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The natural function of PTA is to reversibly convert acetyl-CoA into acetyl phosphate, whereas that of ACK is to reversibly convert acetate into acetyl phosphate. The trait of PTA, together with the reversibility of the conversion catalyzed by ACK, gives them a potential to enhance acetate production when overexpressed. Moreover, this enhancement can be further optimized when the level of its precursor, acetyl-CoA is lifted. Following this strategy, we include fatty acyl-CoA synthetase (FadD), a key enzyme in beta-oxidation of higher fatty acids, to increase the yield of acetate to a further extend.  
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The natural function of phosphotransacetylase (PTA) is to reversibly convert acetyl-CoA into acetyl phosphate, whereas that of acetokinase (ACK) is to reversibly convert acetate into acetyl phosphate. The trait of PTA, together with the reversibility of the conversion catalyzed by ACK, gives them a potential to enhance acetate production when overexpressed. Moreover, this enhancement can be further optimized when the level of its precursor, acetyl-CoA is lifted. Following this strategy, we include fatty acyl-CoA synthetase (FadD), a key enzyme in beta-oxidation of higher fatty acids, to increase the yield of acetate to a further extend.  
  
  

Latest revision as of 03:38, 22 October 2019


Acetic acid overproducing pathway

The natural function of phosphotransacetylase (PTA) is to reversibly convert acetyl-CoA into acetyl phosphate, whereas that of acetokinase (ACK) is to reversibly convert acetate into acetyl phosphate. The trait of PTA, together with the reversibility of the conversion catalyzed by ACK, gives them a potential to enhance acetate production when overexpressed. Moreover, this enhancement can be further optimized when the level of its precursor, acetyl-CoA is lifted. Following this strategy, we include fatty acyl-CoA synthetase (FadD), a key enzyme in beta-oxidation of higher fatty acids, to increase the yield of acetate to a further extend.


Acetate overproducing pathway
Function Acetate overproduction
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 615
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 1848



Properties

The function of this part is validated by comparing it to a negative control, as well as a system that only overexpresses PTA and ACK. As is shown below, the yield of acetate by this part not only remarkably lifted acetate production compared to control group, but also exceed the PTA-ACK system by nearly half fold (Fig. 1).

2019 BNU-China BBa K3036001 fig2.png

Fig. 1 Acetate overproduction by coexpression of ACK, PTA and FadD

Experimental approach

1.Transfer the plasmid into E. coli competent cells.
2.Culture the strains in LB-ampicillin (50 ng/μL) at 37℃ for 5 hours.
3.Add 200 mM oleate to the medium and 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.
[2] ZHANG HanXing. Screening of PoIyhydroxyalkanoates producing bacteria and its expression and metabolic mechanism in E.coli engineered bacteria:[D]. Jinan: Shandong University, 2006.