BRENDA - Enzyme Database

Production of mesaconate in Escherichia coli by engineered glutamate mutase pathway

Wang, J.; Zhang, K.; Metab. Eng. 30, 190-196 (2015)

Data extracted from this reference:

Cloned(Commentary)
EC Number
Commentary
Organism
5.4.99.1
genes mutS and mutE, recombinant expression of His-tagged enzyme in Escherichia coli strain Rosetta2 (DE3) pLysS
Clostridium tetanomorphum
Engineering
EC Number
Amino acid exchange
Commentary
Organism
5.4.99.1
additional information
production of mesaconate in Escherichia coli by engineered glutamate mutase pathway, establishment of mesaconate pathway in Escherichia coli. First, glutamate is synthesized from glucose via glycolysis and TCA cycle.Then glutamate is converted into 3-methylaspartate by glutamate mutase. Finally, mesaconate is formed by elimination of ammonia from 3-methylaspartate via MAL. Since Escherichia coli does not contain glutamate mutase and 3-methylaspartate ammonia lyase, the two enzymes from Clostridium tetanomorphum are heterologously expressed. To increase the flux from glutamate to mesaconate, two effective strategies are employed to optimize the critical enzyme activity in the pathway: one is regenerating inactive mutase. The other is enhancing the availability of glutamate mutase (stability) and coenzyme B12 (regeneration). For the highest mesaconate production strain EM9, the consumed glutamate is 6.91 g/l (40.9 mM) and mesaconate titer is 7.81 g/l (60 mM). The GlmE from Clostridium cochlearium shows best performance in mesaconate titer because GlmE is more stable than MutE
Clostridium tetanomorphum
Natural Substrates/ Products (Substrates)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
5.4.99.1
L-threo-3-methylaspartate
Clostridium tetanomorphum
-
L-glutamate
-
-
r
5.4.99.1
L-threo-3-methylaspartate
Clostridium tetanomorphum ATCC 15920
-
L-glutamate
-
-
r
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
5.4.99.1
Clostridium tetanomorphum
Q05488 AND Q05509
small sigma subunit and large epsilon subunit, respectively
-
5.4.99.1
Clostridium tetanomorphum ATCC 15920
Q05488 AND Q05509
small sigma subunit and large epsilon subunit, respectively
-
5.4.99.1
no activity in Escherichia coli
-
-
-
Purification (Commentary)
EC Number
Commentary
Organism
5.4.99.1
recombinant His-tagged enzyme from Escherichia coli strain Rosetta2 (DE3) pLysS by nickel affinity chromatograpyh and ultrafiltration, recombinant expression in Escherichia coli strain BW25113, coexpression with Clostridium tetanomorphum 3-methylaspartate ammonia lyase
Clostridium tetanomorphum
Substrates and Products (Substrate)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
5.4.99.1
L-threo-3-methylaspartate
-
748550
Clostridium tetanomorphum
L-glutamate
-
-
-
r
5.4.99.1
L-threo-3-methylaspartate
-
748550
Clostridium tetanomorphum ATCC 15920
L-glutamate
-
-
-
r
Subunits
EC Number
Subunits
Commentary
Organism
5.4.99.1
heterodimer
the enzyme contains a small sigma subunit and a large epsilon subunit
Clostridium tetanomorphum
Temperature Optimum [°C]
EC Number
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
5.4.99.1
37
-
assay at
Clostridium tetanomorphum
pH Optimum
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
5.4.99.1
7.5
-
assay at
Clostridium tetanomorphum
Cofactor
EC Number
Cofactor
Commentary
Organism
Structure
5.4.99.1
coenzyme B12
adenosylcobalamin, the concentration of internal coenzyme B12 is crucial for the full activity of B12-dependent enzyme
Clostridium tetanomorphum
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
5.4.99.1
genes mutS and mutE, recombinant expression of His-tagged enzyme in Escherichia coli strain Rosetta2 (DE3) pLysS
Clostridium tetanomorphum
Cofactor (protein specific)
EC Number
Cofactor
Commentary
Organism
Structure
5.4.99.1
coenzyme B12
