Revision as of 14:10, 15 August 2012

Thaumatin

Background and principles

Picture taken from: PDB; ID: 2VHK

Thaumatin is a natural α+β-protein which is synthesized by the katamfe plant (Thaumatococcus daniellii) – a species of tropical flowering plants- and belongs to the thaumatin-like protein family. There exist different varieties of thaumatin, however, thaumatin I und thaumatin II are well characterized and differ only in one position (Position 46 – without signaling sequence; thaumatin I Asn; thaumatin II Lys). Both are said to be 2000 to 100000 times sweeter than sucrose on molar basis, but the sweetness builds slow and lasts long.

Thaumatin is a single chain with 207 amino acids residues and eight disulfide bonds. It is highly water soluble, stable at heating (not for cooking, bakery, etc.) and stable under acidic conditions. The production of thaumatin is induced by an attack upon the plant by viroid pathogens. Thus it is involved in systematically acquired resistance and stress response.

Thaumatin has been approved as a sweetener in the European Union (E957).

The molecular and physiological effects of thaumatin:

The sweet taste receptor is a heterodimeric receptor composed of T1R2 (also TAS1R2) and T1R3 (also TAS1R3) subunits. The large amino-terminal domains (NTD) of the T1R2 and T1R3 subunits have shown to be responsible for the primary ligand binding (E. Maitrepierre et al. [[1]]). In addition these receptors have a transmembrane heptahelical domain. T1R receptors belong to the family of class C G-Protein coupled receptors (GPCRs), which in this case means that through ligand binding an elevation of the cAMP concentration in the taste buds is induced (N. Ide et al.[[2]]; M. Ozeck et al. [[3]]). As a result a decrease in the intracellular cAMP accumulation is measured. Released calcium (Ca2+) seems to be another independent second messenger within the transduction of the taste response (downstream of taste receptors) (KR. Trubey et al; [[4]]).

However, not only sucralose or other sugars can bind with the NTDs of the sweet taste receptor, but also thaumatin (N. Ide et al. [[5]]). It seems to have a longer lasting and stronger effect than sucralose.

Idea

Picture taken from: David P. Clark, Nanette J. Pazdernik (2009); “Molekulare Biotechnologie”: 306 – 309

The general idea is to create via genetic engineering of Saccharomyces cerevisiae a system that expresses thaumatin through a certain environmental stimulus. There are different options for an inducible promotor, however, after being induced the thaumatin gene respectively a precursor (preprothaumatin) should be expressed. One possible construct which could be used contains an ethanol inducible promotor (needs to be strong for the thaumatin expression), possibly an alpha-factor secretion signal plus the thaumatin I gene or the natural preprothaumatin I gene (for the secretion of the recombinant thaumatin) and some kind of resistance gene (e.g. blasticidin resistance). Preferable seems to be the natural preprothaumatin, because of the higher yield (page 377;[[6]]) and the possibility that the pre-sequence is necessary for the correct procession (Ide et al.,Effects of pre- and pro-sequence of thaumatin on the secretion by Pichia pastoris; [[7]]). 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).

General remarks and issues

Secretion of proteins by S. cerevisiae

Recombinant proteins (in this case thaumatin) can be constructed for secretion by S. cerevisiae. The secretion is enabled by addition of the mating factor α upstream of the gene of interest. The signal peptidase of S. cerevisiae recognizes the sequences Lys-Arg after passing the membrane. This means the coding sequence needs to be directly downstream of the codons of Lys-Arg (David P. Clark, Nanette J. Pazdernik (2009); “Molekulare Biotechnologie”: 306 – 309).

The signal peptide of the mating factor α-1 (MF(ALPHA)1) begins with the first amino acid and ends with the 19th [[8]]. Because of the fact that the signal peptidase of S. cerevisiae recognizes only the sequence Lys-Arg (KR) further amino acids until a KR-sequence appears need to be added. Another option is putting a genetically engineered KR-sequence after the signal peptide. It is probably safer to use the sequence until the first KR-sequence appears, because of the fact that afterwards (after the first KR-sequence) the actual mating factor alpha hormones are encoded. This option would mean to use the sequence until the 85th amino acid. [[9]] As mentioned above (-> Idea) just using the preprothaumatin could also cause secretion. Different options should be considered! Through lab work the most effective alternative will be determined and then used in the next experiments.

