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1.
BMC Biotechnol ; 12: 78, 2012 Oct 30.
Article in English | MEDLINE | ID: mdl-23110380

ABSTRACT

BACKGROUND: Natural rubber produced by plants, known as polyisoprene, is the most widely used isoprenoid polymer. Plant polyisoprenes can be classified into two types; cis-polyisoprene and trans-polyisoprene, depending on the type of polymerization of the isoprene unit. More than 2000 species of higher plants produce latex consisting of cis-polyisoprene. Hevea brasiliensis (rubber tree) produces cis-polyisoprene, and is the key source of commercial rubber. In contrast, relatively few plant species produce trans-polyisoprene. Currently, trans-polyisoprene is mainly produced synthetically, and no plant species is used for its commercial production. RESULTS: To develop a plant-based system suitable for large-scale production of trans-polyisoprene, we selected a trans-polyisoprene-producing plant, Eucommia ulmoides Oliver, as the target for genetic transformation. A full-length cDNA (designated as EuIPI, Accession No. AB041629) encoding isopentenyl diphosphate isomerase (IPI) was isolated from E. ulmoides. EuIPI consisted of 1028 bp with a 675-bp open reading frame encoding a protein with 224 amino acid residues. EuIPI shared high identity with other plant IPIs, and the recombinant protein expressed in Escherichia coli showed IPI enzymatic activity in vitro. EuIPI was introduced into E. ulmoides via Agrobacterium-mediated transformation. Transgenic lines of E. ulmoides overexpressing EuIPI showed increased EuIPI expression (up to 19-fold that of the wild-type) and a 3- to 4-fold increase in the total content of trans-polyisoprenes, compared with the wild-type (non-transgenic root line) control. CONCLUSIONS: Increasing the expression level of EuIPI by overexpression increased accumulation of trans-polyisoprenes in transgenic E. ulmoides. IPI catalyzes the conversion of isopentenyl diphosphate to its highly electrophilic isomer, dimethylallyl diphosphate, which is the first step in the biosynthesis of all isoprenoids, including polyisoprene. Our results demonstrated that regulation of IPI expression is a key target for efficient production of trans-polyisoprene in E. ulmoides.


Subject(s)
Butadienes/chemistry , Carbon-Carbon Double Bond Isomerases/metabolism , Eucommiaceae/enzymology , Hemiterpenes/chemistry , Pentanes/chemistry , Polymers/metabolism , Agrobacterium/metabolism , Amino Acid Sequence , Carbon-Carbon Double Bond Isomerases/classification , Carbon-Carbon Double Bond Isomerases/genetics , Cloning, Molecular , Escherichia coli/metabolism , Isomerism , Molecular Sequence Data , Phylogeny , Plant Roots/metabolism , Plants, Genetically Modified/metabolism , Plasmids/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Transformation, Genetic
2.
World J Microbiol Biotechnol ; 28(1): 313-21, 2012 Jan.
Article in English | MEDLINE | ID: mdl-22806807

ABSTRACT

In this study a comparison was made between type 1 and type 2 isopentenyl diphosphate isomerases (IDI) in improving lycopene production in Escherichia coli. The corresponding genes of Bacillus licheniformis and the host (i(Bl) and i(Ec), respectively) were expressed in lycopene producing E. coli strains by pTlyci(Bl) and pTlyci(Ec) plasmids, under the control of tac promoter. The results showed that the overexpression of i(Ec) improved the lycopene production from 33 ± 1 in E. coli Tlyc to 68 ± 3 mg/gDCW in E. coli Tlyci(Ec). In contrast, the expression of i(Bl) increased the lycopene production more efficiently up to 80 ± 9 mg/gDCW in E. coli Tlyci(Bl). The introduction of a heterologous mevalonate pathway to elevate the IPP abundance resulted in a lycopene production up to 132 ± 5 mg/gDCW with i(Ec) in E. coli Tlyci(Ec)-mev and 181 ± 9 mg/gDCW with i(Bl) in E. coli Tlyci(Bl)-mev, that is, 4 and 5.6 times respectively. When fructose, mannose, arabinose, and acetate were each used as an auxiliary substrate with glycerol, lycopene production was inhibited by different extents. Among auxiliary substrates tested, only citrate was an improving one for lycopene production in all strains with a maximum of 198 ± 3 mg/gDCW in E. coli Tlyci(Bl)-mev. It may be concluded that the type 2 IDI performs better than the type 1 in metabolic engineering attempts for isoprenoid production in E. coli. In addition, the metabolic engineering of citrate pathway seems a promising approach to have more isoprenoid accumulation in E. coli.


