bhak,
Binh Nguyen-Quoc, Seon-Won Kim,
Sang Yeol Lee,
Jae-Yean Kim
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ABSTRACT: The ripe fruit of Momordica cochinchinensis Spreng, known as gac, is featured by very high carotenoid content. Although this plant might be a good resource for carotenoid metabolic engineering, so far, the genes involved in the carotenoid metabolic pathways in gac were unidentified due to lack of genomic information in the public database. In order to expedite the process of gene discovery, we have undertaken Illumina deep sequencing of mRNA prepared from aril of gac fruit. From 51,446,670 high-quality reads, we obtained 81,404 assembled unigenes with average length of 388 base pairs. At the protein level, gac aril transcripts showed about 81.5% similarity with cucumber proteomes. In addition 17,104 unigenes have been assigned to specific metabolic pathways in Kyoto Encyclopedia of Genes and Genomes, and all of known enzymes involved in terpenoid backbones biosynthetic and carotenoid biosynthetic pathways were also identified in our library. To analyze the relationship between putative carotenoid biosynthesis genes and alteration of carotenoid content during fruit ripening, digital gene expression analysis was performed on three different ripening stages of aril. This study has revealed putative phytoene synthase, 15-cis-phytone desaturase, zeta-carotene desaturase, carotenoid isomerase and lycopene epsilon cyclase might be key factors for controlling carotenoid contents during aril ripening. Taken together, this study has also made availability of a large gene database. This unique information for gac gene discovery would be helpful to facilitate functional studies for improving carotenoid quantities.
Plant Molecular Biology 05/2012; 79(4-5):413-27. · 4.15 Impact Factor
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ABSTRACT: Sinorhizobium sp., which can convert d-fructose into d-psicose, was isolated from soil. The optimal pH, temperature, and cell concentration for d-psicose production with the isolated strain were 8.5, 40°C, and 60mg/ml, respectively. The toluene-treated cells showed
2.5- and 4.8-fold increases in the d-psicose concentration and productivity compared with untreated washed cells. Under the optimal conditions, the toluene-treated
cells produced 37g d-psicose/l from 70% (w/v) (3.9M) d-fructose after 15h.
World Journal of Microbiology and Biotechnology 04/2012; 23(4):559-563. · 1.53 Impact Factor
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ABSTRACT: Ferulic acid is a phenolic compound that serves as a major biosynthetic precursor of vanillin in higher plants. We investigated
the ability of the 3 commercial enzymes—Ultraflo L, Viscozyme L, and α-Amylase—to induce the release ferulic acid from theIpomoea batatas L. (sweet potato) stem. The rate of release for ferulic acid was optimal when Ultraflo L (1.0%) was used compared with the
other enzymes, whereas Viscozyme L was most effective for the release of vanillic acid and vanillin. Thus, these enzymes may
be useful for the large-scale production of ferulic acid and other phenolic compounds from sweet potato stem.
Biotechnology and Bioprocess Engineering 04/2012; 11(4):372-376. · 1.28 Impact Factor
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ABSTRACT: In an attempt to increase productivity, the effects of the elicitors methyl jasmonate (MJ) and salicylic acid (SA) on the
production of bilobalide (B), ginkgolide A (GA), and ginkgolide B (GB) were studied in cell suspension cultures of Ginkgo biloba. MJ treatments increased the amounts of B, GA, and GB, concomitant with a slight decrease in cell growth. After treatment
of 0.01 mM MJ, levels of GA and GB increased 4.3-and 8.2-fold over controls by 12 h and declined after 24h. The 1.0mM MJ treatment produced a maximal release of B after 12h of exposure and increased the concentration of B in the culture medium
up to 6.25-fold compared with the controls. Treatment with 1.0mM SA transiently enhanced GA and GB production up to 3.1-and 6.1-fold, respectively, compared with the control. However, treatment
1.0 mM SA did not have a significant effect on B production. When treated with 0.01 mM SA, the level of B in the cells was increased 5.4-fold over controls by 12h and declined after 24h. The concentrations and
exposure times of both MJ and SA were factors that strongly affected the production of B, GA, and GB. The results from this
study suggest that MJ and SA directly or indirectly increased the production of B, GA, and GB in cells, and stimulated the
release of these metabolites into the culture medium.
