Information on EC 1.13.12.19 - 2-oxoglutarate dioxygenase (ethylene-forming)

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The expected taxonomic range for this enzyme is: Eukaryota, Bacteria

EC NUMBER
COMMENTARY hide
1.13.12.19
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RECOMMENDED NAME
GeneOntology No.
2-oxoglutarate dioxygenase (ethylene-forming)
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
2-oxoglutarate + O2 = ethylene + 3 CO2 + H2O
show the reaction diagram
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
ethylene biosynthesis II (microbes)
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ethylene biosynthesis IV (engineered)
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ethylene biosynthesis V (engineered)
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SYSTEMATIC NAME
IUBMB Comments
2-oxoglutarate:oxygen oxidoreductase (decarboxylating, ethylene-forming)
This is one of two simultaneous reactions catalysed by the enzyme, which is responsible for ethylene production in bacteria of the Pseudomonas syringae group. In the other reaction [EC 1.14.11.34, 2-oxoglutarate/L-arginine monooxygenase/decarboxylase (succinate-forming)] the enzyme catalyses the mono-oxygenation of both 2-oxoglutarate and L-arginine, forming succinate, carbon dioxide and L-hydroxyarginine, which is subsequently cleaved into guanidine and (S)-1-pyrroline-5-carboxylate. The enzymes catalyse two cycles of the ethylene-forming reaction for each cycle of the succinate-forming reaction, so that the stoichiometry of the products ethylene and succinate is 2:1.
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
metabolism
physiological function
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
2-oxoglutarate + O2
ethylene + 3 CO2 + H2O
show the reaction diagram
2-oxoglutarate + O2
ethylene + ?
show the reaction diagram
presence of oxygen is essential for the ethylene forming reaction by EFE
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-
?
3 2-oxoglutarate + L-arginine + 3 O2
2 C2H4 + succinate + 7 CO2 + 3 H2O + guanidine + L-DELTA1-pyrroline-5-carboxylate
show the reaction diagram
additional information
?
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
2-oxoglutarate + O2
ethylene + 3 CO2 + H2O
show the reaction diagram
3 2-oxoglutarate + L-arginine + 3 O2
2 C2H4 + succinate + 7 CO2 + 3 H2O + guanidine + L-DELTA1-pyrroline-5-carboxylate
show the reaction diagram
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
4,5-dihydroxy-1,3-benzene disulfonic acid
1 mM, 0.8% residual activity
5,5'-dithio-bis(2-nitrobenzoate)
1 mM, 0.7% residual activity
CoCl2
1 mM, 20% residual activity
CuSO4
1 mM, 50% residual activity
EDTA
1 mM, 1% residual activity
H2O2
1 mM, 0.7% residual activity
MnCl2
1 mM, 6% residual activity
N-oxalylglycine
n-propyl gallate
1 mM, 1% residual activity
Sodium azide
1 mM, 90% residual activity
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
ascorbate
D-arginine
3% of the activity with L-arginine
L-arginine
highly specific for cofactor L-arginine, KM value 0.018 mM
L-canavanine sulfate
7% of the activity with L-arginine
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.006 - 0.033
2-oxoglutarate
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.009 - 0.5
2-oxoglutarate
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.27 - 38.5
2-oxoglutarate
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
660
pH 8.0, 25°C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.2
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assay at
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 9
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over 50% of maximal activity within this range
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.9
isoelectric focusing
PDB
SCOP
CATH
UNIPROT
ORGANISM
Pseudomonas savastanoi pv. phaseolicola;
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
39370
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recombinant detagged enzyme, gel filtration
39444
1 * 39444, calculated, 1 * 42000, SDS-PAGE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
additional information
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the overall PsEFE fold comprises 10 alpha-helices and 14 beta-strands, of which eight beta-strands (I-VIII) form the major and minor beta-sheets of the conserved distorted double-stranded beta helix (DSBH): the 2-oxoglutarate oxygenase characteristic fold. beta-Strands beta1 and beta2 at the N-terminus extend the major beta-sheet at the end of the DSBH away from the active site, beta-strands beta3 and beta6 extend the other end of the major beta-sheet close to the active site. alpha-Helices alpha2 and alpha5 bind across the surface of the major beta-sheet and likely stabilize it. A loop region (residues 80-93), located between beta3 and beta6, which harbors beta4 and beta5, acts as a lid partially covering the active site and provides residues that bind the L-Arg cofactor/substrate. Three alpha-helices (alpha8, alpha9, alpha10) at the C-terminus also contribute to the active site
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
analysis of crystal structure of the enzyme in complex with several ligands, detailed overview
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purified recombinant enzyme in three structures: of enzyme in complex with manganese and 2-oxoglutarate, of enzyme in complex with with manganese and bis-Tris-propane buffer, and enzyme in complex with iron, L-Arg, and N-oxalylglycine, hanging drop vapour diffusion method, mixing of 0.003 ml of 12-20 mg/ml protein solution with 0.003 ml of reservoir solution containing 17.5-25% w/v PEG 3350 and PEG 6000, 0.1 M bis-Tris-propane, pH 7.0, 0.04 M sodium/potassium phosphate, 3-5 mM MnCl2, and with or without 10 mM 2-oxoglutarate, and equilibration against 0.5 ml reservoir solution, at 20°, microseeding, method optimization, X-ray diffraction structure determination and analysis at 1.08-1.55 A resolution, single-wavelength anomalous diffraction data obtained from selenomethionine-derivatized PsEFE:Mn:bis-Tris-propane crystals, structure modeling
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
from cell-free extract
recombinant enzyme
