DOI: 10.5061/dryad.zgmsbccmz
title: Compartmentalized sesquiterpenoid biosynthesis and functionalization in the Chlamydomonas reinhardtii plastid

description: 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 efficiencies reaching 80% of accumulated products.
 Carbon source availability influenced product ratios, revealing metabolic
 flexibility in the engineered pathways. Overall sesquiterpenoid yields
 ranged between 250-2500 µg L–1 under screening
 conditions, establishing proof-of-concept for using plastid biochemistry
 in complex terpenoid biosynthesis. Living two-phase terpenoid extractions
 with different perfluorinated solvents revealed variable performances
 based on sesquiterpenoid functionalization and solvent type. This work
 demonstrates that photosynthetic electron transport can drive CYP-mediated
 functionalization in engineered subcellular compartments. However,
 improvements in photobioreactor cultivation concepts will be required to
 facilitate the use of algal chassis for scaled production.
description: <strong>Experimental section </strong>
 <strong>Algae cultivation, plasmid design, transformation,
 and screening</strong> Experiments used a
 <em>C. reinhardtii</em> strain derived from UPN22, modified
 for enhanced terpenoid biosynthesis through squalene synthase knockdown
 and β-carotene ketolase overexpression <sup>[1]</sup>.
 Cultures were maintained in TAPhi-NO<sub>3</sub> medium under
 LED illumination (150 µmol m<sup>–2</sup>
 s<sup>–1</sup>). We selected ten sesquiterpene synthases
 (STPSs) based on documented activity and complete sequence availability,
 including selinene synthase (UniProt: O64404) and vetispiradiene synthase
 (UniProt: A0A411G8M5)<sup>[2, 3]</sup>. All genes underwent
 codon optimization and intron spreading for nuclear genome
 expression <sup>[2]</sup>. We developed three pOpt3-based STPS
 construct designs: cytoplasmic expression with paromomycin selection
 (APHVIII), and two chloroplast-targeted expression with hygromycin
 selection (APHVII)<sup>[4]</sup>. Chloroplast-targeted
 constructs utilized the <em>PsaD</em> promoter and chloroplast
 targeting peptide (CTP), fused to mKOk fluorescent protein, and
 either <em>S. cerevisiae</em> (Erg20) or <em>E.
 coli</em> (ispA) farnesyl diphosphate synthase (FPPS). FPPS
 sequences included C-terminal stop codons to preserve activity
 <sup>[5]</sup>. CYPs were selected based on three criteria:
 (1) previous biochemical characterization, (2) documented activity on
 target sesquiterpenoid scaffolds, and (3) availability of complete
 sequence information. We modified CYPs for plastid targeting by removing
 transmembrane domains, identified through TMHMM-2.0 server analysis and
 AlphaFold structural modeling <sup>[6]</sup>.<strong>
 </strong>The modified sequences were subcloned into pOpt3-based
 constructs containing the <em>PsaD</em> promoter, CTP, mTFP1
 reporter, and zeocin resistance marker (shBle). All constructs were
 synthesized by Genscript (Piscataway, NJ, USA)
 (<strong>Supplementary information, Table S1, File
 S1</strong>). For transformation, we linearized
 plasmid DNA using XbaI and KpnI restriction enzymes and introduced 10 µg
 DNA per transformation via glass-bead protocol <sup>[7]</sup>.
 Following 8-hour recovery in liquid TAPhi-NO<sub>3</sub>
 medium <sup>[8]</sup>under low light, cells were plated on
 selective media containing appropriate antibiotics: spectinomycin (200 µg
 mL<sup>–1</sup>), paromomycin (10 µg
 mL<sup>–1</sup>), hygromycin B (15 µg
 mL<sup>–1</sup>), or zeocin (15
 µg mL<sup>–1</sup>). After 7 days under continuous
 illumination, we employed a PIXL colony-picking robot (Singer Instruments,
 UK) to transfer up to 384 colonies onto fresh
 TAPhi-NO<sub>3</sub> plates. A ROTOR robot (Singer
 Instruments, UK) duplicated colonies after 3 days onto screening plates
 containing amido black (150 µg
 mL<sup>–1</sup>) <sup>[3]</sup>. We selected four
 independent transformants per construct based on fluorescent protein
 signal intensity (<strong>Supplementary information, Figure S1–S4).