In Vitro Cellular & Developmental Biology - Plant 04/2012; 42(1):44-49. · 1.50 Impact Factor
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ABSTRACT: Recombinant Escherichia coli engineered to contain the whole mevalonate pathway and foreign genes for β-carotene biosynthesis, was utilized for production
of β-carotene in bioreactor cultures. Optimum culture conditions were established in batch and pH-stat fed-batch cultures
to determine the optimal feeding strategy thereby improving production yield. The specific growth rate and volumetric productivity
in batch cultures at 37°C were 1.7-fold and 2-fold higher, respectively, than those at 28°C. Glycerol was superior to glucose
as a carbon source. Maximum β-carotene production (titer of 663 mg/L and overall volumetric productivity of 24.6 mg/L × h)
resulted from the simultaneous addition of 500 g/L glycerol and 50 g/L yeast extract in pH-stat fed-batch culture.
Biotechnology and Bioprocess Engineering 04/2012; 14(5):559-564. · 1.28 Impact Factor
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ABSTRACT: Vanillin is one of the world's principal flavoring compounds, and is used extensively in the food industry. The potential
vanillin production of the bacteria was compared to select and clone genes which were appropriate for highly productive vanillin
production byE. coli. Thefcs (feruloyl-CoA synthetase) andech (enoyl-CoA hydratase/aldolase) genes cloned fromAmycolatopsis sp. strain HR104 andDelftia acidovorans were introduced to pBAD24 vector with PBAD promoter and were named pDAHEF and pDDAEF, respectively. We observed 160 mg/L vanillin production withE. coli harboring pDAHEF, whereas 10 mg/L of vanillin was observed with pDDAEF. Vanillin production was optimized withE. coli harboring pDAHEF. Induction of thefcs andech genes from pDAHEF was optimized with the addition of 13.3 mM arabinose at 18 h of culture, from which 450 mg/L of vanillin
was produced. The feeding time and concentration of ferulic acid were also optimized by the supplementation of 0.2% ferulic
acid at 18 h of culture, from which 500 mg/L of vanillin was obtained. Under the above optimized condition of arabinose induction
and ferulic acid supplementation, vanillin production was carried out with four different types of media, M9, LB, 2YT, and
TB. The highest vanillin production, 580 mg/L, was obtained with LB medium, a 3.6 fold increase in comparison to the 160 mg/L
obtained before the optimization of vanillin production.
Biotechnology and Bioprocess Engineering 04/2012; 10(4):378-384. · 1.28 Impact Factor
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ABSTRACT: Lactic acid fermentation of Jerusalem artichoke tuber was performed with strains of Lactobacillus paracasei without acidic or enzymatic inulin hydrolysis prior to fermentation. Some strains of L. paracasei, notably KCTC13090 and KCTC13169, could ferment hot-water extract of Jerusalem artichoke tuber more efficiently compared with other Lactobacillus spp. such as L. casei type strain KCTC3109. The L. paracasei strains could utilize almost completely the fructo-oligosaccharides present in Jerusalem artichoke. Inulin-fermenting L. paracasei strains produced c.a. six times more lactic acid compared with L. casei KCTC3109. Direct lactic fermentation of Jerusalem artichoke tuber extract at 111.6g/L of sugar content with a supplement of 5 g/L of yeast extract by L. paracasei KCTC13169 in a 5L jar fermentor produced 92.5 ce:hsp sp="0.25"/>g/L of lactic acid with 16.8 g/L fructose equivalent remained unutilized in 72 h. The conversion efficiency of inulin-type sugars to lactic acid was 98% of the theoretical yield.
