BRENDA - Enzyme Database show
show all sequences of 1.14.14.151

Production of the sesquiterpenoid (+)-nootkatone by metabolic engineering of Pichia pastoris

Wriessnegger, T.; Augustin, P.; Engleder, M.; Leitner, E.; Mueller, M.; Kaluzna, I.; Schuermann, M.; Mink, D.; Zellnig, G.; Schwab, H.; Pichler, H.; Metab. Eng. 24, 18-29 (2014)

Data extracted from this reference:

Cloned(Commentary)
Commentary
Organism
co-expression of C-terminally FLAG-tagged premnaspirodiene oxygenase of Hyoscyamus muticus (HPO) and the Arabidopsis thaliana cytochrome P450 reductase (CPR) in Pichia pastoris strains ADH-C1 and -C3
Hyoscyamus muticus
Engineering
Amino acid exchange
Commentary
Organism
additional information
production of the sesquiterpenoid (+)-nootkatone by metabolic engineering of Pichia pastoris by generation of a strain co-expressing the premnaspirodiene oxygenase of Hyoscyamus muticus (HPO) and the Arabidopsis thaliana cytochrome P450 reductase (CPR) that hydroxylates extracellularly added (+)-valencene. Intracellular production of (+)-valencene by co-expression of valencene synthase from Callitropsis nootkatensis resolves the phase-transfer issues of (+)-valencene. Bi-phasic cultivations of Pichia pastoris result in the production of trans-nootkatol, which is oxidized to (+)-nootkatone by an intrinsic Pichia pastoris activity. Additional overexpression of a Pichia pastoris alcohol dehydrogenase and truncated hydroxy-methylglutaryl-CoA reductase (tHmg1p) significantly enhances the (+)-nootkatone yield to 208 mg/l cell culture in bioreactor cultivations. After 12 h of biotransformation about 50% of added (+)-valencene is converted to (+)-nootkatone without residual trans-nootkatol or ot herby-products, but with a moderate overall yield of 48% due to high substrate loss overtime. HPO,CPR and ADH-C3 protein levels are only marginally decreased by co-overexpression of tHMG1
Hyoscyamus muticus
Natural Substrates/ Products (Substrates)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
(+)-valencene + [reduced NADPH-hemoprotein reductase] + O2
Hyoscyamus muticus
enzyme HPO-mediated biohydroxylation of (+)-valencene
alpha-nootkatol + beta-nootkatol + [oxidized NADPH-hemoprotein reductase] + H2O
-
-
?
additional information
Hyoscyamus muticus
co-expression of enzyme HPO and cytochrome P450 reductase (CPR) leads to the hydroxylation of externally added (+)-valencene to trans-nootkatol in Pichia pastoris cells
?
-
-
-
Organism
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
Hyoscyamus muticus
A6YIH8
-
-
Substrates and Products (Substrate)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
(+)-valencene + [reduced NADPH-hemoprotein reductase] + O2
enzyme HPO-mediated biohydroxylation of (+)-valencene
745687
Hyoscyamus muticus
alpha-nootkatol + beta-nootkatol + [oxidized NADPH-hemoprotein reductase] + H2O
-
-
-
?
additional information
co-expression of enzyme HPO and cytochrome P450 reductase (CPR) leads to the hydroxylation of externally added (+)-valencene to trans-nootkatol in Pichia pastoris cells
745687
Hyoscyamus muticus
?
-
-
-
-
Cofactor
Cofactor
Commentary
Organism
Structure
NADPH-hemoprotein reductase
A flavoprotein containing both FMN and FAD. This enzyme catalyses the transfer of electrons from NADPH, an obligatory two-electron donor, to microsomal P-450 monooxygenases, EC 1.14.14._
Hyoscyamus muticus
Cloned(Commentary) (protein specific)
Commentary
Organism
co-expression of C-terminally FLAG-tagged premnaspirodiene oxygenase of Hyoscyamus muticus (HPO) and the Arabidopsis thaliana cytochrome P450 reductase (CPR) in Pichia pastoris strains ADH-C1 and -C3
Hyoscyamus muticus
Cofactor (protein specific)
Cofactor
Commentary
Organism
Structure
NADPH-hemoprotein reductase
A flavoprotein containing both FMN and FAD. This enzyme catalyses the transfer of electrons from NADPH, an obligatory two-electron donor, to microsomal P-450 monooxygenases, EC 1.14.14._
Hyoscyamus muticus
Engineering (protein specific)
Amino acid exchange
Commentary
Organism
additional information
production of the sesquiterpenoid (+)-nootkatone by metabolic engineering of Pichia pastoris by generation of a strain co-expressing the premnaspirodiene oxygenase of Hyoscyamus muticus (HPO) and the Arabidopsis thaliana cytochrome P450 reductase (CPR) that hydroxylates extracellularly added (+)-valencene. Intracellular production of (+)-valencene by co-expression of valencene synthase from Callitropsis nootkatensis resolves the phase-transfer issues of (+)-valencene. Bi-phasic cultivations of Pichia pastoris result in the production of trans-nootkatol, which is oxidized to (+)-nootkatone by an intrinsic Pichia pastoris activity. Additional overexpression of a Pichia pastoris alcohol dehydrogenase and truncated hydroxy-methylglutaryl-CoA reductase (tHmg1p) significantly enhances the (+)-nootkatone yield to 208 mg/l cell culture in bioreactor cultivations. After 12 h of biotransformation about 50% of added (+)-valencene is converted to (+)-nootkatone without residual trans-nootkatol or ot herby-products, but with a moderate overall yield of 48% due to high substrate loss overtime. HPO,CPR and ADH-C3 protein levels are only marginally decreased by co-overexpression of tHMG1
Hyoscyamus muticus
Natural Substrates/ Products (Substrates) (protein specific)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
(+)-valencene + [reduced NADPH-hemoprotein reductase] + O2
Hyoscyamus muticus
enzyme HPO-mediated biohydroxylation of (+)-valencene
alpha-nootkatol + beta-nootkatol + [oxidized NADPH-hemoprotein reductase] + H2O
-
-
?
additional information
Hyoscyamus muticus
co-expression of enzyme HPO and cytochrome P450 reductase (CPR) leads to the hydroxylation of externally added (+)-valencene to trans-nootkatol in Pichia pastoris cells
?
-
-
-
Substrates and Products (Substrate) (protein specific)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
(+)-valencene + [reduced NADPH-hemoprotein reductase] + O2
enzyme HPO-mediated biohydroxylation of (+)-valencene
745687
Hyoscyamus muticus
alpha-nootkatol + beta-nootkatol + [oxidized NADPH-hemoprotein reductase] + H2O
-
-
-
?
additional information
co-expression of enzyme HPO and cytochrome P450 reductase (CPR) leads to the hydroxylation of externally added (+)-valencene to trans-nootkatol in Pichia pastoris cells
745687
Hyoscyamus muticus
?
-
-
-
-
Other publictions for EC 1.14.14.151
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)
745627
Nino
-
Overexpression of rice premna ...
Oryza sativa Japonica Group
J. Plant Biotechnol.
43
422-431
2016
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1
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3
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1
3
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1
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1
1
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745687
Wriessnegger
Production of the sesquiterpe ...
Hyoscyamus muticus
Metab. Eng.
24
18-29
2014
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-
1
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1
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2
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10
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2
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1
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1
1
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1
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2
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2
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712360
Takahashi
Functional characterization of ...
Hyoscyamus muticus
J. Biol. Chem.
282
31744-31754
2007
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1
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6
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12
1
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1
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2
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7
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12
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1
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6
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12
1
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1
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7
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12
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12
12