Yeast expression cassette for CaXMT1 and CaMXMT1

Yeast Expression Cassette for the enzymes 1 and 2 of the caffeine biosynthesis pathway Expression of CaXMT1 is controlled by the yeast TEF2 promoter and the yeast ADH1 terminator. Expression of CaMXMT1 is controlled by the yeast TEF1 promoter and the yeast ADH1 terminator. This BioBrick is a generator for the first two enzymes envolved in caffeine biosynthesis: methylxanthosine N-methyltransferase 1 and 7- methylxanthine N-methyltransferase 1. It is made up of the two single generators for CaXMT1 and CaMXMT1, which means that CaXMT1 is regulated by the Tef2 promoter and CaMXMT1 is regulated by the Tef1 promoter. In both cases, the Adh1 terminator was used.

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. Furthermore both 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. 4200bp
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.

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
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 138
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 2385

References