Difference between revisions of "Part:BBa K4711019"

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It was observed that the protein secreted by Escherichia coli BL21 cells, after purification and processing, exhibited a single and clear band on the gel electrophoresis, with a size ranging from 45 kDa to 60 kDa. The OleTJE P450 enzyme used in this study has a size of 50.53 kDa, which aligns well with the position of the band shown on the protein gel.
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Fig 3 (A) Gene circuit (B) SDS-PAGE result image
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Due to the production of medium-chain fatty acids by MEL, with substrate chain lengths ranging from C8-12, we initially determined the in vitro activity of the P450 fatty acid decarboxylase OleTJE through a recombinant in vitro reaction. We conducted experiments using various fatty acids, including C8:0, C9:0, C10:0, C11:0, C12:0, C12:1, and C14:0, as substrates, with the addition of 0.2 μM of the P450 fatty acid decarboxylase OleTJE. The reaction was carried out for two hours, and by measuring the changes in substrate content, we determined whether the purified P450 fatty acid decarboxylase OleTJE possesses the ability to catalyze medium-chain fatty acid substrates in vitro. As a control group, we used C14:0 palmitic acid substrate to confirm the protein activity of P450 fatty acid decarboxylase OleTJE.
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Fig 4 Conversion rate of fatty acid substrates catalyzed by P450 enzyme
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Using the MEL obtained from the experiments described in this study, we prepared medium-chain fatty acids as substrates for the reaction. The reaction system included 0.5 μM of the enzyme, 200 μM of medium-chain fatty acids, and 500 μM of H2O2. The reaction was carried out at 28°C for 2 hours. The products obtained from the reaction were analyzed using GC-MS. Based on the GC-MS analysis results, the actual conversion rates of α-olefins were calculated. For each type of fatty acid in the fatty acid composition, the corresponding content of α-olefins obtained from the measurements was used as the numerator, with the content of each fatty acid type as the denominator, to calculate the actual conversion rate of α-olefins for each medium-chain fatty acid.
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Fig 5 Conversion yield of α-olefins
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Latest revision as of 07:47, 10 October 2023


OleTJE P450

Usage and Biology

We utilize the OleTJE P450 enzyme to catalyze the single-step decarboxylation of fatty acids to produce α-olefins, which is a relatively simple approach in the biosynthesis of α-olefins. It directly uses free fatty acids as substrates to synthesize α-olefins in one step. This process is simple and easy to control, making it highly regarded for its potential in designing biological systems for α-olefin production. The degradation mechanism is shown in the diagram below:

Fig 1 P450 catalytic process and synthesis of α-olefin

Fig 2 The 3D structure of GDH

It was observed that the protein secreted by Escherichia coli BL21 cells, after purification and processing, exhibited a single and clear band on the gel electrophoresis, with a size ranging from 45 kDa to 60 kDa. The OleTJE P450 enzyme used in this study has a size of 50.53 kDa, which aligns well with the position of the band shown on the protein gel.

Fig 3 (A) Gene circuit (B) SDS-PAGE result image

Due to the production of medium-chain fatty acids by MEL, with substrate chain lengths ranging from C8-12, we initially determined the in vitro activity of the P450 fatty acid decarboxylase OleTJE through a recombinant in vitro reaction. We conducted experiments using various fatty acids, including C8:0, C9:0, C10:0, C11:0, C12:0, C12:1, and C14:0, as substrates, with the addition of 0.2 μM of the P450 fatty acid decarboxylase OleTJE. The reaction was carried out for two hours, and by measuring the changes in substrate content, we determined whether the purified P450 fatty acid decarboxylase OleTJE possesses the ability to catalyze medium-chain fatty acid substrates in vitro. As a control group, we used C14:0 palmitic acid substrate to confirm the protein activity of P450 fatty acid decarboxylase OleTJE.

Fig 4 Conversion rate of fatty acid substrates catalyzed by P450 enzyme


Using the MEL obtained from the experiments described in this study, we prepared medium-chain fatty acids as substrates for the reaction. The reaction system included 0.5 μM of the enzyme, 200 μM of medium-chain fatty acids, and 500 μM of H2O2. The reaction was carried out at 28°C for 2 hours. The products obtained from the reaction were analyzed using GC-MS. Based on the GC-MS analysis results, the actual conversion rates of α-olefins were calculated. For each type of fatty acid in the fatty acid composition, the corresponding content of α-olefins obtained from the measurements was used as the numerator, with the content of each fatty acid type as the denominator, to calculate the actual conversion rate of α-olefins for each medium-chain fatty acid.

Fig 5 Conversion yield of α-olefins



Source

Jeotgalicoccus sp. 8456 bacterium

References

[1]Belcher J, McLean KJ, Matthews S, Woodward LS, Fisher K, Rigby SEJ, Nelson DR, Potts D, Baynham MT, Parker DA, Leys D, Munro AW. Structure and biochemical properties of the alkene producing cytochrome P450 OleTJE (CYP152L1) from the Jeotgalicoccus sp. 8456 bacterium. J Biol Chem. 2014 Mar 7;289(10):6535-6550. doi: 10.1074/jbc.M113.527325. Epub 2014 Jan 18. PMID: 24443585; PMCID: PMC3945318.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1003
    Illegal PstI site found at 932
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1003
    Illegal PstI site found at 932
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1003
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1003
    Illegal PstI site found at 932
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1003
    Illegal PstI site found at 932
  • 1000
    COMPATIBLE WITH RFC[1000]