A green alternative to fragrant agarwood sesquiterpenoid production

Certain endangered Thymelaeaceous trees are major sources of the fragrant and highly valued resinous agarwood, comprised of hundreds of oxygenated sesquiterpenoids (STPs). Despite growing pressure on natural agarwood sources, the chemical complexity of STPs severely limits synthetic production. Here, we catalogued the chemical diversity in 58 agarwood samples by two-dimensional gas chromatography–mass spectrometry and partially recreated complex STP mixtures through synthetic biology. We improved STP yields in the unicellular alga Chlamydomonas reinhardtii by combinatorial engineering to biosynthesise nine macrocyclic STP backbones found in agarwood. A bioprocess following green-chemistry principles was developed that exploits ‘milking’ of STPs without cell lysis, solvent–solvent STP extraction, solvent–STP nanofiltration, and bulk STP oxy-functionalisation to obtain terpene mixtures like those of agarwood. This process occurs with total solvent recycling and enables continuous production. Our synthetic-biology approach offers a sustainable alternative to harvesting agarwood trees to obtain mixtures of complex, fragrant, oxygenated STPs.


Introduction
Agarwood is the common name given to the fragrant resinous woods derived from trees in the Thymeleaceae family, including Aquilaria, Enkleia, Aetoxylon, Gonystylus, Wikstroemia, and Gyrinops 1 .Indigenous to Southeast Asian countries, these trees are prized for the complex fragrances of their resins that originate from multiple oxygenated and nonoxygenated sesquiterpenoids [2][3][4] .These resins form after physical wounding of the tree, and their unique chemical profile develops over periods up to several decades 3,4 .The use of agarwood products for incense, perfumes, medicines and ornamentation has been recorded across cultures and centuries [3][4][5] .Common applications are burning wood chips as incense and steam-distillate oils as perfume ingredients 4,6 .In particular, agarwood products were valued at $US 44 billion within the global perfume market 7 .Demand for agarwood has rapidly outpaced the supply of slow-growing natural sources, leading to the inclusion of 13 out of 20 Aquilaria species in the Appendix II of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) as potentially threatened with extinction 8 .Despite global efforts to regulate trade and develop alternative production modes through silviculture or tissue culture, the clandestine sourcing of wild trees remains an issue that further pressures their existence 4,9 .
Both abiotic and biotic factors govern their creation in situ where oxygenation of STPs to derivatives occurs, further shaping their individual scent profiles.From a metabolicengineering standpoint, the tree itself serves only as the vehicle and matrix for chemical biosynthesis and resin accumulation, such that STP biosynthesis by metabolic engineering could be an alternative means for production of fragrant chemicals 4,14 .However, biochemical pathways leading to agarwood STPs are complex, poorly understood, and can be nonenzymatic in nature.So far, only guaiene STP synthases (STPSs) have been identified from Aquilaria crassna and A. sinensis and enzymatic routes to other primary STPs skeletons found in agarwood are not yet identified 11,[14][15][16] .
Although the biochemical pathways to agarwood STPs are not elucidated, the diversity of natural product chemistry found in other organisms can provide alternative starting points to recreate agarwood STP chemical complexity.The primary carbon rings of agarofuran, cadinene, valencene, and selinane mirror STPs biosynthesised by valencene 17 , valerianol 18 , cadinol 19 , and aristolochene 20 synthases, whereas zizaene synthase 21 produces a prezizanelike STP.This existing repertoire of known STP synthases from plant and fungal species can produce STP mixtures that largely replicate the primary STP skeletons in agarwood.
Here, we aimed to reproduce some of the chemical complexity of agarwood STPs using synthetic biology and green bioprocessing.To inform the engineering approach, we first catalogued the chemical complexity of STPs in 58 agarwood samples using highresolution, two-dimensional (2D) gas chromatography-time-of-flight mass spectrometry (GCxGC-TOF/MS).We then engineered unicellular algae to produce nine distinct STP chemical skeletons widely found in agarwood.A low-energy 'microbial-milking' technique was exploited to extract the STPs from growing algal cells, and energy-efficient solvent nanofiltration was used to concentrate the extracted STPs.Finally, we used green chemical reactions on these STP skeletons to generate a complex mixture of 103 oxygenated STPs typically found in agarwood.This process employs an autotrophic host and a low-energy, gentle STP-harvesting technique that enables solvent recycling, thus representing a practical alternative to harvesting agarwood trees to obtain their desirable, fragrant, terpenoid mixtures

