Aryl alcohol oxidase (AAO) is an enzyme containing flavin-adenine-dinucleotide (FAD). This is the version with SpyTag. In order to constructed our multi-enzyme complex, we introduced the SpyTag/SpyCatcher system. We added SpyTag to the N-terminus of each protein through 10 × ELP, which is a oligopeptide linker that does not affect the function of the protein it attaches to, as described in the literature[1]. So this part can be combined with dCas9-SpyCatcher through covalent isopeptide action, thereby being immobilized on dsDNA. His-tag was added to purify the protein. We use BBa_K3853053 to construct the expression system to express and purify the protein.
Biology
Aryl alcohol oxidase (AAO, EC 1.1.3.7), a member of the glucose-methanol-choline oxidase / dehydrogenase (GMC) superfamily, is an enzyme containing flavin-adenine-dinucleotide (FAD) that catalyze the oxidation of aromatic and aliphatic allylic primary alcohols to the corresponding aldehydes while reducing molecular oxygen to H2O2(The mechanism has shown in Fig. 1).
Fig. 1 H2O2 production mechanism of AAO.
Usage
The SpyTag was fused to the N-terminus of AAO. The fusion protein could combined with dCas9-SpyCatcher (which is producted by BBa_K3853055 ) for multi-enzyme complex assembly. we obtained the composite part BBa_K3853052 (Fig. 2) and transformed the constructed plasmid into Pichia pastoris GS115 to verify its expression. The positive clones were cultivated.
Fig. 2 Gene circuit of sfGFP of AAO.
Characterization
1. Identification
The synthetic plasmid was linearized and electrotransformed into Pichia pastoris GS115. Colony PCR was applied to screen monoclonal colonies that had positive transformation results for subsequent gene sequencing verification. The target bands appeared in the normal position, and the result was shown in Fig. 3. Sequencing results (File 1) showed a successful transformation.
Fig. 3 Agarose gel electrophoresis of PCR products of monoclonal colonies of SpyTag-AAO.
2. qRT-PCR
For assaying the mRNA expression level of AAO, qRT-PCRs were performed. After methanol induction for 60 h, samples containing AAO-expressing strain were taken. We did data analysis using a variation of the Livak method. To determine the relative expression level of SpyTag-AAO vs. reference gene ACT1, total RNA was extracted from samples containing equal wet weight of recombinant P.pastoris. The CT values for SpyTag-AAO and the reference gene ACT1 were then used to calculate the fold difference:
The results were shown inFig. 4. The gene of SpyTag-AAO was normally transcribed in the cell.
Fig. 4 RFU change curve with cycle number.
3. Enzyme Activity
Enzyme activity of AAO was determined using veratryl alcohol as substrate, and to continuously detect the formation of the product veratraldehyde (ε310 = 9300 M-1·cm-1) at 310 nm [2] through a 96-well microplate reader. The enzyme activity test should begin immediately after preparing the reaction system. Purified SpyTag-AAO had high degrees of activity outside the living organism (Fig. 5), which proves that AAO works well.
Fig. 5 The detection of veratraldehyde reflected as the changes of A310 in 1 min.
References
[1] Lim, S., Kim, J., Kim, Y., Xu, D. & Clark, D. S. CRISPR/Cas-directed programmable assembly of multi-enzyme complexes. Chemical communications (Cambridge, England)56, 4950-4953, doi:10.1039/d0cc01174f (2020).
[2] Jankowski, N., Koschorreck, K. & Urlacher, V. B. High-level expression of aryl-alcohol oxidase 2 from Pleurotus eryngii in Pichia pastoris for production of fragrances and bioactive precursors. Applied microbiology and biotechnology104, 9205-9218, doi:10.1007/s00253-020-10878-4 (2020).
Sequence and Features
Assembly Compatibility:
10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 699
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 412 Illegal BamHI site found at 1411 Illegal BamHI site found at 1798
