Sclareol Production Driven by Advanced Synthetic Biology Strategies

Abstract

Sclareol, a plant-derived diterpene, holds significant industrial value in perfumery, pharmaceuticals, and agriculture. Traditional production from Salvia sclarea or chemical synthesis has faced challenges such as low yield, high cost, and sustainability concerns. Recent advances in synthetic biology and metabolic engineering have enabled sclareol biosynthesis in microbial chassis cells. This Review systematically examines synthetic biology-driven strategies for improving sclareol production, with particular emphasis on microbial chassis engineering, metabolic pathway optimization, and enzyme engineering. Emerging approaches such as AI-assisted prediction, multiomics analysis, high-throughput screening, and intelligent fermentation technologies are also highlighted. Achievements and challenges in microbial sclareol biosynthesis are critically analyzed, and strategic priorities for future development are proposed. This paper provides a comprehensive reference for science and practice, aiming to advance the sustainable biomanufacturing of high-value plant-derived terpenoids.

1. Introduction

Sclareol is a naturally occurring labdane-type diterpene alcohol (C₂₀H₃₆O₂) characterized by a hydrophobic bicyclic carbon skeleton and two hydroxyl groups. , It is predominantly found in the essential oil of Salvia sclarea, where it is biosynthesized in glandular trichomes via the methylerythritol phosphate (MEP) pathway. , Because of its characteristic bicyclic structure, sclareol exhibits notable chemical stability and diverse biological activities. , In addition, it possesses a distinctive woody and amber-like aroma, making it a highly valued intermediate in the fragrance industry. Notably, sclareol serves as a key precursor for the industrial production of ambroxide, one of the most important substitutes for natural ambergris, which is typically obtained through the chemical modification and cyclization of sclareol. , Consequently, it has been widely applied in perfumes and cosmetic formulations due to its desirable olfactory properties and long-lasting scent. , The major biological functions and industrial applications of sclareol are summarized in Figure .

1.

Functions and applications of sclareol.

Beyond its industrial applications, sclareol has attracted an increasing amount of attention for its pharmacological potential. Previous studies have demonstrated that sclareol exhibits a broad spectrum of biological activities, including antitumor, anti-inflammatory, antibacterial, and antioxidant effects. ,, For instance, in HCT-116 colon cancer cells, sclareol inhibits DNA synthesis, induces G1-phase cell cycle arrest, activates caspase-8 and caspase-9, promotes poly­(ADP-ribose) polymerase (PARP) degradation, and ultimately triggers apoptosis. Toxicological evaluations further indicate that sclareol exhibits relatively low toxicity and is considered safe for use in fragrance and cosmetic applications under standard exposure conditions. In agriculture, sclareol also functions as a bioactive compound with insecticidal and antimicrobial properties. It can induce plant defense responses, including lignification and ethylene-mediated signaling, thereby enhancing resistance against pathogens such as root-knot nematodes. Meanwhile, recent advances in metabolic engineering and synthetic biology have enabled efficient biosynthesis of sclareol in engineered microbial hosts, such as Saccharomyces cerevisiae, through heterologous pathway reconstruction and optimization of microbial precursor supply, providing a sustainable alternative to plant extraction. Collectively, the multifunctional applications of sclareol underscore its significant commercial value and growing scientific interest, thereby driving efforts toward the development of efficient and sustainable production strategies.

Traditionally, sclareol has been sourced mainly through extraction from S. sclarea and related plant species. While this natural extraction approach benefits from renewable biomass, it suffers from several significant drawbacks. Firstly, the yield of sclareol from plants is generally low. Sclareol yield is affected by seasonal and environmental variations, contributing to supply instability. This results in supply instability and high production costs. Additionally, large-scale farming of S. sclarea raises concerns about land use and biodiversity. Although chemical routes for the synthesis of sclareol and its derivatives have been developed, significant challenges remain. The complex bicyclic structure and multiple stereocenters of sclareol make its total synthesis highly challenging. Such syntheses are often laborious, costly, and inefficient. In fact, total syntheses of terpenoids frequently require more than 10–15 steps, with low overall yields and strict stereocontrol requirements. Moreover, classical synthetic routes frequently rely on harsh reagents and generate substantial waste, thereby raising environmental and economic concerns. Consequently, both natural extraction and chemical synthesis face evident bottlenecks in terms of scalability, sustainability, and cost-effectiveness. These limitations restrict the broader application of sclareol in fragrance, pharmaceutical, and agricultural sectors. Hence, the development of novel production technologies is urgently required to overcome these limitations.

Synthetic biology has emerged as a transformative technology capable of addressing the challenges faced by traditional sclareol production. By harnessing the power of engineered microorganisms such as yeast and bacteria, these approaches enable the biosynthesis of complex natural products in controlled, scalable, and eco-friendly processes. With the development of synthetic biology, the modification of host organisms and optimization of biosynthetic pathways are employed for sclareol production. This includes heterologous expression of terpene synthases, microbial precursor pathway engineering, pathway balancing, precursor supply enhancement, and dynamic regulatory controls to maximize yield and productivity. Synthetic biology complements this by providing modular genetic tools, precise regulatory elements, and computational design frameworks that streamline pathway construction and optimization. The microbial production of sclareol offers multiple advantages, including reduced dependence on agricultural land, lower environmental impact, and consistent product quality. Moreover, it allows for the possibility of producing novel derivatives via pathway engineering, expanding the compound’s utility. Given the global push towards sustainable manufacturing and green chemistry, integrating synthetic biology and metabolic engineering represents a promising strategy. This approach may provide a promising route toward scalable and potentially more sustainable sclareol production, although its economic and environmental benefits remain to be validated at larger scales.

Several reviews have previously addressed different aspects of sclareol and its biotechnological production. A dedicated review summarized the occurrence, isolation, biosynthesis, structural modification, and pharmacological properties of sclareol. Recent work has further reviewed the synthetic biology of sclareol, with emphasis on the plant biosynthetic pathway and its production in engineered microbial and photosynthetic chassis. Broader reviews have also summarized microbial biosynthesis of terpenoid compounds, including chassis engineering, pathway optimization, and fermentation strategies for sustainable production of valuable terpenoids. In addition, several reviews have examined sclareol and related labdane diterpenes as precursors for ambroxide and other fragrance compounds. − Recent advances in synthetic biology have expanded the toolbox available for terpenoid biomanufacturing, including microbial chassis engineering, metabolic pathway optimization, enzyme engineering, multiomics-guided analysis, high-throughput screening, AI-assisted prediction and design, and intelligent fermentation. , While several of these strategies have been directly demonstrated in sclareol-producing systems, others have primarily been established in related terpenoid pathways and represent promising approaches for future application to sclareol production. However, an integrated, production-oriented analysis of these emerging strategies for improving sclareol productivity remains limited. Therefore, this Review focuses on sclareol production driven by advanced synthetic biology strategies, systematically covering microbial chassis development, metabolic pathway engineering, enzyme engineering, high-throughput screening, AI- and multiomics-assisted optimization, and intelligent fermentation. The current achievements, technological challenges, and future opportunities for sustainable and scalable sclareol biomanufacturing are discussed.