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recombinant enzyme from Escherichia coli strain BL21(DE3) to near homogeneity by affinityy chromatography, desalting gel filtration, tag cleavage by human SenP2 protease, followed by another step of affinityy chromatography, and gel filtration
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recombinant His-tagged enzyme from Escherichia coli strain BL21 Gold (DE3) by nickel affinity chromatography, tag cleavage by a TEV protease mutant, and another step of nickel affinity chromatography, and dialysis of the flow through
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
DNA and amino acid sequence determination and analysis, sequence comparisons with Pseudomonas digitatum and Pseudomonas chrysogenum, recombinant expression in Saccharomyces cerevisiae
expressed in Saccharomyces cerevisae. Different cultivation factors on ethylene formation in Saccharomyces cerevisiae expressing the EFE in continuous cultures are investigated. Main finding is that oxygen availability is crucial for ethylene production. By employing three different nitrogen sources it is shown that the nitrogen source available can both improve and impair the ethylene productivity. N-Source/yield ethylene (microgram/g glucose): (NH4)2SO4/164, glutamate/233, glutamate+arginine/96.8
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expression in Escherichia coli
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expression in Nicotiana tabacum
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gene efe, DNA and amino acid sequence determination and analysis, sequence comparisons and phylogenetic analysis and tree
gene efe, DNA and amino acid sequence determination and analysis, sequence comparisons and phylogenetic analysis and tree, heterologous expression of the single efe gene from Pseudomonas syringae results in ethylene production in a number of hosts including Escherichia coli, Saccharomyces cerevisiae, Pseudomonas putida, Trichoderma viride, Trichoderma reesei, tobacco, and cyanobacteria. Methods overview
gene efe, large scale expression of His6-tagged enzyme in Escherichia coli strain BL21 Gold (DE3), method optimization and evaluation
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gene efe, recombinant enzyme expression in Synechocystis sp. strain PCC 6803, stable ethylene production through the integration of a codon-optimized version of the efe gene under control of the Ptrc promoter and the core Shine-Dalgarno sequence (5'-AGGAGG-3) as the ribosome-binding site, at the slr0168 neutral site. Increase in ethylene production is achieved twofold by RBS screening, improvement of ethylene production from a single gene copy of efe, using multiple tandem promoters and by putting our best construct on an RSF1010-based broad-hostself-replicating plasmid, which has a higher copy number than the genome
gene efe, recombinant expression in Escherichia coli strain MG1655, importance of promoter strength on the expression of enzyme EFE in Escherichia coli with stronger promoters producing elevated levels of ethylene, e.g. the Amaranthus hybridus chloroplast psbA promoter (PpsbA), expression method optimization, overview
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gene efe, recombinant expression of N-terminally His-tagged or N-terminally His-SUMO-tagged enzyme in Escherichia coli strain BL21(DE3)
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recombinant expression of wild-type and mutant enzymes
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A198V
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site-directed mutagenesis, the mutant produces large amounts of L-DELTA1-pyrroline-5-carboxylate but very little ethylene
A199G
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site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
A281V
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site-directed mutagenesis, the mutant produces low levels of products in comparison to the wild-type enzyme
C280F
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site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
D191A
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site-directed mutagenesis, inactive mutant
E235D
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site-directed mutagenesis, the mutant shows activity similar to the wild-type enzyme
E84D
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site-directed mutagenesis, the mutant does not produce ethylene
E84Q
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site-directed mutagenesis, the mutant does not produce ethylene
F278Y
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site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme
F283A
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site-directed mutagenesis, replacing F283 by tryptophan, tyrosine, arginine, alanine, and valine leads to the near elimination of ethylene production
F283R
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site-directed mutagenesis, replacing F283 by tryptophan, tyrosine, arginine, alanine, and valine leads to the near elimination of ethylene production
F283V
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site-directed mutagenesis, replacing F283 by tryptophan, tyrosine, arginine, alanine, and valine leads to the near elimination of ethylene production
F283Y
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site-directed mutagenesis, replacing F283 by tryptophan, tyrosine, arginine, alanine, and valine leads to the near elimination of ethylene production
H116Q
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site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
H169Q
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site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
H189A
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site-directed mutagenesis, inactive mutant
H233A
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site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme
H233Q
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site-directed mutagenesis, inactive mutant
H268A
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site-directed mutagenesis, inactive mutant
H284Q
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site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
