Coding

Part:BBa_K3036001

Designed by: Zhe Feng   Group: iGEM19_BNU-China   (2019-10-09)


Acetokinase

This part encodes encodes acetokinase (ACK) of E. coli. The natural function of ACK is to convert acetate into acetyl phosphate. However, the reversibility of the reaction confers ACK a potential to produce acetic acid using acetyl phosphate as a precursor. Our team made use of this part, together with phosphotransacetylase (PTA), to overproduce acetic acid.

Biology and Usage

Derived from E. coli DH5alpha genome, the gene ack encodes acetokinase (ACK), of which the natural function is to reversibly convert acetate into acetyl phosphate.

However, the reversibility of the reaction confers ACK a potential to produce acetic acid using acetyl phosphate as a precursor. Together with phosphotransacetylase (PTA), which produces acetyl phosphate out of acetyl-CoA, ACK 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.

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

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 597
  • 1000
    COMPATIBLE WITH RFC[1000]



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.


2019 BNU-China BBa K3036001 fig1.png

Figure 1 Acetate overproduction by coexpression of ACK and PTA

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).

2019 BNU-China BBa K3036001 fig2.png

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.


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 597
  • 1000
    COMPATIBLE WITH RFC[1000]


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Categories
Parameters
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