2. Traditional Production Strategies

The supply of sclareol has traditionally relied on plant extraction and chemical synthesis, both of which have played important roles in science and industry. However, as demand increases, issues such as low yield, high costs, and environmental burdens have become increasingly evident, limiting large-scale utilization. A detailed analysis of these conventional approaches helps to clarify the necessity of alternative production strategies and highlights key directions for future research.

2.1. Plant Extraction

Sclareol is predominantly accumulated in the glandular trichomes of S. sclarea inflorescences and has traditionally been harvested from plant material as the primary industrial source for natural sclareol and its hemisynthetic derivatives. Industrial-scale recovery of sclareol typically involves harvesting the flowering tops of S. sclarea. This is followed by steam distillation or solvent-based solid-liquid extraction, which yields a crude aromatic concrete or oleoresin. The crude product is subsequently fractionated and purified by chromatographic methods or recrystallization to obtain technical- or fragrance-grade sclareol.

The extraction yield and composition are highly dependent on genotype, developmental stage, glandular trichome density, and agronomic factors such as light and environmental stress. These biological variables account for large batch-to-batch variability in raw material quality. To improve process sustainability and selectivity, supercritical carbon dioxide (SC–CO₂) extraction and fractionation have been increasingly adopted and optimized to enrich sclareol fractions while reducing organic solvent usage. , Process parameters such as pressure, temperature, and CO₂ density strongly influence both yield and selectivity, and process modeling approaches have been applied to further optimize the fractionation efficiency. −

Typical yields of sclareol from wild-type or conventionally cultivated clary sage (S. sclarea) remain modest, limiting the feasibility of large-scale production. , Seasonal and environmental variation in essential oil/diterpene content, together with the high costs and technical complexity of downstream purification, pose additional challenges for reliable industrial supply. These limitations have motivated agronomic optimization and elicitor-based strategies aimed at improving the accumulation of specialized metabolites in plants. While some studies have examined agronomic factors affecting clary sage yield and essential oil traits, broader investigations of terpenoid regulation and plant stress responses provide potential strategies that may be explored for enhancing sclareol accumulation. , In addition, alternative production routes such as metabolic engineering in microbial hosts have been actively pursued and in several cases delivered substantially higher sclareol titers than plant extraction.

2.2. Chemical Synthesis

The chemical synthesis of sclareol has long been regarded as challenging due to its complex labdane-type diterpene skeleton containing multiple chiral centers and hydroxyl groups. Traditional synthetic routes mainly rely on semisynthetic transformations starting from naturally isolated sclareol rather than total de novo synthesis. Previously, sclareol could be converted into ambrox through multi-step oxidation and cyclization reactions. However, these processes often result in low overall yields and require complex purification steps, making them unsuitable for industrial-scale production.

Recently, the research focus has gradually shifted from total synthesis to selective oxidation and chemoenzymatic functionalization. For example, in 2015, the development of stereoselective chemoenzymatic oxidation routes for terpenoids was highlighted. These approaches enable site-selective hydroxylation and late-stage functionalization of terpene scaffolds, transformations that are difficult to accomplish by using purely chemical methods.

Nevertheless, the overall chemical synthesis of sclareol remains laborious and cost-inefficient because of its low selectivity, side-product formation, and multi-step nature. These drawbacks have motivated the exploration of synthetic biology and metabolic engineering approaches that can directly produce sclareol in microbial hosts with higher efficiency and environmental sustainability.

2.3. Industrial Bottlenecks of Existing Processes

Despite the long-standing use of S. sclarea as the primary natural source of sclareol, plant extraction has become increasingly recognized as being inefficient and costly. Chemical conversion routes face similar limitations and are now viewed as being environmentally unsustainable.

Sclareol biosynthesis occurs in the glandular trichomes of S. sclarea and is highly sensitive to genotype, developmental stage, and environmental stress, leading to large variability in yield. Although modern supercritical CO₂ extraction and solvent-based recovery techniques have been optimized, the overall yields remain low and require energy-intensive fractionation processes. These biological and technical constraints make plant extraction economically uncompetitive on an industrial scale. On the other hand, the chemical synthesis of sclareol and its derivatives remains highly complex, relying on multi-step reactions and hazardous oxidants. These requirements result in poor atom economy and substantial waste generation.

Recent studies have increasingly explored synthetic biology–driven engineered microbial platforms, such as Yarrowia lipolytica and S. cerevisiae, for high-level sclareol production in fermentation systems. ,,, These advances highlight a promising alternative to overcome the scalability and sustainability bottlenecks inherent in extraction and chemical routes. Unlike native plant biosynthesis, which relies on the MEP pathway for diterpene precursor formation, engineered yeast systems typically optimize endogenous mevalonate (MVA) pathways to isopentenyl diphosphate (IPP)/dimethylallyl diphosphate (DMAPP) and geranylgeranyl diphosphate (GGPP) supply for sclareol production. , Current strategies for sclareol production exhibit distinct advantages and limitations in terms of scalability, sustainability, and production efficiency, as summarized in Table .

1. Strategies for Sclareol Production.

strategy	host/system	production mode	key features	advantages	limitations	refs
Plant extraction	S. sclarea	Natural accumulation in plant tissues	Sclareol is naturally accumulated in aerial tissues, especially in glandular trichomes	Direct access to natural product; no genetic engineering required	Low content; strong environmental dependence; high extraction cost	,,
Chemical synthesis	Chemical system	Multi-step organic synthesis or semi-synthesis (often starting from natural diterpene precursors)	Construction or modification of diterpene scaffolds through stepwise chemical transformations	Enables precise structural control; suitable for derivatization and functional modification	Multi-step complexity; low overall yield; poor sustainability compared to biosynthetic production	,
Synthetic biology	S. cerevisiae; Yarrowia lipolytica	Heterologous biosynthesis via MVA pathway with enhanced precursor supply, pathway optimization, and cellular robustness engineering	Introduction of diterpene synthases and enhancement of GGPP supply through metabolic engineering	High titers; renewable and scalable production; well-established genetic tools	Requires complex pathway optimization and careful balancing of metabolic flux	,,

