Baradwaj Ravi Gopal
Zhen Q. Wang- Department of Biological Sciences, University at Buffalo, The State University of New York, Buffalo, NY, United States
Carotenoids and apocarotenoids constitute a structurally and functionally sundry class of isoprenoids whose significance extends from photosynthetic light capture and photoprotection to phytohormone signaling, flavor and aroma formation, and emerging biomedical applications. While recent appraisals have emphasized quantitative advances in microbial production, this mini-review adopts a pathway module-centric perspective. We examine each biosynthetic stage from precursor supply, condensation to geranylgeranyl diphosphate (GGPP), phytoene synthesis, desaturation/isomerization, cyclization, hydroxylation, ketolation, epoxidation, and oxidative cleavage, highlighting novel enzymatic variants, mutagenesis studies, fusion strategies, and compartmentalization approaches that impart metabolic control. Special emphasis is placed on recently discovered and engineered enzymes, as well as synthetic biology tools. This review integrates diverse enzyme sources, host ranges across plants, fungi, algae, yeasts, and bacteria, as well as pathway modularity, to provide an updated review of recent literature. We conclude by outlining future directions that highlight gaps and potential areas for future work. This focused synthesis aims to equip researchers with a hierarchical understanding of the pathways and strategies to advance carotenoid and apocarotenoid biosynthesis.
1 Introduction
Carotenoids are one of the most diverse and versatile classes of isoprenoids in nature (Gómez-Sagasti et al., 2023). Beyond their roles in photosynthesis, carotenoids contribute to the vivid pigmentation in diverse organisms and serve as precursors to various bioactive apocarotenoids (Nisar et al., 2015; Sun et al., 2018). This duality of structural pigments and bioactive precursors has made carotenoids a key focus of both basic and applied research.
From a commercial standpoint, carotenoids and their oxidative derivatives occupy an exceptionally broad spectrum beyond pigments and perfumes. In nutrition and pharmaceuticals, β-carotene serves as a provitamin A source (Hermanns et al., 2020), lutein and zeaxanthin support eye health (Mrowicka et al., 2022), and apocarotenoids such as crocetin and bixin exhibit antioxidant and anti-inflammatory properties (Imtiaz et al., 2023). In the cosmetics and food industries, carotenoids such as lycopene and astaxanthin are widely used as natural colorants and anti-aging ingredients (Hermanns et al., 2020). In agriculture, apocarotenoids, such as strigolactones and abscisic acid, function as signaling molecules that regulate plant growth and stress responses (Simkin, 2021; Imtiaz et al., 2023).
To meet rising demand, microbial platforms now enable the scalable and sustainable production of high-value carotenoids, such as lycopene, β-carotene, zeaxanthin, and astaxanthin (Jing et al., 2022; Promdonkoy et al., 2024). These systems offer precise control over metabolic flux, avoid the seasonal and environmental limitations of plant cultivation, and allow cost-effective bioprocessing in industrial fermenters. Additionally, apocarotenoids, such as picrocrocin, strigolactones, and vitamin A (retinol), hold immense industrial relevance and are currently being targeted for microbial production (Varghese et al., 2023).
Building upon this industrial momentum, it becomes essential to understand the biochemical logic that generates such structural and functional diversity. This diversity stems from a series of biosynthetic transformations, including condensation, desaturation, isomerization, cyclization, hydroxylation, ketolation, and epoxidation. Beyond these modifications, the oxidative cleavage of specific carotenoids by carotenoid cleavage dioxygenases (CCDs) represents a crucial branching point that gives rise to apocarotenoids. Figure 1 summarizes carotenoid and apocarotenoid biosynthetic modules from precursor supply to late-stage tailoring (panels a–g) and distinguishes native reactions (solid black lines) from heterologous/engineered steps (dotted colored lines). Additionally, we review genetic approaches, enzyme sources, host platforms, and outcomes in recent literature, while highlighting emerging trends, persistent bottlenecks, and future directions that bridge fundamental understanding with applied biotechnology, moving beyond pigments and perfumes.