Chemical characterisation of agarwood and distillates
The Aquilaria sp.wood and distillate samples investigated here originate from various Southeast Asian countries (Fig. 1a, Supplementary Table 1).Wood samples (n = 36) exhibit a range of colours, with darker tones indicating higher resinous content (Fig. 1b), while the distillate oils (n = 22) exhibit varying amber-brown shades (Fig. 1c).Representative 2D GCxGC-TOF/MS analyses of wood (sample 34) and distillate (sample 39) are presented in Fig. 1d and e.Comprehensive data for all samples can be found in Supplementary File 1.
The identified sesquiterpenoid skeletons, spanning from the aromatic agarofurans to the complex prezizanes, offer a detailed insight into agarwood products and their chemistry.
Compounds such as α-guaiene, δ-guaiene, and isolongifolene, expected from earlier research, were confirmed, underscoring the reliability of our analytical approach.Although, many compounds were identified across all agarwood and its distillates, each sample retained a distinct profile.These detailed observations enhance our comprehension of the natural chemical complexity of agarwood samples.

Optimising algal metabolism to biosynthesise heterologous STPs
Chlamydomonas reinhardtii is an emerging host for metabolic engineering and heterologous terpene production 22,23 , as is known to have enhanced heterologous terpenoid yields following metabolic alterations [24][25][26] .These included the knockdown of native squalene synthase (CrSQS-k.d.) increasing farnesyl pyrophosphate pools in the cytoplasm, leading to major improvements in sesquiterpenoid yields when either heterologous bisabolene or patchoulol sesquiterpenoid synthases were co-expressed 24,26 .Volatile engineered heterologous isoprene biosynthesis increased when carotenoid homeostasis was disrupted by introducing a beta-carotene ketolase to biosynthesise ketocarotenoids in the plastid 25 (Fig. 2a, right panel).Here, the effects of these interventions alone or in combination were quantified in algae using the fragrant STP patchoulol as a reporter of biosynthetic activity (Fig. 2b).

Biosynthesis of diverse STPs in C. reinhardtii
Transformation and expression of 21 STPS isoforms in various fusions to the yellow fluorescent protein (YFP) reporter were performed, along with a YFP reporter alone control (23 plasmids in total, Fig. 3a, Supplementary Table 6).Due to non-homologous DNA integration in the algal nuclear genome, random position effects change expression dynamics across a population of transformants and large numbers of colonies must be screened to identify robust over-expressing transformants.Here, high-throughput robotics assisted handling was coupled to plate-level fluorescent protein expression screening for at least 384 transformants per plasmid.This screening allowed identification individual transformants for each target plasmids with robust STPS-FP fusion expression.The STPSs selected for expression were chosen based on their reported ability to make primary STPs skeletons commonly found in our analysis of agarwood samples.These STPSs biosynthesise nine distinct STP products (Fig. 3b).Santalene, zizaene, aristolochene, valencene, δ-guaiene, cadinol, valerianol, patchoulol, and bisabolol, were detectable as new GC-MS/FID peaks in dodecane overlays obtained from transgenic strains compared to overlays from control strains (Fig. 3b,e and Supplementary File 4).Each STPS isoform varied in the level of sesquiterpenoids they biosynthesised (Fig. 3b).Zizaene synthase had higher activity when YFP was placed at the N-terminal (plasmid 8, Fig. 3b), similar to reports of SUMO-tagged variants in E. coli 21 .
The optimal isoform with highest target-compound production was identified for each STPS (Fig. 3b).Subsequently, a second transformation was performed with the same STPS (Fig. 3c) fused to the monomeric teal (cyan) fluorescent protein 1 (mTFP1) reporter with a different selection marker (Fig. 3d).Secondary transformation was used to increase heterologous STPS enzyme titter in the cell to catalyse increased target STP biosynthesis.STP yield improvements were observed in all strains after secondary transformation (Fig. 3f), with an average 5.3-fold improvement in titre for each product (Supplementary Tables 7-9).
Additional STP peaks were also identified in these extracts by GCxGC-TOF/MS, representing known side products of the individual synthases (Supplementary Table 10).