3. Synthetic Biology Strategies for Sclareol Production

The overall synthetic biology route for sclareol production is illustrated in Figure . Compared to traditional approaches, synthetic biology provides a more sustainable and scalable pathway for producing sclareol. By engineering chassis cells, optimizing metabolic flux, and precisely regulating synthetic components, high yield can be achieved using renewable resources. Sclareol, a bicyclic labdane-type diterpene diol, is derived from the universal isoprenoid precursors isopentenyl diphosphate (IPP) and DMAPP. These precursors are generated through the MVA and MEP pathways, which are distributed among different organisms. In native plants such as S. sclarea, diterpene precursors are primarily supplied through the plastidial MEP pathway, whereas engineered microbial hosts, particularly yeasts, commonly utilize and optimize the cytosolic MVA pathway for enhanced IPP/DMAPP and GGPP supply. Both pathways ultimately converge to supply GGPP, the C20 precursor for diterpene biosynthesis. Geranylgeranyl diphosphate synthase (GGPPS) catalyzes the sequential condensation of IPP units with an allylic diphosphate substrate to generate GGPP. This step commits carbon flux to diterpenoid biosynthesis and represents a frequent engineering target to increase GGPP availability. Recently, as a central node, GGPPS was emphasized for redirecting flux toward diterpenes in plant and microbial hosts. , The conversion of GGPP into sclareol is mediated by sclareol-specific diterpene synthases. In S. sclarea, sclareol biosynthesis proceeds through the sequential action of two diterpene synthases (diTPSs). First, a class II diterpene synthase converts GGPP into 8α-hydroxy-copalyl diphosphate (8α-hydroxy-CPP). Subsequently, a class I diterpene synthase catalyzes the conversion of 8α-hydroxy-CPP into sclareol. , These enzymes have been functionally reconstructed in engineered microbial strains, enabling the heterologous production of sclareol at high titers. , These findings demonstrate the successful transfer and functional application of the native sclareol biosynthetic module in alternative microbial hosts. Accordingly, the heterologous biosynthetic pathway engineered for microbial sclareol production can be summarized as IPP/DMAPP → GGPP (via GGPPS) → 8α-hydroxy-CPP (via a class II diterpene synthase)→ sclareol (via a class I diterpene synthase), highlighting precursor supply and diterpene synthase activity as the primary engineering nodes for heterologous sclareol biosynthesis. ,, Cytochrome P450 monooxygenases (P450s) are mainly involved in downstream oxidative modification of diterpenoid scaffolds and contribute to the generation of structural diversity among diterpenoid derivatives.

2.

Route of sclareol production using synthetic biology.

3.1. High-Efficiency Chassis Cell Development

3.1.1. Optimization Strategies for Established Industrial Microbial Chassis

The selection of efficient chassis cells is a fundamental prerequisite for natural product biosynthesis. Among established industrial chassis, S. cerevisiae and Y. lipolytica have been extensively developed for terpene production because of their well-characterized genetic backgrounds, robust fermentation performance, and favorable precursor supply capacities. For sclareol production, S. cerevisiae offers a well-characterized genetic background, high tolerance to fermentation conditions, and a native MVA pathway suitable for terpene precursor synthesis. Optimization strategies for yeast include promoter engineering, dynamic pathway regulation, and cofactor balancing to enhance precursor availability and product titers. In 2022, modular metabolic balancing in S. cerevisiae was applied to improve sclareol production. By overexpressing key MVA pathway enzymes, enhancing precursor and cofactor supply, and dynamically regulating competing pathways, the engineered yeast achieved a sclareol titer of 11.4 g/L in the fermentation. This result demonstrates the effectiveness of promoter engineering, pathway balancing, and cofactor optimization in increasing terpene yields. Similarly, Y. lipolytica, an oleaginous industrial yeast with strong acetyl coenzyme A (acetyl-CoA) flux and lipid accumulation capacity, has recently emerged as a powerful platform for diterpenoid production. Compared with S. cerevisiae, Y. lipolytica exhibits broad substrate utilization, robust stress tolerance, and high metabolic flux toward acetyl-CoA and related precursors, making it particularly suitable for the production of high-value terpenoids, including sesquiterpenes and diterpenes. In 2025, de novo biosynthesis of sclareol was successfully achieved in Y. lipolytica, demonstrating the potential of this industrial yeast chassis for the sustainable production of flavors, fragrances, and other high-value diterpenoids. In 2026, systematic metabolic engineering of Y. lipolytica enabled high-level sclareol production by integrating modular pathway optimization, enhanced GGPP precursor supply, and expression tuning of key biosynthetic enzymes, resulting in a titer of 13.9 g/L in a 5 L bioreactor. More recently, Pichia pastoris has also been engineered as an efficient chassis for sclareol biosynthesis. By systematically rewiring the sclareol biosynthetic pathway and central metabolism to alleviate acetyl-CoA and nicotinamide adenine dinucleotide phosphate (NADPH) limitations, together with regulatory remodeling and fermentation optimization, the engineered strain achieved a sclareol titer of 27.8 g/L in minimal medium, further highlighting the potential of non-conventional yeasts as efficient chassis for high-level sclareol production. Beyond yeast-based chassis, Streptomyces, known for producing complex secondary metabolites, provides a versatile platform for expressing large gene clusters. Systems and synthetic biology approaches provide general frameworks for rational metabolic engineering and pathway optimization across microbial cell factories. In Streptomyces, advanced genome engineering technologies, particularly CRISPR-Cas systems, have been applied for biosynthetic gene cluster refactoring, multiplex genome editing, and regulation of metabolic pathways. Although these strategies have not yet been directly demonstrated for sclareol production in Streptomyces, they provide transferable engineering concepts for the future development of alternative sclareol-producing chassis.

3.1.2. Development and Application Prospects of Emerging Biosystems

Beyond established industrial microbial chassis, emerging biosystems, including microalgae, plant cell cultures, and whole-plant expression systems, provide alternative platforms for the production of complex plant-derived metabolites. Importantly, the level of experimental evidence supporting these systems differs substantially, with microalgal and whole-plant platforms having been directly demonstrated for sclareol production, whereas plant cell culture systems currently represent promising alternative approaches based primarily on their broader applications in plant-specialized metabolite production. Microalgae, particularly Chlamydomonas reinhardtii, have emerged as promising photoautotrophic chassis for diterpenoid biosynthesis. Direct evidence for sclareol production was first provided by chloroplast engineering of C. reinhardtii, in which the introduction of a plant-derived labdane-type diterpene cyclase enabled the production of several labdane diterpenes, including sclareol. Subsequent metabolic and synthetic biology engineering further improved the performance of this microalgal chassis. By redirecting metabolic flux through the MEP pathway and optimizing key enzymes involved in precursor supply and diterpene formation, engineered C. reinhardtii achieved photoautotrophic production of sclareol up to 656 mg/L in a 2.5 L photobioreactor. These studies demonstrate that microalgae can serve as directly validated alternative chassis for light-driven sclareol biosynthesis.

In addition to pathway and metabolic engineering, advances in algal synthetic biology have expanded the available genetic engineering toolbox. Rational promoter engineering and the development of synthetic expression elements have significantly improved heterologous gene expression and terpene production in C. reinhardtii. Although the promoter engineering strategy in this study was demonstrated using the sesquiterpene (E)-α-bisabolene rather than sclareol, the resulting genetic tools provide transferable approaches that may facilitate further optimization of sclareol-producing microalgal strains.

Plant cell culture systems, particularly hairy root cultures, also provide alternative platforms for the production of complex plant-derived secondary metabolites. These systems offer advantages including genetic and biochemical stability, relatively rapid growth, and the capacity to support plant-specific metabolic pathways. Various metabolic engineering strategies, including precursor enhancement, regulation of biosynthetic genes, multigene engineering, elicitation, and transcription-factor engineering, have been applied to improve secondary metabolite production in transformed root cultures. However, direct high-level sclareol production in plant cell culture systems has not yet been extensively demonstrated. Therefore, these systems should currently be regarded as prospective platforms whose potential for sclareol production remains to be experimentally established. Whole-plant metabolic engineering provides another emerging platform that has been directly demonstrated for sclareol biosynthesis. In 2024, the two S. sclarea genes responsible for sclareol biosynthesis, labda-13-en-8-ol diphosphate synthase and sclareol synthase, were introduced and overexpressed in Nicotiana tabacum, enabling the accumulation of sclareol in transgenic tobacco leaves. The best-performing lines accumulated 4.1 μg/cm² of sclareol, demonstrating that plant-based heterologous expression can provide an alternative route for sclareol production. This strategy may benefit from the native plant metabolic environment and the capacity to accommodate complex plant-derived biosynthetic pathways.

