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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, 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's 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?

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]. 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_FRASCHENSCHORSCH!). This expression casset was subsequently cloned into the integration vector (BBa_K300001 ) to assure expression in the brewing medium.

Expression of Thaumatin

The BioBrick for Thaumatin in our yeast vector pTUM104 was expressed in YPD media in a 2l scale and the cells were harvested and disrupted using glass beads. On the SDS-Page showing the cell lysate there was no additional band at the size 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.

Ion exchange chromatography

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 desintegrated yeast cells using ion exchange chromatography to have a proove 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 colum 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)
  • nealy 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 arround 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 propperties 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 frowthrough and eluted protein was less favourable 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 proteinbands 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 in order to load more than 10 ml of cell lysate on the column in order to purify sufficient protein for a mass spectrum of the produced thaumatin. Another aspect that should be investigated is the transport process that could be monitored in an ELISA or via quantitative western blotting using a polyclonal serum against thaumatin.

Mass Spectrometry data

Toxicity Assay

Die Grafik ist falsch! Ist bestimmt nicht 1M. Wir hatten abgesprochen 1mg/ml anzusetzen. Außerdem müsste man hier noch die Daten von der Hefe ohne Protein als Negativkontrolle zeigen. ich habe auch 1 mg/ml angesetzt - ich kümmer mich darum (Mary). - die richtige Graphik + Negativkontrolle kommt noch!

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 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.

The general idea is to create via genetic engineering of Saccharomyces cerevisiae a system that expresses thaumatin, respectively the direct precursor (preprothaumatin). The N-terminal "pre" part is the internal signalling sequence for secretion, the C-terminal "pro" sequence supports the correct and functional folding of thaumatin. Alternatively one could have used a fusion product of prothaumatin and the Mat-α-factor to achieve secretion.

Preferable seems to be the natural preprothaumatin, because of the expected higher yield http://www.ncbi.nlm.nih.gov/pubmed?term=21636903 Masuda et al., 2011 and the possibility that the pre-sequence is necessary for the correct procession http://www.ncbi.nlm.nih.gov/pubmed?term=17897626 Ide et al., 2007. A similar construct was used by the Kyoto University (Ide et al., submitted) in Pichia pastoris with a the pPIC6α expression vector with a high yield (especially with the preprothaumatin I gene and without the α-factor secretion signal).