A green bioprocess for functionalised STPs
To achieve sustainable production of complex STP mixtures present in agarwood, we developed a bioprocess design that minimised waste and could be operated at room temperature with only non-hazardous reagents (Fig. 4a).STP-producing algal cells were subjected to culture-solvent two-phase cultivations with a fluorocarbon underlay that allowed simple gravity separation of the fluorocarbon liquids containing STPs.Algal cultivation was followed by liquid-liquid extraction of STPs from the fluorocarbon solutions with ethanol, yielding individual STP-ethanol solutions, which were combined to form a STP mixture in ethanol (Fig. 4a,b).Low-energy, pressure-driven, organic-solvent nanofiltration (OSN) was then applied at room temperature, using only the pressure derived from a gas cylinder to drive ethanol through a nanofiltration membrane (Fig. 4a).OSN enabled concentration of STPs in ethanol by 53 times without evaporative loss (Fig. 4b).
The hydroboration-oxidation technique is commonly used in organic synthesis for alkenes conversion to alcohols.Compared to halogenation it results in consistent stereochemistry and minimal waste generation.The non-functionalised cyclic primary STP structures produced by our algae were then subjected to hydroboration reactions to cause bulk hydroxy-functionalisation of double-bond locations within the STP backbone (Fig. 4a).

Discussion
Agarwoods have captivated human interest for centuries due to their complex and diverse fragrances 27 .However, the increasing demand for their resinous wood has placed an unprecedented pressure on their natural sources, particularly older trees native to Southeast Asian forests, and tissue wounding to promote resin formation further increases this pressure 3,8 .In this sample set, we observed that each agarwood and distillate sample contained hundreds of unique STPs and share a subset of these chemicals in varying abundances (Supplementary Files 1-3; Supplementary Tables 2-4).There was no obvious correlation between agarwood prices, origin locations, or sample type, indicating that their market value is subjective, largely based on perceived fragrance or rarity (Supplementary Table 1).
We sought to develop a sustainable approach using metabolically engineered algae to heterologously biosynthesise the STP backbones found in agarwood, followed by oxyfunctionalisation using green chemistry as an alternative to tree sources of STPs.Our goal was to generate not only a microbial production alternative to agarwood STPs, but also to develop a sustainable extraction process that generates minimal waste and uses only gentle reagents within the principles of green chemistry and bio-process design 28,29 .We opted for a microalgal host as our bioengineering platform due to its ability to thrive on the nutrients found in wastewaters and CO2 as a sole carbon source 30 .This not only enhances the sustainability of the process but also potentially facilitates the conversion of wasted materials into valuable resources, aligning with the principles of circular economy.Our downstream bioprocess uses only low-energy methods for STP extraction and concentration; that is, microbial-milking of STPs during cultivation, liquid-liquid STP transfer to ethanol, and nanofiltration for STP concentration.Using boron and H2O2, we generated complex, functionalised STP mixtures that resemble those found in agarwood samples in nature.
Endogenous isoprenoids in C. reinhardtii are supplied exclusively by the methyl-Derythritol phosphate (MEP) pathway localised to the plastid (Fig. 2a) 31 .That algae rely on this pathway for essential photosynthetic carotenoids implies an inherent capacity to channel carbon fixed by photosynthesis to isoprenoids, which could be tapped for heterologous terpene biosynthesis.The first reports of micrograms of heterologous terpenes per litre of algal culture 32 have now been superseded by hundreds of milligrams per litre 25,33 , owing to improved transgene design 34,35 , improved metabolic channelling and high-cell-density cultivation strategies 26,36 .Here, we demonstrate for the first time a combinatorial modification of two metabolic pathways simultaneously in C. reinhardtii by perturbing plastid carotenoid and cytoplasmic sterol biosynthesis in the same strain (Fig. 2).This combination improved target STP production by up to 25-fold over wild-type levels, and enabled us to use this strain to produce other STPs of interest.Improvements in transgene optimisation and DNA synthesis capacity together with improved robotic handling and fluorescent screening methods of transformants has facilitated STP isoform benchmarking directly in alga (Fig. 3).
This is a key step as isoform-performance testing is a role previously conducted only in fermentative microbes.Screening directly in alga enabled nuances of expression in this host to be ruled out directly when choosing a target isoform.
Terpene extraction from engineered microbes is a challenge in bioprocess design.
Microbes lack the specialised tissues of multicellular organisms to accumulate hydrophobic terpene products, necessitating a physical hydrophobic contactor be used as sink for terpene extraction during cultivation.This can be achieved in numerous ways: using resinous particles 37 , membrane contactors 38 , and through biocompatible solvent-culture two-phase milking 17,39 .We previously found that perfluorinated heat-transfer fluids (namely fluorocarbons) can be used as heavy solvents that sit underneath microbial cultures as an underlay for terpene milking, and that terpenes accumulated in the fluorocarbons rapidly partitioned into ethanol in subsequent liquid-liquid extractions 39 .We leveraged this property to first milk STP skeletons from algal cultures and subsequently pool the ethanol extracts to make a starting mixture of STPs (Fig. 4).This extraction method is advantageous for its simplicity, operating at standard atmospheric conditions, and not requiring complex machinery to yield STPs in ethanol.
Although not yet demonstrated at larger scales, the process presented here could readily be incorporated into tubular photobioreactor or even raceway pond algal production facilties.
To create alternatives to agarwood-derived STPs, synthases for the central seven classes of common STP skeletons were required.However, only a handful of δ-guaiene synthase isoforms from Aquilaria trees have been reported 11,15,40 .We overcame this limitation by using substitute terpene synthases from different plants and fungi that could reproduce the chemical skeletons we detected in agarwood (Table 1).However, the fragrances of agarwood largely comprise oxy-functionalised derivative STPs.Terpene functionalisation in metabolic engineering is traditionally conducted by the concerted action of highly regio-and stereo-specific enzymes, such as P450s 41 .The chemical complexity of agarwood terpene profiles is both the product of time and likely non-enzymatic chemistry, however, some P450s may be implicated in certain classes of functionalisation observed within 42 .These P450s have yet to be biochemically characterised and would require expression optimisation geneby-gene to participate in single-STP functionalisation in a microbe.By starting with a mixture of STP skeletons and subjecting them to a chemical process that catalyses oxygenation reactions at sterically accessible double bonds, we could bypass biological functionalisation to obtain complex STP mixtures (Fig. 4).In practice, this procedure mimics parts of the natural maturation process of STPs subjected to abiotic factors in resins over time and successfully reproduced numerous functionalised STPs typical of agarwood samples (Figs.

