Composite

Part:BBa_K801077

Designed by: Dennis Hell   Group: iGEM12_TU_Munich   (2012-09-24)
Revision as of 19:47, 26 October 2012 by Nadine1990 (Talk | contribs) (→‎LC/MS)

Caffeine Synthesis Pathway pTEF2-XMT1-tADH1-pTEF1-MXMT1-tADH1-pTEF2-DXMT1-tADH1

This BioBrick is the final "caffeine synthesis device". It contains all three necessary enzymes: CaXMT1, CaMXMT1 and CaDXMT1, i.e. it is made up of the three single expression cassettes for each enzyme (see also BBa_K801073, BBa_K801074 and BBa_K801075). It can be directly transformed into competent yeast cells or cloned in an adequate yeast genome integration vector. The expression of the enzymes CaXMT1 (Coffea arabica xanthosine-N-methyl-transferase 1, BBa_K801070) and CaDXMT1 (Coffea arabica dimethyl-xanthine N-methyl transferase 1, = caffeine synthase, BBa_K801072) is controlled by the TEF2-promoter (BBa_K801010) and the ADH1-terminator (BBa_K801012) The expression of CaMXMT1 (Coffea arabica methyl-xanthine N-methyl transferase 1, BBa_K801071) is controlled by the TEF1 promoter (BBa_K319003) and the ADH1-terminator (BBa_K801012)

Background and principles

Structure of Caffeine.

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 CaXMT1 (xanthosine N-methyltransferase 1 of coffea arabica) catalyses the first reaction step of the caffeine biosynthesis pathway and convertes the sustrate Xanthosine into 7-Methylxanthosine. It uses SAM als methyl-donor and is located in the cytoplasm of the plants. 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. The enzyme CaDXMT1 (3,7-dimethylxanthine N-methyltransferase of coffea arabica) catalyses the fourth reaction step of the caffeine biosynthesis, leading to caffeine. It uses SAM (S-Adenosyl-Methionine) als methyl-donor and is located in the cytoplasm of the plants. Furthermore all three exists as homodimer, being also able to form heterodimers with the other enzymes of the caffeine pathway (see [http://www.brenda-enzymes.info Brenda]).

Biosynthesis Pathway

Caffeine biosynthesis pathway







Characterization

Cloning into pSB1C3

Anal. digest

In order to submit the enzyme xanthosine methyltransferase (CaXMT1) 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.

The expected lengths of the fragments were:

  • Insert (CaXMT1): ca. 6400bp
  • Backbone (pSB1C3): ca. 2050 bp


The picture on the left shows the analytical digest of BioBrick BBa_K801070 with Xba1 and Pst1, separated by gel-electrophoresis on 1% agarose gel upon usage of ethidium bromide.

LC/MS

We have been successful in the expression of the three single enzymes making up the caffeine biosynthesis metabolic pathway. Therefore, we used this composite part BBa_K801077 for expression of the enzymes, followed by enzymatic analyses, in order to prove the functionality of our enzymes. After having cloned it into our vector pTUM100, we performed an enzyme assay as described in [Uefuji et al., 2003], with the enzymes having been harvested after 20 hours. The reaction conditions were 27 °C at a pH of 8.0. Detection of single metabolites of the caffeine biosynthesis pathway was performed by LC/MS analysis, using multiple reaction monitoring (MRM). The picture below shows that we were in fact able to perform an in vitro synthesis of theobromine, which is the immediate precursor of caffeine. However, we failed in the detection of caffeine, because the negative control also showed the corresponding signal with an identical retention time and the amount was not significant, i.e. the e-value was not comparable to the one of the reference. We assumed, that the reason for the failed detection could be the fact, that there was to less produced theobromine in the reaction mixture. Thus, we started another experiment, in which we added theobromine directly to the isolated crude extract.

Used substrate: Xanthosine; A: Reference chromatogram of theobromine; B and C: Chromatograms of two solutions obtained from the enzyme assay; Reacton conditions: T= 27°C, pH= 8.0; Reaction time: 16h; Each 100µl reaction batch consisted of: 50mM Tris/HCl (pH 8.0), 500 µM Xanthosine, 1,5 mM SAM, 200 µM MgCl2, 500 µg protein (crude extract); Reaction products were extracted with 1 ml chloroform, dried at 60°C and subjected to LC/MS analysis after having been resolved in 100µl 70% methanol;

























Usage and Biology

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 3613
    Illegal BglII site found at 5829
    Illegal BglII site found at 5925
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 138
    Illegal NgoMIV site found at 4317
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 2385



References

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