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

Abstract

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.

Biosynthesis of unnatural polyisoprenes by engineered prenyltransferases on rubber particles

“…Lipid droplets (LDs) are ER-derived lipid monolayer membrane organelles that enclose triacylglycerols 50 , which are also suitable storage organelles for the overproduction of valuable hydrophobic compounds to avoid growth defects in host cells 51 . Metabolic engineering approaches to introduce enzymes into artificial LDs increase the production of target molecules, such as squalene 52 ; however, there are no reports of increased hydrocarbon polymer production achieved by compartmentalisation in LDs. Development of polymer synthesis systems by introducing foreign enzymes into LDs is the future challenge.…”

Engineering Terpene Production Pathways in Methylobacterium extorquens AM1

“…To investigate the production by a native, lanthanide-inducible promoter, the gene encoding the patchoulol synthase was inserted into a pES503, a plasmid containing the xox1 methylotrophic promoter [ 26 ], to generate pAH2 ( Table 2 ). Analogously, the gene encoding casbene synthase DgTPS1 (accession number: MZ485349.1) [ 21 ] was amplified using the 9 and 10 primers, resulting in pAH3 ( Table 2 ). A construct containing instead the mTac promoter was generated by using primers 11 and 10, resulting in pAH4 with casbene synthase ( Table 2 ).…”

“…Constructs were verified by Sanger sequencing. For the E. coli expression system, previously established plasmids for increasing the precursors needed, pIRS and pGG, were utilized [ 21 , 28 ]. An overview of the MEP pathway and vectors is given in Figure 1 .…”

“…Casbene is a diterpene with antimicrobial effects, formed through a one-step cyclization of geranylgeranyl diphosphate (GGDP) [ 20 ]. Casbene synthase, DgTPS1 , is natively found in the Daphne genkwa plant [ 21 ]. The scaffold can be further modified to afford important precursors in drug discovery [ 22 ].…”

Engineered poplar for bioproduction of the triterpene squalene

“…This is complemented by the gene encoding the Lipid Droplet Surface Protein from Nannochloropsis oceanica ( No LDSP), which inserts into, and aids in the formation of lipid droplets (Sadre et al ., 2019; Vieler et al ., 2012). Previous work has shown co‐expressing AtWRI1 1‐397 and NoLDSP with the soluble squalene pathway increased squalene yields in a Nicotiana benthamiana transient expression system, with even further increases observed when fusing At FDPS and Ma SQS CΔ17 to No LDSP, scaffolding the pathway on the surface of lipid droplets (Bibik et al ., 2022).…”