Team:WrightState/Engineering
Engineering
Our approach in biomanufacturing α-Terpineol was a two plasmid system that would be able to utilize a native yeast pathway to convert Acetyl-CoA all the way to α-Terpineol . This native yeast pathway is called the Mevalonate pathway and is normally used for momoterpene production. However, we decided to utilize this system in E. coli rather than yeast. This decision was made due to natural inhibitors that exist in yeast that may cause a low yield. The thought was to remove the inhibitors and get a much higher production of our monoterpene.
Design of Dual Plasmid System
The first plasmid (JBEI-3085) contains the MEV pathway and produces IPP and DMAPP
The second plasmid (pTrc_GppsGb_VvTs) was purchased from Addgene (#50603) and modified to produce α -Terpineol
*Addgene plasmid (#50603) prior to being digested and recloned with newly designed part (BBa_K4078003)
*Cloned to include GPPS/αTS geneblock from IDT
*Our GPPS (BBa_K2753002) was used from a past iGEM team (Great Bay). This newly constructed plasmid contains a LacI repressor gene and is inducible by IPTG*
Build
To build our plasmids we used transformation methods with MG1655 competent cells and sequenced the resulting mini-preps to confirm prior to moving forward. The building process took the most time as we were having issues with digests and verifying what we were sent is correct. Numerous gels were run, and many digests were performed but after that was figured out we were ready for production assays.
Test
In order to test production, we ran production assays where 5mL cultures were grown to an OD600 of 0.8. 1mL of dodecane was added along with the given concentration of IPTG and allowed to grow for 72 hours. To extract, 4mL of ethyl acetate was added and the organic layer was harvested, the samples were then quantified with GCMS.
Quantification by GC/MS
We believe that the production levels that we obtained may be too low to be detected by GC/MS. Though as it is being shown, there is no production of α -Terpineol at the moment and something that can be optimized and worked on in the years to come.
Learn
The experiments that followed the discovery of zero production included a few different steps. The first was varying the concentrations in which we induced production with IPTG to see if the problem was the use of note enough or far too much. We also spoke to an expert in the field, Dr. Michael Miller, who worked with terpene production for several years, and he suggested that switching the order of the GPPS and the αTS may affect production.
In the future, we plan to use different enzymes, try out different RBS as well as Bacterial strains that may be more effective. Though it is still a work in progress, we are continually learning from our results and reevaluating.
Sources
Sarria, S., Wong, B., Martín, H. G., Keasling, J. D., & Peralta-Yahya, P. (2014). Microbial synthesis of pinene. ACS Synthetic Biology, 3(7), 466–475. https://doi.org/10.1021/sb4001382
Alonso-Gutierrez, J., Chan, R., Batth, T. S., Adams, P. D., Keasling, J. D., Petzold, C. J., & Lee, T. S. (2013). Metabolic engineering of escherichia coli for limonene and perillyl alcohol production. Metabolic Engineering, 19, 33–41. https://doi.org/10.1016/j.ymben.2013.05.004