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.

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.

Figure 2. Identification of BFD-M7 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.

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