4, 5).
Traditional methods of oxidation or hydroxylation of STPs have often involved the use of heavy-metal catalysts 43 (such as chromium and mercury), strong oxidising agents 44 (i.e., osmium tetroxide), toxic solvents 45 (i.e., benzene), or hazardous reagents 46 (i.e., chlorine)all of which pose serious environmental and health risks.We instead used organoboron reagents to introduce hydroxyl groups to the most accessible double bonds of the STP skeletons, and eliminated use of heavy-metal reagents and hazardous by-products associated with conventional methods (Fig. 4b-d, Fig. 5 and Supplementary Table 11).
Because mono-and di-terpenes are also present in agarwood (Fig. 1, Supplemental File 1), using more complex mixtures of STPs obtained by additional metabolic engineering will undoubtedly increase the complexity achievable in synthetically functionalised mixtures.
Here, we did not express specific α-humulene, β-eudesmol 14 and vetispiradiene synthases, which could broaden the complexity of the starting mixtures.The combination of possibilities for these mixtures is immense, offering wide scope for additional combinations of microbial engineered terpene production and synthetic chemistry to yield fragrant mixtures.Having said that, this work also should not be considered to over-promise the potential of syntheticbiology alternatives to natural agarwood.Previous hype around novel terpene production techniques has created instability in the natural-product market, leading to economic challenges within legitimate supply chains 47 .This process should instead initially be considered complementary to traditional agarwood production, especially silviculture or tissue-culture alternatives, as one pathway to replicate some of the aromatic functionalised STPs.
In summary, we have developed a strategy following green-bioprocess design principles that combines metabolic engineering, terpene milking, and pharmaceutical-grade chemical processing to obtain terpene mixtures that mimic some of the natural chemical diversity found in agarwood.While our set of 103 semi-synthetic engineered sesquiterpenoids does not fully mirror the diversity and yields of agarwood, our approach presents a sustainable complement to legitimate sources of these products, and offers a potential solution to the current demand for agarwood that has surpassed natural supply for these culturally important goods.We demonstrate the feasibility of a cyclic, synthetic-biologymediated and chemically complemented process to generate functionalised STP mixtures.

