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Literature summary for 2.3.3.21 extracted from
- 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.
Application
| Application | Comment | Organism |
|---|---|---|
| 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 |
| energy production | the citratmalate pathway is enhanced for the production of 1-propanol and 1-butanol | Methanocaldococcus jannaschii |
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| expressed in Escherichia coli | Methanocaldococcus jannaschii |
| expression in Escherichia coli BL21 Star DE3 cells | Methanocaldococcus jannaschii |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| I47V/D86G/H126Q/T204A | CimA3.5, mutant generated by directed evolution | Methanocaldococcus jannaschii |
| 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 |
| I47V/E111K/E121V/H126Q/T204A/M250V | CimA3.8, mutant generated by directed evolution | Methanocaldococcus jannaschii |
| 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 |
| I47V/E114V/H126Q/T204A/L238S | CimA3.7, mutant generated by directed evolution | Methanocaldococcus jannaschii |
| 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 |
| I47V/H126Q/D141E/T204A/I286V/L327H | CimA3.4, mutant generated by directed evolution | Methanocaldococcus jannaschii |
| 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 |
| I47V/H126Q/E183K/T204A/L253S | CimA3.2, mutant generated by directed evolution | Methanocaldococcus jannaschii |
| 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 |
| I47V/H126Q/T204A | CimA2delta, mutant generated by directed evolution | Methanocaldococcus jannaschii |
| 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 |
| I47V/H126Q/T204A/D328V | CimA3.6, mutant generated by directed evolution | Methanocaldococcus jannaschii |
| 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 |
| I47V/H126Q/T204A/K265R/F349C | CimA3.3, mutant generated by directed evolution | Methanocaldococcus jannaschii |
| 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 |
| 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 |
| I47V/H126Q/T204A/V373STOP | CimA2, mutant generated by directed evolution | Methanocaldococcus jannaschii |
| 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 |
| I47V/K435N/V441A | CimA1, mutant generated by directed evolution | Methanocaldococcus jannaschii |
| I47V/R53S/H126Q/T204A | CimA3.1, mutant generated by directed evolution | Methanocaldococcus jannaschii |
| 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 |
| K32N/I47V/H126Q/T204A | CimA3.9, mutant generated by directed evolution | Methanocaldococcus jannaschii |
| 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 |
| 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 |
| WTDELTA | wild-type delta, mutant generated by directed evolution | Methanocaldococcus jannaschii |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.105 | - |
acetyl-CoA | I47V/E114V/H126Q/T204A/L238S mutant | Methanocaldococcus jannaschii |  |
| 0.105 | - |
acetyl-CoA | mutant CimA3.7 | Methanocaldococcus jannaschii | |
| 0.184 | - |
pyruvate | wild-type | Methanocaldococcus jannaschii | |
| 0.184 | - |
pyruvate | wild type enzyme | Methanocaldococcus jannaschii | |
| 0.303 | - |
acetyl-CoA | wild-type | Methanocaldococcus jannaschii | |
| 0.303 | - |
acetyl-CoA | wild type enzyme | Methanocaldococcus jannaschii | |
| 0.342 | - |
pyruvate | I47V/E114V/H126Q/T204A/L238S mutant | Methanocaldococcus jannaschii | |
| 0.342 | - |
pyruvate | mutant CimA3.7 | Methanocaldococcus jannaschii | |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| (2S)-2-hydroxy-2-methylbutanedioate | Methanocaldococcus jannaschii | - |
acetate + pyruvate | - |
r | |
| (2S)-2-hydroxy-2-methylbutanedioate | Methanocaldococcus jannaschii BW25113 | - |
acetate + pyruvate | - |
r | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Methanocaldococcus jannaschii | - |
- |
- |
| Methanocaldococcus jannaschii | - |
and JCL16, KS145, SA405, SA408 | - |
| Methanocaldococcus jannaschii BW25113 | - |
and JCL16, KS145, SA405, SA408 | - |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| by using Ni-nitrilotriacetic spin columns | Methanocaldococcus jannaschii |
| with Ni-nitrilotriacetic acid spin columns | Methanocaldococcus jannaschii |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| (2S)-2-hydroxy-2-methylbutanedioate | - |
Methanocaldococcus jannaschii | acetate + pyruvate | - |
r | |
| (2S)-2-hydroxy-2-methylbutanedioate | - |
Methanocaldococcus jannaschii BW25113 | acetate + pyruvate | - |
r | |
| acetyl-CoA + pyruvate + H2O | - |
Methanocaldococcus jannaschii | CoA + (R)-citramalate | - |
? | |
| acetyl-CoA + pyruvate + H2O | - |
Methanocaldococcus jannaschii BW25113 | CoA + (R)-citramalate | - |
? | |
| acetyl-CoA + pyruvate + H2O | - |
Methanocaldococcus jannaschii | CoA + (2R)-2-hydroxy-2-methyl-butanedioate | - |
? | |
| acetyl-CoA + pyruvate + H2O | - |
Methanocaldococcus jannaschii BW25113 | CoA + (2R)-2-hydroxy-2-methyl-butanedioate | - |
? | |
| acetyl-CoA + pyruvate + H2O | - |
Methanocaldococcus jannaschii | (2S)-2-hydroxy-2-methylbutanedioate + CoA | - |
r | |
| acetyl-CoA + pyruvate + H2O | - |
Methanocaldococcus jannaschii BW25113 | (2S)-2-hydroxy-2-methylbutanedioate + CoA | - |
r | |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| CimA | - |
Methanocaldococcus jannaschii |
| citramalate lyase | - |
Methanocaldococcus jannaschii |
| citramalate synthase | - |
Methanocaldococcus jannaschii |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 30 | - |
- |
Methanocaldococcus jannaschii |
Temperature Range [°C]
| Temperature Minimum [°C] | Temperature Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 30 | 70 | - |
Methanocaldococcus jannaschii |
| 30 | 70 | directed evolution of CimA enhances the specific activity over a wide temperature range | Methanocaldococcus jannaschii |
Turnover Number [1/s]
| Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.36 | - |
acetyl-CoA | wild-type | Methanocaldococcus jannaschii | |
| 0.36 | - |
pyruvate | wild-type | Methanocaldococcus jannaschii | |
| 0.36 | - |
pyruvate | wild type enzyme | Methanocaldococcus jannaschii | |
| 0.84 | - |
acetyl-CoA | I47V/E114V/H126Q/T204A/L238S mutant | Methanocaldococcus jannaschii | |
| 0.84 | - |
pyruvate | I47V/E114V/H126Q/T204A/L238S mutant | Methanocaldococcus jannaschii | |
| 0.84 | - |
pyruvate | mutant CimA3.7 | Methanocaldococcus jannaschii | |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 7.5 | - |
citrat malate synthase assay | Methanocaldococcus jannaschii |
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