BRENDA - Enzyme Database show
show all sequences of 4.2.3.119

Bacterial production of pinene by a laboratory-evolved pinene-synthase

Tashiro, M.; Kiyota, H.; Kawai-Noma, S.; Saito, K.; Ikeuchi, M.; Iijima, Y.; Umeno, D.; ACS Synth. Biol. 5, 1011-1020 (2016)

Data extracted from this reference:

Cloned(Commentary)
Commentary
Organism
gene PT1, recombinant expression of His-tagged enzyme from codon-optimized gene in Escherichia coli cytosol, coexpression of prenyltransferases GPP synthase and FPP synthase
Pinus taeda
Engineering
Amino acid exchange
Commentary
Organism
H346Y
site-directed mutagenesis, mutation in the alpha-domain (catalytic domain), no phenotype, similar to wild-type
Pinus taeda
additional information
estalishment of a pinene production system in recombinant Escherichia coli by coexpression of (-)-alpha-pinene synthase from Pinus paeda and Abies grandis GPPS, as well as farnesyl diphosphate synthase mutant IspA(S80F) from Escherichia coli. The isolated alpha-pinene synthase variant PSmut outperforms the wild-type (parent) enzyme in multiple contexts in Escherichia coli and cyanobacteria. The purified variant exhibits drastically altered metal dependency, enabling to keep the activity in the cytosol that is manganese-deficient. Coexpression of this variant with mevalonate pathway enzymes, isopentenyl diphosphate isomerase, and GPP synthase yield 140 mg/l pinene in a flask culture. Screening for PS mutants with higher cellular activity and production method optimization, overview
Pinus taeda
Q456L
site-directed mutagenesis, mutation in the alpha-domain (catalytic domain), the mutant shows a reduction in pigmentation (PSmut) but shows improved catalytic activity
Pinus taeda
KM Value [mM]
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
0.0014
-
geranyl diphosphate
pH and temperature not specified in the publication
Pinus taeda
Metals/Ions
Metals/Ions
Commentary
Organism
Structure
Mg2+
required
Pinus taeda
Natural Substrates/ Products (Substrates)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
geranyl diphosphate
Pinus taeda
-
(-)-alpha-pinene + diphosphate
-
-
?
Organism
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
Pinus taeda
Q84KL6
-
-
Substrates and Products (Substrate)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
geranyl diphosphate
-
746601
Pinus taeda
(-)-alpha-pinene + diphosphate
-
-
-
?
Cloned(Commentary) (protein specific)
Commentary
Organism
gene PT1, recombinant expression of His-tagged enzyme from codon-optimized gene in Escherichia coli cytosol, coexpression of prenyltransferases GPP synthase and FPP synthase
Pinus taeda
Engineering (protein specific)
Amino acid exchange
Commentary
Organism
H346Y
site-directed mutagenesis, mutation in the alpha-domain (catalytic domain), no phenotype, similar to wild-type
Pinus taeda
additional information
estalishment of a pinene production system in recombinant Escherichia coli by coexpression of (-)-alpha-pinene synthase from Pinus paeda and Abies grandis GPPS, as well as farnesyl diphosphate synthase mutant IspA(S80F) from Escherichia coli. The isolated alpha-pinene synthase variant PSmut outperforms the wild-type (parent) enzyme in multiple contexts in Escherichia coli and cyanobacteria. The purified variant exhibits drastically altered metal dependency, enabling to keep the activity in the cytosol that is manganese-deficient. Coexpression of this variant with mevalonate pathway enzymes, isopentenyl diphosphate isomerase, and GPP synthase yield 140 mg/l pinene in a flask culture. Screening for PS mutants with higher cellular activity and production method optimization, overview
Pinus taeda
Q456L
site-directed mutagenesis, mutation in the alpha-domain (catalytic domain), the mutant shows a reduction in pigmentation (PSmut) but shows improved catalytic activity
Pinus taeda
KM Value [mM] (protein specific)
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
0.0014
-
geranyl diphosphate
pH and temperature not specified in the publication
Pinus taeda
Metals/Ions (protein specific)
Metals/Ions
Commentary
Organism
Structure
Mg2+
required
Pinus taeda
Natural Substrates/ Products (Substrates) (protein specific)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
geranyl diphosphate
Pinus taeda
-
(-)-alpha-pinene + diphosphate
-
-
?
Substrates and Products (Substrate) (protein specific)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
geranyl diphosphate
-
746601
Pinus taeda
(-)-alpha-pinene + diphosphate
-
-
-
?
General Information
General Information
Commentary
Organism
malfunction
mutation Q456L in the alpha-domain (catalytic domain) causes a reduction in pigmentation (PSmut)
Pinus taeda
additional information
Q457 is located on helix F, which supports a part of the catalytic core and is proximate to the highly conserved residues Y455 and E458
Pinus taeda
General Information (protein specific)
General Information
Commentary
Organism
malfunction
mutation Q456L in the alpha-domain (catalytic domain) causes a reduction in pigmentation (PSmut)
Pinus taeda
additional information
Q457 is located on helix F, which supports a part of the catalytic core and is proximate to the highly conserved residues Y455 and E458
Pinus taeda
Other publictions for EC 4.2.3.119
No.
1st author
Pub Med
title
organims
journal
volume
pages
year
Activating Compound
Application
Cloned(Commentary)
Crystallization (Commentary)
Engineering
General Stability
Inhibitors
KM Value [mM]
Localization
Metals/Ions
Molecular Weight [Da]
Natural Substrates/ Products (Substrates)
Organic Solvent Stability
Organism
Oxidation Stability
Posttranslational Modification
Purification (Commentary)
Reaction
Renatured (Commentary)
Source Tissue
Specific Activity [micromol/min/mg]
Storage Stability
Substrates and Products (Substrate)
Subunits
Temperature Optimum [°C]
Temperature Range [°C]
Temperature Stability [°C]
Turnover Number [1/s]
pH Optimum
pH Range
pH Stability
Cofactor
Ki Value [mM]
pI Value
IC50 Value
Activating Compound (protein specific)
Application (protein specific)
Cloned(Commentary) (protein specific)
Cofactor (protein specific)
Crystallization (Commentary) (protein specific)
Engineering (protein specific)
General Stability (protein specific)
IC50 Value (protein specific)
Inhibitors (protein specific)
Ki Value [mM] (protein specific)
KM Value [mM] (protein specific)
Localization (protein specific)
Metals/Ions (protein specific)
Molecular Weight [Da] (protein specific)
Natural Substrates/ Products (Substrates) (protein specific)
Organic Solvent Stability (protein specific)
Oxidation Stability (protein specific)
Posttranslational Modification (protein specific)
Purification (Commentary) (protein specific)
Renatured (Commentary) (protein specific)
Source Tissue (protein specific)
Specific Activity [micromol/min/mg] (protein specific)
Storage Stability (protein specific)
Substrates and Products (Substrate) (protein specific)
Subunits (protein specific)
Temperature Optimum [°C] (protein specific)
Temperature Range [°C] (protein specific)
Temperature Stability [°C] (protein specific)
Turnover Number [1/s] (protein specific)
pH Optimum (protein specific)
pH Range (protein specific)
pH Stability (protein specific)
pI Value (protein specific)
Expression
General Information
General Information (protein specific)
Expression (protein specific)
KCat/KM [mM/s]
KCat/KM [mM/s] (protein specific)
747323
Tashiro
Tweezing the cofactor prefere ...
Pinus taeda
Biosci. Biotechnol. Biochem.
82
1058-1061
2018
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4
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2
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1
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2
2
