Team:JNFLS/Design
Project Design:
1.The xylulose synthesis pathway from formaldehyde
To construct the key pathway for converting formaldehyde to xylulose, two enzymes are involved, Benzoylformate decarboxylase (BFD) mutant (from Pseudomonas putida) and transaldolase (TalB) mutant (from Escherichia coli). First, formaldehyde is condensed to glycolaldehyde by BFD mutant(BFD-M7), consuming two molecules of formaldehyde and therefore glycolaldehyde contains 2 carbon atoms. Subsequently, the formaldehyde and glycolaldehyde are converted to dihydroxyacetone (DHA) containing 3 carbons, catalyzed by another active site of BFD-M7, which contains 7 amino acids mutations (W86R-N87T-L109G-L110E-H281V-Q282F-A460M). In the following reaction, TalB mutant (TalB-F178Y) catalyzes glycolaldehyde and DHA to form xylulose which is a pentose sugar. This pathway can not only eliminate formaldehyde indoors, but also generate xylulose with formaldehyde.
Figure 1. The BFD mutant (BFD-M7) catalyzes the synthesis of glycolaldehyde and dihydroxyacetone (DHA) from formaldehyde.
Figure 2. Xylulose synthesis catalyzed by TalB-F187Y, using glycolaldehyde and dihydroxyacetone (DHA).
2.The construction and expression of BFD-M7 and TalB-F187Y vectors
(1) In order to find the best promoter to express BFD-M7 and TalB-F187Y, we constructed 3 GFP generators using different promoters to detect the one with best efficiency under the existence of formaldehyde. Two promoters (PfrmR and PhxlR) are induced by formaldehyde, and one promoter (Plac) is induced under IPTG existence. In this way, the promoters can be controlled by formaldehyde or IPTG.
Figure 3. Using GFP as reporter to detect the best promoter.
(2) Next, we set experiments to detect the best concentration of formaldehyde or IPTG for inducing expression. Also, we need to know the best original OD600 at which the inducer are added to the culture medium.
(3) To further check out the best way to express the enzymes we require, the arrangement of BFD-M7 and TalB-F187Y genes would be taken into consider. Distinguishing check the expression vectors with single BFD-M7 or TalB-F187Y, and also to investigate the level of expression the hybrid ones with both of the genes.
3.The Purification and activity test of expressed protein
Purification and activity test for BFD-M7 and TalB-F187Y is important step in our project. We use the plasmid pET-28a as the vector containing the his tag, which is convenient to purify the expressed protein. HPLC (High performance Liquid Chromatography) is used to test production catalyzed by the two enzymes, indicating the enzyme activity.
Figure 4. The pET-28a vector map containing BFD-M7 and TalB-F187Y.
Hardware Design and Application:
1.The demand for a device collecting formaldehyde
Since our project aim at both eliminating formaldehyde indoors and converting it to xylulose, we need to construct a device to collect formaldehyde indoors used for xylusose production. So the device that could capture more polluted air with higher density of formaldehyde had become a significant challenge. In order to put it into practice, among with our past ideas, we finally came out with this machine (Figure 5).
Figure 5. The real machine we constructed to collect formaldehyde indoors.
2.The principle of this machine
Due to its special characteristics, formaldehyde is soluble in water, we decide to use this feature to absorb formaldehyde through the interaction with water. The principle of this machine is to pump the water from the tank and transport through the filings to the tank, while to collect the air with formaldehyde and pump it upward though the fillings to get it attached with water. After that, formaldehyde in the air will dissolve in the water and the solution will be collected into the tank below, where the enzyme catalyzed reaction will take place.
Figure 6. The mechanism skeleton of real machine collecting formaldehyde indoors (longitudinal section).
3.How to use this machine?
(1)While using, turn on the pump, blower and open the cork.
(2)put the system in use for 1 hour to absorb the formaldehyde in door. The gas will rise through the fillings, while the water will drop from the top of the machine. They will encounter at the fillings. As a result, the time of reacting will be prolonged.
(3)After absorbing, turn off the pump and the blower. Cork the machine to avoid the volatilization of the formaldehyde collected.
(4)Wait for the system to take reactions to transform the formaldehyde.
Notice:
(1)The time for absorption can be adjusted due to the situation and the amount of pollution.
(2)If the assimilation is prolonged, the same period of time should be prolonged in the transversion process.
(3)If there is needed to add or exchange the water, check the height of the water through the sight glass and add water through the hole.
Figure 7. The mechanism skeleton of real machine collecting formaldehyde indoors (outside view).
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.