Best Basic Part

This part is the coding sequence of geraniol synthase (GES) from the species Ocimum basilicum (sweet basil)^[1] 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 and is commercially important in favour and fragrance industries^[2], and it’s an intermediate to the biosynthesis of nepetalactol which is an active ingredient in catnip.

Characterization

In our study, we aim to achieve geraniol synthesis in E. coli, so at the beginning, we expressed a geraniol synthesis operon (BBa_K2753016) containing an Abies Grandis geranyl pyrophosphate synthase (BBa_K2753002) and an Ocimum basilicum geraniol synthase (BBa_K2753003) regulated by a pTac promoter in E. coli. Assuming that higher expression leads to higher yield, the operon was assembled on a high copy vector pUC20 whose copy number is approximately 500 per cell. However, when carrying out shake-flask fermentation with this strain, after induced by 25uM IPTG for 24 hours^[2], no geraniol production was detected using gas chromatography. (See detailed methods in our Notebook)

We infer that the failure to produce geraniol was due to the lack of substrates IPP and DMAPP. Thus we decided to co-express an MVA pathway in addition to the geraniol synthesis operon. The pathway can either be added by cotransformation of a plasmid containing only the MVA pathway and the other plasmid with only GPPS and GES, or by constructing an all-in-one plasmid containing the MVA pathway, GPPS, and GES. We obtained a plasmid pMVA-only which was a gift from Taek Soon Lee, and have added GPPS and GES onto pMVA-only to acquire a pMVA-GPPS-GES.

Under the same fermentation condition, pMVA-GPPS-GES gave a geraniol titer of 11.4mg/L while the negative control pMVA-only as well produced geraniol titer of about 5.65mg/L. Then we are assured that a stable supply of precursors IPP and DMAPP are crucial for geraniol production. But still, there is room for higher geraniol yield. Therefore, to gain maximum flux to geraniol, we tuned the expression level of GPPS and GES using three promoters:

• TALE sp1 (BBa_K2753030), copy number-independent promoter of medium strength
• TALE sp2 (BBa_K2753018), copy number-independent promoter of low strength
• PTac (BBa_K180000), IPTG inducible promoter

And three vectors of unique copy number:

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

Having transferred the constructs above along with pMVA-only into strain E. coli DH5α, the same shake-flask fermentation experiment was conducted. The result shows detectable geraniol production by each strain.

With pTac promoters, geraniol yield increased with the copy number of the vector, showing positive correlation. However, when TALE stabilized promoter (TALEsp) was used, higher the copy number of the vector, lower the production of geraniol, being the opposite of pTac. And the stronger TALEsp promoter, TALE1sp1 gave generally reduced yield compared to its weaker counterpart TALE2sp1. Interestingly, the graphs of the two TALEsp promoters appeared to be seemingly parallel to each other.

We surmised that the yield of geraniol was affected by two factors: the expression level of enzymes and the cellular burden. As for enzyme expression, there exists an optimal gene expression level that produces just enough enzyme to metabolize all the substrate. The yield would be the greatest at this level. But if lower than this level, production would increase with enzyme expression since there is a surplus of substrates. And if the enzyme expression is higher, the more enzymes now becomes a cellular burden as it no longer contributes to more product.

In the case of pTac, it fits into the scenario when the gene expression is lower than the optimum: higher copy produced more enzymes to catalyze geraniol synthesis reaction. As for TALE stabilized promoters, the expression level of enzymes would remain unchanged regardless of copy number due to the stabilization nature of TALE (More about TALE). So in low copy vector where the cellular burden isn’t significant, the product yield is only related to the strength of the promoter, explaining why TALE sp2 has better performance than TALE sp1 as it’s closer to the optimum. But when the copy number increases, the expression level is unaffected while the cellular burden rises sharply because much more TALE would be made to stabilize expression, leaving less energy available for geraniol synthesis. And it in turns answers why a negative trend of production is shown when regulated by TALE.

References

1. Fischer, M. J. C., Meyer, S., Claudel, P., Perrin, M., Ginglinger, J. F., Gertz, C., … Karst, F. (2013). Specificity of Ocimum basilicum geraniol synthase modified by its expression in different heterologous systems. Journal of Biotechnology, 163(1), 24–29. https://doi.org/10.1016/j.jbiotec.2012.10.012
2. 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