Part:BBa_K2906016
T7 promoter- RBS- COMT- T7 terminator
comt is from the plant Arabidopsis thaliana and is codon optimised by GeneArt GeneOptimiser for expression in E.coli.. A His-tag is fused to it after cloning into pETDuet-1 plasmid. It encodes catechol O-methyltransferase (COMT), which converts caffeic acid to ferulic acid, the third step in the vanillin biosynthesis pathway.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 102
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 141
Illegal AgeI site found at 324 - 1000COMPATIBLE WITH RFC[1000]
Background:
Figure 1. Vanillin biosynthesis pathway from L-tyrosine consisting of five enzymes.
Characterisation:
To show the functionality of the gene after cloning into pETM-11, we performed SDS-PAGE and Western blot to prove expression of the protein C3H (Figure 2).
Figure 2. SDS-gel (a) and western blot (b) of TAL, C3H and COMT, the first three enzymes in the vaniliin biosynthesis pathway encoded by sam8, sam5 and comt. The expected sizes of the proteins are 57.4 kDa, 60.49 kDa and 41.39 kDa for TAL, C3H and COMT respectively. The negative control (-ve) is the pETM-11 (empty vector) and the positive control (+ve) is the pETM-11-ABU58587 expressing a 36 kDa His-tagged protein.
As sam5 and comt were cloned to separate incompatible plasmids and due to the low stability of caffeic acid, we decided to make a pETM-11-sam5 and pETDuet-1-comt BL21 (DE3) co-culture (1:1) to detect and quantify caffeic acid and ferulic acid by UPLC-MS/MS providing 3 mM p-coumaric acid as a substrate for C3H (Figure 3). A 10 mL culture (1/100) of the respective strain in 2xYT media, was induced by 100 µM IPTG and grown for 72 hours at 26°C and 180 rpm which was then processed for analysis by UPLC-MS/MS; 1.207 µM and 3,914 µM of caffeic acid and ferulic acid were produced.
Figure 3. UPLC–MS/MS chromatograms of caffeic acid (a) and ferulic acid (b), the products of C3H and COMT respectively, from pETM-11-sam5 (c) and pETDuet-1-comt (d) BL21 (DE3) bacterial providing 3 mM of the substrate p-coumaric acid. X-axis show retention time.
References:
Gruz, J., Novák, O. and Strnad, M. (2008). Rapid analysis of phenolic acids in beverages by UPLC–MS/MS. Food chemistry, 111(3), pp.789-794. DOI: https://doi.org/10.1016/j.foodchem.2008.05.014.
Ni, J., Tao, F., Du, H. and Xu, P. (2015). Mimicking a natural pathway for de novo biosynthesis: natural vanillin production from accessible carbon sources. Scientific reports, 5: 13670. DOI: https://doi.org/10.1038/srep13670.
Kim, Y.H., Kwon, T., Yang, H.J., Kim, W., Youn, H., Lee, J.Y. and Youn, B. (2011). Gene engineering, purification, crystallization and preliminary X-ray diffraction of cytochrome P450 p-coumarate-3-hydroxylase (C3H), the Arabidopsis membrane protein. Protein expression and purification, 79(1), pp.149-155. DOI: https://doi.org/10.1007/s10811-013-0113-5.
| None |