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Literature summary extracted from

  • Atsumi, S.; Liao, J.C.
    Directed evolution of Methanococcus jannaschii citramalate synthase for biosynthesis of 1-propanol and 1-butanol by Escherichia coli (2008), Appl. Environ. Microbiol., 74, 7802-7808.
    View publication on PubMedView publication on EuropePMC

Application

EC Number Application Comment Organism
2.3.3.21 biofuel production advantage of the growth phenotype associated with 2-keto acid deficiency to construct a hyperproducer of 1-propanol and 1-butanol by evolving citramalate synthase (CimA) from Methanococcus jannaschii Methanocaldococcus jannaschii
2.3.3.21 energy production the citratmalate pathway is enhanced for the production of 1-propanol and 1-butanol Methanocaldococcus jannaschii

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.3.3.21 expressed in Escherichia coli Methanocaldococcus jannaschii
2.3.3.21 expression in Escherichia coli BL21 Star DE3 cells Methanocaldococcus jannaschii

Protein Variants

EC Number Protein Variants Comment Organism
2.3.3.21 I47V/D86G/H126Q/T204A CimA3.5, mutant generated by directed evolution Methanocaldococcus jannaschii
2.3.3.21 I47V/D86G/H126Q/T204A CimA3.5. 1-Propanol and 1-butanol production with CimA wild-type is 302 mg/l for 1-propanol and 18 mg/l for 1-butanol, production increased with CimA3.5 mutant 7.7fold for 1-propanol and 7.8fold for 1-butanol Methanocaldococcus jannaschii
2.3.3.21 I47V/E111K/E121V/H126Q/T204A/M250V CimA3.8, mutant generated by directed evolution Methanocaldococcus jannaschii
2.3.3.21 I47V/E111K/E121V/H126Q/T204A/M250V CimA3.8. 1-Propanol and 1-butanol production with CimA wild-type is 302 mg/l for 1-propanol and 18 mg/l for 1-butanol, production increased with CimA3.8 mutant 8.2fold for 1-propanol and 21.7fold for 1-butanol Methanocaldococcus jannaschii
2.3.3.21 I47V/E114V/H126Q/T204A/L238S CimA3.7, mutant generated by directed evolution Methanocaldococcus jannaschii
2.3.3.21 I47V/E114V/H126Q/T204A/L238S CimA3.7, shows higher activity at all temperatures tested relative to wild-type CimA, although the difference of the specific activity is larger at lower temperature, possibly because the mutant is screened at 30°C. 1-Propanol and 1-butanol production with CimA wild-type is 302 mg/l for 1-propanol and 18 mg/l for 1-butanol, production increased with CimA3.7 mutant 9.2fold for 1-propanol and 21.9fold for 1-butanol Methanocaldococcus jannaschii
2.3.3.21 I47V/H126Q/D141E/T204A/I286V/L327H CimA3.4, mutant generated by directed evolution Methanocaldococcus jannaschii
2.3.3.21 I47V/H126Q/D141E/T204A/I286V/L327H CimA3.4. 1-Propanol and 1-butanol production with CimA wild-type is 302 mg/l for 1-propanol and 18 mg/l for 1-butanol, production increased with CimA3.4 mutant 8.1fold for 1-propanol and 6.2fold for 1-butanol Methanocaldococcus jannaschii
2.3.3.21 I47V/H126Q/E183K/T204A/L253S CimA3.2, mutant generated by directed evolution Methanocaldococcus jannaschii
2.3.3.21 I47V/H126Q/E183K/T204A/L253S CimA3.2. 1-Propanol and 1-butanol production with CimA wild-type is 302 mg/l for 1-propanol and 18 mg/l for 1-butanol, production increased with CimA3.2 mutant 8.5fold for 1-propanol and 14.1fold for 1-butanol Methanocaldococcus jannaschii
2.3.3.21 I47V/H126Q/T204A CimA2delta, mutant generated by directed evolution Methanocaldococcus jannaschii
2.3.3.21 I47V/H126Q/T204A CimA2DELTA. 1-Propanol and 1-butanol production with CimA wild-type is 302 mg/l for 1-propanol and 18 mg/l for 1-butanol, production increased with CimA2DELTA mutant 8.0fold for 1-propanol and 5.9fold for 1-butanol Methanocaldococcus jannaschii
