Difference between revisions of "Part:BBa K2753003"

Revision as of 11:58, 1 October 2018

pSB1C3-obGES cds

This part is the coding sequence of geraniol synthase (GES) from the species Ocimum basilica (sweet basil) and it’s codon-optimised for the use in E. coli. GES catalyses the conversion of geranyl diphosphate (GPP) into geraniol, an acyclic monoterpene alcohol that has been widely used in many industries^[1], and it’s an intermediate to the biosynthesis of nepetalactol which is an active ingredient in catnip.

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 30℃, 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

Reference

[1]: Chen, F., Li, W., Jiang, L., Pu, X., Yang, Y., & Zhang, G. (2016). Functional characterization of a geraniol synthase ‑ encoding gene from Camptotheca acuminata and its application in production of geraniol in Escherichia coli. Journal of Industrial Microbiology & Biotechnology. https://doi.org/10.1007/s10295-016-1802-2