3.1.3. Metabolic Flux Rewiring and Prospective Chassis Engineering

Metabolic flux rewiring represents an important strategy for developing a high-efficiency microbial chassis for sclareol biosynthesis. By reinforcing the precursor supply, redirecting carbon flux, and reducing competing metabolic pathways, cellular resources can be more effectively channeled toward the production of the target diterpenoid.

Several metabolic engineering strategies have been directly demonstrated for the production of sclareol. In S. cerevisiae, a modular metabolic engineering approach combined with global metabolic rewiring was used to improve sclareol production. Metabolic flux analysis showed that balanced cellular metabolism promoted carbon flux toward the target pathway, enabling a sclareol titer of 11.4 g/L. In Y. lipolytica, construction of a two-layer GGPP accumulation pathway and scaffold-free multienzyme complexes redirected metabolic flux toward sclareol biosynthesis, reduced by-product formation, and enabled a titer of 12.9 g/L in a 5 L bioreactor. More recently, systematic metabolic engineering in Y. lipolytica, including improvement of key enzyme activity, optimization of GGPP supply and consumption, transporter engineering, and elimination of citrate overflow metabolism, further increased sclareol production to 13.9 g/L in a 5 L bioreactor. Collectively, these studies demonstrate that precursor reinforcement, metabolic flux redistribution, and elimination of competing metabolic processes are directly applicable strategies for improving sclareol biosynthesis. Beyond these strategies directly demonstrated for sclareol, advanced chassis engineering approaches may provide additional opportunities for future developments. Genome minimization, which involves the systematic reduction of non-essential genetic elements, has been explored as a strategy to simplify cellular systems and improve desirable chassis properties such as genetic stability and transformation efficiency. Although genome minimization has not yet been directly demonstrated for sclareol production, it represents a prospective strategy for developing streamlined and robust microbial chassis that could support future diterpenoid biosynthesis. Together, strategies directly demonstrated for sclareol and prospective chassis engineering approaches provide complementary directions for the development of more efficient and robust microbial cell factories.

3.1.4. Enhancing Fermentation Performance and Stress Tolerance of Industrial Chassis

For industrial-scale sclareol production, chassis cells must achieve high titers and withstand the stresses associated with large-scale fermentation. Improving the fermentation performance involves increasing substrate utilization, enhancing oxygen transfer efficiency, and boosting product secretion. At the same time, engineering cellular robustness is just as important since the buildup of terpenoids inside cells can be toxic to the host. Recent studies have further shown that aligning metabolic rewiring with the regulation of cellular aging can significantly enhance both the system robustness and production performance. By modulating nutrient-sensing pathways and enhancing mitophagy, engineered yeast achieved significantly improved chronological lifespan together with elevated sclareol titers up to 25.9 g/L, indicating a direct coupling between cellular longevity and terpenoid biosynthetic capacity. Strategies such as pathway compartmentalization to spatially isolate toxic intermediates, modification of membrane properties, and transporter engineering to facilitate product export and reduce intracellular stress have been widely explored in terpenoid-producing cell factories. In addition, synthetic regulatory circuits can dynamically adjust pathway expression in response to environmental or metabolic cues, maintaining cell viability and productivity under fluctuating conditions. For example, in 2024, metabolic flux redirection combined with chassis engineering in the oleaginous yeast Y. lipolytica significantly enhanced the fermentation performance for de novo sclareol production. Reinforced GGPP supply together with bioprocess optimization efficiently redirected carbon flux toward the diterpene pathway, reduced by-product formation, and enabled reactor-scale production. In 2025, a high-throughput promoter replacement strategy was developed in the industrial yeast Y. lipolytica to fine-tune gene expression and improve production-related phenotypes, providing a scalable and transferable tool for future strain optimization. These strategies jointly support the development of chassis strains capable of high-density cultivation, thereby reducing production costs while maintaining consistency and quality in industrial bioprocesses. As sclareol titers increase, cellular robustness and genetic stability become increasingly important for a prolonged fermentation. High intracellular accumulation of terpenoids may cause cytotoxicity and impose limitations on intracellular storage capacity, while compartmentalization and transporter engineering can help alleviate these constraints. In Y. lipolytica, ATP-binding cassette transporter engineering enhanced extracellular sclareol accumulation and contributed to high-level production without an in situ solvent extractant. In addition, the 25.9 g/L S. cerevisiae process demonstrated that chronological lifespan engineering and reinforcement of central metabolism can sustain productive metabolism during long-term cultivation. However, systematic evaluation of long-term genetic and production stability remains limited in current sclareol-producing strains and should be addressed in future industrial studies. Representative microbial engineering strategies and reported sclareol titers achieved in different chassis cells are summarized in Table .

2. Microbial Synthetic Biology and Fermentation Performance for Sclareol Production.

host	engineering strategy	key modifications	carbon source	feeding strategy	fermentation scale	fermentation duration	titer (g/L)	yield (as reported)	productivity	downstream recovery	refs
Escherichia coli	Heterologous pathway reconstruction and MEP-pathway engineering	Plant-derived diterpene synthases; basic pathway assembly	Glycerol	pO2-controlled glycerol fed-batch feeding	Bench-scale bioreactors	∼48 h	1.46 ± 0.17	Not reported	Not reported	Organic solvent/dodecane overlay; GC-MS quantification
S. cerevisiae	Modular metabolic engineering and global metabolic rewiring	MVA pathway overexpression; enhanced acetyl-CoA and NADPH supply; dynamic pathway regulation	Glucose	Exponential glucose-limited fed-batch	1.0 L bioreactor, 0.4 L initial volume	192 h	11.4	10.9% theoretical yield	0.059 g/L/h	Hexane extraction; precipitate resuspension & rotary evaporation
Y. lipolytica	Combinatorial metabolic engineering	Enhanced GGPP supply; precursor flux optimization; pathway balancing	Glucose	Intermittent glucose fed-batch	5 L bioreactor, 2 L initial volume	240 h	12.9	53.2 mg/g glucose	0.054 g/L/h	20% v/v dodecane two-phase extraction
Y. lipolytica	Systematic metabolic engineering	Synthase site-directed mutagenesis; GGPP flux redistribution; competing pathway elimination; transporter engineering	Glucose	Glucose fed-batch, residual glucose ≤5 g/L	5 L bioreactor, 2 L working volume	156 h	13.9	Not reported	Not reported	Spontaneous precipitation; whole-broth ethyl acetate extraction
S. cerevisiae	Chronological lifespan engineering and metabolic rewiring	Nutrient-sensing attenuation; enhanced mitophagy; ACS1/2 overexpression; ERG9 downregulation	Glucose	Variable-rate glucose fed-batch	1.0 L bioreactor, 0.3 L initial volume	Not reported	25.9	0.051 g/g glucose	Not reported	Hexane extraction with internal standard
P. pastoris	Systematic pathway and central metabolic rewiring	Acetyl-CoA and NADPH bottleneck relief; regulatory remodeling; transcriptional reprogramming; fermentation optimization	Glycerol	Glycerol fed-batch with pH-coupled ammonia feeding	1.5 L bioreactor, 0.5 L initial volume	Not reported	27.8	Not reported	Not reported	Spontaneous precipitation/crystallization; resuspension and hexane extraction

