Part:BBa_K4927044
A2
We constructed a methanol dehydrogenase labeled with CL2 (BBa_K4927004). 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
Methanol dehydrogenase (MDH) is divided into 3 types, namely PQQ dependent MDH, oxygen-dependent alcohol oxidase (AOX), and NAD+ dependent MDH. PQQ-ependent MDH is a kind of MDH that has been well studied and is located in the peritroplasmic region of Gram-negative methyltrophic bacteria.
It is mainly divided into MxaFI and XoxF. Among them, the structure and function of MxaFI type MDH have been extensively studied. Oxygen-dependent alcohol oxidase (AOX) is a special enzyme of methylotrophic yeast that catalyzes the oxidation of methanol to formaldehyde. Similar to PQQ-dependent MDH, AOX also needs to function under aerobic conditions. It uses oxygen as an electron acceptor to oxidize methanol to formaldehyde and hydrogen peroxide. The toxic hydrogen peroxide is then decomposed into O2 and H2O by catalase. Compared with other types of MDH, AOX has the lowest Gibbs free energy and the highest methanol oxidation efficiency. NAD+ dependent MDH is commonly found in thermophilic Gram-positive bacteria. This enzyme is located in the cytoplasm and uses NAD+ as a cofactor. Compared with PQQ-dependent MDH, it has a simple structure and only requires the participation of one gene to catalyze the entire oxidation reaction.
Uses and Biology
We selected methanol dehydrogenase (ADH) derived from Stearothermophilus to obtain the whole genome of the strain from NCBI, which was synthesized by Wuhan Jinkailui Company, and then PCR was used to obtain the ADH fragment of the target gene by corresponding bidirectional primers. The expression plasmid PET28A-Cl2-ADH (2A) and PET28A-ADH-CL2 (A2) were formed by split-connecting CL2(BBa_K4927004) to the N-terminal and C-terminal of ADH through a T5 exonuclidenase mediated linkage system, and then assembled with the vector pET28a.
Figure 1. (A)CL2 protein is fused to the C terminal of methanol dehydrogenase; (B)CL2 protein fuses at the N terminal of methanol dehydrogenase
We will use methanol and NAD+ as substrates to generate formaldehyde and NADH through the catalytic reaction of methanol dehydrogenase. NADH will produce hydrogen gas through hydrogenase in the final step of our reaction.
Characterization
We transferred the constructed pET28a-CL2-ADH (2A) and pET28a-ADH-CL2 (A2) expression plasmids into the receptive state BL21, and after 8h expansion culture, 0.1Mm IPTG was used to induce 16h. The target protein was obtained by crushing the bacteria at low temperature and high pressure, and purified by nickel column, and the activities of the two enzymes were tested.Finally, we found that the activity of A2 was significantly higher than 2A under the same conditions, so we chose A2 in the subsequent experiment.
Figure 2. (A) 2A SDS-PAGE test results; (B)A2 SDS-PAGE test result. The red arrow marks the target protein
Figure 2. (A) 2A activity test results; (B)A2 activity test results"
References
[1] Keltjens JT, Pol A, Reimann J, et al. PQQ-dependent methanol dehydrogenases: rare-earth elements make a difference. Appl Microbiol Biotechnol, 2014, 98(14): 6163-6183.
[2] Zhang WM, Zhang T, Wu SH, et al. Guidance for engineering of synthetic methylotrophy based on methanol metabolism in methylotrophy. RSC Adv, 2017, 7(7): 4083-4091.
[3] Whitaker WB, Sandoval NR, Bennett RK, et al. Synthetic methylotrophy: engineering the production of biofuels and chemicals based on the biology of aerobic methanol utilization. Curr Opin Biotechnol, 2015, 33: 165-175.
[4] Krog A, Heggeset TMB, Muller JEN, et al. Methylotrophic Bacillus methanolicus encodes two chromosomal and one plasmid born NAD+ dependent methanol dehydrogenase paralogs with different catalytic and biochemical properties. PLoS ONE, 2013, 8(3): e59188.
[5] Fan LW, Wang Y, Zheng P, et al. Methanol dehydrogenase, a key enzyme of one-carbon metabolism: a review. Chinese Journal of Biotechnology, 2021, 37(2): 530-540.
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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