Part:BBa_K3209000

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.

Experiment Results:

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

Figure 2. Identification of BDD-M7 and TalB-F187Y 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.

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.

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