Agarwood sample collection and processing
Agarwood samples were procured from the old-town market "Al Balad" in Jeddah, Saudi Arabia (21.481°N, 39.187° E) in Winter 2023.The collection includes 36 different samples of agarwood chips, dried Aquilaria spp.wood (also known as "bahkour"), and 22 agarwood steam-distillate oils, known as "oudh".The origins of these samples cannot be accurately traced; however, many were labelled with their country of origin.The samples varied in price at the time of purchase, reflecting their purported rarity, the density of fragrant compounds, and complexity of aromatic notes (Supplementary Table 1).It was determined that the appropriate organic solvent for extraction was acetone based on preliminary analysis with different solvents, and each agarwood sample was then diluted in order to obtain chromatograms with clear peaks for product detection and identification.The wood samples were weighed (1 g) and ground into a fine powder using a combination of freeze-thaw cycles with liquid nitrogen and mechanical grinding.The homogenised samples were immersed in

Algal strain cultivation
Chlamydomonas reinhardtii strain UPN22 26 was used for all experiments.C. reinhardtii UPN22 is a derivative of the UVM4 strain 48 .It has been genetically enhanced to use phosphite and nitrate as a sole source of phosphorous and nitrogen, respectively, to minimise contamination and maximise cell densities in cultivation 26 .Strains were cultured in Trisacetate phosphite nitrate (TAPhi-NO3, 26 ) liquid medium with shaking at 120 rpm or on solid agar and 150 µmol m -2 s -1 light intensity from a combination of cool-and warm white LED tubes (light spectra as reported in 26 ).500 mL algal cultures were agitated with stirring in Erlenmeyer flasks under the same growth conditions.

Plasmid construction, algal transformation, and screening for sesquiterpenoid synthase expression
Heterologous expression of sesquiterpenoid synthases (STPSs) in C. reinhardtii was achieved through synthetic transgene redesign based on the amino-acid sequences of each STPS, following previously established protocols 34,49 .We selected 21 STPSs from various species encoding isoforms that yield nine different STP skeletons (Table 1).Targeted STPSs for aristolochene, δ-guaiene, santalene, valencene, valerianol, zizaene, τ-cadinol, bisabolol, and patchoulol were designed; all accession numbers are listed in Table 1.Their amino-acid sequences were used to generate algal-adapted nucleotide coding sequences using the Intronserter programme 34 .This programme back-translates amino-acid sequences to frequently used codons, removes unwanted restriction sites, and systematically integrates the first intron of CrRBCS2i1 at a set distance to enable expression from the C. reinhardtii nuclear genome, as previously reported 49,50 .Algal-adapted coding sequences were synthesised and sub-cloned into pOptimized_3 expression plasmids 35 by Genscript (Piscataway, NJ, USA).Ketocarotenoid biosynthesis in alga was achieved by transformation with the pOpt2_CrBKT_aadA plasmid 51 , and knockdown of C. reinhardtii squalene synthase (Uniprot: A8IE29) was achieved using the previously reported luciferase-artificial microRNA expression plasmid pOpt2_ cCA_gLuc-TAA_i3_ami_Spec 24 .Both plasmids confer resistance to spectinomycin as a selectable marker.Combining both cassettes into a single plasmid was unsuccessful.Co-transformation of both plasmids and selection on spectinomycin was combined with robotics assisted colony picking and plate-level screening of 768 transformants to find those with luciferase activity (indicative of SQS knockdown) and a brown-colony phenotype from CrBKT-mediated ketocarotenoid biosynthesis.All plasmid constructs used are listed in Supplementary Table 6 and the complete annotated plasmid sequences are available in Supplementary File 5.
The glass-bead protocol was used to transform the nuclear genome of C. reinhardtii with a plasmid DNA 52 .Each plasmid was linearised using restriction enzymes (XbaI + KpnI, Thermo Scientific FastDigest), and 10 µg of DNA was used for each transformation.Following an ~8 h recovery period in liquid TAPhi-NO3 medium under low light, algal cells were plated on a selective medium with paromomycin (10 µg mL -1 ), spectinomycin (200 µg mL -1 ) or zeocin (15 µg mL -1 ) antibiotics either individual or combinations of selection agents relative to each target plasmid.Plates were illuminated continuously for ~7 d before colony picking.
A PIXL robot (Singer Instruments, Watchet, UK) transferred up to 384 colonies per transformation event to TAPhi-NO3 agar plates.After an additional 3 d, a ROTOR robot (Singer Instruments) was used to replicate colonies onto new medium and plates containing amido black (150 µg mL -1 ) for fluorescence screening as previously described 35 .
All algal-optimised STPSs were expressed as fusions with mVenus (yellow fluorescent protein) or the monomeric teal (cyan) fluorescent protein 1 (mTFP1).Transgene expression of each STPS was determined by fluorescence imaging at the agar-plate level, as previously described 32,35 .Chlorophyll fluorescence was observed with 2 sec of 475/20 nm excitation and 640/160 nm emission to show colony presence/absence on amido black-containing plates.

