Difference between revisions of "Part:BBa K3209000"

 
 
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<partinfo>BBa_K3209000 short</partinfo>
 
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This is benzoylformate decarboxylase mutant(BFD-M7) from Pseudomonas putida. It contains 7 amino acids mutations, which is different from BFD (BBa_K2155001). The mutation information of BFD-M7 is as followings: W86R, N87T, L109G, L110E, H281V, Q282F and A460M. It can catalyze glycolaldehyde formation from formaldehye, then the glycolaldehyde reacts with formaldehyde to form dihydroxy acetone, also catalyzed by this enzyme.
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This is benzoylformate decarboxylase mutant (BFD-M7) from Pseudomonas putida. It contains 7 amino acids mutations, which sequence is different from BFD (BBa_K2155001). The mutation information of BFD-M7 is as follows: W86R, N87T, L109G, L110E, H281V, Q282F and A460M. BFD-M7 can catalyze glycolaldehyde synthesis from formaldehye, then the glycolaldehyde reacts with formaldehyde to form dihydroxyacetone (DHA), also catalyzed by this enzyme.
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[[File:K3209000-1-1.jpg|center]]
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Figure 1. The synthesis of Glycolaldehyde and Dihydroxyacetone (DHA) from formaldehyde, catalyzed by BFD-M7. This enzyme can catalyze the synthesis of two compounds Glycolaldehyde and Dihydroxyacetone, using formadehyde.
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[[File:K3209000-2-1.jpg|center]]
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Figure 2. Identification of BFD-M7 (BBa_K3209000) and TalB-F187Y (BBa_K3209002) expression vectors construction.
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1:BFD-M7 plasmid; 2:Digestion of BFD-M7 plasmid by EcoRI and PstI; 3:TalB-F187Y plasmid; 4:Digestion of TalB-F187Y plasmid by EcoRI and PstI; M: Marker.
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[[File:K3209000-3-1.jpg|center]]
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Figure 3. Expression of BFD-M7 and purification by Ni-NTA affinity chromatography.
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M:protein marker; 1:precipitation samples in the cell lysates; 2:supernatant samples in the cell lysates; 3:50 mM imidazole eluent; 4:100 mM imidazole eluent; 5:200 mM imidazole eluent. This figure showed that 200mM imidazole eluent is the best concentration for elution expressed BFD-M7.
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[[File:K3209000-4-1.jpg|center]]
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Figure 4. HPLC analysis of the products catalyzed by BFD-M7.
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A: Standard glycolaldehyde; B: Standard DHA; C: The products catalyzed by BFD-M7 in vitro. The results showed that our expression vector expressed active BFD-M7, which catalyzed the synthesis of glycolaldehyde and DHA.
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==References:==
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# Bo Cui, Bingzhao Zhuo, Xiaoyun Lu, et al. Enzymatic synthesis of xylulose from formaldehyde. Chinese Journal of Biotechnology,2018, 34(7): 1128-1136.
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# Xiaoyun Lu, Yuwan Liu, Yiqun Yang, et al. Constructing a synthetic pathway for acetyl coenzyme A from one-carbon through enzyme design. Nature communications, 2019 Mar 26;10(1):1378.
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Latest revision as of 00:21, 21 October 2019


Benzoylformate decarboxylase mutant (BFD-M7)

This is benzoylformate decarboxylase mutant (BFD-M7) from Pseudomonas putida. It contains 7 amino acids mutations, which sequence is different from BFD (BBa_K2155001). The mutation information of BFD-M7 is as follows: W86R, N87T, L109G, L110E, H281V, Q282F and A460M. BFD-M7 can catalyze glycolaldehyde synthesis from formaldehye, then the glycolaldehyde reacts with formaldehyde to form dihydroxyacetone (DHA), also catalyzed by this enzyme.

K3209000-1-1.jpg

Figure 1. The synthesis of Glycolaldehyde and Dihydroxyacetone (DHA) from formaldehyde, catalyzed by BFD-M7. This enzyme can catalyze the synthesis of two compounds Glycolaldehyde and Dihydroxyacetone, using formadehyde.



K3209000-2-1.jpg

Figure 2. Identification of BFD-M7 (BBa_K3209000) and TalB-F187Y (BBa_K3209002) expression vectors construction. 1:BFD-M7 plasmid; 2:Digestion of BFD-M7 plasmid by EcoRI and PstI; 3:TalB-F187Y plasmid; 4:Digestion of TalB-F187Y plasmid by EcoRI and PstI; M: Marker.



K3209000-3-1.jpg

Figure 3. Expression of BFD-M7 and purification by Ni-NTA affinity chromatography. M:protein marker; 1:precipitation samples in the cell lysates; 2:supernatant samples in the cell lysates; 3:50 mM imidazole eluent; 4:100 mM imidazole eluent; 5:200 mM imidazole eluent. This figure showed that 200mM imidazole eluent is the best concentration for elution expressed BFD-M7.


K3209000-4-1.jpg

Figure 4. HPLC analysis of the products catalyzed by BFD-M7. A: Standard glycolaldehyde; B: Standard DHA; C: The products catalyzed by BFD-M7 in vitro. The results showed that our expression vector expressed active BFD-M7, which catalyzed the synthesis of glycolaldehyde and DHA.

References:

  1. Bo Cui, Bingzhao Zhuo, Xiaoyun Lu, et al. Enzymatic synthesis of xylulose from formaldehyde. Chinese Journal of Biotechnology,2018, 34(7): 1128-1136.
  2. Xiaoyun Lu, Yuwan Liu, Yiqun Yang, et al. Constructing a synthetic pathway for acetyl coenzyme A from one-carbon through enzyme design. Nature communications, 2019 Mar 26;10(1):1378.



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 771
    Illegal NgoMIV site found at 1233
    Illegal AgeI site found at 235
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 340