Comparison of the available fermentation data shows that final titer alone does not fully reflect process performance. The early E. coli process reached 1.46 ± 0.17 g/L sclareol in almost 2 days using controlled glycerol fed-batch cultivation. In S. cerevisiae, 11.4 g/L was obtained after approximately 192 h with a productivity of 0.059 g/L/h and a reported yield of 10.9% of the theoretical maximum, while Y. lipolytica reached 12.9 g/L after 240 h with a yield of 53.2 mg/g glucose and a productivity of 0.054 g/L/h. A later Y. lipolytica process increased the titer to 13.9 g/L within 156 h, whereas more recent S. cerevisiae and P. pastoris systems achieved 25.9 and 27.8 g/L, respectively, although complete productivity data were not reported. , Feeding strategies also differed substantially, ranging from exponential glucose-limited feeding and residual-glucose-controlled feeding to glycerol fed-batch combined with pH-coupled ammonia feeding in P. pastoris. These comparisons highlight the importance of considering fermentation time, yield, productivity, substrate, and feeding strategy together with final titer.

Downstream recovery has also evolved with increasing titers. Early E. coli and Y. lipolytica processes relied on organic solvent overlays for in situ product extraction, , whereas later high-titer Y. lipolytica and P. pastoris processes enabled spontaneous precipitation or crystallization of sclareol without a solvent overlay. , This transition may simplify product recovery, although scalable isolation and purification of precipitated sclareol still require further process development.

3.2. Metabolic Pathway Design and Dynamic Regulation

To achieve high-level sclareol biosynthesis, a rational metabolic pathway design must go beyond simple gene overexpression. This requires the systematic optimization of precursor supply, regulatory circuits, and pathway coordination. In this context, multiple engineering strategies have been developed to enhance the flux efficiency, reduce cellular stress, and enable dynamic adaptation to metabolic demands. The following sections summarize key advances in pathway construction and regulation that support the efficient production of diterpenoids such as sclareol.

3.2.1. Integration and Comparison of MVA, MEP, and Hybrid Pathways

The contribution of these pathways is organism-dependent: plant diterpene biosynthesis generally relies on plastidial MEP-derived GGPP, whereas engineered yeast platforms commonly exploit and optimize the cytosolic MVA pathway for precursor enhancement. , The MVA and MEP pathways represent two primary routes for the biosynthesis of isoprenoid precursors in nature. Early radiolabeling studies demonstrated that sclareol originates from the isoprenoid precursor pool, as the incorporation of 14C-labeled mevalonic acid into sclareol was observed in plant systems. However, subsequent studies of diterpenoid biosynthesis have established that plastidial MEP pathway-derived GGPP is the major precursor source for native diterpenes in plants such as S. sclarea. , The MVA pathway is typically native to eukaryotes and some archaea. It has been extensively engineered in S. cerevisiae and other microbial hosts for terpenoid production due to its compatibility and robustness under industrial conditions. In contrast, the MEP pathway, which is common in bacteria and plant plastids, offers higher theoretical carbon efficiency but is more sensitive to oxygen and redox imbalances. For sclareol production, the engineering of the MVA pathway has been directly demonstrated in microbial hosts. For example, in 2023, the MVA pathway in S. cerevisiae was engineered to achieve a sclareol titer of 11.4 g/L, demonstrating that strengthening MVA-derived precursor flux together with cofactor balancing can markedly enhance diterpenoid production. Metabolic flux analysis showed that balanced cellular metabolism promoted carbon flux toward the target biosynthetic pathway, demonstrating the importance of MVA-derived precursor supply and global metabolic rewiring for efficient sclareol production. Similarly, in 2024, the endogenous MVA pathway of Y. lipolytica was optimized and combined with a scaffold-free multienzyme complex, which effectively redirected the metabolic flux toward GGPP and sclareol, resulting in a titer of 12.9 g/L in a 5 L bioreactor.

In contrast, although MEP-pathway engineering and hybrid MVA–MEP strategies have been explored in other isoprenoid and terpenoid production systems, their direct application to microbial sclareol production has not yet been reported. Nevertheless, these approaches may provide transferable strategies for improving precursor supply, balancing cofactor utilization, and enhancing carbon flux toward diterpenoid biosynthesis. Future comparative studies of MVA, MEP, and hybrid pathways under identical chassis conditions will be important for identifying context-specific advantages and guiding rational pathway selection or combination for improved sclareol production. Integrated metabolic engineering strategies for microbial sclareol biosynthesis are summarized in Figure .

3.

Integrated metabolic engineering strategies for sclareol biosynthesis in microbial systems.

The abbreviations of enzymes are as follows: AACT (acetyl-CoA acetyltransferase); HMGS (3-hydroxy-3-methylglutaryl-CoA synthase); HMGR (3-hydroxy-3-methylglutaryl-CoA reductase); MVK (mevalonate kinase); PMK (phosphomevalonate kinase); MVD (mevalonate diphosphate decarboxylase); DXS (1-deoxy-D-xylulose-5-phosphate synthase); DXR (1-deoxy-D-xylulose-5-phosphate reductoisomerase); IspD (2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase); IspE (4-diphosphocytidyl-2-C-methyl-D-erythritol kinase); IspF (2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase); IspG (4-hydroxy-3-methylbut-2-enyl diphosphate synthase); IspH (4-hydroxy-3-methylbut-2-enyl diphosphate reductase); IDI (isopentenyl diphosphate isomerase); FPPS (farnesyl diphosphate synthase); GGPPS (geranylgeranyl diphosphate synthase); diTPS (diterpene synthase).

3.2.2. Synthetic Promoters, Regulatory Elements, and Dynamic Control Networks

To achieve fine-tuned, high-efficiency sclareol production, it is essential to dynamically regulate the engineered metabolic pathways. Synthetic biology provides diverse regulatory tools, including tunable promoters, transcriptional regulators, and dynamic control systems, which enable the precise adjustment of gene expression and pathway activity.

However, most sophisticated promoter engineering and dynamic regulatory strategies have so far been demonstrated in other microbial or terpenoid production systems rather than directly in sclareol-producing hosts. For example, rational promoter engineering has enabled robust terpene production in microalgae, while high-throughput gene expression tuning has been applied to systematically optimize metabolic pathways in Y. lipolytica. These studies provide transferable strategies for fine-tuning pathway enzyme expression and balancing metabolic flux in future sclareol-producing cell factories.