Subject(s)
Carbon-Carbon Double Bond Isomerases/metabolism , Carotenoids/biosynthesis , Escherichia coli/metabolism , Amino Acid Sequence , Bacillus/enzymology , Bacillus/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Base Sequence , Carbon/metabolism , Carbon-Carbon Double Bond Isomerases/classification , Carbon-Carbon Double Bond Isomerases/genetics , DNA, Bacterial/genetics , Escherichia coli/genetics , Genes, Bacterial , Hemiterpenes , Isoenzymes/genetics , Isoenzymes/metabolism , Kinetics , Lycopene , Metabolic Engineering , Metabolic Networks and Pathways , Mevalonic Acid/metabolism , Molecular Sequence Data , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Substrate Specificity
3.
Biochimie ; 94(8): 1621-34, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22503704

ABSTRACT

Even if the isopentenyl diphosphate (IPP) isomerases have been discovered in the 50s, it is only in the last decade that the genetical, enzymatical, structural richness and cellular importance of this large family of crucial enzymes has been uncovered. Present in all living kingdoms, they can be classified in two subfamilies: type 1 and type 2 IPP isomerases, which show clearly distinct characteristics. They all perform the regulatory isomerization of isopentenyl diphosphate into dimethylallyl diphosphate, a key rate-limiting step of the terpenoid biosynthesis, via a protonation/deprotonation mechanism. Due to their importance in the isoprenoid metabolism and the increasing interest of industry devoted to terpenoid production, it is foreseen that the biotechnological development of such enzymes should be under intense scrutiny in the near future.


Subject(s)
Carbon-Carbon Double Bond Isomerases/chemistry , Carbon-Carbon Double Bond Isomerases/genetics , Plants/enzymology , Terpenes/chemistry , Amino Acid Sequence , Animals , Carbon-Carbon Double Bond Isomerases/classification , Carbon-Carbon Double Bond Isomerases/metabolism , Catalysis , Hemiterpenes/chemistry , Humans , Molecular Conformation , Molecular Sequence Data , Organophosphorus Compounds/chemistry , Phylogeny , Terpenes/metabolism
4.
J Biol Chem ; 284(14): 9160-7, 2009 Apr 03.
Article in English | MEDLINE | ID: mdl-19158086

ABSTRACT

Using FMN and a reducing agent such as NAD(P)H, type 2 isopentenyl-diphosphate isomerase catalyzes isomerization between isopentenyl diphosphate and dimethylallyl diphosphate, both of which are elemental units for the biosynthesis of highly diverse isoprenoid compounds. Although the flavin cofactor is expected to be integrally involved in catalysis, its exact role remains controversial. Here we report the crystal structures of the substrate-free and complex forms of type 2 isopentenyl-diphosphate isomerase from the thermoacidophilic archaeon Sulfolobus shibatae, not only in the oxidized state but also in the reduced state. Based on the active-site structures of the reduced FMN-substrate-enzyme ternary complexes, which are in the active state, and on the data from site-directed mutagenesis at highly conserved charged or polar amino acid residues around the active site, we demonstrate that only reduced FMN, not amino acid residues, can catalyze proton addition/elimination required for the isomerase reaction. This discovery is the first evidence for this long suspected, but previously unobserved, role of flavins just as a general acid-base catalyst without playing any redox roles, and thereby expands the known functions of these versatile coenzymes.