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747887
Wang
An integrative volatile terpe ...
Wurfbainia villosa
Front. Plant Sci.
9
846
2018
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1
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3
3
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746601
Tashiro
Bacterial production of pinen ...
Pinus taeda
ACS Synth. Biol.
5
1011-1020
2016
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1
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3
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1
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2
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747387
Ma
Characterization of a monoter ...
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3
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1
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1
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2
2
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729473
Kang
Biosynthesis of pinene from gl ...
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2014
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1
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2
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3
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3
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Isolation and characterization ...
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46-56
2013
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1
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5
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1
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1
1
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Identification and characteriz ...
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Plant Physiol. Biochem.
73
302-308
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1
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1
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1
1
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1
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663770
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Mutational analysis of a monot ...
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439
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12
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12
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1
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694598
Huber
Characterization of four terpe ...
Pseudotsuga menziesii
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66
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649494
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Altering product outcome in Ab ...
Abies grandis
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425
65-76
2004
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1
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717151
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cDNA isolation, functional exp ...
Pinus taeda
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411
267-276
2003
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1
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1
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4
1
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5
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1
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1
1
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1
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1
1
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1
2
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718229
McKay
Insect attack and wounding ind ...
Picea sitchensis
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133
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2003
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1
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6
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653533
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Cloning and functional charact ...
Artemisia annua
Plant Physiol.
130
477-486
2002
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1
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1
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368
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1999
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1
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717150
Phillips
Monoterpene synthases of loblo ...
Pinus taeda
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372
197-204
1999
-
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1
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1
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2
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1
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McGeady
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2
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1
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1
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6
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210762
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Stereochemistry of the proton ...
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308
488-496
1994
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2
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210760
Lewinsohn
Wound-inducible pinene cyclase ...
Abies grandis
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293
167-173
1992
-
-
-
-
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5
1
-
3
2
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1
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1
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1
1
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1
1
1
1
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1
2
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4
-
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4
5
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1
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3
2
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1
1
1
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1
1
1
1
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1
2
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210754
Gijzen
Characterization of the consti ...
Abies grandis
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289
267-273
1991
1
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1
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Biosynthesis of monoterpenes: ...
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1989
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4
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5
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5
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Biosynthesis of monoterpenes: ...
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1988
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9
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9
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210756
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Isotopically sensitive branchi ...
Salvia officinalis
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1987
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1
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717749
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Pinene cyclases I and II. Two ...
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3
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