Team:Wisconsin-Madison/lemon

• Team

  • Team Members
  • Advisors
  • Sponsors

• Projects

  • Engineering Limonene Production in E.coli
  • Translational Coupling Cassette (TCC)

• Parts

  • Translational Coupling Cassette
  • Negative TCC Control
  • Positive TCC Control
  • Codon-optomized LIMS1

• Modeling

• Protocol

• Safety

• Human practice

Engineering a limonene production pathway of E.coli

Why Produce Limonene?

The goal of this project is to engineer Escherichia coli to produce a compound called limonene. Limonene is a 10 carbon monoterpene and is found naturally in the oils of citrus fruits. It is used as a cleaning agent, solvent, food additive and is even finding a place in new medical applications. Limonene also possesses the chemical properties of an ideal biofuel, sparking interest specifically in its application as a jet fuel due to its low freezing point. Currently, we are limited to extracting limonene directly from citrus fruits which prevents its collection in quantities large enough to be useful as a biofuel. E coli. could be used to produce limonene more effectively and efficiently.

In this research, a strain of E. coli will be bioengineered to produce limonene by inserting the genes for the necessary biochemical pathways. Our strain contains the genes coding for the Mevalonate pathway ,a synthesized Geranyl Diphosphate Synthase (GPPS) gene, and a synthesized and codon optimized Limonene Synthase gene. The plasmid, pBba5c, contains the genes for the mevalonate pathway in an operon under a lac inducible promoter. During our research we used the Gibson cloning method to take out the ispA gene and put in the GPPS gene into the pBba5c plasmid, creating pBba5c-GPPS for better production of Limonene. This strain would theoretically be able to produce Limonene using common media.

Mevalonate Pathway

5 mL cultures of each strain (listed below) were grown overnight at 37 C in LB, then normalized to an OD600 of 1 and diluted 1:100 into 40 mL cultures. They were then grown up to an OD600 of 0.2 and the promoters in the pBba5C vector were induced with 1 mM IPTG. 10 mL of dodecane was used to overlay the culture. This overlay was used as an organic layer for the limonene to diffuse into. These cultures were then grown for 18 hours, spun down, and 1mL of the dodecane overlay was diluted in ethyl acetate and sampled in a GC/MS.

In this research, a strain of E. coli will be bioengineered to produce limonene by inserting the genes for the necessary biochemical pathways. Our strain contains the genes coding for the Mevalonate pathway ,a synthesized Geranyl Diphosphate Synthase (GPPS) gene, and a synthesized and codon optimized Limonene Synthase gene. The plasmid, pBba5c, contains the genes for the mevalonate pathway in an operon under a lac inducible promoter. During our research we used the Gibson cloning method to take out the ispA gene and put in the GPPS gene into the pBba5c plasmid, creating pBba5c-GPPS for better production of Limonene. This strain would theoretically be able to produce Limonene using common media.

The Production Assay

5 mL cultures of each strain (listed below) were grown overnight at 37 C in LB, then normalized to an OD600 of 1 and diluted 1:100 into 40 mL cultures. They were then grown up to an OD600 of 0.2 and the promoters in the pBba5C vector were induced with 1 mM IPTG. 10 mL of dodecane was used to overlay the culture. This overlay was used as an organic layer for the limonene to diffuse into. These cultures were then grown for 18 hours, spun down, and 1mL of the dodecane overlay was diluted in ethyl acetate and sampled in a GC/MS.

As seen in this GC/MS data, we were not able to produce any limonene. There was a small peak located where limonene was supposed to be, but unfortunately that peak also showed up in our empty promoter strain as well. In order to troubleshoot the lack of production of limonene, we tried to produce amorphadiene, which shares the same precursor molecules as limonene up until GPP. The ispA gene synthesizes (FPP), the precursor to amorphadiene. When we created the modified pBba5C gene, the one with GPP synthase, we swapped out this ispA gene. So by using these two pBba5C strains, we could troubleshoot the production of limonene by testing the mevalonate pathway located within the pBba5C vector. In the GC/MS data, our pathway does create amorphadiene when amorphadiene synthase is included in the bacterial strain with pBba5C. Thus, our mevalonate pathway seems to be functioning correctly. However, we also tested pBba5C-GPPS with the amorphadiene synthase, which should create no amorphadiene, as a negative control. There was still a peak found in the GC/MS data, but this is because the Escherichia coli genome naturally contains the ispA gene. This peak was much smaller than the peak made by the strain containing the ispA gene in the plasmid. This tells us that our pBba5C-GPPS construct seems to be correctly functioning as well.

Conclusions

As stated in the last section, it seems as though the mevalonate pathway is functioning correctly. This leads us to think that something is wrong with our limonene synthase gene, or the extraction protocol. Neither the codon optimized or classic versions are producing limonene, and we are unsure why. There are a number of things that can go wrong from transcription through a functioning protein, and we needed a way to troubleshoot. This led us to an idea that our PI’s lab had engineered upstairs: a translational coupling cassette. With this cassette we could determine if limonene synthase (codon and non-codon optimized) was being translated.

References

“Engineering a mevalonate pathway in Escherichia coli for production of terpenoids”

Keasling et al., 2003

“Monoterpene biosynthesis in lemon”

Verhoeven et al., 2002

“Biosynthesis of plant isoprenoids: perspectives for microbial engineering”

Keasling et al., 2009