Compartmentalized sesquiterpenoid biosynthesis and functionalization in the Chlamydomonas reinhardtii plastid

Terpenoids play key roles in cellular metabolism, with some organisms having evolved expanded terpenoid profiles for specialized functions such as signaling and defense. While heterologous production in microbial hosts offers an alternative to natural extraction, the development of efficient biosynthetic platforms remains challenging. Here, we developed a subcellular engineering approach in the model green alga Chlamydomonas reinhardtii by targeting both sesquiterpenoid synthases and cytochrome P450s (CYPs) to the plastid, exploiting its photosynthetic electron transport chain to drive CYP-mediated oxidation without reductase partners. Nuclear-encoded sesquiterpenoid synthases were expressed with farnesyl pyrophosphate synthase fusions and targeted to the plastid, while CYPs were modified for soluble localization in the plastid stroma by removing transmembrane domains. The plastid environment supported hydroxylation, epoxidation, and oxidation reactions, with functionalization efficienc..., Experimental section
Algae cultivation, plasmid design, transformation, and screening
Experiments used a C. reinhardtii strain derived from UPN22, modified for enhanced terpenoid biosynthesis through squalene synthase knockdown and β-carotene ketolase overexpression [1]. Cultures were maintained in TAPhi-NO3 medium under LED illumination (150 µmol m–2 s–1). We selected ten sesquiterpene synthases (STPSs) based on documented activity and complete sequence availability, including selinene synthase (UniProt: O64404) and vetispiradiene synthase (UniProt: A0A411G8M5)[2, 3]. All genes underwent codon optimization and intron spreading for nuclear genome expression [2]. We developed three pOpt3-based STPS construct designs: cytoplasmic expression with paromomycin selection (APHVIII), and two chloroplast-targeted expression with hygromycin selection (APHVII)[4]. Chloroplast-targeted constructs utilized the PsaD promoter and chloroplast targeting peptide (CTP), fused to mKOk fluorescent protein,..., , # Compartmentalized sesquiterpenoid biosynthesis and functionalization in the *Chlamydomonas reinhardtii* plastid

**Generated on:** 2024-11-21 by Sergio Gutiérrez

\**Date of Data Collection: **2023–2024

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**Contributors: Sergio Gutiérrez**, **Sebastian Overmans**, **Gordon B. Wellman**, and **Kyle J. Lauersen***\
Bioengineering Program, BESE Division, King Abdullah University of Science and Technology (KAUST), Thuwal, Saudi Arabia.

*Corresponding Author: *[[e-mail]](mailto:[e-mail])*

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## **Overview**

This dataset provides comprehensive information on the sesquiterpenoid biosynthesis pathway engineering and functionalization in *Chlamydomonas reinhardtii*. The dataset includes genetic constructs and chromatographic data generated during the study. These files support studies of metabolic engineering, biochemistry, and bioinformatics, offering detailed insights into the design and analysis of engineered strains, their product profiles, and ...