Figure 1. Overview of recent works on carotenoid and apocarotenoid biosynthesis. (a) IPP and DMAPP Pathway Engineering, (b) Initial Condensation Steps, (c) Desaturation and Isomerization, (d) Cyclization Reactions, (e) Hydroxylation Reactions, (f) Ketolation Reactions, (g) Epoxidation and Isomerization. (Reactions: Solid black line - native reactions, Dotted colored lines - heterologous reactions. Abbreviations: IP, isopentenyl monophosphate; ISO, isopentenol; IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; GPP, geranyl diphosphate; FPP, farnesyl diphosphate; GGPP, geranylgeranyl diphosphate).
2 Engineering strategies by biosynthetic steps
2.1 Precursor engineering for carotenoid and apocarotenoid production
Precursor engineering defines the fluxes of carotenoid and apocarotenoid pathways that can be channeled to the final products in any host. Engineering the upstream supply of C5 isoprenoid precursors, isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), is essential for boosting carotenoid and apocarotenoid yield. The two main biosynthetic routes, the methylerythritol (MEP) and the mevalonate (MVA) pathways, have been extensively engineered in various organisms. The following studies illustrate the control points and how relieving them increases carotenoid precursor levels.
Khana et al. analyzed the bottlenecks of the MEP pathway in the bacterium Zymomonas mobilis. They identified 1-deoxy-D-xylulose 5-phosphate synthase (dxs) as the first major bottleneck, and 4-hydroxy-3-methylbut-2-enyl diphosphate (HMBDP) synthase (ispG) and HMBDP reductase (ispH) as the subsequent bottlenecks. Overexpressing dxs, ispG, and ispH alleviated these blocks, while the introduction of a heterologous isoprene synthase provided an effective carbon sink to channel flux (Khana et al., 2023). Similar bottlenecks were identified in the Escherichia coli MEP pathway. Raghavan et al. showed that dxs, ispG, and ispH constrain flux through the MEP pathway. Chromosomal integration of the whole MEP pathway genes significantly increased the IPP/DMAPP supply compared with episomal expression (Raghavan et al., 2024).
Extensive MVA pathway engineering has been conducted in Saccharomyces cerevisiae in recent years. Mukherjee et al. targeted the MVA pathway to both the cytosol and peroxisomes, resulting in a 94-fold increase in the titer of monoterpene geraniol (Mukherjee et al., 2022). Additionally, they revealed that mevalonate kinase (ERG12) is another bottleneck, along with HMG-CoA reductase (HmgR) and isopentenyl-diphosphate isomerase (IDI). Yanagibashi et al. targeted the mevalonate (MVA) pathway to mitochondria, where the substrate acetyl-CoA is abundant, resulting in a 15-fold increase in IPP and DMAPP. Furthermore, mutants with enlarged mitochondria showed a 1.3-fold increase in IPP/DMAPP and terpenoids, including squalene and β-carotene, which were increased by 2.8- and 1.4-fold, respectively (Yanagibashi et al., 2024).
Beyond the MEP and the MVA pathways, the isopentenol utilization pathway (IUP) presents a robust alternative to boost IPP/DMAPP production. In Chlamydomonas reinhardtii, a choline kinase from S. cerevisiae (ScCK) converts isoprenol to isopentenyl monophosphate (IP), followed by isopentenyl phosphate kinase from Arabidopsis thaliana (AtIPK) converting IP to IPP. Isoprenol feeding increased IPP 8.6-fold and limonene production 23-fold (Zhao et al., 2022). Kinase-driven phosphorylation of C5 alcohols (isopentenol and prenol) bypasses native regulation to directly supply IPP and DMAPP. Ma et al. (2023) combined IUP with the native MVA pathway in S. cerevisiae, creating a universal IPP/DMAPP platform for mono-, di-, and tetraterpene synthesis. Other microbial studies optimized IUP modules, engineered better kinases, and extended the pathway to E. coli and non-conventional hosts like Yarrowia lipolytica (Ma X. et al., 2022; Zhao et al., 2022; Ma et al., 2023; Pan et al., 2023; Roth and Ward, 2023; Li G. et al., 2024).
Together, these approaches demonstrate that precursor engineering lays the groundwork for subsequent carotenoid pathway optimizations, as summarized in Figure 1a.
