Part:BBa_K4011017

pRPL8B-AeAT9-tSSA1

pRPL8B-AeAT9-tSSA1 is an expression cassette in S. cerevisiae capable of expressing AeAT9, which will synthesize ethyl-acetate from acetyl-CoA and ethanol. This is the second step into our pathway to turn acetate and ethanol into ethyl-acetate. pRPL8B BBa_K4011020, AeAT9 BBa_K4011006, and tSSA1 BBa_K4012020 were used to construct pRPL8B-AeAT9-tSSA1 using Golden Gate assembly. This part was used to construct pTEF1-SaACS2-tADH1-pRPL8B-AeAT9-tSSA1 BBa_K4011018.

Usage and Biology

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). pRPL8B is a medium strength promoter and tADH1 is a relatively strong terminator both characterized by Lee et al in 2015 (Lee et al, 2015).

Source

pRPL8B and tSSA1 all come from S. cerevisiae, while AeAT9 comes from Actinidia eriantha.

Design Considerations

1. All codons were optimized for S. cerevisiae based on S. cerevisiae codon bias.

2. BsaI restriction sites were added to the three basic parts used to construct pRPL8B-AeAT9-tSSA1 for Golden Gate assembly.

3. BsmB1 sites flank the composite part for further Golden Gate assembly construction.

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. 2A).

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 (Fig. 1), and pTEF1-SaACS2-tADH1-pRPL8B-AeAT9-tSSA1 (Fig. 2C). 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. 2B).

Figure 1: Gel electrophoresis results for pRPL8B-AeAT9-tSSA1

After transformation, we attempted to do fermentation but we have yet to receive optimal fermentation results due to time constraints.

Figure 1: Production of ethyl-acetate using engineered S. cerevisiae BY4741. A). Production pathway of ethyl-acetate from acetate and ethanol using ACS2 and AT9. B) Schematic representing engineeredS. cerevisiae BY4741 expressing ACS2 and AT9. C) Gel electrophoresis results of pTEF1-SaACS2-tADH1-pRPL8B-AeAT9-tSSA1.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 705
  Illegal BglII site found at 912
  Illegal BglII site found at 1530
  Illegal XhoI site found at 2029
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 939
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 2015
  Illegal BsaI.rc site found at 2265
  Illegal SapI.rc site found at 1777
  

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.