Issues

Possible differences between P. pastoris and S. cerevisiae which might influence the production of recombinant thaumatin I should be researched. Codon-usage could become a problem. There are no thaumatin parts in the Registry of Standard Biological Parts. Because of the fact, that a “mating factor α-1 precursor secretion signal” [[10]] is needed upstream of the gene of interest, I am not sure whether this means just the signal peptide or a further part of the amino acid sequence. The MF(ALPHA)1 signal peptide is incompatible to the assembly standard 10 (has PstI).

Proof of principle

First of all the existence of the preprothaumatin I / thaumatin I gene within the gene construct (an inducible promotor could be more useful for the proof) needs to be proofed (e.g. determination of DNA-length by gel electrophoresis and staining by EtBr; sequencing). In the next step gene expression needs to be measured (e. g. through gel electrophoresis and Northern blotting).
Then protein secretion through mating factor α can be proofed by adding GFP or YFP gene downstream of the mating factor α gene. If there is fluorescence within the solution secretion can be proofed.
Afterwards the successful thaumatin gene can replace the fluorescence gene. Through SDS-PAGE and Western blotting thaumatin can be proofed.
If safety regulations permit the consumption of our product it can be proofed by taste in the end.

References

Reviews (PMID)

3167035 [[11]]
17897626 [[12]]
14991654 [[13]]
10049878 [[14]]
21636903 [[15]]
19489607 [[16]]
22450161 [[17]]
15087236 [[18]]
16510847 [[19]]

Others

Masuda and Kitabatake (2006), Development of Biotechnological Production of Sweet Proteins [[20]]
Thaumatococcus daniellii mRNA forpreprothaumatin I, comeplete cds [[21]]
Preprothaumatin I [Thaumatococcus daniellii], FASTA [[22]]
Masuda, Ohta, Mikami, Kitabatake (2011), High-resolution structure of the recombinant sweet-tasting protein thaumatin I [[23]]
Nobuyuki Ide, Tetsuya Masuda, Naofumi Kitabatake (2007), Effects of pre- and pro-sequence of thaumatin on the secretion by Pichia pastoris [[24]]
David P. Clark, Nanette J. Pazdernik (2009); “Molekulare Biotechnologie”: 306 – 309
yeastgenome.org [[25]]

