Part:BBa_K1587004

Butyrate synthesis pathway regulated by constitutive promoter p(Bla)

BioBrick Description

Constitutive promoter p(Bla): BBa_I14018. Crotonyl-coA reductase ccr coding region. 3-hydroxybutyryl-CoA dehydrogenase hbd coding region. 3-hydroxybutyryl-CoA dehydratase crt coding region. Acyl-CoA thioesterase 2 tesB coding region. acetyl-CoA acetyltransferase atoB coding region. Every genes are framed by strong RBS based on Ron Weiss thesis:BBa_B0030. This operon ends with an artificial terminator according to BBa_B1006.
Due to a lack of time we decided to have this construction synthesised instead of using the assembly protocol.

Figure 1: Genetic construct to produce butyrate via glucose

Usage and Biology

Synthesis of butyrate has interested the scientific community because it is the precursor of butanol. Indeed, butanol would be an interesting alternative fuel especially because it could be transported by present pipeline.[1]
In our project we want to produce butyrate to attract mites which infected bees but thanks to this energy interest, some metabolic pathways have been designed to produce butyrate.
An Escherichia coli strain is used for its known simplicity of genetic manipulation and its adequacy with butyrate synthesis [2]. Indeed, among the five enzymes we selected to produce butyrate from acetyl-CoA, two enzymes are naturally produced by the bacteria. The following engineered butyrate pathway has been designed:

Figure 2: Metabolic pathway to produce butyrate via glucose using BBa_K1587004 in E. coli

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 36
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1717
  Illegal BamHI site found at 3634
  Illegal XhoI site found at 1407
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 332
  Illegal AgeI site found at 1254
  Illegal AgeI site found at 2900
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1752
  Illegal BsaI.rc site found at 2169
  Illegal BsaI.rc site found at 3237
  Illegal SapI site found at 123
  Illegal SapI.rc site found at 3249
  

Sequencing has been made thanks to 7 forward specific primers for genes contained in BBa_K1587004 in order to have the complete sequence:
VF2, CCR, hbd, crt, testB, atoB and VR
Sequence is therefore validated.

Experiments

In order to test BBa_K1587004 for butyrate production we made [http://2015.igem.org/Team:Toulouse/Experiments#erlencult micro-aerobic culture]. Samples were analyzed by [http://2015.igem.org/Team:Toulouse/Experiments#NMR NMR].

Unfortunatly NMR analysis did not yield a significant over production of butyrate in E. coli. Nevertheless, we obtained [http://2015.igem.org/Team:Toulouse/Results#butyproduct results] as the figure 3 below:

Figure 3: NMR analysis of butyrate production in E. coli with BBa_K1587004 and wt

We observed a statistically significant change in the production of other fermentation products such as ethanol or acetate.

References

[1] Mukesh Saini, Min Hong Chen, Chung-Jen Chiang, Yun-Peng Chao. Potential production platform of n-butanol in Escherichia coli. Metabolic Engineering 27 (2015) 76–82
[2] Atsumi S, Cann AF, Connor MR, Shen CR, Smith KM, Brynildsen MP, Chou KJY, Hanai T & Liao JC (2008) Metabolic engineering of Escherichia coli for 1-butanol production. Metabolic Engineering 10: 305–311