Part:BBa_K2753002

pSB1C3-GPPS cds

GPPS: Geranyl diphosphate (GPP), the entry point to the formation of terpene moiety, is a product of the condensation of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) by GPP synthase (GPPS)^[1]. IPP and DMAPP are supplied by either MVA pathway or MEP pathway or both depending on species. This parts originated from the species Abies Grandis, and it’s codon-optimized for the use in E. coli.

Fig.1 The biosynthesis pathway for GPP ^[2].

Characterization

In our study, to investigate the optimal expression level of GPPS and GES for geraniol production, we assembled GPPS and obGES to make a geraniol-generating operon without a promoter(Part#), We employed three different promoters:

• pTALE sp1 (https://parts.igem.org/Part:BBa_K2753000), copy number-independent promoter of medium strength
• pTALE sp2 (https://parts.igem.org/Part:BBa_K2753001), copy number-independent promoter of low strength
• pTac (https://parts.igem.org/Part:BBa_K180000), IPTG inducible promoter

And three vectors of unique copy number:

• pUC20: ~500
• pR6K: ~15
• pSC101: ~1

In total, we obtained nine combinations of promoters and vectors which regulate the expression of GPPS with GES.

We co-expressed a heterologous yeast MVA pathway with the nine synthetic constructs in E. coli, obtaining nine E. coli strain potentially able to produce geraniol. Shake-flask fermentation is carried out with all strains along with the negative control strain containing only the MVA pathway. E. coli was incubated into 50ml LB supplemented with corresponding antibiotics, and the initial OD₆₀₀ was adjusted to 0.01. When OD₆₀₀ reached 1, 25uM of IPTG was added into the culture, and the culture was shaken at 37℃, 250rpm for 24h. Then 2ml of dodecane phase was added into 5ml of culture. The mixture was vortexed for 2min, and the organic phase was analyzed by gas chromatography to identify geraniol production.

The results indicate all combinations except for pTALEsp1 with high copy number generate significantly higher titer of geraniol compared to the negative control. Moreover, the combinations of pTac with high copy number and pTALE with lower copy number are more advantageous.

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
  COMPATIBLE WITH RFC[1000]

Referece

[1]: Rai, A., Smita, S. S., Singh, A. K., Shanker, K., & Nagegowda, D. A. (2013). Heteromeric and Homomeric Geranyl Diphosphate Synthases from Catharanthus roseus and Their Role in Monoterpene Indole Alkaloid Biosynthesis. Molecular Plant, 6(5), 1531–1549. doi:10.1093/mp/sst058

[2]: Zebec, Z., Wilkes, J., Jervis, A. J., Scrutton, N. S., Takano, E., & Breitling, R. (n.d.). Towards synthesis of monoterpenes and derivatives using synthetic biology. Current Opinion in Chemical Biology, 34, 37–43. https://doi.org/10.1016/j.cbpa.2016.06.002