2.2 Initial condensation steps—GGPP and phytoene synthesis
Iterative condensation of IPP and DMAPP yields C20 geranylgeranyl diphosphate (GGPP); two GGPP molecules then condense to form C40 phytoene, the first committed step in carotenoid biosynthesis. Expressing bacterial GGPP synthase (GGPPS) crtE and phytoene synthase crtB, along with downstream enzymes, enabled zeaxanthin production in Yarrowia lipolytica at 21.98 mg/L (Xie et al., 2021). Additionally, bifunctional enzymes CarRP (phytoene synthase + lycopene cyclase) and phytoene dehydrogenase CarB from the fungus M. circinelloides efficiently convert GGPP to lycopene (Xie et al., 2021).
Recent studies identified novel GGPPS enzymes from higher plants Liriodendron tulipifera and Withania somnifera, oleaginous yeast Rhodosporidium toruloides, and bacterium Elizabethkingia meningoseptica, expanding the GGPP biosynthesis toolbox (Zhang et al., 2021; Satta et al., 2022; Srivastava et al., 2022; Yang et al., 2022; Song et al., 2023; Adusumilli et al., 2024). A pentuple mutant of NtGGPPS from Nicotiana tabacum showed enhanced catalytic efficiency and carotenoid output (Dong et al., 2022).
Recent studies also expanded the potential phytoene synthases (PSY) that could be employed for metabolic engineering. In tomato, fruit-specific expression of the autumn olive PSY (EutPSY), combined with the suppression of lycopene ε-cyclase (SlLCYe), enhanced the accumulation of lycopene and β-carotene (Wang et al., 2020). In the green alga Dunaliella salina, DsPSY1 was confirmed to be catalytically active, and its stabilization by the Orange (OR) protein boosted carotenoid levels (Liang M. H. et al., 2023). Expression of tea PSY1 (CsPSY1) in carrot callus increased α- and β-carotene to about 400 and 1250 μg/g dry weight, respectively (Li J. W. et al., 2024). In the archaeon Haloferax volcanii, the phytoene synthase (HVO-PSY) was identified as the rate-limiting step for bacterioruberin (C50 carotenoid) biosynthesis (Lin et al., 2025). Finally, in Pyropia yezoensis, the first bona fide phytoene synthase (PyPSY) from a red alga was functionally validated (Li C. L. et al., 2024).
Fusion of GGPPS with PSY also demonstrated promise. The GGPS11-PSY chimera (PYGG) in Arabidopsis thaliana allowed efficient GGPP channeling to phytoene. Despite a lower turnover rate, this fusion protein showed high substrate utilization and minimal GGPP leakage (Camagna et al., 2019). This strategy may be extendable to other GGPPS and PSY enzymes.
The initial condensation steps determine how effectively the C5 precursor carbons can commit to carotenoid backbones, as depicted in Figure 1b, which connects IPP/DMAPP condensation to phytoene formation.
2.3 Desaturation and isomerization—phytoene to lycopene conversion
Desaturation and isomerization of phytoene to lycopene are key bottlenecks in carotenoid biosynthesis. Engineering across diverse hosts has utilized both one-step bacterial or fungal enzymes, such as CrtI, and multi-enzyme plant modules including the phytoene desaturase (PDS), the 15-cis-ζ-carotene isomerase (ZISO), the ζ-carotene desaturase (ZDS), and the carotenoid isomerase (CRTISO).
Notably, CrtI from bacterium Pantoea ananatis and red-pigmented yeast Xanthophyllomyces dendrorhous enables a streamlined, high-yield conversion of phytoene to lycopene in E. coli and S. cerevisiae, respectively, when codon-optimized and paired with FAD support (Yoon et al., 2007; Schaub et al., 2012; Hong et al., 2019). Diversified crtI homologs across evolutionary clades demonstrated broad variability in desaturation and could be used for bioengineering (Fournié and Truan, 2020). Integrative pathway designs, such as expression of heterologous Pantoea ananatis crtE, Pantoea agglomerans crtB, and Blakeslea trispora crtI, along with membrane efflux transporters, further enhance yield by relieving product inhibition in S. cerevisiae systems, resulting in a lycopene yield of 343.7 mg/L (Huang et al., 2024).
Plant-based strategies emphasize modular control, as demonstrated by overexpressing Lycium chinense PDS, ZDS, and CRTISO in Nicotiana tabacum, which increased total carotenoid content and enhanced stress tolerance (Li et al., 2020). Other recently identified plant-based phytoene desaturases and isomerases include PDS, ZISO, ZDS, and CRTISO from microalga Dunaliella salina (Chen et al., 2023).