Heterologous sesquiterpenoid biosynthesis analyses
For each transformant, biosynthesis of heterologous STPs was assessed by gas chromatography-mass spectrometry (GC-MS/FID).Four individual transformants containing each plasmid with the highest fluorescence signals were selected, and solvent-culture twophase living extractions were performed using a 10% v/v dodecane-culture overlay in 6-well plates, as previously reported 17,53 .Cultivations were performed in biological triplicate for 6 d in 4.5 mL TAPhi-NO3 media with 500 µL dodecane overlay.Phases were separated, and culture samples were taken for cell-density analysis by flow cytometry as previously described 26 , and dodecane was spun at 20,000 x g for 3 min, then 150 µL clarified solvent was transferred into amber GC vials in triplicate prior to analysis.
Samples were analysed as previously described 26,38 using an Agilent 7890A gas chromatograph equipped with a mass spectrometer and a flame-ionisation detector (GC-MS/FID).The gas chromatograph comprises a 5975C inert MSD with a triple-axis detector and a DB-5MS column (30 m × 0.25 mm i.d., 0.25 μm film thickness).The injector, interface, and ion-source temperature profiles were set to 250°C, 250°C, and 220°C, respectively.In splitless mode, 1 μL of the sample was injected using an autosampler (G4513A, Agilent).The column flow was constant at 1 mL min −1 , with helium as carrier gas.The initial GC oven temperature was set to 80°C for 1 min, increased to 120°C at 10°C min −1 , raised to 160°C at 3°C min −1 , and to 240°C at 10°C min −1 , holding for 3 min.After a 12-min solvent delay, mass spectra were recorded using a scanning range of 50-750 m/z at 20 scans per second.4).For compound identification, retention-time acquisition, internal digital-library calibration, and method development, we used a set of 12 microampules containing a standard terpene mixture, which covered 98 terpenes at 1 mM in methanol (CAT# MSITPN101, MetaSci, ON, Canada, Supplementary Table 4).

Two-dimensional Gas chromatography time-of-flight mass spectrometry (GCxGC-TOF/MS) analysis
Comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry (GCxGC-TOF/MS) analysis of agarwood and distillate extracts in acetone was performed using an Agilent 7890B gas-chromatography system equipped with a Zoex ZX1 cryogenic thermal modulator and a JEOL TOF MS (AccuTOF GCx-plus, JEOL, Japan).The GCxGC system featured a normal (non-polar x mid-polar) two-dimensional column configuration, comprising a first-dimension column with a 30 m non-polar HP-5MS UI capillary column (5%-phenyl-methylpolysiloxane) and a second-dimension column with a 2 m midpolar BPX-50 capillary column (50% phenyl polysilphenylene-siloxane).We used helium (99.999%) as the carrier gas at a constant flow rate of 0.8 mL min −1 .The GCxGC-TOF/MS injector temperature was maintained at 300°C with a 10:1 split ratio.The oven temperature was initially held at 80°C for 1 min, then increased to 325°C at 2°C min −1 .The modulation period was set at 6 s with a pulse time of 0.35 ms.The mass spectrometer operated in electron ionisation (EI+) mode at 70 eV.Both the transfer-line and ion-source temperatures were maintained at 250°C.The detector voltage of TOF was set to 2,500 Volts, and data were acquired at a rate of 50 Hz.Mass spectra were obtained within a mass-to-charge ratio (m/z) from 50 to 600.

Gas chromatography data analysis
The analysis of terpenoid extracts followed a procedure similar to previously reported methods 26,38,39 , and qualitative analyses primarily relied on the retention index and match factor.The GC-MS/FID data were processed using the MassHunter Workstation software version B.08.00 (Agilent Technologies, USA).The identification of compounds was assisted by the NIST Mass Spectral Library Version 2.3 (National Institute of Standards and Technology, Gaithersburg, MD, USA).The mass-spectral data, derived from the GCxGC-TOF/MS analysis, were evaluated with GC Image TM Version 2.9 software (Lincoln, NE, USA) and referenced against the NIST2020/EPA/NIH EI Mass Spectral Library.The spectral data were cross-referenced with library spectra to identify potential chemical structures, facilitated by calculating the match factor and probability, thereby generating a list of probable compound matches.The match factor directly compares the unknown mass spectrum peaks with those in the library spectra, indicating their similarity.In contrast, the probability determines the relative likelihood of the list of hits being accurate, assuming that the unknown spectrum is present in the library.Using these metrics provides the relative assuredness of matching chemical structures within the library spectra.

