Synthetic Design of Pathways and Organelles for Photosynthetic Terpene Production

Synthetic Design of Pathways and Organelles for Photosynthetic Terpene Production
YongKyoung Kim, Hong Ma, Yu Wang, Sheba Goklany, Eiji Takahashi, Yi-cheng
Liu, Susie Y. Dai, Don Ort, Joseph Chappell, Xinguang Zhu, Joshua S. Yuan
One of the fundamental challenges in renewable energy is the efficient harnessing and transformation of sunlight energy for reducing inorganic carbon toward the production of fuels and chemicals. Photosynthetically-driven terpene production represents one of the most straightforward and energy-efficient route for converting sunlight and CO2 to fuel molecules. Moreover, terpene has a diverse range of industrial applications including special chemicals and nutraceuticals. We have designed and implemented synthetic pathways and organelles to achieve record-level photosynthetic production of squalene, a triterpene for biofuel, nutriceutical and other applications. First, the synthetic pathways were designed and implemented to redirect photorespiration by-product glycolate to pyruvate for terpene biosynthesis. The pathway design directly channeled a photosynthate into terpene production and has the potential to also reduce the energy consumed for recycling glycolate. Kinetics-based computational modeling was first carried out to evaluate how carbon re-partitioning will impact photosynthesis and terpene production. The results indicated that high terpene level via C2 (glycolate) redirection can be achieved without significant reduction of photosynthesis rate. The implementation of two alternative pathways clearly indicated that redirecting glycolate to terpene biosynthesis will lead to significant increase of terpene production. In particular, the synergistic engineering of C2 redirection with terpene biosynthesis has led to the production of 2700ug/g FW squalene, which is over four-fold increase of the level achieved by engineering terpene pathway alone. The partial redirection led to a relatively less significant increase of squalene production at about 1200ug/g FW. The further pathway channeling assay and photosynthesis measurement all indicated that C2 redirection is functional and can lead to a high level of terpene production without comprising plant growth. The research thus established a novel approach to directly couple photosynthesis process with terpene production. Second, in addition to pathway design to couple photosynthesis with terpene synthesis, we designed and implemented a Chloroplast-originated organelle to further increase terpene production by enhancing carbon sink. Redesigned lipid droplet forming proteins were used to generate the chloroplast originated droplet organelle. The organelle design has led to an increased level of squalene production at 2500ug/g FW. The novel organelle represents a unique approach to enhance the carbon sink for high value compounds or fuels. Overall, the two strategies enabled both the better integration of photosynthesis to terpene synthesis and the enhancement of carbon sink in terpene droplet. Combining the two technologies together will lead to a â??push and pullâ?? engineering strategy to maximize the photosynthetic terpene production.

co,

o,

RuBisCo P-glycolate

Glyc ate

Glyoxylate

ME MS

G3P Pyruvate +---- Malate

\ / OXPS DXP

DXR

MEP

OMAPP IPP FPPS \ /

FPP

Squalene ----7 E::> Lipid

Figure 1. Proposed photorespiration rechanneling pathway.