In short, desaturation/isomerization is often the first place where tuning redox balance and enzyme expression have a substantial impact on pathway throughput, as shown in Figure 1c, which traces the phytoene-to-lycopene transition catalyzed by CrtI and plant PDS/ZDS modules.
2.4 Cyclization reactions—formation of α and β-carotene
Cyclization reactions are key steps in carotenoid biosynthesis, converting lycopene into cyclic carotenoids like β-carotene (two β-ionone rings) and α-carotene (one β- and one α-ionone ring). In plants, LCYB and LCYE catalyze these two reactions, respectively. In microbes, CrtY and CrtL-b form β-carotene, while CrtL-e with CrtL-b/CrtY yields α-carotene (Williamson, 1994; Cunningham Jr and Gantt, 2001). Modulating LCYB and LCYE expression via overexpression, mutagenesis, and promoter tuning regulates carotenoid flux partitioning. Overexpressing IbLCYB2 in sweet potato boosted β-carotene and stress tolerance (Kang et al., 2018), while bifunctional enzyme DbLCYB (lycopene β-cyclase and weak lycopene ε-cyclase activity) from Dunaliella bardawil enabled dual cyclization in E. coli (Liang et al., 2019). Ma Y. et al. (2022) demonstrated high-yield of β-carotene production in Yarrowia lipolytica through expression of the phytoene dehydrogenase (CarB) (Velayos et al., 2000a) and bifunctional phytoene synthase and lycopene cyclase carRP genes (Velayos et al., 2000b) from Mucor circinelloides. It involved overcoming CarRP’s lycopene substrate inhibition by introducing a specific point mutation, Y27R, within its lycopene cyclase domain, alleviating this feedback inhibition. Co-expressing CarB and X. dendrorhous GGPP synthase crtE further improved flux after relieving CarRP inhibition. The engineered strain produced 39.5 g/L β-carotene at a rate of 0.165 g/L/h, which was 1,441-fold higher than that of the parent, highlighting the industrial promise of fungal β-cyclization (Ma Y. et al., 2022).
Recent advances highlight the utility of other fungal systems for lycopene cyclization. In the fungus Trichoderma reesei, introduction of the Mucor circinelloides carB and the bifunctional carRP enabled concurrent cellulase production and β-carotene accumulation (Li et al., 2023). Similarly, in Yarrowia lipolytica, expression of crtE, crtB, crtI, and carRP, followed by the addition of bacterial β-carotene hydroxylase crtZ, achieved efficient β-cyclization and zeaxanthin formation (∼22 mg/L), demonstrating the usefulness of fungal β-cyclases in microbial platforms (Xie et al., 2021).
In Chlamydomonas reinhardtii, expressing crtY from the bacterium Pantoea agglomerans increased β-carotene more than the Dunaliella salina lycopene β-cyclase (DsLCYB1), showing bacterial cyclases outperform algal ones (Huang et al., 2023). Cross-kingdom enzyme substitution can enhance carotenoid yields in microalgae.
Functionally, this step is the main lever for steering α/β-branch allocation in both plants and engineered microbes, as illustrated in Figure 1d, showing the bifurcation of lycopene into α- and β-carotene via LCYE and LCYB.
2.5 Hydroxylation reactions—zeaxanthin, cryptoxanthin, and lutein formation
Hydroxylation increases polarity, converting carotenes into nutritionally and industrially relevant xanthophylls (e.g., zeaxanthin, cryptoxanthins, and lutein) and priming them for further tailoring steps (Cunningham Jr and Gantt, 1998; Cunningham Jr and Gantt, 2001). Key enzymes include non-heme, di-iron type β-carotene hydroxylase (CrtZ/BCH) and cytochrome P450s (CYP97 family), acting on β- or α-ionone rings (Tian et al., 2004; Kim and DellaPenna, 2006; Quinlan et al., 2012). The bacterial crtZ gene is preferred for microbial engineering. Enzyme localization, when targeting the bacterial Pantoea ananatis CrtZ to peroxisomes or the ER, increased titers, reaching up to 412 mg/L zeaxanthin in Y. lipolytica (Soldat et al., 2024). Engineering a β-carotene pathway using carRP and carB from Mucor circinelloides, along with optimized enzyme variants and enhancements to the MVA pathway, increased the supply of the precursor β-carotenoid. The crtZ gene from Pantoea ananatis enabled zeaxanthin synthesis from β-carotenoid. Further, co-expression of RFNR1 (ferredoxin–NADP+ reductase 1) improved hydroxylation via optimizing electron transfer. This modular strategy achieved a zeaxanthin titer of 775.3 mg/L, among the highest reported in Yarrowia lipolytica (Zhang et al., 2023).