2.3.3.21 I47V/H126Q/T204A/D328V CimA3.6, mutant generated by directed evolution Methanocaldococcus jannaschii
2.3.3.21 I47V/H126Q/T204A/D328V CimA3.6. 1-Propanol and 1-butanol production with CimA wild-type is 302 mg/l for 1-propanol and 18 mg/l for 1-butanol, production increased with CimA3.6 mutant 7.8fold for 1-propanol and 8.0fold for 1-butanol Methanocaldococcus jannaschii
2.3.3.21 I47V/H126Q/T204A/K265R/F349C CimA3.3, mutant generated by directed evolution Methanocaldococcus jannaschii
2.3.3.21 I47V/H126Q/T204A/K265R/F349C CimA3.3. 1-Propanol and 1-butanol production with CimA wild-type is 302 mg/l for 1-propanol and 18 mg/l for 1-butanol, production increased with CimA3.3 mutant 7.9fold for 1-propanol and 6.9fold for 1-butanol Methanocaldococcus jannaschii
2.3.3.21 I47V/H126Q/T204A/V373STOP CimA2, contains a frameshift mutation at bp 1117, creating a CimA variant missing the C-terminal domain from the 373rd residue. 1-Propanol and 1-butanol production with CimA wild-type is 302 mg/l for 1-propanol and 18 mg/l for 1-butanol, production increased with CimA2 mutant 3.9fold for 1-propanol and 4.3fold for 1-butanol Methanocaldococcus jannaschii
2.3.3.21 I47V/H126Q/T204A/V373STOP CimA2, mutant generated by directed evolution Methanocaldococcus jannaschii
2.3.3.21 I47V/K435N/V441A CimA1, leads to the highest 1-propanol production, and thus the largest 2-ketobutyrate pool and highest cimA activity. CimA1 mutant contains 3 amino acid substitutions (I47V, K435N, and V441A). 1-Propanol and 1-butanol production with CimA wild-type is 302 mg/l for 1-propanol and 18 mg/l for 1-butanol, production increased with CimA1 mutant 2.3fold for 1-propanol and 1.2fold for 1-butanol Methanocaldococcus jannaschii
2.3.3.21 I47V/K435N/V441A CimA1, mutant generated by directed evolution Methanocaldococcus jannaschii
2.3.3.21 I47V/R53S/H126Q/T204A CimA3.1, mutant generated by directed evolution Methanocaldococcus jannaschii
2.3.3.21 I47V/R53S/H126Q/T204A CimA3.1. 1-Propanol and 1-butanol production with CimA wild-type is 302 mg/l for 1-propanol and 18 mg/l for 1-butanol, production increased with CimA3.1 mutant 8.2fold for 1-propanol and 20.7fold for 1-butanol Methanocaldococcus jannaschii
2.3.3.21 K32N/I47V/H126Q/T204A CimA3.9, mutant generated by directed evolution Methanocaldococcus jannaschii
2.3.3.21 K32N/I47V/H126Q/T204A CimA3.9. 1-Propanol and 1-butanol production with CimA wild-type is 302 mg/l for 1-propanol and 18 mg/l for 1-butanol, production increased with CimA3.9 mutant 8.1fold for 1-propanol and 11.0fold for 1-butanol Methanocaldococcus jannaschii
2.3.3.21 wild-typeDELTA 1-Propanol and 1-butanol production with CimA wild-type is 302 mg/l for 1-propanol and 18 mg/l for 1-butanol, production increased with wild-typeDELTA mutant 0.2fold for 1-propanol, production for 1-butanol is not detected Methanocaldococcus jannaschii
2.3.3.21 WTDELTA wild-type delta, mutant generated by directed evolution Methanocaldococcus jannaschii

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
2.3.3.21 0.105
-
 acetyl-CoA I47V/E114V/H126Q/T204A/L238S mutant Methanocaldococcus jannaschii
2.3.3.21 0.105
-
 acetyl-CoA mutant CimA3.7 Methanocaldococcus jannaschii
2.3.3.21 0.184
-
 pyruvate wild-type Methanocaldococcus jannaschii
2.3.3.21 0.184
-
 pyruvate wild type enzyme Methanocaldococcus jannaschii
2.3.3.21 0.303
-
 acetyl-CoA wild-type Methanocaldococcus jannaschii
2.3.3.21 0.303
-
 acetyl-CoA wild type enzyme Methanocaldococcus jannaschii
2.3.3.21 0.342
-
 pyruvate I47V/E114V/H126Q/T204A/L238S mutant Methanocaldococcus jannaschii
2.3.3.21 0.342
-
 pyruvate mutant CimA3.7 Methanocaldococcus jannaschii

