Part:BBa_K3036002

Phosphotransacetylase

This part encodes encodes phosphotransacetylase (PTA) of E. coli. The natural function of ACK is to convert acetyl-CoA into acetyl phosphate, giving it a potential to enhance acetate production by providing its precursor. Our team made use of this part, together with acetokinase (ACK), to overproduce acetic acid.

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
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 583
  

Characterized by BNU-China 2019

Biology and Usage

Derived from E. coli DH5alpha genome, the gene pta encodes phosphotransacetylase (PTA), of which the natural function is to reversibly convert acetyl-CoA into acetyl phosphate.

The natural trait of PTA gives it a potential to produce acetic acid by lifting the level of its precursor acetyl phosphate. Together with acetokinase (ACK), which converts acetyl phosphate into acetic acid, PTA notably enhances yield of acetate through a two-step enzymatic reaction[1]. In our project, we take advantage of the pathway to overexpress acetate using an intestinal microbe inside human intestine, where acetate signals consumption of human white fat tissue.

pta
Function	Phosphotransacetylase
Use in	Prokaryotes
RFC standard	RFC10 compatible
Backbone	pSB1C3
Derived from	Escherichia. coli DH5alpha

Properties

The function of this part is validated in an acetate-overproducing system, where ACK and PTA are coexpressed in a polycistron system under control of a Plac promoter. Using Megazyme acetic acid assay kit, we verified that the yield of acetate is enhanced by a remarkable 9 fold, as is shown in Fig. 1.

Figure 1 Acetate overproduction by coexpression of PTA and ACK

This overproduction can be further enhanced by coexpressing enzymes involved in acetyl-CoA-yielding processes, such as fatty acyl-CoA synthetase (FadD), key enzyme in beta-oxidation of higher fatty acids (Fig. 2).

Fig. 2 Acetate overproduction by coexpression of ACK, PTA and FadD

Experimental approach

1.Transfer the plasmid into E. coli competent cells.
2.Culture the strain in LB-ampicillin (50 ng/μL) at 37℃ for 5 hours, use a strain containing pUC19 as control group.
3.Induce both groups by addition of IPTG to a final concentration of 5 mM.
4.Keep culturing at 37℃ and take samples at 0 hr, 2 hr and 4 hr after induction.
5.Measure acetate content using Megazyme acetic acid assay kit.
6.Three replicas are tested in each group.

Reference

[1] Kakuda H, Shiroishi K, Hosono K, Ichihara S. Construction of Pta-Ack Pathway Deletion Mutants of Escherichia coli and Characteristic Growth Profiles of the Mutants in a Rich Medium. Biotech. Biochem., 58 (12), 2232~2235, 1994.