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 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 even new medical applications. Limonene also possesses the chemical properties of an ideal biofuel, specifically as a jet fuel due to its low freezing point, combustibility and high energy density. Currently, industrial production is restricted to direct extraction from citrus fruits. This prevents collection in quantities large enough to be useful as a biofuel. Other organisms, such as E. coli, could be used to produce limonene more effectively and efficiently.

The Mevalonate Pathway

The mevalonate pathway is a series of enzymes used to take Acetyl-CoA to 3-isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) through several chemical reactions. IPP and DMAPP are the building blocks for a family of molecules called Isoprenoids which are a group of organic molecules with a wide array of functions. It was used in the Jay Keasling’s lab to create an antimalarial drug precursor, amorphadiene, much cheaper and faster than was previously possible.

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 replace the ispA gene with the GPPS gene into the pBba5c plasmid. This created pBba5c-GPPS for better theoretical 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 were 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, centrifuged, and 1mL of the dodecane overlay was diluted in ethyl acetate and sampled in a GC/MS.

pBba5c + J23102-RFP (J23102 promoter only as a negative control)

pBba5c + J23102-LimS1 (Production strain)

pBba5c + J23102-CO_LimS (Production Strain)

pBba5c + pTRC-ADS (pTRC-ADS is the amorphadiene synthase)

pBba5c-GPPS + J23102-RFP (Negative control)

pBba5c-GPPS + J23102-LimS1 (Production strain)

pBba5c-GPPS + J23102-CO_LimS (Production strain

pBba5c-GPPS + J23102-pTRC-ADS (Testing the GPPS-ispA swap with gibson)

---Insert GC/MS graphs here, (J23-lims, J23-COlims, both with pbb and pbb-gpps)
---(empty promoters [rfp] with pbb and pbb-gps)
---(pADS with pbb and pbb-gpps)
---(limonene standards)

As seen in this GC/MS data, limonene was not produced. There was a small peak located where limonene was supposed to be, but unfortunately that peak also showed up in the empty promoter strain as well. In order to troubleshoot the lack of production, we tried to produce amorphadiene, which shares the same precursor molecules as limonene up until the GPP step of the pathway. The ispA gene synthesizes Farnesyl Pyrophosphate (FPP), the precursor to amorphadiene. Both the pBbA5c with the ispA and with the GPPS were used to troubleshoot the mevalonate pathway by attempting to generate amorphadiene. In the GC/MS data, both strains created amorphadiene when amorphadiene synthase was included in the bacterial strain. Thus, the mevalonate pathway seems to be functioning correctly. However, the pBba5C-GPPS strain with the amorphadiene synthase should not create amorphadiene because ispA has been replaced with GPPS. There was still an amorphadiene peak found in the GC/MS data, but this is because the E. coli genome naturally contains the ispA gene. The amorphadiene peak associated with the pBbA5c-GPPS was much smaller than the peak generated by the pBbA5c-ispA construct. This implies that ispA was replaced correctly (also confirmed by sequencing data), but does not prove that the GPPS is working as anticipated.

The Conclusion

As stated in the last section, the data shows that the mevalonate pathway is functioning correctly. This means that something is either wrong with the limonene synthase gene or the GPPS. Neither the codon optimized nor the classic versions of limonene synthase are producing limonene. To further troubleshoot the production strains, a growth curve was run to determine if cell viability was being affected by the synthetic pathway.

These growth curves present two important conclusions. The classic limonene synthase shows a growth defect in comparison to the synthesized codon-optimized limonene synthase. More dramatically, the pBbA5c with ispA greatly inhibits cell growth. A possible explanation for this is the toxicity of the FPP to the cell as the pBbA5c-GPPS did not inhibit cell growth in comparison to the pBbA5c-ispA. Since ispA is native to E. coli, it should be translated more efficiently than the other enzymes in the mevalonate pathway, including GPPS. To further interpret this growth curve data it would be beneficial to insert GPPS and ispA into the Translation Coupling Cassette (TCC) and compare their translational efficiency. The rationale for the focus on GPPS is due to the fact that we now know the codon-optimized limonene synthase is being translated because of our TCC data (See TCC page for further explanation). However, even though limonene synthase is being translated, it may not be functioning correctly. This leads us to the in vitro assays described on the TCC project page. With additional TCC data and in vitro assays, we would hope to determine why no limonene is being produced.

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