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"Why? O WHY???" is the reaction we usually face when we tell our friends, that we genetically engineer yeast to secrete a sweetener. "Kruzifixnochemol" is another, more cryptic one.

We must admit, even we don't think that a "Münchner Hell" or a "Pils" would benefit from the distinct sweetness of Thaumatin, originally produced by Thaumacoccus daniellii.
So, why, o why have we chosen Thaumatin? There are a variety of arguments that have to be taken into consideration:

• Since experiments in the 1980ies, nobody tried to express Thaumatin via S. cerevisiae - so we saw the possibility to enhance the production and make it more efficient (as one of the biggest problems for industrial use of Thaumatin so far is the low yield efficiency) by codon usage optimization.

• If you think of the beers of the Anglo-Saxon world - stouts and ales - these are the sorts that could directly profit from the licorice-like sweetness, as they are generally more full-bodied and show a variety of caramel flavors.

• It opens the world of brewage to a whole new generation of lifestyle drinks - perhaps a bit too innovative for the conservative german beer market, but nevertheless there is quite a potential there for cutting-edge beverages with the soul of beer and the savor of all the fruits you can imagine. Thaumatin might serve as a low-carb ingredient to balance out the bitter and sour flavors of guava, grapefruits, currant - or even horseradish.

Background and principles

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Theoretical consideration: How much thaumatin is needed in foodstuff?

The molar weight of sucrose is 342,30 g/mol, respectively 22194,13 g/mol for thaumatin. This means, that although the relative sweetness of thaumatin is 2 000-10 000 times the sweetness of sucrose on a molar basis, it's only ~30-150 times the sweetness when measured in mass.

Thus means you'd have to put 0,07-0,35 g of thaumatin into 100 ml generic soft drink to create an equal level of sweetness.

Results

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BioBricks planning

The BioBricks designed encode the Prepro-Thaumatin ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K801080 BBa_K801080]) that is believed to be exported on it's own http://www.ncbi.nlm.nih.gov/pubmed/6327079 Edens et al., 1984. The original gene sequence was optimized for best yeast codon usage using GeneArt® GeneOptimizer®. We ordered the gene synthesis and cloned this BioBrick into pSB1C3 and our galactose inducible Yeast expression vector pTUM104. Furthermore we constructed an expression cassette with TEF1-promoter and the TEF1-terminator, which is also available as a protein generator (BBa_K801081). This expression casset was subsequently cloned into the integration vector (BBa_K300001 ) to assure expression in the brewing medium.

Expression of Thaumatin

We plan to do a western blot of the supernatant to prove proper secretion. SDS page and coomassie/silver staining have proven to be too insensitive.

Preprothaumatin becomes posttranslationally modified by cleaving a part of the N- and the C-terminal polypeptide. Therefore it was not possible to add a tag for affinity chromatography. For this reason it was necessary to purify the protein from the cytoplasm of the disintegrated yeast cells using ion exchange chromatography to have a proof of principle.

Experimental details:

• Samples: cell lysate, supernatant from culture, reference for thaumatin (MedHerbs)
• Dialysis against 20mM MES Buffer pH 6.0 (twice) using a 12-16 kDa dialysis membrane
• Chromatography with a Äkta purifier equipped with a Ressource S 6ml (S: Methyl sulfonate (strong cation exchanger))
• Sample was applied using a super-loop
• Wash with two column volumes 20mM MES Buffer pH 6.0
• Elution with a gradient of 0 - 500 mM NaCl over 5 column volumes
• Fractions of 1 ml were collected during the elution
  

Experimental results:

• Reference thaumatin (see figure A and B)
  • nearly no protein in the flow through during sample application (see figure A)
  • a major peak eluting at ~14 mS*cm-1 which corresponds to fraction Nr. 14 see figure A)
  • The corresponding SDS-PAGE showed a clear band around fraction Nr. 14 corresponding matching the expected 22 kDa
• Cell lysate
  • high concentration of protein in the flow through (see figure C and D)
  • no clear peak around fraction 14 could be detected
  • The SDS-PAGE performed from the reference and the fractions 12 to 15 showed a weak band at the height of the reference. This band having the same size as the reference (see running properties on SDS-PAGE in figure D) and the same isoelectric point (both eluted in fraction 14) is very likely to be thaumatin which was produced by the yeast cells.
• Supernatant of yeast culture (see figure E and F)
  • Export of the protein in high concentrations was unlikely to happen, therefore 48 ml of supernatant were loaded on the column
  • Ratio between flow through and eluted protein was less favorable compared to the cell lysate.
  • Total protein quantities were to low to be detected by Coomassie stain, therefore a silver stain was performed (see figure F)
  • Beside the reference no additional protein bands could be detected on the silver stained SDS-PAGE
    

Conclusion from this experiment:

A proof of principle for the expression of thaumatin was achieved using ion exchange chromotography and comparison of bands optained on an SDS-PAGE relative to a standard of thaumatin.
Further goals would include to increase the expression of the thaumatin and to investigate the secretion. This could be achieved by improving the purification protocol to load more than 10 ml of cell lysate on the column in order to purify a sufficient amount of protein for a mass spectrum of the produced thaumatin.

Toxicity Assay

To check if thaumatin has an effect on yeast cells , we inoculated three different yeast strains with YPD including 45 mM of thaumatin and measured their growth rate after a defined period of time. The used yeast strains were the laboratory strain INVSc1, a strain which is used for brewing beer and a strain which can be purchased in a supermarket. The growth rate of yeast cells which were incubated with thaumatin does not show a difference compared to the negative control (incubation of analogous strains with YPD without thaumatin). The small differences in doubling time are errors in measurement.

As expected, thaumatin does not have an effect on the growth rate of yeast cells. Hence we are not limited in over-expressing the sweetener in our beer.