Team:JNFLS/Results
Results:
Our project intended to produce xylulose while removing formaldehyde indoors. In this process, two main genes are involved in our project, they are BBa_K3209000 and BBa_K3209002 parts which can express 2 important enzymes: BFD-M7 and TalB-F178Y.
BFD-M7 is Benzoylformate decarboxylase mutant 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.
TalB-F178Y is transaldolase mutant 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 1. The pathway of xylulose synthesis from formaldehyde, including the synthesis of Glycolaldehyde and Dihydroxyacetone (DHA) from formaldehyde, catalyzed by BFD-M7, and the xylulose production, catalyzed by TalB-F187Y.
In order to achieve these two enzymes, we constructed the enzymes expression vectors: pET-28a-BFD-M7 and pET-28a-TalB-F187Y. And we identified these plasmids using electrophoresis, the results showed as Fig.2.
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.
We also expressed these two enzymes in E.coli BL21, and purified them using Ni-NTA affinity chromatography. Results showed in Fig.3.
Figure 3. Expression of BFD-M7and TalB-F187Y enzymes, and purified by Ni-NTA affinity chromatography. A. BFD-M7 expression and purification; B. TalB-F187Y expression and purification.
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.
After success of expression and purification of BFD-M7and TalB-F187Y, we prepared the reaction system in which glycolaldehyde and dihydroxyacetone (DHA) from formaldehyde, followed by xylulose generation. HPLC was used to analyze the products catalyzed by BFD-M7and TalB-F187Y. Results showed in figure 4 and figure 5.
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.
Figure 5. 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.