Team:UNebraska-Lincoln/Design

UNL 2018 Improving Early Detection of The Emerald Ash Borer

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Project design

Our project design process started with literature search to find relevant information about the state-of-the-art strategy to engineer mavalonate pathway for sesquiterpene biosynthesis in E. coli. The team then had brainstorming sessions to go over various ideas about how to implement the strategy in our project. Next, we moved on to detailed experimental design, where we also outlined goals that we wanted to ultimately accomplish.

Literature survey and brainstorming:

The 7-epi-sesquithujene is a natural sesquiterpene. Its biosynthesis as an efficient attractant of Emerald Ash Borer initially appeared to be no easy task. The team came across a research paper on the production of another sesquiterpene, amorphadiene (amorpha-4,11-diene), which is a precursor to the potent antimalarial drug artemisinin, through the engineering of the mevalonate pathway in E. coli (Martin et. al, 2003). The mevalonate pathway is native to S. cerevisiae, but not to E. coli. A second paper published by the same research group further examined the optimization of the mevalonate pathway in E. coli in order to increase the production of amorphadiene (Anthony et. al, 2009). The first paper used a a two-plasmid system, where one plasmid supports the expression of mevalonate pathway enzymes and the second plasmid supports the expression of enzymes for the biosynthesis of FPP (farnesyl pyrophosphate) from mevalonate and its conversion to amorphadiene. The second paper used a different two-plasmid system, which showed improve production of amorphadiene. In this paper, the first plasmid encodes all the enzymes that are required for the biosynthesis of FPP (Addgene, 100167), while the second plasmid (Addgene 100169) encodes the ADS (amorphadiene synthase) and mevalonate kinase, which was identified as the rate-limiting enzyme. The team decided to moved forward with the design in the second paper. We obtained plasmid 100167 and 100169 from Addgene.

Although it was reported that plasmids 100167 and 100169 can be used together in E. coli. The team still did literature search to confirm that double transformation of the two plasmids can be easily performed in the lab. Initial research on the topic of double transformation of plasmids through chemical transformation showed that efficiencies were likely to be low when the plasmids had the same anitbiotic resistance gene and/or when the plasmids have the same origin of replication (Goldsmith et. al, 2007). Plasmid 100167 has chloramphenicol resistance marker and p15A origin of replication. Plasmid 100169 has ampicillin resistance marker and pBR322 origin of replication. The two plasmids can be simultaneously stably maintained by E. coli.

During brainstorming, the team also identified the enzyme that can synthesize 7-epi-sesquithujene from FPP. Gene TPS4-B73 was first isolated from Zea mays. We discussed strategy to enable the expression of the plant enzyme in E. coli host. After comparing the codon usage preference of Z. mays and E. coli, we decided to examine both the native and a codon-optimized version of TPS4-B73 gene and compare which one can support high production of 7-epi-sesquithujene.

Experimental design:

The team first determined cloning strategies to replace the amorphadiene synthase (ADS) gene in plasmid 100169 with either the native or the codon-optimized TPS4-B73 gene for the biosynthesis of 7-epi-sesquithujene. The team decided to use two methods in order to expand our lab skills. For the cloning of the native TPS4-B73 gene, we used sequence and ligation independent cloning (SLIC) method. For the case of the the codon optimized TPS4-B73 gene, we decided to use conventional cloning method, which requires restriction enzymes and DNA ligase. More details on the methods used are available in the lab notebook, experiments, and parts sections.

We next designed protocols for culturing the E. coli cells that express pathway for the biosynthesis of 7-epi-sesquithujene. Following the procedure of an article that describes the purification of volatile sesquiterpenes produced from unidentified terpene synthase genes (Kazutoshi et. al, 2018), we decided to use a culturing condition that requires a two-layer culture media consisting of Terrific Broth and octane. The E. coli cells were first cultured to desirable cell densities, IPTG was added for induction of the enzyme expression and octane was then added to culture media. The cultures were shaken for 2-4 days. The octane was then separated from the Terrific Broth via centrifugation and collected in an Erlenmeyer flask. The resulting cell pellets were stored in a -80 C freezer for ensuing protein analysis. More detailed explanation of these procedures can be found in the lab notebook, experiments and results sections.

Future Outlooks: Where From Here?:

  1. The next step would be a scale-up operation to see if the cells would be viable in a larger bioreactor with such a large amount of organic solvent in the media
  2. For verification of production, further purifying 7-epi-sesquithujene for analytical techniques such as NMR and IR would be beneficial. A procedure has been outlined in Kazutoshi et. al for a wide range of different sesquiterpenes, and preliminary trials were ran with purification of amorphadiene.
  3. Discussing with trap manufactures and further researching the amounts required for effective baiting in modern Emerald Ash Borer traps.
  4. Further collaboration with the Nebraska Forest Service and potentially collaborating with the U.S. Forest Service to see if our initial research could be helpful in any way.

Works Cited

  • Köllner, T. G.; Schnee, C.; Gershenzon, J.; Degenhardt, J. The Variability of Sesquiterpenes Emitted from Two Zea mays Cultivars Is Controlled by Allelic Variation of Two Terpene Synthase Genes Encoding Stereoselective Multiple Product Enzymes. http://www.plantcell.org/content/16/5/1115 (accessed Oct 12, 2018).
  • Martin, V. J. J.; Pitera, D. J.; Withers, S. T.; Newman, J. D.; Keasling, J. D. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. https://www.nature.com/articles/nbt833 (accessed Oct 12, 2018).
  • Anthony, J. R.; Anthony, L. C.; Nowroozi, F.; Kwon, G.; Newman, J. D.; Keasling, J. D. Optimization of the mevalonate-based isoprenoid biosynthetic pathway in Escherichia coli for production of the anti-malarial drug precursor amorpha-4,11-diene. https://www.sciencedirect.com/science/article/pii/S1096717608000438?via=ihub (accessed Oct 12, 2018).
  • Goldsmith; Moshe; Csaba; Bradbury; R.M., A.; Tawfik; S., D. Avoiding and controlling double transformation artifacts | Protein Engineering, Design and Selection | Oxford Academic. https://academic.oup.com/peds/article/20/7/315/1547087 (accessed Oct 12, 2018).
  • Kazutoshi, Shindo.; Jun-ichiro, Hattan.; Mariko, Kato.; Miho, Sato.; Tomoko, Ito.; Arisa, Watanabe; Maki, Sugiyama.; Yuri, Nakamura.; Norihiko, Misawa. Purification and structural analysis of volatile sesquiterpenes produced by Escherichia coli carrying unidentified terpene synthase genes from edible plants of the family Araliaceae | Researcher Information | J-GLOBAL. https://jglobal.jst.go.jp/en/detail?JGLOBAL_ID=201802282950523084 (accessed Oct 12, 2018).


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