Part:BBa_K4011016

pTEF1-SaACS2-tADH1

TEF1-SaACS2-tADH1 is an expression cassette in S. cerevisiae capable of expressing SaACS2, which will synthesize acetyl-CoA from acetate. Our final goal is to synthesize ethyl-acetate in yeast. pTEF1 BBa_K319003, SaACS2 BBa_K4011007, and tADH1 BBa_K4012022 were used to construct pTEF1-SaACS2-tADH1 using Golden Gate assembly. This part was used to construct pTEF1-SaACS2-tADH1-pRPL8B-AeAT9-tSSA1 BBa_K4011018.

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. pTEF1 is a relatively strong promoter and tADH1 is a medium strength terminator both characterized by Lee et al in 2021 (Lee et al, 2021).

Source

pTEF1 and tADH1 all come from S. cerevisiae, while SaACS2 comes from Salmonella enterica.

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 pTEF1-SaACS2-tADH1 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 (Fig. 1), pRPL8B-AeAT9-tSSA1, 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 pTEF1-SaACS2-tADH1

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

Figure 2: 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 XhoI site found at 2426
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1987
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 2412
  Illegal BsaI.rc site found at 205
  Illegal BsaI.rc site found at 2662
  

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.