H309Q
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site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
I254M
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site-directed mutagenesis, the mutant shows activity similar to the wild-type enzyme
I304N
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site-directed mutagenesis, inactive mutant
I322V
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site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme
L22M
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site-directed mutagenesis, the mutant shows activity similar to the wild-type enzyme
R171K
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site-directed mutagenesis, the mutant does not produce ethylene
R236S
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site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
R277A
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site-directed mutagenesis, the mutant is expressed in inclusion bodies
R316A
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site-directed mutagenesis, the mutant shows reduced ethylene production compared to the wild-type enzyme
R316K
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site-directed mutagenesis, the mutant shows reduced ethylene production compared to the wild-type enzyme
V172T
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site-directed mutagenesis, the mutant shows activity similar to the wild-type enzyme
V196F
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site-directed mutagenesis, the mutant is expressed in inclusion bodies
V212Y/E213S
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site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
Y172F
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site-directed mutagenesis, the mutant shows reduced ethylene production compared to the wild-type enzyme
A198V
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site-directed mutagenesis, the mutant produces large amounts of L-DELTA1-pyrroline-5-carboxylate but very little ethylene
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F283Y
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site-directed mutagenesis, replacing F283 by tryptophan, tyrosine, arginine, alanine, and valine leads to the near elimination of ethylene production
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H309Q
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site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
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R171A
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site-directed mutagenesis, the mutant is soluble, it produces no detectable ethylene
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V196F
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site-directed mutagenesis, the mutant is expressed in inclusion bodies
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H116Q
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kcat value decreases to 2.4% of wild-type. Mutant is more thermolabile than wild-type
H168Q
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kcat value decreases to 3% of wild-type. Mutant is more thermolabile than wild-type
H169Q
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kcat value decreases to 9.3% of wild-type. Mutant is more thermolabile than wild-type
H189Q
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complete loss of activity
H233Q
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complete loss of activity
H268Q
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kcat value decreases to 1.8% of wild-type
H284Q
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kcat value decreases to 2% of wild-type. Mutant is more thermolabile than wild-type
H305Q
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kcat value decreases to 40% of wild-type
H309Q
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kcat value decreases to 3.3% of wild-type. Mutant is more thermolabile than wild-type
H335Q
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kcat value decreases to 60% of wild-type
A199G
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
C280F
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
D191A
site-directed mutagenesis, inactive mutant
E235D
site-directed mutagenesis, the mutant shows activity similar to the wild-type enzyme
F278Y
site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme
H189A
site-directed mutagenesis, inactive mutant
H233A
site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme
H268A
site-directed mutagenesis, inactive mutant
I254M
site-directed mutagenesis, the mutant shows activity similar to the wild-type enzyme
I304N
site-directed mutagenesis, inactive mutant
I322V
site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme
L22M
site-directed mutagenesis, the mutant shows activity similar to the wild-type enzyme
R236S
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
V172T
site-directed mutagenesis, the mutant shows activity similar to the wild-type enzyme
V212Y/E213S
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
agriculture
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introduction of a gene encoding a chimeric protein consisting of EFE and beta-glucuronidase GUS into the tobacco genome using a binary vector which directs expression of the EFE-beta-glucuronidase fusion protein under the control of constitutive promoter of cauliflower mosaic virus 35S RNA. Transgenic plants produce ethylene at consistently higher rates than the untransformed plant, and their beta-glucuronidase activities are expressed in different tissues. A significant dwarf morphology observed in the transgenic tobacco displaying the highest ethylene production resembles the phenotype of a wild-type plant exposed to excess ethylene
biotechnology
synthesis
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biological ethylene production can be achieved via expression of the ethylene-forming enzyme (EFE), found in some bacteria and fungi. It has the potential to provide a sustainable alternative to steam cracking. Ethylene is an important industrial compound for the production of a wide variety of plastics and chemicals
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