@@ Line 46: / Line 46: @@
 * If safety regulations permit the consumption of our product it can be proofed by taste in the end.
-==Biobricks and sequences==
-=== Preprothaumatin I===
-==== Cds-pequence of preprothaumatin I (708bp) ====
-atggccgcca ccacttgctt cttcttcctc ttccccttcc tcctcctcct cacgctctcc
-cgcgctgcca ccttcgagat cgtcaaccgc tgctcctaca ccgtgtgggc ggccgcctcc
-aaaggcgacg ccgccctgga cgccggcggc cgccagctca actcgggaga gtcctggacc
-atcaacgtag aacccggcac caacggtggc aaaatctggg cccgcaccga ctgctatttc
-gacgacagcg gcagcggcat ctgcaagacc ggcgactgcg gcggcctcct ccggtgcaag
-cgcttcggcc ggccgcccac cacgctggcg gagttctcgc tcaaccagta cggcaaggac
-tacatcgaca tctccaacat caaaggcttc aacgtgccga tggacttcag cccgaccacg
-cgcggctgcc gcggggtgcg gtgcgccgcc gacatcgtgg ggcagtgccc ggcgaagctg
-aaggcgccgg ggggtggttg caacgatgcg tgcaccgtgt tccagacgag cgagtactgc
-tgcaccacgg ggaagtgcgg gccgacggag tactcgcgct tcttcaagag gctttgcccg
-gacgcgttca gttatgtcct ggacaagcca accaccgtca cctgccccgg cagctccaac
-tacagggtca ctttctgccc tactgccctt gaacttgaag acgagtaa
-{| class="wikitable" cellpadding="10" border=1px
-| Name || Length || RFC10 || RFC25 ||  Codon Usage || NCBI
-|-
-| Preprothaumatin I || 708bp || 2x NotI (109-117/146-152) || 2x NgoMIV (142-148/308-314) || 3AS<10% || [http://www.ncbi.nlm.nih.gov/nuccore/121945717 NP_015137.1]
-|}
-==== Designed primer with ''QuikChange Primer Designer'' (if not synthesized) ====
-Primer sequences:
-==== Improved Cds-pequence of preprothaumatin I (708bp) (if synthesized) ====
-The first NotI site can be avoided through changing the codon GCG (Ala; pos. 109-111) with GCT. GCT has a higher codon usage in yeast anyway [[http://downloads.yeastgenome.org/unpublished_data/codon/ysc.orf.cod]]
-The second NotI site can be avoided through changing GGC (Gly; pos. 145-147) with GGT. With this change the NgoMIV site can be changed, too.
-The three condons which have a usage smaller than ten percent are all codons coding for arginin (R) with the triplet CGG. It can be changed to AGG. One of these codons definately needs to be changed (Pos. ), because simultaineously the second NgoMIV site will be avoided.
-The resulting sequence would be (changed codons are in capitol letters):
-atg gcc gcc acc act tgc ttc ttc ttc ctc ttc ccc ttc ctc ctc ctc ctc acg ctc tcc
-cgc gct gcc acc ttc gag atc gtc aac cgc tgc tcc tac acc gtg tgg GCT gcc gcc tcc
-aaa ggc gac gcc gcc ctg gac gcc GGT ggc cgc cag ctc aac tcg gga gag tcc tgg acc
-atc aac gta gaa ccc ggc acc aac ggt ggc aaa atc tgg gcc cgc acc gac tgc tat ttc
-gac gac agc ggc agc ggc atc tgc aag acc ggc gac tgc ggc ggc ctc ctc AGG tgc aag
-cgc ttc ggc AGG ccg ccc acc acg ctg gcg gag ttc tcg ctc aac cag tac ggc aag gac
-tac atc gac atc tcc aac atc aaa ggc ttc aac gtg ccg atg gac ttc agc ccg acc acg
-cgc ggc tgc cgc ggg gtg AGG tgc gcc gcc gac atc gtg ggg cag tgc ccg gcg aag ctg
-aag gcg ccg ggg ggt ggt tgc aac gat gcg tgc acc gtg ttc cag acg agc gag tac tgc
-tgc acc acg ggg aag tgc ggg ccg acg gag tac tcg cgc ttc ttc aag agg ctt tgc ccg
-gac gcg ttc agt tat gtc ctg gac aag cca acc acc gtc acc tgc ccc ggc agc tcc aac
-tac agg gtc act ttc tgc cct act gcc ctt gaa ctt gaa gac gag taa
-==== FASTA-Sequence of Preprothaumatin I ====
-<pre style="color:red"> MAATTCFFFLFPFLLLLTLSRA</pre>
-ATFEIVNRCSYTVWAAASKGDAALDAGGRQLNSGESWTINVEPGTNGGKIWARTDCYFDDSGSGICKTGDCGGLLRCKRFGRPPTTLAEFSLNQYGKDYIDISNIK
-GFNVPMDFSPTTRGCRGVRCAADIVGQCPAKLKAPGGGCNDACTVFQTSEYCCTTGKCGPTEYSRFFKRLCPDAFSYVLDKPTTVTCPGSSNYRVTFCPTA
-<pre style="color:red">LELEDE</pre>
-Pre- and prosequence are labeled in red
-=== mating factor α-1===
-==== Cds-sequence of the mating factor α-1 (498 bp)====
-   atgagatttc cttcaatttt tactgcagtt ttattcgcag catcctccgc attagctgct
-  ccagtcaaca ctacaacaga agatgaaacg gcacaaattc cggctgaagc tgtcatcggt
-tacttagatt tagaagggga tttcgatgtt gctgttttgc cattttccaa cagcacaaat
