Engineered poplar for bioproduction of the triterpene squalene.

Building sustainable platforms to produce biofuels and specialty chemicals has become an increasingly important strategy to supplement and replace fossil fuels and petrochemical-derived products. Terpenoids are the most diverse class of natural products that have many commercial roles as specialty chemicals. Poplar is a fast growing, biomassdense bioenergy crop with many species known to produce large amounts of the hemiterpene isoprene, suggesting an inherent capacity to produce significant quantities of other terpenes. Here we aimed to engineer poplar with optimized pathways to produce squalene, a triterpene commonly used in cosmetic oils, a potential biofuel candidate, and the precursor to the further diversified classes of triterpenoids and sterols. The squalene production pathways were either re-targeted from the cytosol to plastids or co-produced with lipid droplets in the cytosol. Squalene and lipid droplet co-production appeared to be toxic, which we hypothesize to be due to disruption of adventitious root formation, suggesting a need for tissue specific production. Plastidial squalene production enabled up to 0.63 mg/g fresh weight in leaf tissue, which also resulted in reductions in isoprene emission and photosynthesis. These results were also studied through a technoeconomic analysis, providing further insight into developing poplar as a production host.

Pathway Engineering, Re-targeting, and Synthetic Scaffolding Improve the Production of Squalene in Plants.

Plants are increasingly becoming an option for sustainable bioproduction of chemicals and complex molecules like terpenoids. The triterpene squalene has a variety of biotechnological uses and is the precursor to a diverse array of triterpenoids, but we currently lack a sustainable strategy to produce large quantities for industrial applications. Here, we further establish engineered plants as a platform for production of squalene through pathway re-targeting and membrane scaffolding. The squalene biosynthetic pathway, which natively resides in the cytosol and endoplasmic reticulum, was re-targeted to plastids, where screening of diverse variants of enzymes at key steps improved squalene yields. The highest yielding enzymes were used to create biosynthetic scaffolds on co-engineered, cytosolic lipid droplets, resulting in squalene yields up to 0.58 mg/gFW or 318% higher than a cytosolic pathway without scaffolding during transient expression. These scaffolds were also re-targeted to plastids where they associated with membranes throughout, including the formation of plastoglobules or plastidial lipid droplets. Plastid scaffolding ameliorated the negative effects of squalene biosynthesis and showed up to 345% higher rates of photosynthesis than without scaffolding. This study establishes a platform for engineering the production of squalene in plants, providing the opportunity to expand future work into production of higher-value triterpenoids.

Standards for plant synthetic biology: a common syntax for exchange of DNA parts.

Inventors in the field of mechanical and electronic engineering can access multitudes of components and, thanks to standardization, parts from different manufacturers can be used in combination with each other. The introduction of BioBrick standards for the assembly of characterized DNA sequences was a landmark in microbial engineering, shaping the field of synthetic biology. Here, we describe a standard for Type IIS restriction endonuclease-mediated assembly, defining a common syntax of 12 fusion sites to enable the facile assembly of eukaryotic transcriptional units. This standard has been developed and agreed by representatives and leaders of the international plant science and synthetic biology communities, including inventors, developers and adopters of Type IIS cloning methods. Our vision is of an extensive catalogue of standardized, characterized DNA parts that will accelerate plant bioengineering.