Part:BBa_K2027040:Design

DXS Synthase protein (G3P +Pyr->DXP)

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 318
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 25
  Illegal SapI site found at 195
  Illegal SapI.rc site found at 749
  

Design Notes

Although IPP and DMAPP could be supplied to the cell by a media supplement, a more appealing and system incorporated approach is to produce both compounds within the host organism using only glucose. Since IPP and DMAPP are endogenous to many species, we identified the two IPP biosynthesis pathways: the mevalonate (MVA) pathway and methylerythritol phosphate (MEP/DOXP) pathway. While pathways generate IPP and DMAPP from ; however, only the MEP/DOXP pathway is endogenous in E. coli.[1] Through a six step process, the MEP/DOXP pathway converts molecules of pyruvate and glyceraldehyde 3-phosphate (G3P) into IPP and DMAPP. While incorporating the MVA pathway into E. coli would further enhance IPP and DMAPP output, we opted for optimizing the MEP/DOXP pathway to minimize the step count from converting a substrate into IPP.[2, 3] Additionally, since G3P and pyruvate are products of glycolysis, providing a glucose supplement to E. coli would allow for G3P and pyruvate use by the MEP/DOXP pathway without increasing cell stress.[4]

To optimize the MEP/DOXP pathway, we overexpressed reaction rate limiting enzymes in the pathway, namely DXP synthase (DXS). The source of DXS is from E. coli K12 (EG13612). Although we could have overexpressed all of the enzymes in the MEP/DOXP pathway, from prior research DXS was determined to be the rate limiting step in the metabolic pathway.[5] To control for DXS expression, we codon optimized and reintroduced the DXS enzyme into E. coli under an IPTG inducible constitutive promoter (DXS plasmid), allowing for regulated production of DXS. A supplement of IPTG would then activate high level expression of DXS, which in turn would override the pathway bottleneck at the beginning of the MEP/DOXP pathway. In accelerating the process limiting chemical conversion of G3P and pyruvate to 1-Deoxy-D-xylulose 5-phosphate (DXP or DOXP), we can subsequently receive a greater output of IPP and DMAPP downstream.

In order to design our optimized DXS synthase, we obtained the sequence for the DXS gene from EcoGene (EG13612). To allow for future protein purification and characterization, we also attached a FLAG, tetracysteine, and hexahistidine tag to the DXS gene. A double terminator, (BBa_B0010 and BBa_B0012) was also used to prevent RNA polymerase leak through.[6]

The DXS synthase has been biobricked into BBa_K2027056 (https://parts.igem.org/Part:BBa_K2027056).

Additionally, this construct also includes the following ribosome binding sites, terminators, tags, and promoters:

• T7 Strong promoter (https://parts.igem.org/Part:BBa_Z0251)
• Elowitz RBS (https://parts.igem.org/Part:BBa_B0034)
• FLAG-Lumio His Tag (https://parts.igem.org/Part:BBa_K2027052)
• Terminator from E. coli rrnB (https://parts.igem.org/Part:BBa_B0010)
• Terminator from coliphage T7 (https://parts.igem.org/Part:BBa_B0012)

Sequencing

Because the construct was too large to sequence in its entirety, we sequenced it in ~800-1000 bp stretches.

Characterization

DXS Synthase is a 152.47 kDa protein.

Source

• http://www.ecogene.org/gene/EG13612/dnasequence?q=gene/EG13612/dnasequence&sites[0]=BamHI&sites[1]=EcoRI&sites[2]=HindIII&left_end=433315&right_end=443315&type=fasta

References

1. Wiemer, AJ; Hsiao, CH; Wiemer, DF (2010). "Isoprenoid metabolism as a therapeutic target in gram-negative pathogens.". Current topics in medicinal chemistry 10 (18): 1858–71.
2. Buhaescu I, Izzedine H. Mevalonate pathway: A review of clinical and therapeutical implications. 2007. ClinBiochem 40:575–584.
3. Martin VJ, Pitera DJ, Withers ST, Newman JD, Keasling JD. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nat Biotechnol. 2003;21(7):796-802.
4. Zhao Y, Yang J, Qin B, et al. Biosynthesis of isoprene in Escherichia coli via methylerythritol phosphate (MEP) pathway. Appl Microbiol Biotechnol. 2011;90(6):1915-22.
5. “World Butadiene supply to tighten.” Oil and Gas Journal. 22 Jan 2001. Web.
6. Nielsen, Alec. “Genetic Circuits.” BioE 393. Stanford University. 2 May 2016. Conference.