 </strong>Each transformant underwent analysis in biological
 triplicate (n=12) to account for expression variability due to random
 nuclear integration. Selected colonies were cultured in 12-well plates
 containing 2 mL TAPhi-NO<sub>3</sub> medium at 160 rpm for
 subsequent two-phase cultivation and solvent analysis <sup>[2,
 5]</sup>. <strong>Capture and analysis of
 algal-produced sesquiterpenoids by GC-MS/FID</strong>
 We employed a two-phase cultivation system for sesquiterpenoid
 extraction and quantification <sup>[2, 9]</sup>. For each
 construct, four independent transformants showing strong fluorescence
 signals were analyzed in biological triplicate (n=12 total samples).
 Cultures were grown in 6-well microtiter plates containing 4.5 mL
 TAPhi-NO<sub>3 </sub>medium with a 500 µL dodecane overlay
 (10% total volume) for 7 days <sup>[2, 3]</sup>. To
 systematically evaluate extraction efficiency, we compared ten
 perfluorocarbon solvents (FCs) against dodecane: CFL7160, CXFL-68,
 CFL3000A, CXFL-3288, FC-770, FC-3284, FC-43, FC-72, FC-40, and FC-3283
 (sourced from Sigma-Aldrich, Germany; Acros Organics, Belgium; Hunan
 Chemfish Pharmaceutical Co., Ltd, China). FC extractions used 1000 μL
 solvent (20% total volume) with 4 mL cultures, with dodecane forming an
 upper phase and FCs forming lower phases (<strong>Supplementary
 information, Table S5</strong>). For these extractions, we used 1000
 μL of solvent (20% of total volume) with 4 mL cultures. Dodecane formed an
 upper 'overlay' while FCs formed 'underlays'. We
 monitored culture volumes throughout cultivation to account for
 evaporation effects. Phases were separated by centrifugation (3500
 × <em>g</em>, 5 min), and both solvent fractions were
 collected for GC analysis. Cell density was determined using flow
 cytometry (<strong>Supplementary information, File
 S7</strong>) <sup>[9]</sup>.
 GC-MS/FID analysis followed established protocols
 <sup>[5]</sup> and processed chromatograms using MassHunter
 software (Agilent, Germany, version B.08.00). Compound identification
 involved three complementary approaches: (1) Comparison of mass spectra
 against the NIST Mass Spectral Library (National Institute of Standards
 and Technology, USA); (2) Analysis of retention indices; (3) Matching
 against authenticated standards. For absolute quantification, we generated
 calibration curves (1–500 μM) using purified standards in both dodecane
 and FCs: δ-guaiene, patchoulol, α-santalene, valerianol, α-bisabolol,
 valencene, and cedrene (Toronto Research Chemicals, Canada)
 (<strong>Supplementary information, Figure S5</strong>).
 Compound identification reliability was evaluated using three metrics: (1)
 mass spectral match factor ("P%" in <strong>Supplementary
 information, Tables S7-S8</strong>), calculated as the mathematical
 similarity between sample and reference spectra on a scale of 0-100; (2)
 retention index comparison with literature values; and (3)<strong>
 </strong>comparison against a standard terpene mixture containing 98
 compounds at 1 mM in methanol (MetaSci, Canada)  (<strong>Figure 6,
 Supplementary information, Table S6</strong>). Mass spectral matches
 below 50% were considered tentative identifications. For functionalized
 products without available standards, identification relied on the
 presence of expected mass shifts corresponding to specific chemical
 modifications (e.g., +16 m/z for hydroxylation) and fragmentation patterns
 characteristic of the predicted structures. These identifications are in
 the data tables (<strong>Supplementary information, Tables
 S7-S9</strong>). <strong>Data
 analysis</strong> The experimental design
 included four independent transformants per construct, each analyzed in
 biological triplicate (n=12 total samples per construct). We included two
 types of controls: the parental non-transformed <em>C.