For instance, transcription-factor-based dynamic sensor–regulator systems (DSRS) that sense pathway intermediates and automatically adjust pathway gene expression significantly improved fatty-acid-derived product titers and stability. Although these sophisticated dynamic control systems have not yet been directly demonstrated for microbial sclareol production, they provide promising regulatory frameworks for dynamically balancing pathway activity and cellular metabolism. In contrast, metabolic rewiring and balanced pathway regulation have been directly demonstrated for sclareol production. In 2023, S. cerevisiae was engineered by globally rewiring its central metabolism to improve acetyl-CoA and NADPH supply, achieving a sclareol titer of 11.4 g/L. This study provides direct evidence that balanced metabolism and improved precursor allocation can enhance sclareol biosynthesis. However, unlike the dynamic regulatory strategies described above, this approach primarily relied on systematic metabolic rewiring rather than a synthetic sensor-based dynamic control network.

3.2.3. Multipathway Synergy and Strategies for Balancing Metabolic Burden

Engineering multipathway systems to coordinate precursor supply is an effective strategy for enhancing sclareol biosynthesis. However, such approaches can impose a metabolic burden due to competition for cellular resources. Modular pathway design has been widely demonstrated to improve the balance between microbial growth and terpenoid production, as highlighted in several recent reviews of isoprenoid metabolic engineering. , Although these strategies have not been extensively demonstrated in microbial sclareol-producing systems, they provide transferable engineering principles for balancing precursor supply and metabolic burden in future sclareol cell factories.

In addition to intracellular pathway optimization, intercellular pathway division has emerged as an alternative strategy to alleviate the metabolic burden. For example, an artificial microbial consortium combining engineered S. cerevisiae and H. roseonigra enabled the conversion of glucose to sclareol-derived intermediates through distributed metabolic modules, achieving 644.2 mg/L of ambradiol in shake flasks. Notably, this study focused on the downstream conversion of microbially produced sclareol to ambradiol rather than the direct enhancement of sclareol biosynthesis. Nevertheless, it demonstrates the potential of pathway division among multiple hosts for reducing the metabolic burden associated with the complex diterpenoid production. Such strategies may therefore provide a transferable framework for future sclareol biosyntheses.

Spatial organization of enzymes using synthetic scaffold systems further improves the pathway efficiency. Protein scaffolds can colocalize sequential enzymes, promote metabolite channeling, and reduce diffusion losses of hydrophobic intermediates, leading to enhanced flux through isoprenoid pathways. , These strategies have been demonstrated in other metabolic and terpenoid-related systems rather than directly in microbial sclareol production but may provide useful approaches for improving enzyme coordination and pathway efficiency in future sclareol-producing cell factories.

In addition, computational approaches integrating genome-scale metabolic models (GEMs) and machine learning enable rational prediction and dynamic adjustment of pathway fluxes. , While these approaches have not yet been specifically applied to optimize microbial sclareol production, they provide prospective systems-level tools for identifying metabolic bottlenecks, balancing pathway fluxes, and improving the strain performance. Future integration of computational modeling with experimental metabolic engineering may, therefore, facilitate the rational optimization of complex sclareol biosynthetic pathways.

3.3. Key Enzyme Engineering and Synthetic Module Optimization

3.3.1. Directed Evolution and Rational Design to Enhance Enzyme Activity and Specificity

The strategies discussed in this section include approaches directly demonstrated in sclareol-producing systems, transferable strategies supported by studies of related terpenoids, and prospective strategies that require further validation for sclareol biosynthesis. Key enzymes in sclareol biosynthesis, particularly diterpene synthases, critically determine the pathway efficiency and product selectivity. The diterpene synthases responsible for sclareol biosynthesis were first identified and functionally characterized from Salvia sclarea, establishing the enzymatic basis for sclareol formation from GGPP. Directed evolution has become an effective approach for enhancing enzyme activity, stability, and substrate specificity via repeated rounds of mutagenesis and screening, and it has been extensively used for terpene-modifying enzymes. In particular, extensive engineering of terpene synthases has demonstrated that cyclization specificity and product profiles can be substantially reprogrammed. These findings highlight the plasticity of terpene-forming enzymes and provide a potential methodological basis for engineering sclareol synthases. In parallel, directed evolution and rational enzyme engineering have been used to modify enzyme activity and selectivity in related biocatalytic systems, providing potential strategies for engineering enzymes involved in terpenoid biosynthesis. More recently, the integration of computational modeling with enzyme engineering has further accelerated the identification of beneficial mutations. Together, these complementary approaches provide a potential framework for overcoming enzymatic bottlenecks in microbial sclareol biosynthesis, although their direct application to sclareol-producing systems requires further validation.

3.3.2. Multienzyme Synergy and Expression Balancing

Efficient sclareol biosynthesis requires the coordinated action of multiple enzymes spanning the precursor supply, diterpene cyclization, and downstream oxidation steps. A major challenge is achieving a balanced expression of these enzymes to prevent metabolic bottlenecks and the accumulation of toxic intermediates. Common strategies include promoter engineering, gene copy number adjustment, and modular pathway optimization to match enzymatic capacities and improve the overall metabolic flux. Coordinated multienzyme expression has been shown to enhance diterpenoid and secondary metabolite biosynthesis in related systems. For example, simultaneous engineering of a diterpene synthase and a precursor-supplying enzyme resulted in an increased level of accumulation of secondary metabolites through improved metabolic flux and pathway coordination.

In engineered yeast systems for sclareol production, the coordinated balancing of the GGPP synthesis module with sclareol biosynthetic enzymes is critical for efficient pathway performance. In particular, systematic pathway reconstruction and metabolic optimization in Y. lipolytica enabled synchronized flux through upstream and downstream reactions, significantly enhancing de novo sclareol accumulation under fed-batch fermentation conditions.

More broadly, multienzyme synergy has been achieved through spatial organization and compartmentalization strategies in microbial biosynthesis. For example, cytoplasmic–peroxisomal pathway partitioning in Candida tropicalis markedly improved the production of fatty-acid-derived compounds by separating precursor generation and downstream conversion steps. Similarly, modular balancing of the mevalonate pathway in S. cerevisiae demonstrated that fine-tuning expression levels across multiple enzymes can improve flux toward high-value terpenoids, such as farnesene. Collectively, these studies highlight that expression balancing and multienzyme coordination are important considerations for scalable microbial sclareol production and provide transferable strategies for further pathway optimization.

3.3.3. Application of Synthetic Modular Design

Synthetic modular design is a fundamental strategy in advanced synthetic biology for organizing and optimizing complex biosynthetic routes such as microbial sclareol production. Under this paradigm, biosynthetic pathways are conceptually partitioned into functionally distinct units: precursor supply, core carbon skeleton formation, and subsequent modification, allowing independent tuning and recombination to alleviate metabolic imbalance and enhance flux. Although sclareol-specific studies do not always explicitly describe their strategies as “modular design”, current yeast and microbial cell factory engineering approaches inherently reflect such principles. These approaches coordinate precursor supply with core diterpene biosynthetic modules and downstream pathway components, allowing independent tuning of different pathway segments. Modular pathway balancing and systematic metabolic reconstruction have been widely demonstrated to enhance terpenoid production efficiency in engineered yeast systems, enabling scalable biosynthesis. , These well-established principles provide a conceptual basis for further applying synthetic modular design to sclareol biosynthesis. Integrated metabolic engineering strategies for enhancing sclareol biosynthesis are summarized in Table .