CYP97 family members are known to catalyze the hydroxylation of carotenoid ionone rings. Engineering of CYP97H1 and CYP97A3/C1 by strategies such as codon optimization, truncation, cytochrome P450 reductase (CPR) screening, and co-expression enabled hydroxylation of the β-ring of β-carotene and β-/ε-rings of α-carotene, respectively, enabling the production of lutein, zeinoxanthin, α- and β-cryptoxanthin in E. coli (Niu et al., 2020; Lautier et al., 2023). Meanwhile, the recently identified CYP97B family enzymes from the diatom Phaeodactylum tricornutum and the red alga P. umbilicalis can also hydroxylate β-carotene to zeaxanthin (Yang et al., 2014; Cui et al., 2019). Hydroxylases from bacterium Chondromyces crocatus (CcBCH), higher plant Arabidopsis thaliana (CYP97A3, CYP97C1), and algae Euglena gracilis (CYP97H1), have also been engineered into E. coli, Y. lipolytica, and S. cerevisiae (Tian et al., 2004; Yang et al., 2014; Cui et al., 2019; Tamaki et al., 2019; Wang et al., 2023; Soldat et al., 2024).
Thus, hydroxylation is the step that converts pigment backbones into market-relevant xanthophylls and expands the palette for downstream chemistry, as detailed in Figure 1e, which highlights the CrtZ/BCH- and CYP97-mediated conversions to zeaxanthin, lutein, and cryptoxanthin.
2.6 Ketolation reactions—astaxanthin formation
Ketocarotenoids (astaxanthin, canthaxanthin, echinenone) have vivid coloration and exceptional antioxidant properties, which are central to aquaculture pigmentation, cosmetic applications, and nutraceutical/pharmaceutical uses. Ketolation modifies the ionone rings of β-carotene, yielding ketocarotenoids (Tran and Kaldenhoff, 2020). Engineering efforts focus on β-carotene ketolases (e.g., crtW and BKT) and β-hydroxylases (crtZ) to divert flux toward astaxanthin (Martín et al., 2008; Allen et al., 2022; Abdullah et al., 2025; Hou et al., 2025).
Bacterial CrtW enzymes, especially from Brevundimonas sp., showed robust ketolation activities in E. coli and yeast, with mutagenesis boosting astaxanthin titers to 81 mg/L in 5-L bioreactors (Ye et al., 2006; Wang R. et al., 2017). Co-expression of crtZ and crtW enabled 1.82 g/L astaxanthin production in fed-batch fermentation (Zhang et al., 2022). Expressing bacterium Pantoea agglomerans β-carotene hydroxylases (crtZ) and alga Haematococcus pluvialis β-carotene ketolases (HpBKT) in Synechocystis sp. PCC 6803 converted β-carotene to astaxanthin via echinenone and canthaxanthin. Balanced expression of ketolases and hydroxylases, achieved by tuning promoter strength and expression ratios to harmonize their catalytic activities, was essential for efficient flux (Liang H. et al., 2023).
Taken together, precise tuning between hydroxylases and ketolases is the crux of industrial astaxanthin pathways, as shown in Figure 1f, where balanced CrtW/BKT and CrtZ activities channel β-carotene toward astaxanthin.
2.7 Epoxidation and isomerization reactions—violaxanthin and downstream carotenoids
The zeaxanthin–antheraxanthin–violaxanthin route links pigment composition with photoprotection. Engineering this node can modulate light stress responses. Epoxidation and de-epoxidation reactions form the xanthophyll cycle, crucial for balancing photoprotection and carotenoid flux in plants and algae. The epoxidation of zeaxanthin to violaxanthin, catalyzed by zeaxanthin epoxidase (ZEP), is a critical step in the xanthophyll cycle, essential for responding to light stress and maintaining a balance of carotenoids.
