Part:BBa_K4011006

AeAT9

AeAT9 is an alcohol acyltransferase, and is responsible for the production of ethyl-acetate from acetyl-CoA and ethanol in our fermentation process. We will express AeAT9 in yeast cells to synthesize ethyl-acetate from acetyl-CoA and ethanol, the second step into our pathway to turn acetate and ethanol into ethyl-acetate. We hope to give our yeast culture a fruit smell. This part will also be used to construct yeast expression plasmids through golden gate assembly into BBa_K4011017 and 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.

It was first characterized and expressed in yeast to produce ethyl-acetate by Shi et al in 2021.

Source

Source: AeAT9 is from Actinidia eriantha.

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).

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

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).

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 BglII site found at 199
  Illegal BglII site found at 817
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 226
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 1064