Part:BBa_K4940001
P450BM-3 QM-his
P450BM-3 QM is a coding sequence of a type of Cytochrome P450 monooxygenases, which contains a quintuple mutant (V26T/R47F/A74G/F87V/L188K). In our projects, the enzyme can degrade α-pinene to α-pinene oxidation in a NADPH-dependent manner.
Usage and Biology
In contrast to other bacterial P450 monooxygenases, the catalytically self-sufficient cytosolic P450 monooxygenase CYP102A1 from Bacillus megaterium (P450BM-3) contains both, an amino-terminal heme domain and a carboxy-terminal flavin reductase in a single polypeptide. The P450BM-3 wild-type enzyme naturally catalyzes subterminal hydroxylation of saturated long-chain fatty acids up to reaction rates which are the highest reported for P450s (turnover frequencies >1,000 min−1). Furthermore, the enzyme, which has a wide substrate spectrum, has been shown to be a very suitable candidate for rational and evolutionary engineering approaches by which its substrate spectrum has been shifted towards a variety of other compounds, e.g., more hydrophobic ones, such as aromatics and terpenoids (1). A quintuple mutant (V26T/R47F/A74G/F87V/L188K) of P450BM-3 from B. megaterium (P450BM-3 QM) was used to oxidize α-pinene, which can generate α-pinene oxide, verbenol, and myrtenol in a ratio of 5:2:1 (2).
Plasmid design and construction
We inserted P450BM-3 QM and GlcDH-II into pCDFDUET-1 vector to form pCDFDUET-1 p450bm-3qm glcdh, achieving efficient α-pinene oxidation.
Figure 1. The construction of plasmid pCDFDUET-1 p450bm-3qm glcdh.
Cultivation, Purification and SDS-PAGE
The recombinant E.coli was cultured and induced by IPTG to allow GLF protein expression. Since the protein was fused to a his tag, we purified the protein via affinity chromatography. The protein samples were tested by SDS-PAGE. We have already confirmed the expression of P450BM-3 QM based on the corresponding bands.
Figure 2. Transformation results of three plasmids in the LB agar plate. Three plasmids were all successfully transformed into bacteria, indicated by the colonies on the agar plate. (A) E. coli BL21 star (DE3) with pQE-80L-Kan glf; (B) E. coli BL21 star (DE3) with pCDFDUET-1 p450bm-3qm glcdh-II; (C) E. coli BL21 star (DE3) pET-28a prα-pol.
Figure 3. The results of the SDS-PAGE. The corresponding protein bands were indicated by the red box.
References
1. Lentz O, Li QS, Schwaneberg U, Lutz-Wahl S, Fischer P, Schmid RD. Modification of the fatty acid specificity of cytochrome P450 BM-3 from Bacillus megaterium by directed evolution: a validated assay. J Mol Catal B Enzym. 2001 Nov 1;15(4):123–33.
2. Schewe H, Kaup BA, Schrader J. Improvement of P450BM-3 whole-cell biocatalysis by integrating heterologous cofactor regeneration combining glucose facilitator and dehydrogenase in E. coli. Appl Microbiol Biotechnol. 2008 Feb 1;78(1):55–65.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1373
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1373
- 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1373
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1373
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1373
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 1219
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