Bioresource technology 04/2012; 114:745-7. · 4.25 Impact Factor
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ABSTRACT: The optimal temperature and pH for retinal production using metabolically engineered Escherichia coli in a 7-l fermentor were found to be 30°C and 7.0, respectively. The agitation speed was a critical factor for retinal production. The optimal agitation speed was 400 rpm (oxygen transfer coefficient, k(L)a, = 92 1/h) in batch culture and 600 rpm (k(L)a=148 1/h) in a fed-batch culture of glycerol. Span 80 was selected as a surfactant for retinal production in metabolically engineered E. coli because Span 80 had proven the most effective for increased retinal production among the tested surfactants. Under the optimal conditions in the fed-batch culture with 5 g/l Span 80, the cell mass and the concentration, content, and productivity of retinal were 24.7 g/l, 600 mg/l, 24.3mg/g-cells, and 18 mg l(-1)h(-1) after 33 h, respectively. They were 1.2-, 2.7-, 2.3-, and 2.7-fold higher than those in the fed-batch culture without Span 80, respectively. The concentration and productivity of retinal in this study were the highest ever reported. The hydrophilic portion of Span 80 (sorbitan) did not affect cell growth and retinal production, but the hydrophobic portion (oleic acid) stimulated cell growth. However, oleic acid plus sorbitan did not stimulate retinal production. Thus, Span 80, as a linked compound of oleic acid and sorbitan produced by esterification, proved to be an effective surfactant for the enhancement of retinal production.
Journal of Bioscience and Bioengineering 12/2011; 113(4):461-6. · 1.79 Impact Factor
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ABSTRACT: Sesquiterpenes are important materials in pharmaceuticals and industry. Metabolic engineering has been successfully used to produce these valuable compounds in microbial hosts. However, the microbial potential of sesquiterpene production is limited by the poor heterologous expression of plant sesquiterpene synthases and the deficient FPP precursor supply. In this study, we engineered E. coli to produce α-farnesene using a codon-optimized α-farnesene synthase and an exogenous MVA pathway. Codon optimization of α-farnesene synthase improved both the synthase expression and α-farnesene production. Augmentation of the metabolic flux for FPP synthesis conferred a 1.6- to 48.0-fold increase in α-farnesene production. An additional increase in α-farnesene production was achieved by the protein fusion of FPP synthase and α-farnesene synthase. The engineered E. coli strain was able to produce 380.0 mg/L of α-farnesene, which is an approximately 317-fold increase over the initial production of 1.2 mg/L.
Metabolic Engineering 09/2011; 13(6):648-55. · 5.61 Impact Factor
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ABSTRACT: Retinoids are lipophilic isoprenoids composed of a cyclic group and a linear chain with a hydrophilic end group. These compounds include retinol, retinal, retinoic acid, retinyl esters, and various derivatives of these structures. Retinoids are used as cosmetic agents and effective pharmaceuticals for skin diseases. Retinal, an immediate precursor of retinoids, is derived by β-carotene 15,15'-mono(di)oxygenase (BCM(D)O) from β-carotene, which is synthesized from the isoprenoid building blocks isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Retinoids are chemically unstable and biologically degraded via retinoic acid. Although extensive studies have been performed on the microbial production of carotenoids, retinoid production using microbial metabolic engineering has not been reported. Here, we report retinoid production using engineered Escherichia coli that express exogenous BCM(D)O and the mevalonate (MVA) pathway for the building blocks synthesis in combination with a two-phase culture system using a dodecane overlay.