 reinhardtii</em> strain and vector-only constructs with matched
 fluorescent reporters. GC-MS/FID measurements followed a rigorous quality
 control protocol including: (1) Manual review of all chromatograms for
 peak quality and integration accuracy; (2)<strong>
 </strong>Triplicate technical measurements of each biological
 sample; (3) Monitoring of internal standards for instrument performance;
 (4) Regular blank injections to detect potential carryover. Compound
 identification utilized three criteria: (1) Retention index comparison
 with reference standards; (2) Mass spectral match factors (reported as P%
 in <strong>Supplementary information, Tables S7-S8</strong>);
 (3) NIST library comparison with match quality threshold &gt;50%. For
 quantitative analysis, we calculated means and standard deviations of
 sesquiterpenoid yields from biological replicates. Due to random nuclear
 transgene integration in <em>C. reinhardtii</em>, expression
 levels can vary substantially between transformants <sup>[2, 9,
 10]</sup>. Our multi-transformant approach captures this inherent
 variability, providing a realistic assessment of production potential.
 High standard deviations in yield data reflect this biological variation
 rather than technical measurement uncertainty. Statistical analysis
 employed two-way ANOVA with Tukey's post-hoc test to evaluate
 differences between subcellular localization strategies
 (<strong>Supplementary information, Table S3</strong>). We
 used JMP v.16 (SAS Institute, NC) and R v.3.6.2 (R Foundation for
 Statistical Computing, Austria) for statistical computations. Data
 visualization utilized JMP v.16 and GraphPad Prism v.10.3 (GraphPad
 Software, USA). Figures were prepared using Affinity Designer v.2.5.3
 (Serif Ltd., UK) for diagrams, ChemDraw v.20.1 (PerkinElmer, MA, USA) for
 chemical structures, and Affinity Publisher v.2.5.3 (Serif Ltd., WB, UK)
 for layout integration.
description: # Compartmentalized sesquiterpenoid biosynthesis and functionalization in
 the *Chlamydomonas reinhardtii* plastid **Generated on:** 2024-11-21 by
 Sergio Gutiérrez \**Date of Data Collection: **2023–2024 ---
 **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:
 *[kyle.lauersen@kaust.edu.sa](mailto:kyle.lauersen@kaust.edu.sa)* --- ##
 **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 experimental parameters. This dataset
 is an excellent resource for researchers in synthetic biology, microbial
 metabolic engineering, and plastid biology. --- ## **Data Formats** The
 dataset is organized into two major file types: 1. **Genetic construct
 sequences**: GenBank files (`.gb`) * Contain complete plasmid sequences,
 annotations, and regulatory details. 2. **Chromatographic data**: CSV
 files (`.csv`) * Include raw data from GC-FID experiments and processed
 output, documenting sesquiterpenoid product profiles. All files are
 provided in open formats to ensure compatibility with widely available
 tools and maximum usability. --- ## **Number Suffix (Enzyme)** The
 engineered strains are identified using a **number suffix** to specify the
 sesquiterpenoid synthase expressed: * `01`: Negative control (fluorescent
 protein only) * `02`: Aristolochene synthase * `03`: Valencene synthase *
 `04`: Selinene synthase * `05`: Vetispiradiene synthase * `06`: Santalene
 synthase * `07`: Bisabolol synthase * `08`: Cadinol synthase * `09`:
 Guaiene synthase * `10`: Valerianol synthase * `11`: Patchoulol synthase
 These suffixes are used consistently across all files to identify the
 specific sesquiterpenoid synthase expressed in each strain. --- ## **Data
 Organization and Description** ### **Genetic Constructs** * **File
 Format**: GenBank (`.gb`) * **Content Overview**: * **Plasmid sequences**:
 Complete nucleotide sequences of the genetic constructs used in the study.
