Part:BBa_K4825057
pTDH3-ARO10-tTDH1-pPGK1-ADH7-tPGK1-pTEF2-ATF1-tSSA1
This composite part contains the parts ARO10 BBa_K4825015, ADH7 BBa_K4825016 and ATF1 BBa_K4825017. In the pathway of producing isoamyl acetate in S. cerevisiae, ARO10 encodes for 2-oxoacid decarboxylase, which is reponsible for converting 2-ketoisocaproate (KIC) to isopentaldehyde. ADH7, which encodes for alcohol dehydrogenase 7, is responsible for converting isopentaldehyde to isopantanol. ATF1 encodes for alcohol acetyltransferase, which catalyzes the conversion of isopenanol to isopentyl acetate (isoamyl acetate). By overexpression of ARO10 and ADH7, the supply of precursors isopenaldehyde and isopentanol is increased, and the overexpression of ATF1 directly enhances the corresponding conversion to isoamyl acetate. Therefore, The composite part increases the production of isoamyl acetate in S. cerevisiae.
Usage and Biology
The composite part contains pTDH3(BBa_K124002), ARO10(BBa_K4825015), tTDH1(BBa_K2753052), pPGK1(BBa_K4012003), ADH7(BBa_K4825016), tPGK1(BBa_K3126012), pTEF2(BBa_K165037), ATF1(BBa_K4825017) and tSSA1(BBa_K4012020).
ARO10 is derived from the genome of S.cerevisiae, encoding for a 2-ketoacid decarboxylase. During the fermentation of yeast, it terminates the recursive pathway, which catalyzes the elongation of a 2-ketoacid by one carbon atom, by converting the 2-ketoacid to an aldehyde. ADH7 is derived from the genome of S.cerevisiae encoding for an alcohol dehydrogenase (ADH). During the fermentation of yeast, it catalyzes the production of n-carbon alcohols by converting n-carbon ketoacids to their corresponding (n − 1)-carbon alcohols along with KDCs (ketoacid decarboxylases) and terminates the recursive pathway which catalyzes the elongation of a 2-ketoacid by one carbon atom. ATF1 is a DNA sequence derived from the genome of S.cerevisiae that encodes for the enzyme alcohol acetyltransferase 1 (AATase-1). It catalyzes the conversion from acetyl-CoA and alcohols to acetate esters. During fermentation, the rates of producing acetate esters demonstrate a correlation to AATase activity. We used pTDH3 for expressing ARO10 gene, pPGK1 for ADH7, and pTEF2 for ATF1; we used tTDH1, tPGK1, and tSSA1 for the genes respectively.
Characterization
This year, GreatBay-SCIE confronts the issue of the invasive species, Achatina Fulica, and its parasitic worm, A.cantonensis. We developed the ALERTS solution, in which we are determined to use fruit odors to attract the snails and use a protein to eliminate the nematodes. For attraction, we carried out a test at a private yard to determine the attraction effects of different fruits. The result shows that the banana odor can attract the snails. Therefore, we decided to synthesize isoamyl acetate, which is a compound that has a smell resembling to banana odor, by engineering S.cerevisiae.
Table.1 Number of giant African snails attracted by different dough at different time durations.
Fig.2 Three fermented doughs mixed with different fruits are placed to attract the giant African snails in the yard.
In order to increase the production of isoamyl acetate, we are determined to increase the accumulation of isopentanol by overexpression of ARO10 and ADH7. (Fig.3A) The overexpression of ATF1 enables the conversion from the alcohol to isoamyl acetate. The endogenous DNA strands were extracted and amplified from CEN.PK2-1C genome and integrated simultaneously to site 106 of CEN.PK2-1C using lithium acetate transformation, in which CRISPR-Cas9 is utilized to cut the site and the donor DNA strands, which were our coding sequences, would be inserted through homologous recombination. (Fig.3B) The construction was then verified by conducting yeast colony PCR followed by gel electrophoresis, which shows the target strands were integrated into the genome successfully (Fig.3C). The constructed strain was named SCIE L1. The fermentation was carried out and lasted for 48 hours using YPD+2% glucose medium and dodecane as solvent. We collected the product, and detected it by GC-MS analysis. The result of the analysis demonstrates that we have successfully produced isoamyl acetate through our engineered S. cerevisiae using isoamyl acetate as the control. (Fig.4)(Fig.5)
Fig.3 Expression of isoamyl acetate in S. cerevisiae. (A) Genetic pathway of producing isoamyl acetate. LEU4S547∆, encoding for 2-isopropylmalate synthase (2-IPMS), in which the codon of the 547th amino acid S is deleted. LEU1, encoding isopropylmalate isomerase. LEU2, encoding 3-IPM dehydrogenase. ARO10, encoding 2-oxoacid decarboxylase. ADH7, encoding for alcohol dehydrogenase 7. ATF1, encoding for alcohol acetyl-coenzyme A (acetyl-CoA) transferase (AATase) (B) Genetic circuit construction for producing isoamyl acetate. ARO10, ADH7, and ATF1 are transformed into site 106 through homologous recombination using 106 upstream (106 US) and 106 downstream (106 DS). (C) Gel electrophoresis analysis of integrated sequence ARO10-ADH7-ATF1
Fig.4 GC-MS analysis of the product isoamyl acetate of SCIE L1 fermentation, using Standard isoamyl acetate as control
Fig.5 Testing the production of isoamyl acetate (A) standard curve of isoamyl acetate (B) the production of isoamyl acetate of SCIE L1.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 2478
Illegal EcoRI site found at 6138
Illegal SpeI site found at 1429
Illegal PstI site found at 1307 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 2478
Illegal EcoRI site found at 6138
Illegal NheI site found at 4461
Illegal SpeI site found at 1429
Illegal PstI site found at 1307 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 2478
Illegal EcoRI site found at 6138
Illegal BglII site found at 3967
Illegal BglII site found at 5825
Illegal BamHI site found at 4222
Illegal BamHI site found at 7152
Illegal XhoI site found at 2611
Illegal XhoI site found at 7329 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 2478
Illegal EcoRI site found at 6138
Illegal SpeI site found at 1429
Illegal PstI site found at 1307 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 2478
Illegal EcoRI site found at 6138
Illegal SpeI site found at 1429
Illegal PstI site found at 1307 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 2865
Illegal BsaI site found at 7315
Illegal BsaI.rc site found at 3977
Illegal BsaI.rc site found at 7565
Illegal SapI.rc site found at 6252
Illegal SapI.rc site found at 7260
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