Among the BCM(D)O tested in E. coli, the synthetic retinoid synthesis protein (SR), based on bacteriorhodopsin-related protein-like homolog (Blh) of the uncultured marine bacteria 66A03, showed the highest β-carotene cleavage activity with no residual intracellular β-carotene. By introducing the exogenous MVA pathway, 8.7 mg/L of retinal was produced, which is 4-fold higher production than that of augmenting the MEP pathway (dxs overexpression). There was a large gap between retinal production and β-carotene consumption using the exogenous MVA pathway; therefore, the retinal derivatives were analyzed. The derivatives, except for retinoic acid, that formed were identified, and the levels of retinal, retinol, and retinyl acetate were measured. Amounts as high as 95 mg/L retinoids were obtained from engineered E. coli DH5α harboring the synthetic SR gene and the exogenous MVA pathway in addition to dxs overexpression, which were cultured at 29°C for 72 hours with 2YT medium containing 2.0% (w/v) glycerol as the main carbon source. However, a significant level of intracellular degradation of the retinoids was also observed in the culture. To prevent degradation of the intracellular retinoids through in situ extraction from the cells, a two-phase culture system with dodecane was used. The highest level of retinoid production (136 mg/L) was obtained after 72 hours with 5 mL of dodecane overlaid on a 5 mL culture.
In this study, we successfully produced 136 mg/L retinoids, which were composed of 67 mg/L retinal, 54 mg/L retinol, and 15 mg/L retinyl acetate, using a two-phase culture system with dodecane, which produced 68-fold more retinoids than the initial level of production (2.2 mg/L). Our results demonstrate the potential use of E. coli as a promising microbial cell factory for retinoid production.
Microbial Cell Factories 01/2011; 10:59. · 3.55 Impact Factor
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ABSTRACT: In the fed-batch culture of glycerol using a metabolically engineered strain of Escherichia coli, supplementation with glucose as an auxiliary carbon source increased lycopene production due to a significant increase in cell mass, despite a reduction in specific lycopene content. L-Arabinose supplementation increased lycopene production due to increases in cell mass and specific lycopene content. Supplementation with both glucose and L-arabinose increased lycopene production significantly due to the synergistic effect of the two sugars. Cell growth by the consumption of carbon sources was related to endogenous metabolism in the host E. coli. Supplementation with L-arabinose stimulated only the mevalonate pathway for lycopene biosynthesis and supplementation with both glucose and L-arabinose stimulated synergistically only the mevalonate pathway. In the fed-batch culture of glycerol with 10 gl(-1) glucose and 7.5 gl(-1) L-arabinose, the cell mass, lycopene concentration, specific lycopene content, and lycopene productivity after 34 h were 42 gl(-1), 1,350 mgl(-1), 32 mg g cells(-1), and 40 mgl(-1)h(-1), respectively. These values were 3.9-, 7.1-, 1.9-, and 11.7-fold higher than those without the auxiliary carbon sources, respectively. This is the highest reported concentration and productivity of lycopene.
Applied Microbiology and Biotechnology 01/2011; 90(2):489-97. · 3.42 Impact Factor
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ABSTRACT: Farnesol (FOH) production has been carried out in metabolically engineered Escherichia coli. FOH is formed through the depyrophosphorylation of farnesyl pyrophosphate (FPP), which is synthesized from isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) by FPP synthase. In order to increase FPP synthesis, E. coli was metabolically engineered to overexpress ispA and to utilize the foreign mevalonate (MVA) pathway for the efficient synthesis of IPP and DMAPP. Two-phase culture using a decane overlay of the culture broth was applied to reduce volatile loss of FOH produced during culture and to extract FOH from the culture broth. A FOH production of 135.5 mg/L was obtained from the recombinant E. coli harboring the pTispA and pSNA plasmids for ispA overexpression and MVA pathway utilization, respectively. It is interesting to observe that a large amount of FOH could be produced from E. coli without FOH synthase by the augmentation of FPP synthesis. Introduction of the exogenous MVA pathway enabled the dramatic production of FOH by E. coli while no detectable FOH production was observed in the endogenous MEP pathway-only control.
Biotechnology and Bioengineering 10/2010; 107(3):421-9. · 3.95 Impact Factor
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ABSTRACT: The phloem is the major transport route for both small substances and large molecules, such as proteins and RNAs, from their sources to sink tissues. To investigate the proteins present in pumpkin phloem sap, proteome analysis using multidimensional protein identification technology was carried out. Pumpkin phloem peptides obtained by liquid chromatography/mass spectrometry/mass spectrometry were searched against pumpkin protein data derived from the National Center for Biotechnology Information. A total of 47 pumpkin phloem proteins were identified. The identified proteins mainly corresponded to enzymes involved in gibberellin biosynthesis, antioxidation processes, or defense mechanisms. Interestingly, seven enzymes required for gibberellin biosynthesis were identified for the first time by this proteomics approach. In summary, the new phloem proteins identified in this study provide strong evidence for stress and defense signaling and new insights regarding the role of gibberellin in the developmental programming of higher plants through the phloem.
