Cytosolic lipid droplets as engineered organelles for production and accumulation of terpenoid biomaterials in leaves

Abstract

Cytosolic lipid droplets are endoplasmic reticulum-derived organelles typically found in seeds as reservoirs for physiological energy and carbon to fuel germination. Here, we report synthetic biology approaches to co-produce high-value sesqui- or diterpenoids together with lipid droplets in plant leaves. The formation of cytosolic lipid droplets is enhanced in the transient Nicotiana benthamiana system through ectopic production of WRINKLED1, a key regulator of plastid fatty acid biosynthesis, and a microalgal lipid droplet surface protein. Engineering of the pathways providing the universal C5-building blocks for terpenoids and installation of terpenoid biosynthetic pathways through direction of the enzymes to native and non-native compartments boost the production of target terpenoids. We show that anchoring of distinct biosynthetic steps onto the surface of lipid droplets leads to efficient production of terpenoid scaffolds and functionalized terpenoids. The co-produced lipid droplets “trap” the terpenoids in the cells.

A Review of Diatom Lipid Droplets

“…Unlike in plant leaf tissues, metabolic engineering of diatom TAG accumulation benefits from the existence of LD biogenesis and regulatory systems already in place. Various steps within the "push, pull, package, protect" paradigm have already has been accomplished in diatoms by over-expression of enzymes implicated in fatty acid and TAG biosynthesis [114,117,[240][241][242], modulating the acyl-CoA pool [243] or repression of lipid catabolism [124,125,244]. A deeper understanding of the mechanisms by which proteins are targeted to LDs could facilitate the engineering of LD-localized enzymes or metabolic pathways, which could allow for the customization of LD contents.…”

Harnessing evolutionary diversification of primary metabolism for plant synthetic biology

“…It will be also important to couple with improved metabolic sinks (e.g. vacuole transport and storage) (240) and down-regulation of competing and catabolic pathways. Unlike the exploration of unknown specialized metabolic pathways, that of primary metabolism may not lead to novel gene and enzyme discoveries.…”

Genetic elucidation of complex biochemical traits mediating maize innate immunity

“…For transient expression in N. benthamiana , pLIFE33 constructs carrying individual target genes and pEarleyGate100 with ElHMGR 159–582 construct 35 were electroporated into Agrobacterium tumefaciens strain GV3101. To ensure detectable production of sesquiterpenoid pathway products, all assays utilized co-expression of the coding sequence for truncated cytosolic Euphorbia lathyris 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR; ElHMGR 159–582 , JQ694150.1) 71 . An A. tumefaciens strain encoding the P19 protein was also equally added in order to suppress host gene silencing.…”