Subject(s)
Acids , Alkalies , Biocatalysis , Carbon-Carbon Double Bond Isomerases/chemistry , Carbon-Carbon Double Bond Isomerases/metabolism , Flavins/chemistry , Flavins/metabolism , Amino Acid Sequence , Carbon-Carbon Double Bond Isomerases/classification , Carbon-Carbon Double Bond Isomerases/genetics , Catalytic Domain , Conserved Sequence , Crystallography, X-Ray , Hemiterpenes , Hydrogen-Ion Concentration , Ligands , Models, Molecular , Molecular Sequence Data , Mutation/genetics , Oxidation-Reduction , Protein Binding , Protein Structure, Quaternary , Protein Structure, Tertiary , Sequence Alignment , Substrate Specificity , Sulfolobus/enzymology
5.
Proteins ; 71(4): 1699-707, 2008 Jun.
Article in English | MEDLINE | ID: mdl-18076031

ABSTRACT

In the first step of this study, type 2 isopentenyl diphosphate isomerase (IDI2) from Pyrococcus furiosus (pf-IDI2), a hyperthermophilic microorganism, was cloned and overexpressed in E. coli. After purification, hyperthermophilic behavior of this protein was approached by means of enzymatic assays and thermal denaturation studies. Compared with the mesophilic Streptococcus pneumoniae IDI2, which unfolds and looses activity above 50 degrees C, pf-IDI2 is still folded and active at 80 degrees C. Molecular modeling was applied, in a parallel step, to understand the molecular basis of thermal stability. Comparison of IDI2 from S. pneumoniae, T. thermophilus, and P. furiosus suggested that additional charged residues present in the hyperthermophilic enzyme might contribute to its higher thermal stability. This could increase the number of salt bridges between monomers of IDI2 in P. furiosus enzyme and, hence, decrease flexibility of loops or N-terminal segment, thereby enhancing its thermal stability.


Subject(s)
Carbon-Carbon Double Bond Isomerases/chemistry , Carbon-Carbon Double Bond Isomerases/classification , Carbon-Carbon Double Bond Isomerases/metabolism , Models, Molecular , Pyrococcus furiosus/enzymology , Amino Acid Sequence , Base Sequence , Binding Sites , Carbon-Carbon Double Bond Isomerases/analysis , Carbon-Carbon Double Bond Isomerases/genetics , Carbon-Carbon Double Bond Isomerases/isolation & purification , Cloning, Molecular , Conserved Sequence , Crystallography, X-Ray , Enzyme Stability , Escherichia coli/genetics , Hemiterpenes , Hot Temperature , Hydrogen Bonding , Molecular Sequence Data , Molecular Weight , Protein Binding , Protein Denaturation , Protein Folding , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid
6.
Bioorg Chem ; 32(5): 292-308, 2004 Oct.
Article in English | MEDLINE | ID: mdl-15381396

ABSTRACT

A mevalonate-independent pathway for the biosynthesis of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) that has been elucidated during the last decade is essential in plants, many eubacteria and apicomplexan parasites, but is absent in Archaea and animals. The enzymes of the pathway are potential targets for the development of novel antibiotic, antimalarial and herbicidal agents. This review is focused on the late steps of this pathway. The intermediate 2C-methyl-D-erythritol 2,4-cyclodiphosphate is converted into IPP and DMAPP via 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate by the consecutive action of the iron-sulfur proteins IspG and IspH. IPP and DMAPP can be interconverted by IPP isomerase which is essential in microorganisms using the mevalonate pathway, whereas its presence is optional in microorganisms using the non-mevalonate pathway. A hitherto unknown family of IPP isomerases using FMN as coenzyme has been discovered recently in Archaea and certain eubacteria.


Subject(s)
Anti-Infective Agents/metabolism , Drug Design , Hemiterpenes/biosynthesis , Organophosphorus Compounds/metabolism , Terpenes/metabolism , Anti-Infective Agents/chemistry , Bacterial Proteins/metabolism , Carbon-Carbon Double Bond Isomerases/chemistry , Carbon-Carbon Double Bond Isomerases/classification , Carbon-Carbon Double Bond Isomerases/metabolism , Hemiterpenes/chemistry , Molecular Conformation , Molecular Sequence Data , Organophosphorus Compounds/chemistry , Phylogeny , Terpenes/chemistry
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