Part:BBa_K4011018
pTEF1-SaACS2-tADH1-pRPL8B-AeAT9-tSSA1
pTEF1-SaACS2-tADH1-pRPL8B-AeAT9-tSSA1 is an expression cassette in S. cerevisiae capable of expressing AeAT9 and SaACS2, which will synthesize ethyl-acetate from acetate and ethanol. SaACS2 will convert acetate into acetyl-CoA, and AeAT9 will convert acetyl-CoA and ethanol to ethyl-acetate. pTEF1-SaACS2-tADH1 BBa_K4011016 and pRPL8B-AeAT9-tSSA1 BBa_K4011017 were used to construct pTEF1-SaACS2-tADH1-pRPL8B-AeAT9-tSSA1.
Usage and Biology
SaACS2 is an acetyl-CoA synthase from Salmonella enterica. Its natural function is to convert acetate into acetyl-CoA under anaerobic conditions. Its enzymatic rate is around 50 times that of ACS1 found in S. cerevisiae. AeAT9 is an alcohol acyltransferase from Actinidia eriantha, otherwise known as kiwifruit. Its general function is to convert alcohols into esters. The fruity smell one smells from kiwifruit it largely a result of AeAT9 (Shi et al, 2021). pTEF1 is a relatively strong promoter, tADH1 is a medium strength terminator, pRPL8B is a medium strength promoter, and tADH1 is a relatively strong terminator all characterized by Lee et al in 2015 (Lee et al, 2015).
Source
pTEF1, tADH1, pRPL8B, and tSSA1 all come from S. cerevisiae, while SaACS2 comes from Salmonella enterica and AeAT9 comes from Actinidia eriantha.
Design Considerations
1. All codons were optimized for S. cerevisiae based on S. cerevisiae codon bias.
2. Homologous recombination of S. cerevisiae was avoided by choosing different promoters and terminators for each expression casette (SaACS2 and AeAT9).
Characterization
We observed an accumulation of acetate in SCOBY, which contains an odorous smell and can interfere with secreted proteins such as Mα-CBM3-2Rep-CBM3. Therefore, in order to utilize the accumulated acetate and to convert it into something useful, we decided on a simple metabolic pathway to turn ethanol (also produced by yeast fermentation in SCOBY) and acetate into ethyl acetate, which contains a fruity smell. This pathway can be completed with two enzymes: SaACS2 (acetyl-CoA synthase) from Salmonella enterica and AeAT9 (acyltransferase) from Actinidia eriantha (kiwifruit). SaACS2 converts acetate into acetyl-CoA under anaerobic condition. AeAT9 will transfer the acetyl group from acetyl-CoA to ethanol to form ethyl acetate (Fig. 1A).
To construct plasmids capable of expression in yeast, we utilized a yeast genetic toolkit first characterized by Lee et al. to construct three plasmids: pTEF1-SaACS2-tADH1, pRPL8B-AeAT9-tSSA1, and pTEF1-SaACS2-tADH1-pRPL8B-AeAT9-tSSA1 (Fig. 1C). The whole construction process was done in close contact and collaboration with AISSU_Union. After our final plasmid (pTEF1-SaACS2-tADH1-pRPL8B-AeAT9-tSSA1) was constructed, we transformed it into yeast (Fig. 1B).
After transformation, we attempted to do fermentation but we have yet to receive optimal fermentation results due to time constraints.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 3375
Illegal BglII site found at 3582
Illegal BglII site found at 4200
Illegal XhoI site found at 2426
Illegal XhoI site found at 4699 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 3609
Illegal AgeI site found at 1987 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 2412
Illegal BsaI site found at 4685
Illegal BsaI.rc site found at 205
Illegal BsaI.rc site found at 2662
Illegal BsaI.rc site found at 4935
Illegal SapI.rc site found at 4447
References
Lee, M., DeLoache, W., Cervantes, B., & Dueber, J. (2015). A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. ACS Synthetic Biology, 4(9), 975-986.
Shi W, Li J, Chen Y, Liu X, Chen Y, Guo X, Xiao D. Metabolic Engineering of Saccharomyces cerevisiae for Ethyl Acetate Biosynthesis. ACS Synth Biol. 2021 Mar 19;10(3):495-504. doi: 10.1021/acssynbio.0c00446. Epub 2021 Feb 12. PMID: 33576609.
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