3. Integrated Metabolic Engineering Strategies for Sclareol Production Enhancement.

step	strategy	core engineering targets	representative approaches	effect	refs
Upstream carbon supply	Precursor supply enhancement	Acetyl-CoA, IPP/DMAPP	MVA pathway enhancement; acetyl-CoA supply improvement	Increased precursor availability	,,
Key branch point	GGPP pool enhancement	GGPP biosynthesis	GGPPS overexpression; multi-copy integration	Increased diterpene precursor flux	,,,
Flux control	Carbon flux redistribution	Competing pathways (sterol biosynthesis)	ERG9 downregulation; pathway balancing	Improved carbon partitioning toward sclareol	,
Product formation	Enzyme engineering	diTPS (SsLPPS, SsSS)	Enzyme engineering; heterologous expression	Improved sclareol biosynthesis	,
System balance	Cofactor and pathway coordination	NADPH supply; pathway balance	NADPH regeneration; modular pathway optimization	Improved pathway efficiency
Host optimization	Chassis engineering	Acetyl-CoA flux; lipid metabolism	Y. lipolytica as production host	Enhanced terpene accumulation capacity	,
Strain optimization	Strain screening and optimization	Strain performance	High-throughput screening; adaptive evolution in related terpenoid systems	Potential for accelerated strain improvement	,

3.4. High-Throughput Screening, Intelligent Modeling, and Process Scale-Up

Advances in automation, high-throughput screening, machine learning, and multiomics technologies are transforming the development of microbial cell factories for diterpenoid production. These tools enable rapid exploration of enzyme and strain diversity and data-driven prediction of optimal pathway configurations, while process engineering and scale-up strategies facilitate the translation of laboratory performance to larger-scale fermentation systems. Although applications specifically targeting sclareol remain relatively limited, emerging studies of engineered diterpenoid pathways provide valuable insights. The examples discussed below therefore represent either strategies demonstrated in sclareol-related systems, transferable approaches developed for other terpenoids, or prospective tools that remain to be validated for sclareol production. High-throughput screening of terpene synthases and related pathway components has been shown to accelerate the enzyme and pathway optimization in terpene-producing systems. In parallel, intelligent modeling of metabolic flux and fermentation parameters further enhances strain development and the overall production performance. Collectively, these approaches establish an integrated framework to shorten engineering cycles and facilitate the scalable biomanufacturing of sclareol and related diterpenoids.

3.4.1. Automated Screening Platforms and Phenotype-Based Strain Selection

High-throughput screening (HTS) and phenotype-based strain selection have become enabling tools for accelerating the development of microbial platforms for sclareol biosynthesis. Automated and semiautomated screening approaches allow parallel evaluation of large genetic libraries and cultivation conditions, significantly shortening strain optimization cycles. Although direct biosensors or fluorescent reporters for sclareol are not yet available, several screening strategies developed for terpenes and related isoprenoid pathways provide transferable options.

Genetically encoded biosensors responsive to isoprenoid-derived metabolites have been successfully applied to guide strain optimization in microbial terpene production and provide transferable screening strategies for sclareol biosynthesis. For example, transcription-factor-based biosensors coupled with fluorescence-activated cell sorting (FACS) enabled rapid enrichment of high-producing terpene strains, thereby accelerating design-build-test-learn cycles in metabolic engineering. In parallel, standardized analytical screening pipelines based on heterologous expression and gas chromatography-mass spectrometry (GC-MS) product profiling have been developed to systematically evaluate monoterpene synthase variant libraries, enabling efficient identification of enzymes with altered or improved product profiles.

In addition, droplet-based microfluidic technologies have enabled ultrahigh-throughput, single-cell-level functional screening of large enzyme libraries, allowing rare high-performing variants to be isolated from highly heterogeneous populations. For complex diterpenoids such as sclareol, where direct product sensors remain unavailable, phenotype-based screening strategies, such as growth-coupled selection, tolerance to pathway intermediates, or reduced precursor toxicity, may provide practical alternatives. Collectively, these approaches provide a potential and adaptable framework for future high-throughput screening and strain optimization in microbial sclareol production, although their direct application to sclareol has yet to be validated.

3.4.2. AI- and Multiomics-Driven Pathway Prediction and Optimization

Artificial intelligence (AI) and machine learning, when integrated with multiomics data sets, are increasingly used to support predictive design and optimization of complex biosynthetic pathways. By combining genomic, transcriptomic, proteomic, and metabolomic data, AI-driven models can predict metabolic flux distributions, identify hidden bottlenecks, and propose non-intuitive engineering targets, thereby reducing empirical trial and error. , Recent studies in other microbial systems have demonstrated that machine-learning-augmented GEMs can significantly improve prediction accuracy for gene expression, growth behavior, and metabolite synthesis across conditions.

Although direct applications of these approaches to sclareol biosynthesis remain limited, they provide a potential conceptual and technical basis for future pathway optimizations. These AI- and multiomics-driven frameworks may help identify rate-limiting steps and guide rational pathway optimization in future sclareol-producing microbial chassis.

4. Techno-Economic Feasibility and Challenges

Sclareol is a high-value labdane-type diterpene that is widely used as a precursor for ambroxide in the fragrance industry, underscoring its significant commercial importance. , However, its current supply relies predominantly on plant extraction from S. sclarea, where sclareol is present at relatively low levels in glandular trichomes, resulting in limited productivity and contributing to high extraction costs. In addition, plant-based production is constrained by seasonal variability, land use, and labor requirements, making a large-scale supply both unstable and economically challenging.

Techno-economic data specific to sclareol production are limited. Therefore, insights into the cost structure and process feasibility are commonly inferred from studies on structurally related terpenoids and microbial production systems. In these related terpene production systems, the production cost is strongly influenced by key bioprocess parameters, including titer, yield, and productivity, as demonstrated by techno-economic analyses of microbial terpene biosynthesis. For plant-derived natural products, extraction processes such as supercritical CO₂ extraction can incur substantial operational costs, providing a useful benchmark for evaluating alternative production strategies for sclareol.

Recent advances in metabolic engineering have substantially improved microbial sclareol production. For example, in engineered S. cerevisiae, global metabolic rewiring combined with enhanced precursor supply enabled sclareol titers of approximately 11.4 g/L. Furthermore, by integrating chronological lifespan engineering with global metabolic rewiring, sclareol production was further increased to 25.9 g/L, representing one of the highest titers reported to date in microbial systems. More recently, host-specific pathway, regulatory, and process engineering in P. pastoris achieved a sclareol titer of 27.8 g/L in a 1.5 L bioreactor using fed-batch fermentation with pH-coupled ammonia feeding, representing the highest reported microbial sclareol titer to date. These results demonstrate the technical potential of microbial platforms for high-level sclareol production and provide useful benchmarks for future techno-economic assessments.

Therefore, industrial feasibility should not be evaluated according to the final titer alone. Yield, volumetric productivity, fermentation duration, substrate, aeration requirements, and downstream recovery collectively influence process efficiency and production cost. , Long-term genetic and production stability should also be considered when evaluating sclareol-producing strains for industrial fermentation. Future studies should report these parameters more systematically to enable a meaningful comparison among microbial production platforms.