Sesquiterpenoid production and concentration
Transformants of best-performing STPS isoforms that accumulated the most of each heterologous sesquiterpenoid in screening conditions, were subjected to solvent-culture twophase cultivations at 300 mL scale in TAPhi-NO3 medium using FC-3283 as a solvent underlay phase, as previously described 39 .FC-3283 is a perfluorinated amine that is inert and denser than water, forming an underlay to the culture and accumulating heterologous terpene products from the algae.After 6 days of cultivation, gravity and gentle centrifugation separated the fluorocarbon and cultures.After centrifugation to clarify, fluorocarbons were subjected to liquid-liquid extraction to partition accumulated STPs into an equal volume of 96% ethanol.The mixture was shaken for 16 h at room temperature at 200 rpm.Next, samples were again centrifuged gently to further separate the phases.FC-3283, after ethanol extraction, can be reused on algal cultures and is effectively recycled in this process.500 μL aliquots from each phase were sampled and stored in separate GC vials at -20°C for subsequent analysis and separation performance quantification.
For each of the nine STPs biosynthesised by the algae, 20 mL 96% ethanol-containing STOs were generated.The ethanol fractions were pooled and subjected to organic solvent nanofiltration (OSN) in a dead-end cell to concentrate the terpenes in ethanol without evaporative losses.A Duramem solvent-resistant membrane (Evonik, Germany) with a nominal molecular weight cut-off value of 300 g mol -1 suitable for STP retention and chemical compatibility with ethanol was selected.The 200 mL ethanol-sesquiterpenoid mixture was loaded into the OSN chamber containing a 16 mm membrane disc and subjected to 20 bar pressure delivered by CO2 as an inert gas, to drive the ethanol phase through the nanofiltration membrane at 2.84 L m -2 h -1 flux.While ethanol permeated the membrane, STPs were kept in the retentate due to their molecular weight and consequently concentrated in the ethanol.The permeate ethanol is suitable for recycling and can be reused in subsequent liquid-liquid extraction processes.

Bulk hydroxylation of sesquiterpenoid backbones
To selectively introduce hydroxyl groups at the double bonds present in the terpenes, hydroboration-oxidation reactions were performed using two distinct organoboron reagents: borane-tetrahydrofuran complex (BH3•THF) (CAT#176192, Sigma-Aldrich) and 9borabicyclo[3.3.1]nonane(151076, Sigma-Aldrich).Three different stoichiometric ratios (0.5, 1.0, and 2.0) were explored for each reagent to investigate different degrees of hydroxylation within the STP mixture.The sterically hindered 9-BBN was anticipated to selectively react with the least sterically demanding sites of the terpenes, while the borane-tetrahydrofuran complex was expected to facilitate hydroxylation even at the more challenging endocyclic positions.To remove residual ethanol and water content from the concentrated terpene mixture, a rotary evaporator was used under reduced pressure at 40°C, ensuring effective removal without compromising the integrity of the terpenes.To ensure the complete removal of water, molecular sieves (CAT#105734, Sigma-Aldrich) were added to the terpene-THF solvent mixture.The reactions were carried out in anhydrous tetrahydrofuran (THF, Sigma-Aldrich) to enable removal by evaporation after completion.Hydrogen peroxide (H2O2, 30%, VWR Chemicals) and sodium hydroxide aqueous solution (NaOH, Sigma-Aldrich) were used to convert the organoboron intermediates into non-toxic, water-soluble boric acid after the reaction.All subsequent terpene derivatives were extracted from the aqueous mixture with ethyl acetate.

Data analysis
To evaluate the impact of engineered modifications on STP biosynthesis and growth characteristics in C. reinhardtii strains, one-way analysis of variance (ANOVA) was performed to compare mean STP (patchoulol) titres and growth rates among different transformants and the parental control strain.This statistical approach allowed for the simultaneous analysis of differences between multiple groups, to provide a robust assessment of the experimental manipulations.ANOVAs were performed separately for STP titres and growth rates under mixotrophic and phototrophic conditions.Post-hoc pairwise comparisons using Tukey's HSD were conducted to identify which groups were statistically significantly different.A one-way ANOVA was used to assess the effects of differences in STP biosynthesis among tested strategies (single or double transformation), followed by a post hoc Tukey's HSD test for specific pairwise data-set comparisons.Mean production values (in fg cell -1 and mg L -         1. Supplementary Tables 2 and 3 provide summaries of common STPs identified in agarwoods and distillates, respectively.Supplementary Files 1-3 contain the supporting data for GC-TOF/MS, GC-MS/FID, and GCxGC-TOF/MS, respectively.5. Detailed calculations of productivity and cell concentrations are in Supplementary Tables 6-8.Supplementary Files 5 and 6 provide supporting data for GC-MS/FID and genetic constructs, respectively.11 and Supplementary File 5.

