Team:JNFLS/Basic Parts
Basic Parts:
In 2019 iGEM year, we created 5 basic parts which are involved in the metabolic pathway of formaldehyde. Here we are going to focus on 2 parts, which can catalyze the xylulose synthesis form formaldehyde.
I. BBa_K3209000:
It 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 both Glycolaldehyde and Dihydroxyacetone (DHA), using formadehyde.
Figure 2. Identification of BDD-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.
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.
II. BBa_K3209002:
This transaldolase mutant (TalB-F178Y) is derived from E.coli, which is different from the part BBa_K2155003, containing mutation F178Y. It catalyzes xylulose synthesis from glycolaldehyde and dihydroxyacetone (DHA). Along with the BFD-M7, TalB-F178Y catalyzes the xylulose production which is a precursor of some rare sugars.
Figure 5. The synthesis of xylulose from Glycolaldehyde and Dihydroxyacetone, catalyzed by TalB-F187Y.
Figure 6. Expression of TalB-F287Y 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. The results showed that 200 mM imidazole eluent is the best concentration for elution expressed TalB-F187Y.
Figure 7. HPLC analysis of the xylulose product catalyzed by TalB-F187Y. A: Standard Xylulose; B: The xylulose product catalyzed by TalB-F187Y in vitro. The results showed that our expression vector expressed active TalB-F187Y, which catalyzed the synthesis of xylulose from 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.