Journal: bioRxiv
Article Title: Compartmentalized sesquiterpenoid biosynthesis and functionalization in the Chlamydomonas reinhardtii plastid
doi: 10.1101/2024.08.21.608953
Figure Lengend Snippet: Sesquiterpene synthases (STPS) and cytochrome P450 enzymes (CYPs) were targeted to the algal chloroplast. Each STPS construct included C-terminal ScErg20 FPPS, while CYPs were co-expressed through plastid targeting and without transmembrane domains. (A) GC-MS chromatograms of dodecane extracts from C. reinhardtii strains expressing plastid localized STPS and CYP combinations, numbers indicate specific compounds identified by MS. (B) Quantitative analysis of sesquiterpenoid production based on GC-FID data. Circles: sum of peak areas for sesquiterpenoids; triangles: sum of peak areas for modified sesquiterpenoids. Data represent mean ± SD (n=12, 4 transformants × 3 biological replicates). (C) Functionalization efficiency (%) of each CYP, calculated as the fraction of functionalized sesquiterpenoids from total sesquiterpenoids. Circles represent individual transformants. Compounds: [1] Aristolene, [2] Aristolochene, [3] Aristolochone, [4] Valencene, [5] Nootkatone, [6] α-Santalene, [7] Bergamotol, [8] Santalol, [9] α-Cadinene, [10] δ-Cadinene, [11] β-Cadinene, [12] Muurolene, [13] Muurolol, [14] τ-Cadinol, [15] α-Guaiene, [16] β-Guaiene, [17] α-Humulene, [18] δ-Guaiene, [19] Alloaromadendrene, [20] α-Guaiol, [21] β-Guaiol, [22] Globulol, [23] Rotundone, [24] Alloaromadendrene oxide. GC-MS/FID data in SI Appendix File S5, Tables S3, and Fig. S6–S7.
Article Snippet: For quantification, we used calibration curves (1 – 500 μM) of purified standards in dodecane or FCs: δ-guaiene, patchoulol, α-santalene, valerianol, α-bisabolol, valencene, and cedrene (Toronto Research Chemicals, Canada) ( SI Appendix, Fig. S5 ).
Techniques: Construct, Gas Chromatography-Mass Spectrometry, Expressing, Modification
![Sesquiterpene synthases (STPS) and cytochrome P450 enzymes (CYPs) were targeted to the algal chloroplast. Each STPS construct included C-terminal ScErg20 FPPS, while CYPs were co-expressed through plastid targeting and without transmembrane domains. (A) GC-MS chromatograms of dodecane extracts from C. reinhardtii strains expressing plastid localized STPS and CYP combinations, numbers indicate specific compounds identified by MS. (B) Quantitative analysis of sesquiterpenoid production based on GC-FID data. Circles: sum of peak areas for sesquiterpenoids; triangles: sum of peak areas for modified sesquiterpenoids. Data represent mean ± SD (n=12, 4 transformants × 3 biological replicates). (C) Functionalization efficiency (%) of each CYP, calculated as the fraction of functionalized sesquiterpenoids from total sesquiterpenoids. Circles represent individual transformants. Compounds: [1] Aristolene, [2] Aristolochene, [3] Aristolochone, [4] <t>Valencene,</t> [5] Nootkatone, [6] α-Santalene, [7] Bergamotol, [8] Santalol, [9] α-Cadinene, [10] δ-Cadinene, [11] β-Cadinene, [12] Muurolene, [13] Muurolol, [14] τ-Cadinol, [15] α-Guaiene, [16] β-Guaiene, [17] α-Humulene, [18] δ-Guaiene, [19] Alloaromadendrene, [20] α-Guaiol, [21] β-Guaiol, [22] Globulol, [23] Rotundone, [24] Alloaromadendrene oxide. GC-MS/FID data in SI Appendix File S5, Tables S3, and Fig. S6–S7.](https://bio-rxiv-images-cdn.bioz.com/dois_ending_with_53/10__1101_slash_2024__08__21__608953/10__1101_slash_2024__08__21__608953___F2.large.jpg)
![Illustration of sesquiterpenoid chemical structures identified in this work. Colored sections depict distinct sesquiterpenoid classes produced through the action of specific sesquiterpene synthases and cytochrome P450 enzymes. Compounds are labeled with unique identifiers corresponding to their structures: Aristolene [01], Aristolochene [02], Aristolochone [03], Valencene [04], Nootkatone [05], α-Santalene [06], Bergamotol [07], Santalol [08], α-Cadinene [09], δ-Cadinene [10], β-Cadinene [11], Muurolene [12], Muurolol [13], τ-Cadinol [14], α-Guaiene [15], β-Guaiene [16], α-Humulene [17], δ-Guaiene [18], Alloaromadendrene [19], α-Guaiol [20], β-Guaiol [21], Globulol [22], Rotundone [23], and Alloaromadendrene oxide [24]. GC-MS/FID data in SI Appendix File S5, Tables S7–S8 .](https://bio-rxiv-images-cdn.bioz.com/dois_ending_with_53/10__1101_slash_2024__08__21__608953/10__1101_slash_2024__08__21__608953___F4.large.jpg)
