CaMXMT1 expression cassette for yeast
Yeast expression cassette for CaMXMT1 (BBa_K801071) controlled by the yeast TEF1 promoter (BBa_K319003) and the yeast ADH1 terminator (BBa_K801012).
This BioBrick is the generator for the enzyme 7-methylxanthine N-methyltransferase 1(CaMXMT1) (= theobromine synthase) and part of the caffeine synthesis pathway. It is regulated by the constitutive promoter Tef1, which is one of the strongest yeast promoters. The used terminator is Adh1, as it is among the other expression cassettes. The choice of the strong Tef1 promoter was made, because the in vivo biosynthesis of caffeine is an irreversible reaction, which is ensured by continously increasing Km and vmax values of the single enzymes. As soon as a certain amount of theobromine is available, the caffeine synthesis can go on. To support this irreversible, stepwise caffeine synthesis, we used the strongest promoter for this enzyme, to establish high concentrations of the caffeine precursor theobromine.
Background and principles
Caffeine is a purine-alkaloid and its biosynthesis occurs in coffee plants and tea plants. Its chemical structure is similar to that of the ribonucleoside adenosine. Hence it can block specific receptors in the hypothalamus. Adenosine binding leads to decreased neurotransmitter-release and therefore decreased neuron activity. This induces sleep and thus avoids overexertion of the brain. Since caffeine antagonizes adenosine and increases neuronal activity, it is used as a means to stay awake. On average, one cup (150 ml) of coffee contains about 50 - 130 mg caffeine and one cup of tea 25 - 90 mg. At higher doses (1g), however, caffeine leads to higher pulse rates and hyperactivity. Moreover, caffeine was shown to decrease the growth of E. Coli and yeast reversibly as of a concentration of 0.1 % by acting as a mutagen (Putrament et al., On the Specificity of Caffeine Effects, MGG, 1972), but previous caffeine synthesis experiments (see below) have only led to a concentration of about 5 µg/g (per g fresh weight of tobacco leaves), so it is not expected to reach critical concentrations and the amounts of caffeine in coffee or tea (leading to physiological effects) is usually a little bit lower.
Usage and Biology
The enzyme CaMXMT1 (7-methylxanthine N-methyltransferase of coffea arabica) catalyses the third reaction step of the caffeine biosynthesis pathway and convertes the sustrate 7-Methylxanthine into 3,7-Dimethylxanthine. It uses SAM (S-Adenosyl-Methionine) als methyl-donor and is located in the cytoplasm of the plants. Furthermore it exists as homodimer, being also able to form heterodimers with the other enzymes of the caffeine pathway (see Brenda).
- the 5' UTR and 3' UTR of the original sequences were removed
- the yeast consensus sequence for improved ribosome binding (TACACA) was added 5' of the start codon ATG
- according to N- end rule and the yeast consensus sequence for improved ribosome binding, the first triplet after ATG (GAG) was exchanged with TCT (serine), to optimize both, protein stability and mRNA translation. This decision was made after proofing the 3D- structure of the enzyme CaDXMT1. Due to the fact, that the first two residues of the amino acid sequence are not shown in the crystalized structure (probably because of high flexibility), we chose to exchange this amino acid, because it is probably not necessary for the uptake of the ligands (uniprot entry further shows, that it is not immediately involved in ligand binding). Because of the high similarity of the enzyme sequences, we also exchanged this amino acid.
- we added a c- terminal strep-tag for purification and detection
- the remaining coding sequence was extended with the standard RFC10 prefix and suffix, respectively
- at last we made an optimization of the coding sequences with respect to the codon usage and mRNA structures
- remove of all critical restriction sites (RFC10 and RFC25)
Note: Because of the yeast consensus sequence, this coding part does not start with ATG!
Cloning into pSB1C3
In order to submit the enzyme 7-methylxanthine methyltransferase (CaMXMT1) to the parts.igem, we cloned the generated sequence into pSB1C3, making the system RFC10 compatible. However, since the sequence does not contain any restriction sites of the RFC25 standard (NgoMIV and Age1), RFC25 compatibility can easily be reached without required quick changes by the use of PCR upon usage of appropriate primers.
To check the successful cloning, we performed an analytical digest with XbaI and PstI and Eco53kI. Because the backbone of pSB1C3 has the same length as the Insert, to analyze, we digest a second time in the backbone with Eco53kI.
The expected lengths of the fragments were:
- Insert (CaMXMT1): ca. 2000bp
- Backbone fragment 1 : ca. 1150bp
- Backbone fragment 2 : ca. 900bp
The picture on the left shows the analytical digest of BioBrick BBa_K801071 (red box) with Xba1 and Pst1, separated by gel-electrophoresis on 1% agarose gel upon usage of ethidium bromide (with the other bands being control digests from different clones).
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21Illegal BglII site found at 1433
- 23COMPATIBLE WITH RFC
- 25COMPATIBLE WITH RFC
- 1000Illegal BsaI.rc site found at 205
- [Ashihara et al., 2008] Ashihara, H., Sano, H., and Crozier, A. (2008). Caffeine and related purine alkaloids: biosynthesis, catabolism, function and genetic engineering. Phytochemistry, 69(4):841–56.
- [Franco et al., 2012] Franco, L., Sánchez, C., Bravo, R., Rodriguez, A., Barriga, C., and Juánez, J. C. (2012). The sedative effects of hops (humulus lupulus), a component of beer, on the activity/rest rhythm. Acta Physiol Hung, 99(2):133–9.
- [Kim and Sano, 2008] Kim, Y.-S. and Sano, H. (2008). Pathogen resistance of transgenic tobacco plants producing caffeine. Phytochemistry, 69(4):882–8.
- [Kuranda et al., 2006] Kuranda, K., Leberre, V., Sokol, S., Palamarczyk, G., and François, J. (2006). Investigating the caffeine effects in the yeast Saccharomyces cerevisiae brings new insights into the connection between TOR, PKC and Ras/cAMP signalling pathways. Mol Microbiol, 61(5):1147–66.
- [Uefuji et al., 2003] Uefuji, H., Ogita, S., Yamaguchi, Y., Koizumi, N., and Sano, H. (2003). Molecular cloning and functional characterization of three distinct n-methyltransferases involved in the caffeine biosynthetic pathway in coffee plants. Plant Physiol, 132(1):372–80.
- [Uefuji et al., 2005] Uefuji, H., Tatsumi, Y., Morimoto, M., Kaothien-Nakayama, P., Ogita, S., and Sano, H. (2005). Caffeine production in tobacco plants by simultaneous expression of three coffee n-methyltrasferases and its potential as a pest repellant. Plant Mol Biol, 59(2):221–7.