Part:BBa_K4011007

SaACS2

SaACS2 is an acetyl-CoA synthase, responsible for synthesizing acetyl-CoA from a host of different substrates. This year, we will express SaACS2 in yeast cells to turn acetate into acetyl-CoA, providing the first step for our metabolic pathway which converts acetate and ethanol into ethyl-acetate. The part will be used to construct yeast expression plasmids through golden gate assembly into pTEF-SaACS2-tADH1 BBa_K4011016 and 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.

Source

SaACS2 is from Salmonella enterica.

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
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1537
  
• 1000
  COMPATIBLE WITH RFC[1000]