Metabolic engineering of Phaeodactylum tricornutum for heterologous production of the plant triterpenoid friedelin

Abstract

Background: Friedelin is a pharmacologically valuable pentacyclic triterpenoid with anti-inflammatory, anticancer, antiviral, and antiobesity properties. Conventional methods of friedelin production rely on solvent-intensive extraction from plant biomass, which is often expensive, inefficient, and environmentally unsustainable. Phaeodactylum tricornutum, a model marine diatom with a unique chimeric sterol biosynthetic pathway and native oxidosqualene accumulation, presents a promising platform for heterologous triterpenoid biosynthesis. Results: In this study, the TwOSC4 gene from Tripterygium wilfordii, which encodes friedelin synthase, was successfully expressed in P. tricornutum via biolistic transformation. As a result, two transgenic strains, Pt-OSC4-20 and Pt-OSC4-48, were confirmed to express TwOSC4 at both transcript and protein levels. Liquid chromatography–quadrupole time-of-flight mass spectrometry (LC-QTOF/MS) analysis validated friedelin production in these strains, with a baseline accumulation of 26 ng/mL under standard conditions. Upon treatment with Ro 48-8071, an oxidosqualene cyclase inhibitor that suppresses endogenous sterol biosynthesis, friedelin production increased up to 55 ng/mL. Nile red staining revealed increased lipid droplet formation in the transgenic strains, suggesting the possible intracellular storage of friedelin. Importantly, transgene expression did not impair cell growth, indicating the metabolic compatibility of the host with exogenous triterpenoid synthesis. Conclusions: This is the first study to demonstrate successful biosynthesis of friedelin in a microalgal system, highlighting the potential of P. tricornutum as a sustainable phototrophic chassis for the production of plant-derived triterpenoids. Unlike yeast-based systems, which require extensive metabolic amplification, P. tricornutum enables simpler genetic engineering and simultaneous coproduction of valuable compounds, such as fucoxanthin and Omega-3 Eicosapentaenoic Acid (EPA). These findings lay the groundwork for further strain optimization aimed at increasing friedelin yield and broadening the scope of triterpenoid biosynthesis in microalgae.