Metabolic Engineering to Enhance Terpene Yield in Nicotiana Tabacum
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Terpenoids provide a diverse range of applications in fuels, chemicals, specialty materials, nutraceuticals, and pharmaceuticals. Terpenes also provide unconventional sinks for the carbon fixed by photosynthesis. However, it is highly challenging to increase terpene production in higher plants, partially due to downstream conversion, toxicity to the cell and feedback regulations. My thesis research has mainly focused on developing strategies to address the challenges of terpene production in Nicotiana tabacum. First, a co-compartmentation strategy was design and implemented, in which biosynthesis and storage were co-compartmentalized via a synthetic droplet as an effective new strategy to improve the bioproduct yield, with squalene as a model compound. Instability of squalene was demonstrated by mathematic modeling and dark treatment experiments. Thus, a hydrophobic protein was designed and introduced into the tobacco chloroplast to generate a synthetic droplet for terpene storage. Simultaneously, squalene biosynthesis enzymes were introduced to chloroplasts together with the droplet-forming protein to co-compartmentalize the biosynthesis and storage of squalene. The strategy has enabled a significant increase of squalene yield without compromising plant growth. Confocal fluorescent microscopy imaging, Stimulated Raman Scattering microscopy, and droplet composition analysis confirmed the formation of synthetic storage droplets in chloroplasts. Second, an inter-compartmental ‘pull and block’ strategy was established to re-balance squalene biosynthesis and degradation via silencing squalene epoxidases. An actively expressed squalene epoxidase SQE3 was identified in the leaf of N. tabacum. An artificial microRNA was designed to target the consensus coding sequence of SQE3 to reduce the downstream conversion of squalene. Simultaneously, the squalene biosynthesis pathway was over-expressed in N. tabacum chloroplasts. Suppression of SQE3 was confirmed by RT-PCR. Squalene conversion decrease in SQE3-suppressed plants was confirmed by measuring sterol levels by GC-MS. The squalene yield in SQE3 suppressed plants has achieved 3.98 mg/g fresh weight without comprising plant growth. Overall, these studies established efficient photosynthetic platforms for terpene production. A sufficient sink capacity and a minimized downstream conversion was emphasized in the studies for high yield of bioproducts. The technology advances also provided new strategies for stabilizing high value bioproducts.
Zhao, Cheng (2018). Metabolic Engineering to Enhance Terpene Yield in Nicotiana Tabacum. Doctoral dissertation, Texas A & M University. Available electronically from