Oxygen transfer may become an important constraint during the scale-up of high-cell-density sclareol fermentation. The reported high-titer S. cerevisiae, Y. lipolytica, and P. pastoris processes relied on active control of dissolved oxygen through increased agitation and/or aeration. In particular, oxygen mass transfer at ultrahigh cell density has been identified as an important challenge for larger-scale production. In parallel, the hydrophobic nature of sclareol complicates downstream recovery. Although spontaneous precipitation or crystallization can reduce the dependence on solvent overlays, , solid deposition and the handling of crystalline products may complicate reactor operation and downstream processing. ,

In microbial terpene production systems, fermentation-related expenses can represent a major fraction of total production costs, including costs associated with carbon sources, aeration, sterilization, and downstream processing. As pathway efficiency improves and metabolic flux is more precisely regulated, these systems may provide advantages over plant-based extraction in terms of production controllability and resource utilization, although their economic competitiveness requires further validation through comprehensive techno-economic analysis.

Moreover, advanced bioprocessing systems incorporating real-time monitoring, automated control, and data-driven optimization could further improve process robustness and reproducibility at an industrial scale. Integrated process design combining fermentation control with efficient product separation may be particularly important for improving the overall process efficiency. Environmentally friendly downstream processing strategies, including solvent-free recovery, supercritical CO₂ extraction, and membrane-based separation, represent potential approaches for improving the sclareol recovery efficiency and reducing the environmental burden associated with downstream processing. However, their economic feasibility and performance at an industrial scale require further validation.

Emerging carbon utilization strategies, such as microbial electrosynthesis, provide a promising route for producing value-added chemicals from CO₂-derived intermediates. , Notably, a two-stage microbial electrosynthesis–yeast cultivation process has been demonstrated for sclareol production from CO₂-derived acetate, yielding 2.54 ± 0.91 mg/L sclareol, although substantial optimization will be required for practical application. Although still at an early stage of development, such approaches highlight the potential for integrating renewable electricity with microbial cell factories, offering a potential pathway toward more sustainable and carbon-efficient sclareol production in the future.

Despite these advances, several technological and economic barriers remain. On the basis of techno-economic analyses of microbial terpene production, achieving sufficiently high titers, yields, and productivities will be critical for microbial sclareol production to offset fermentation and downstream processing costs. In addition, production systems must meet regulatory requirements for bio-based products and maintain genetic and metabolic stability during long-term industrial fermentation. Addressing these challenges will require coordinated advances in synthetic biology, metabolic engineering, and bioprocess optimization. Nevertheless, continued improvements in pathway design, chassis engineering, and intelligent process control could further advance microbial sclareol production toward industrial applicability. However, comprehensive techno-economic analyses, life-cycle assessments, and large-scale fermentation validations are still required to determine whether microbial production can achieve cost competitiveness and sustainability advantages compared with traditional plant-based supply.

5. Conclusion and Future Perspectives

Recently, synthetic biology has enabled significant progress in sclareol production. Plant-derived biosynthetic pathways have been successfully reconstructed in microbial hosts, particularly yeast systems such as S. cerevisiae and Y. lipolytica, enabling de novo sclareol biosynthesis from simple carbon sources. Optimization of the endogenous MVA pathway in engineered microbial hosts has enhanced the precursor supply in sclareol-producing systems, while hybrid MVA-MEP strategies represent transferable approaches with potential for further improvement. Meanwhile, enzyme engineering through directed evolution and rational design has improved the activity and specificity of terpene biosynthetic enzymes in related terpene systems, providing transferable strategies for the engineering of sclareol-forming enzymes. In addition, modular pathway design has improved flux control in sclareol-producing systems, while synthetic scaffolding and dynamic regulatory strategies provide transferable and prospective tools for further pathway optimization. These engineering strategies have the potential to reduce the metabolic burden and improve production performance in engineered microbial systems. High-throughput screening platforms, multiomics-guided modeling, and smart fermentation technologies have accelerated the development of related microbial production systems and provide promising tools for future sclareol strain development and process optimization.

Despite these advances, several challenges remain. Future efforts should focus on developing robust chassis cells with enhanced stress tolerance, improved carbon utilization efficiency, and compatibility with low-cost feedstocks. In particular, longevity and metabolic robustness engineering has emerged as a promising sclareol-specific strategy for improving cellular robustness during high-density fermentation. By integrating the regulation of cellular aging, nutrient-sensing pathways, and organelle quality control mechanisms, future chassis optimization may achieve enhanced cellular longevity, sustained metabolic activity, and improved tolerance toward toxic terpenoid accumulation. The integration of AI-driven pathway design with adaptive dynamic control systems may provide valuable tools for fine-tuning metabolic networks under industrial conditions. Expanding the synthetic biology toolkitincluding orthogonal regulators, programmable promoters, and synthetic organellesmay further enable precise pathway engineering. In parallel, green downstream processing technologies, such as solvent-free or low-energy extraction methods, should be further developed and evaluated for their feasibility in sclareol recovery. Collectively, continued innovation across synthetic biology, bioprocess engineering, and data-driven optimization may allow microbial sclareol production to become a scalable alternative to traditional plant-based supply. Nevertheless, comprehensive techno-economic and life-cycle evaluation will be essential before its economic competitiveness and environmental-sustainability benefits can be confirmed.

Glossary

Abbreviations

Acetyl-CoA

Acetyl coenzyme A

AI

Artificial intelligence

Cas

CRISPR-associated protein

CPP

Copalyl diphosphate

CRISPR

Clustered Regularly Interspaced Short Palindromic Repeats

CRISPRa

CRISPR activation

CRISPR-Cas

CRISPR–Cas system

CRISPR-Cas9

CRISPR-associated protein 9

CRISPRi

CRISPR interference

diTPSs

Diterpene synthases

DNA

Deoxyribonucleic acid

DMAPP

Dimethylallyl diphosphate

DSRS

Dynamic sensor–regulator system

FACS

Fluorescence-activated cell sorting

G1

Gap 1 phase

GC-MS

Gas chromatography–mass spectrometry

GEMs

Genome-scale metabolic models

GGPP

Geranylgeranyl diphosphate

GGPPS

Geranylgeranyl diphosphate synthase

HCT-116

Human colon cancer cell line 116

HTS

High-throughput screening

IPP

Isopentenyl diphosphate

MEP

Methylerythritol phosphate

MVA

Mevalonate

NADPH

Nicotinamide adenine dinucleotide phosphates

P450s

Cytochrome P450 monooxygenase

PARP

Poly­(ADP-ribose) polymerase

SC–CO₂

Supercritical carbon dioxide

SsLPPS

S. sclarea labda-13-en-8-ol diphosphate synthase

SsSS

S. sclarea sclareol synthase

Y.Y.: Conceptualization, methodology, formal analysis, data curation, writingoriginal draft. L.W.: Visualization, investigation, writingoriginal draft, formal analysis. Y.Z.: Methodology, formal analysis. R.Z.: Investigation, writingreview and editing. A.A.S.: Conceptualization, writingreview and editing. C.G.: Conceptualization, supervision, funding acquisition, writingreview and editing. All authors read and approved the final manuscript.

The authors declare no competing financial interest.