5 mL of 1 :
1 hexane:acetone and subjected to ultrasonic agitation at 40°C for 8 h to facilitate terpenoid extraction.The samples were then passed through a 0.2 µm filter to obtain clear solvent extracts that were evaporated under a nitrogen stream for 20 min to concentrate the terpenoids in the samples.Concentrated samples were resuspended in 500 µL acetone and stored at -20°C until gas chromatography-flame-ionisation detector and mass spectrometry (GC-MS/FID) and two-dimensional gas chromatography with time-of-flight mass spectrometry (GCxGC-TOF/MS) analyses.All photographs were captured with a Canon EOS RP camera using a Canon RF 24-105 mm f/4-7.1 IS STM lens (Canon, Tokyo, Japan) and ColorChecker Passport (CCPP2, Calibrite LLC, DE, USA) used for colour calibration.
5 min exposure.Yellow fluorescent signals were captured with 504/10 nm excitation and 530/10 nm emission filters with 30-sec exposures.Transformants displaying strong fluorescent-protein signals were selected and inoculated into 12-well plates containing 2 mL of liquid TAPhi-NO3 medium and grown with shaking at 160 rpm.Predicted molecular masses of the expressed heterologous STPS-FP fusions were verified using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) in-gel fluorescence against the fusion-protein fluorescent reporter.

Figure 2 .
Figure 2. Native and engineered terpenoid pathway in C. reinhardtii for enhanced sesquiterpenoid biosynthesis.

Figure 3 .
Figure 3. Biosynthesis of diverse STPs in C. reinhardtii through sesquiterpenoid synthase isoform expression.

Figure 4 .
Figure 4.A green bioprocess workflow for the generation of agarwood-related sesquiterpenoids.

Figure 3 .
Figure 3. Biosynthesis of diverse STPs in C. reinhardtii through sesquiterpenoid synthase isoforms expression.a.The research workflow depicts the progression from selecting sesquiterpenoid synthases across various species for expression in C. reinhardtii.Isoform integration location into the pOpt3 in fusion to fluorescent reporter and robotics-assisted colony screening.Top-right box: Depicted is a transformant plate one week after transformation, organised transformant colonies and YFP signal indicating synthase-reporter fusion expression.Brightest colonies were screened for expression in replica-plate assays with dodecane solvent overlay, which could be separated from the culture by gravity and directly used for analysis in GC-MS/FID.b.Isoform performance was quantified from the most highly expressed transformants per STP (n = 25).Those chosen for further analysis are underlined c.An illustrated vector structure of the STPS-YFP with paromomycin resistance and (d) -mTFP1 with bleomycin resistance.e. GC-MS/FID chromatograms for each target STP.Annotated peaks include: A santalene, Bzizaene, Caristolochene, Dvalencene, E -δ-guaiene, Fcadinol, Gvalerianol, Hpatchoulol, Ibisabolol.f.STP titres of single and double transformations.Abbreviations: YFP: mVenus yellow fluorescent protein; TFP: mTFP1 cyan fluorescent protein; paroR and bleR: paromomycin and bleomycin resistance, respectively; STPS: terpene synthase; Tag: strepII epitope.Chevrons i1/i2: intron addition to codon-optimised transgenes to enable strong algal expression.For a comprehensive list of plasmids, refer to Supplementary Table5.Detailed calculations of productivity and cell concentrations are in
1) for each STP were compared, considering standard deviations to evaluate data variability.Mean values were considered statistically significantly different at a level of p < 0.05.For data analyses, JMP v.16 (SAS Institute Inc, NC, USA) and Rstudio 3.6.2(PositSoftware,Boston, USA) were used.Data visualisation was done using JMP v.16and GraphPad Prism v.10 (GraphPad Software, MA, USA).Image adjustments involved ColourChecker calibration (Calibrite LLC, DE, USA) paired with Adobe Lightroom (Adobe Inc., CA, USA) for colour accuracy.Images were cropped and organised in Affinity Photo v.1.10.6 (Serif Ltd., WB, UK).Diagrams and illustrations were made using Affinity Designer