Team:Wisconsin-Madison/lemon

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• 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

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Engineering a limonene production pathway of E.coli

Why Produce Limonene?

Limonene is a 10-carbon monoterpene found in the oils of citrus fruits. It is used as a cleaning agent, solvent, and food additive, and recent research has shown it has potential ant-cancer applications. Limonene also possesses the chemical properties of an ideal biofuel; its low freezing point, combustibility, and high energy density make it a potential jet fuel replacement. 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 Escherichia coli , could be used to produce limonene more effectively and efficiently.

The Mevalonate Pathway

The mevalonate pathway, found in plants and fungi, produces 3-isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) from acetyl-CoA. IPP and DMAPP are the building blocks of the isoprenoids, a group of organic molecules with a wide array of functions. These compounds are normally synthesized in small amounts, preventing large-scale production; by moving the mevalonate pathway into E. coli this limitation can be circumvented. The Keasling lab at UC-Berkeley used this approach to produce the isoprenoid amorphadiene, which can be converted into the antimalarial drug artemisinin. This breakthrough brought the price of artemisinin down dramatically for treatment in the developing world.

We have constructed a strain containing the mevalonate pathway, a codon-optimized geranyl diphosphate synthase (GPPS), and a codon-optimized limonene synthase gene. The plasmid pBba5c contains the genes for the mevalonate pathway under a lactose-inducible promoter. The plasmid originally contained the unnecessary ispA gene; we used the Gibson cloning method to replace ispA with the GPPS gene. The resulting plasmid, pBba5c-GPPS, should theoretically allow higher production of limonene.

The Production Assay

Five milliliter cultures of each strain (listed below) were grown overnight at 37°C in LB, then normalized to an OD_{600 of 1 and diluted 1:100 into 40 mL cultures. These were then grown up to an OD600 of 0.2 and the promoters in the pBba5C vector induced with 1 mM IPTG. A 10 mL overlay of dodecane was used to trap any limonene produced. 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.}

Strain	Purpose
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

In figures A, C, and D, the large black limonene peak is a doped limonene standard included as a positive control for the GC/MS assay. The blue and purple lines cannot be seen, because they are along the baseline; i.e. no limonene is being produced. J23102 (empty) in figures A, B, and D is the promotoer run as a negative control. Figure B demonstrates the decrease in amorphadiene between the regular pBbA5c and the pBbA5c with the GPPS swapped in for the ispA gene.

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

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 natural version 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 6 curves are organized by either base mevalonate vector or by a version of limonene synthase. pBad33 mimics pBbA5c, by acting as an empty vector with no operons. The error bars come from the standard deviation of 4 different turbidity measurements from 96-well plate data.

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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