adenosylcobalamin, the concentration of internal coenzyme B12 is crucial for the full activity of B12-dependent enzyme
Clostridium tetanomorphum
Engineering (protein specific)
EC Number
Amino acid exchange
Commentary
Organism
5.4.99.1
additional information
production of mesaconate in Escherichia coli by engineered glutamate mutase pathway, establishment of mesaconate pathway in Escherichia coli. First, glutamate is synthesized from glucose via glycolysis and TCA cycle.Then glutamate is converted into 3-methylaspartate by glutamate mutase. Finally, mesaconate is formed by elimination of ammonia from 3-methylaspartate via MAL. Since Escherichia coli does not contain glutamate mutase and 3-methylaspartate ammonia lyase, the two enzymes from Clostridium tetanomorphum are heterologously expressed. To increase the flux from glutamate to mesaconate, two effective strategies are employed to optimize the critical enzyme activity in the pathway: one is regenerating inactive mutase. The other is enhancing the availability of glutamate mutase (stability) and coenzyme B12 (regeneration). For the highest mesaconate production strain EM9, the consumed glutamate is 6.91 g/l (40.9 mM) and mesaconate titer is 7.81 g/l (60 mM). The GlmE from Clostridium cochlearium shows best performance in mesaconate titer because GlmE is more stable than MutE
Clostridium tetanomorphum
Natural Substrates/ Products (Substrates) (protein specific)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
5.4.99.1
L-threo-3-methylaspartate
Clostridium tetanomorphum
-
L-glutamate
-
-
r
5.4.99.1
L-threo-3-methylaspartate
Clostridium tetanomorphum ATCC 15920
-
L-glutamate
-
-
r
Purification (Commentary) (protein specific)
EC Number
Commentary
Organism
5.4.99.1
recombinant His-tagged enzyme from Escherichia coli strain Rosetta2 (DE3) pLysS by nickel affinity chromatograpyh and ultrafiltration, recombinant expression in Escherichia coli strain BW25113, coexpression with Clostridium tetanomorphum 3-methylaspartate ammonia lyase
Clostridium tetanomorphum
Substrates and Products (Substrate) (protein specific)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
5.4.99.1
L-threo-3-methylaspartate
-
748550
Clostridium tetanomorphum
L-glutamate
-
-
-
r
5.4.99.1
L-threo-3-methylaspartate
-
748550
Clostridium tetanomorphum ATCC 15920
L-glutamate
-
-
-
r
Subunits (protein specific)
EC Number
Subunits
Commentary
Organism
5.4.99.1
heterodimer
the enzyme contains a small sigma subunit and a large epsilon subunit
Clostridium tetanomorphum
Temperature Optimum [°C] (protein specific)
EC Number
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
5.4.99.1
37
-
assay at
Clostridium tetanomorphum
pH Optimum (protein specific)
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
5.4.99.1
7.5
-
assay at
Clostridium tetanomorphum
General Information
EC Number
General Information
Commentary
Organism
5.4.99.1
additional information
identification of the reactivating factor of glutamate mutase, MutL. MutL regenerates glutamate mutase and releases inactive coenzyme from the inactive glutamate mutase-adenosylcobalamine complex. Reactivation of the spontaneous inactive mutase (mutase-X-Cbl) and inactive form B12 binding with mutase (mutase-CNCbl) in presence of ATP and coenzyme B12 by MutL
Clostridium tetanomorphum
General Information (protein specific)
EC Number
General Information
Commentary
Organism
5.4.99.1
additional information
identification of the reactivating factor of glutamate mutase, MutL. MutL regenerates glutamate mutase and releases inactive coenzyme from the inactive glutamate mutase-adenosylcobalamine complex. Reactivation of the spontaneous inactive mutase (mutase-X-Cbl) and inactive form B12 binding with mutase (mutase-CNCbl) in presence of ATP and coenzyme B12 by MutL
Clostridium tetanomorphum