Part:BBa_K4927052
M8
We constructed a Formate dehydrogenase Dehydrogenase labeled with CL8 (BBa_K4927006). It will form an artificial multi enzyme complex with a protein scaffold designed by us to improve the reaction efficiency of the entire system.
Introduction
Formate dehydrogenase can be divided into 2 categories[1]: the first type of enzyme protein contains metal ions, such as the active center of tungsten, iron, sulfur, molybdenum, selenium, such as FDH in the protein structure, molecular weight and stability there are great differences; The second category is NAD+ dependent FDH (EC 1.17.1.9) and NADP+ dependent FDH, this type of enzyme does not contain metal ions, can oxidize formate for CO2, while NAD(P)+ reduced to NAD(P)H, is the most important class of FDH. At present, FDH has been found in a variety of organisms, such as bacteria[2], fungi [3-4]and so on.
Usage and Biology
We selected formate dehydrogenase (FDH) derived from Methylomonas koyama and obtained the target gene fragment from NCBI, which was synthesized by Wuhan Jinkairui Company. Then, through the plasmid construction system[5] mediated by T5 nuclease, CL8 was fused with the 3 'end of M.koFDH gene to construct the pET28a-M.ko-FDH-CL8 expression plasmid.
Figure1: pET28a-M.ko-FDH-CL8 plasmid map
M.koFDH will use formaldehyde and NAD+ as substrates to generate NADH and carbon dioxide through catalytic oxidation. NADH will produce hydrogen gas in the final step of the reaction we designed, with the action of hydrogenase.
Figure2: M8 Genetic circuit
Characterization
We transferred the constructed pET28a-M.ko-FDH-CL8 (M8) expression plasmid into the receptive state BL21, and after 8h expansion culture, 0.5mM IPTG was used to induce 16h. Formate dehydrogenase (M8) was obtained by crushing the bacteria at low temperature and high pressure, purification by nickel column.
Figure3: M8 SDS-PAGE Test result. Lane 1 is the mixed liquid after breaking bacteria, lane 2 is the supernatant after breaking bacteria, lane 3 is the precipitation after breaking bacteria , lane 4 is the flow-through liquid, lane 5 is 10mM imidazole eluent, lane 6 is 10mM imidazole eluent, lane 7 is 50mM imidazole eluent, lane 8 is 80mM imidazole eluent, lane 9 is 100mM imidazole eluent, and lane 10 is 150mM imidazole eluent,lane 11 is 300mM imidazole eluent,lane 12 is 600mM imidazole eluent,lane 13 is marker. The red arrow marks the target protein band
We investigated the activity of M8 at different PH values by UV spectrophotometer at room temperature, and recorded the change of light absorption value at 340nm. We found that 8F showed the highest activity at PH=6.43.
Figure 4:Activity test results of M8 under different pH conditions
In addition, we also compared the activity of two designed methanol dehydrogenase enzymes M8 and 8F (BBa_K4297050). 8F has the best activity at pH=5.59, and M8 has the best activity at pH=6.43. But after reacting with the substrate for 60 seconds, the absorbance change at 340nm of 8F is about four times that of M8. Therefore, in subsequent experiments, we used 8F.
Figure 5:Comparison results of optimal activity between M8 and 8F
References
[1] Tishkov VI, Popov VO. Catalytic mechanism and application of formate dehydrogenase. Biochemistry (Mosc), 2004, 69(11): 1252-1267.
[2] Galkin A, Kulakova L, Tishkov V, et al. Cloning of formate dehydrogenase gene from a methanol-utilizing bacterium Mycobacterium vaccae N10. Appl Microbiol Biotechnol, 1995, 44(3/4): 479-483.
[3] Chow CM, RajBhandary UL. Developmental regulation of the gene for formate dehydrogenase in Neurospora crassa. J Bacteriol, 1993, 175(12): 3703-3709.
[4] Allen SJ, Holbrook JJ. Isolation, sequence and overexpression of the gene encoding NAD-dependent formate dehydrogenase from the methylotrophic yeast Candida methylica. Gene, 1995, 162(1): 99-104.
[5]Yongzhen Xia, Kai Li, Jingjing Li, Tianqi Wang, Lichuan Gu, Luying Xun, T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis, Nucleic Acids Research, Volume 47, Issue 3, 20 February 2019, Page e15.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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