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2.3.3.21 (2S)-2-hydroxy-2-methylbutanedioate Methanocaldococcus jannaschii
-
 acetate + pyruvate
-
 r
2.3.3.21 (2S)-2-hydroxy-2-methylbutanedioate Methanocaldococcus jannaschii BW25113
-
 acetate + pyruvate
-
 r

Organism

EC Number Organism UniProt Comment Textmining
2.3.3.21 Methanocaldococcus jannaschii
-
-
-
2.3.3.21 Methanocaldococcus jannaschii
-
 and JCL16, KS145, SA405, SA408
-
2.3.3.21 Methanocaldococcus jannaschii BW25113
-
 and JCL16, KS145, SA405, SA408
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.3.3.21 by using Ni-nitrilotriacetic spin columns Methanocaldococcus jannaschii
2.3.3.21 with Ni-nitrilotriacetic acid spin columns Methanocaldococcus jannaschii

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.3.3.21 (2S)-2-hydroxy-2-methylbutanedioate
-
 Methanocaldococcus jannaschii acetate + pyruvate
-
 r
2.3.3.21 (2S)-2-hydroxy-2-methylbutanedioate
-
 Methanocaldococcus jannaschii BW25113 acetate + pyruvate
-
 r
2.3.3.21 acetyl-CoA + pyruvate + H2O
-
 Methanocaldococcus jannaschii CoA + (R)-citramalate
-
 ?
2.3.3.21 acetyl-CoA + pyruvate + H2O
-
 Methanocaldococcus jannaschii BW25113 CoA + (R)-citramalate
-
 ?
2.3.3.21 acetyl-CoA + pyruvate + H2O
-
 Methanocaldococcus jannaschii CoA + (2R)-2-hydroxy-2-methyl-butanedioate
-
 ?
2.3.3.21 acetyl-CoA + pyruvate + H2O
-
 Methanocaldococcus jannaschii BW25113 CoA + (2R)-2-hydroxy-2-methyl-butanedioate
-
 ?
2.3.3.21 acetyl-CoA + pyruvate + H2O
-
 Methanocaldococcus jannaschii (2S)-2-hydroxy-2-methylbutanedioate + CoA
-
 r
2.3.3.21 acetyl-CoA + pyruvate + H2O
-
 Methanocaldococcus jannaschii BW25113 (2S)-2-hydroxy-2-methylbutanedioate + CoA
-
 r

Synonyms

EC Number Synonyms Comment Organism
2.3.3.21 CimA
-
 Methanocaldococcus jannaschii
2.3.3.21 citramalate lyase
-
 Methanocaldococcus jannaschii
2.3.3.21 citramalate synthase
-
 Methanocaldococcus jannaschii

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
2.3.3.21 30
-
-
 Methanocaldococcus jannaschii

Temperature Range [°C]

EC Number Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
2.3.3.21 30 70
-
 Methanocaldococcus jannaschii
2.3.3.21 30 70 directed evolution of CimA enhances the specific activity over a wide temperature range Methanocaldococcus jannaschii

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
2.3.3.21 0.36
-
 acetyl-CoA wild-type Methanocaldococcus jannaschii
2.3.3.21 0.36
-
 pyruvate wild-type Methanocaldococcus jannaschii
2.3.3.21 0.36
-
 pyruvate wild type enzyme Methanocaldococcus jannaschii
2.3.3.21 0.84
-
 acetyl-CoA I47V/E114V/H126Q/T204A/L238S mutant Methanocaldococcus jannaschii
2.3.3.21 0.84
-
 pyruvate I47V/E114V/H126Q/T204A/L238S mutant Methanocaldococcus jannaschii
2.3.3.21 0.84
-
 pyruvate mutant CimA3.7 Methanocaldococcus jannaschii

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
2.3.3.21 7.5
-
 citrat malate synthase assay Methanocaldococcus jannaschii