Journal of plant physiology 02/2010; 167(10):771-8. · 2.50 Impact Factor
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International journal of systematic and evolutionary microbiology 01/2010; 60(Pt 1):267. · 2.27 Impact Factor
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International journal of systematic and evolutionary microbiology 01/2010; 60(Pt 1):267. · 2.27 Impact Factor
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ABSTRACT: A bacterial strain designated YC6842T, isolated from the rhizosphere of rice (Oryza sativa L.) managed under no-tillage practice in Jinju, Korea, was characterized using polyphasic taxonomic approach. Cells of the strain were Gram-negatively stained, aerobic, rod-shaped and motile by multiple polar flagella. It grew at a temperature of 20 to 40 degrees C (optimum at 28 degrees C). Growth occurred between pH 6.0 and 10.0, with an optimum of pH 7.0-8.0. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the strain represented a separate clade within the family Xanthomonadaceae. It showed the highest 16S rRNA gene sequence similarity with Dyella yeojuensis R2A16-10T (93.6 %). The DNA G+C content was 62.6 mol%. Q-8 was the major quinone. Major polar lipids were diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylcholine (PC) and phosphatidylglycerol (PG). Phylogenetic analysis, biochemical and physiological characteristics strongly supported the genotypic and phenotypic differentiation of the strain YC6842T from the validly published genera of the family Xanthomonadaceae. Therefore, it is proposed that the strain YC6842T represents a novel species within a novel genus, with the name Gynumella flava gen. nov., sp. nov. The type strain is YC6842T (= KCTC 22443T = DSM 21636T).
International journal of systematic and evolutionary microbiology 08/2009; · 2.27 Impact Factor
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ABSTRACT: The taxonomic position of a novel bacterial strain, YC6267(T) isolated from a field of rice (Oryza sativa L.) managed under a no-tillage regime in Jinju, Korea, was studied using a polyphasic taxonomic approach. Cells of the strain were Gram-stain-negative, rod-shaped and aerobic. It grew at 15-37 degrees C (optimum at 28 degrees C). Growth of the strain occurred between pH 5.0 and 10.0, with an optimum of pH 7.0-8.0. The G+C content of the total DNA was 65.8 mol%. The 16S rRNA gene sequence of the strain was most closely related to species of the genera Arenimonas (95.6-94.4 %) and Aspromonas (95.1 %), with <95.0 % similarity to species of the genus Lysobacter and other genera of the family Xanthomonadaceae. Chemotaxonomic data (major quinone Q-8; major polar lipids phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol; and major fatty acids iso-C(15 : 0), iso-C(14 : 0), iso-C(16 : 0), and iso-C(17 : 1)omega9c) supported the affiliation of strain YC6267(T) to the genus Arenimonas. Phylogenetic analysis based on 16S rRNA gene sequences and biochemical and physiological characteristics strongly supported the genotypic and phenotypic differentiation of strain YC6267(T) from described species of the genus Arenimonas. Strain YC6267(T), therefore, represents a novel species, for which the name Arenimonas oryziterrae sp. nov. is proposed. The type strain is YC6267(T) (=KCTC 22247(T) =DSM 21050(T)). In addition, the reclassification of Aspromonas composti as Arenimonas composti comb. nov. is proposed (type strain TR7-09(T) =KCTC 12666(T) =DSM 18010(T)). A common line of descent and a number of shared phenotypic traits support this reclassification.
International journal of systematic and evolutionary microbiology 08/2009; 59(Pt 12):2967-72. · 2.27 Impact Factor