 * **Gene annotations**: Information on gene insertions, promoter regions,
 regulatory elements, and fluorescent protein sequences. * **Regulatory
 details**: Cloning sites, antibiotic resistance markers, and other
 functional sequences included in the constructs. * **Purpose**: Enables
 replication of genetic engineering efforts, understanding of construct
 design, and further experimentation. --- ### **Chromatographic Data** The
 chromatographic data are organized into separate CSV files based on
 experimental setups, including cytoplasmic expression, chloroplast
 targeting, and functionalization experiments. #### **Supplementary
 File\_02: Cytoplasmic Expression (A-Series)** * **File Format**: CSV
 (`.csv`) * **Content**: * GC-FID data for cytoplasmic expression of
 sesquiterpenoid synthases (A-series strains). * **Structure**: *
 **Columns**: * `Time (min)`: Retention time during gas chromatography. *
 `Peak Area (response)`: Detector response corresponding to sesquiterpenoid
 product peaks. * **Rows**: Individual time points recorded during analysis
 for each replicate. * **Samples**: * A01: Negative control (fluorescent
 protein only). * A02–A11: Strains expressing the following sesquiterpenoid
 synthases: * `02`: Aristolochene synthase * `03`: Valencene synthase *
 `04`: Selinene synthase * `05`: Vetispiradiene synthase * `06`: Santalene
 synthase * `07`: Bisabolol synthase * `08`: Cadinol synthase * `09`:
 Guaiene synthase * `10`: Valerianol synthase * `11`: Patchoulol synthase *
 **Experimental Conditions**: * Temperature gradient: 60–300°C. * GC
 column: HP-5MS. * Carrier gas: Helium. * **Purpose**: Provides raw data to
 analyze product formation in cytoplasmic strains. --- #### **Supplementary
 File\_03: Chloroplast-Targeted Erg20 Fusion (B-Series)** * **File
 Format**: CSV (`.csv`) * **Content**: * GC-FID data for
 chloroplast-targeted sesquiterpenoid synthase strains fused with
 *Saccharomyces cerevisiae*Erg20. * **Structure**: * **Columns**: * `Time
 (min)`: Retention time during gas chromatography. * `Peak Area
 (response)`: Detector response corresponding to sesquiterpenoid product
 peaks. * **Rows**: Individual time points recorded during analysis for
 each replicate. * **Samples**: * B01: Negative control (fluorescent
 protein only). * B02–B11: Strains expressing sesquiterpenoid synthases as
 detailed in the **Number Suffix** list above. * **Experimental
 Conditions**: * Temperature gradient: 60–300°C. * GC column: HP-5MS. *
 Carrier gas: Helium. * **Purpose**: Provides data on sesquiterpenoid
 biosynthesis using the chloroplast-targeted Erg20 fusion. --- ####
 **Supplementary File\_04: Chloroplast-Targeted ispA Fusion (C-Series)** *
 **File Format**: CSV (`.csv`) * **Content**: * GC-FID data for
 chloroplast-targeted sesquiterpenoid synthase strains fused with
 *Escherichia coli* ispA. * **Structure**: * **Columns**: * `Time (min)`:
 Retention time during gas chromatography. * `Peak Area (response)`:
 Detector response corresponding to sesquiterpenoid product peaks. *
 **Rows**: Individual time points recorded during analysis for each
 replicate. * **Samples**: * C01: Negative control (fluorescent protein
 only). * C02–C11: Strains expressing sesquiterpenoid synthases as detailed
 in the **Number Suffix** list above. * **Experimental Conditions**: *
 Temperature gradient: 60–300°C. * GC column: HP-5MS. * Carrier gas:
 Helium. * **Purpose**: Provides data on sesquiterpenoid biosynthesis using
 the chloroplast-targeted ispA fusion. --- #### **Supplementary File\_05:
 CYP Co-Expression Analysis** * **File Format**: CSV (`.csv`) *
 **Content**: * GC-FID data from co-expression of sesquiterpenoid synthases
 and cytochrome P450 enzymes (CYPs). * **Structure**: * **Columns**: *
 `Time (min)`: Retention time during gas chromatography. * `Peak Area
 (response)`: Detector response for modified sesquiterpenoid products. *
 **Rows**: Individual time points recorded during analysis for each
 replicate. * **Samples**: * Controls: B01–B09 strains without CYP enzymes.
 * Modified: Strains co-expressing CYPs with sesquiterpenoid synthases as
 detailed in the **Number Suffix** list above. --- #### **Supplementary
 Files\_06–08: Carbon Source Effect Studies** * **File Format**: CSV
 (`.csv`) * **Content**: * Data from experiments testing different carbon
 sources on sesquiterpenoid biosynthesis. * **Samples**: * Strains
 expressing sesquiterpenoid synthases as detailed in the **Number Suffix**
 list. * Carbon Sources: CO2, acetate, and CO2+acetate conditions. --- ##
 **Usage and Accessibility** * All files are in open, non-proprietary
 formats, ensuring accessibility without requiring specialized software. *
 Biological triplicates are included for all strains to ensure statistical
 rigor.