-aacgggttat tgtttataaa tactactatt gccagcattg ctgctaaaga agaaggggta
-tctttggata aaagagaggc tgaagcttgg cattggttgc aactaaaacc tggccaacca
-atgtacaaga gagaagccga agctgaagct tggcattggc tgcaactaaa gcctggccaa
-ccaatgtaca aaagagaagc cgacgctgaa gcttggcatt ggctgcaact aaagcctggc
-caaccaatgt acaaaagaga agccgacgct gaagcttggc attggttgca gttaaaaccc
-ggccaaccaa tgtactaa
-{| class="wikitable" cellpadding="10" border=1px
-| Name || Length || RFC10 || RFC25 || Codon Usage || NCBI
-|-
-| MF&alpha;1 || 498bp/255bp || 1x PstI (23-29) || ok after RFC10 || 0AS<10% || [http://www.ncbi.nlm.nih.gov/nuccore/NM_001184001.1 NM_001184001.1]
-|}
-[[http://www.yeastgenome.org/cgi-bin/getSeq?map=amap&seq=YPL187W&flankl=&flankr=&rev=]] [[http://www.ncbi.nlm.nih.gov/nuccore/NM_001184001.1]]
-==== Designed primer with ''QuikChange Primer Designer'' (if not synthesized) ====
-Primer sequences:
-Primer Name	Primer Sequence (5' to 3')
-a27t	5'-atttccttcaatttttactgctgttttattcgcagcatcctcc-3'
-a27t_antisense	5'-ggaggatgctgcgaataaaacagcagtaaaaattgaaggaaat-3'
-==== Improved and '''necessary''' cds-pequence mating factor α-1 (255 bp) (if synthesized) ====
-This signal sequence is incompatible to the assembly standard 10 (has PstI: CTGCAG).
-  ATG AGA TTT CCT TCA ATT TTT A'''CT GCA G'''TT TTA TTC GCA GCA TCC TCC GC
-However, GCA (Ala) can be changed to GCT (changed nucleotide is not in capitol letters):
-ATG AGA TTT CCT TCA ATT TTT ACT gct GTT TTA TTC GCA GCA TCC TCC GCA TTA GCT GCT CCA GTC AAC ACT ACA ACA GAA GAT GAA ACG GCA CAA ATT CCG GCT GAA GCT GTC ATC GGT TAC TTA GAT TTA GAA GGG GAT TTC GAT GTT GCT GTT TTG CCA TTT TCC AAC AGC ACA AAT AAC GGG TTA TTG TTT ATA AAT ACT ACT ATT GCC AGC ATT GCT GCT AAA GAA GAA GGG GTA TCT TTG GAT AAA AGA
-==== FASTA-Sequence of the mating factor α-1====
-<pre style="color:red">MRFPSIFTAVLFAASSALA</pre>APVNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTN
-NGLLFINTTIASIAAKEEGVSLDKREAEAWHWLQLKPGQPMYKREAEAEAWHWLQLKPGQ
-PMYKREADAEAWHWLQLKPGQPMYKREADAEAWHWLQLKPGQPMY*
-[[http://www.yeastgenome.org/tmp/protein_fasta.21250.txt]]
-The Signal sequence is labeled in red
-=== If synthesized: Final product!: ===
-This is the final product of the secretion signal sequence of mating factor alpha-1 (sequence until base 255; amino acid 85) plus the sequence for preprothaumatin I. After secretion the yeast endoprotease Kex2 is suppost to cut right after the Lys-Arg-sequence at the end of the mating factor alpha-1, resulting in the hopefully correct processed preprothaumatin I.
-ATG AGA TTT CCT TCA ATT TTT ACT GCT GTT TTA TTC GCA GCA TCC TCC GCA TTA GCT GCT CCA GTC AAC ACT ACA ACA GAA GAT GAA ACG GCA CAA ATT CCG GCT GAA GCT GTC ATC GGT TAC TTA GAT TTA GAA GGG GAT TTC GAT GTT GCT GTT TTG CCA TTT TCC AAC AGC ACA AAT AAC GGG TTA TTG TTT ATA AAT ACT ACT ATT GCC AGC ATT GCT GCT AAA GAA GAA GGG GTA TCT TTG GAT AAA AGA
-atg gcc gcc acc act tgc ttc ttc ttc ctc ttc ccc ttc ctc ctc ctc ctc acg ctc tcc
-cgc gct gcc acc ttc gag atc gtc aac cgc tgc tcc tac acc gtg tgg gct gcc gcc tcc
-aaa ggc gac gcc gcc ctg gac gcc ggt ggc cgc cag ctc aac tcg gga gag tcc tgg acc
-atc aac gta gaa ccc ggc acc aac ggt ggc aaa atc tgg gcc cgc acc gac tgc tat ttc
-gac gac agc ggc agc ggc atc tgc aag acc ggc gac tgc ggc ggc ctc ctc agg tgc aag
-cgc ttc ggc agg ccg ccc acc acg ctg gcg gag ttc tcg ctc aac cag tac ggc aag gac
-tac atc gac atc tcc aac atc aaa ggc ttc aac gtg ccg atg gac ttc agc ccg acc acg
-cgc ggc tgc cgc ggg gtg agg tgc gcc gcc gac atc gtg ggg cag tgc ccg gcg aag ctg
-aag gcg ccg ggg ggt ggt tgc aac gat gcg tgc acc gtg ttc cag acg agc gag tac tgc
-tgc acc acg ggg aag tgc ggg ccg acg gag tac tcg cgc ttc ttc aag agg ctt tgc ccg
-gac gcg ttc agt tat gtc ctg gac aag cca acc acc gtc acc tgc ccc ggc agc tcc aac
-tac agg gtc act ttc tgc cct act gcc ctt gaa ctt gaa gac gag taa
 ==References==

Team:TU Munich/Project/Thaumatin

From 2012.igem.org