Zeaxanthin epoxidase (ZEP) converts zeaxanthin to violaxanthin. In the microalga Nannochloropsis oceanica, NoZEP1/2 modulate violaxanthin pools and non-photochemical quenching (Liu et al., 2023). Expressing the alga Ulva prolifera ZEP in S. cerevisiae and Chlamydomonas reinhardtii improved salt tolerance, consistent with enhanced xanthophyll-cycle activity and increased violaxanthin formation (He et al., 2024). The engineering of violaxanthin de-epoxidase (VDE) remains limited, although strategies such as redox circuit optimization and balancing ZEP/VDE expression could be explored (Xinrui et al., 2023).
A review of violaxanthin biosynthesis and heterologous production in E. coli and yeast has been published (Takemura et al., 2021). Expressing bamboo VDE shifted the xanthophyll equilibrium toward zeaxanthin, lowering violaxanthin content, and enhanced quenching and stress resilience in Arabidopsis (Lou et al., 2022), while ZEP mutations (a single recessive splicing mutation) altered carotenoid balance in pepper (Capsicum annuum) (Lee et al., 2021). In E. coli, expressing the crtY, crtZ, and ZEP genes and optimizing redox balance resulted in violaxanthin production of 25.3 mg/g DCW (Xinrui et al., 2023). Expression of Capsicum annuum capsanthin/capsorubin synthase CsCCS yielded 6.77 mg/g capsanthin and 2.18 mg/g capsorubin from antheraxanthin and violaxanthin (Chen et al., 2025).
Hence, epoxidation engineering serves both physiology (light tolerance) and product diversification (epoxy- and keto-xanthophylls), as summarized in Figure 1g, which situates ZEP within the violaxanthin cycle.
2.8 Carotenoid cleavage enzymes in apocarotenoid biosynthesis
Cleavage defines the branching from pigments to signaling molecules. Carotenoid cleavage dioxygenases (CCDs) and 9-cis-epoxycarotenoid dioxygenases (NCEDs) generate apocarotenoids such as β-ionone and abscisic acid (ABA). CCDs and the specialized subfamily, NCEDs, are key enzymes in apocarotenoid biosynthesis, producing compounds like β-ionone, crocetin dialdehyde, and ABA. CCDs are non-heme iron-dependent enzymes that cleave carotenoid polyenes via a dioxygenase mechanism, forming keto or aldehyde cleavage products (Ahrazem et al., 2016; Wang et al., 2022). NCEDs, a CCD subfamily, specifically cleave 9-cis-epoxycarotenoids to xanthoxin—the direct ABA precursor—thereby linking carotenoid turnover to stress-responsive hormone biosynthesis (Daruwalla and Kiser, 2020). Overexpressing Oryza sativa OsNCED3 in Arabidopsis and rice increased ABA and stress tolerance (Hwang et al., 2010; Feng et al., 2024). Their catalytic mechanisms, active site architecture, and substrate specificity have been elucidated through structural and biochemical studies. CCD clade diversity and their bond-cleaving roles across carotenoids have been extensively reviewed (Harrison and Bugg, 2014; Ahrazem et al., 2016; Daruwalla and Kiser, 2020).
Plant CCD1 and CCD4 enzymes cleave β-carotene to produce β-ionone and other volatiles. Dendrobium officinale CCD1 forms β-ionone and pseudoionone from β-carotene and lycopene, respectively (Wang et al., 2022). Expressing Nicotiana tabacum NtCCD1-3 in yeast, with a K38A mutation and redox balancing, boosted β-ionone yields (Gong et al., 2022). In S. cerevisiae, precursor pool size and membrane access were key bottlenecks (López et al., 2020). Microbial hosts, such as Yarrowia lipolytica and Candida tropicalis, have been engineered for β-ionone production, yielding 358 mg/L and 400.5 mg/L, respectively, in shake flasks through enhanced carotenogenic flux and CCD1 expression (Lu et al., 2020; Xu et al., 2024). Enhancements include membrane anchoring, NADPH/ferredoxin boosting, and modular cassette design. CCDs paired with enoate reductases also enable chemo-enzymatic conversion to dihydro-β-ionone (Qi et al., 2022).
Additionally, CCD4 can cleave zeaxanthin to crocetin dialdehyde, initiating crocin biosynthesis. Gardenia jasminoides CCD4a exhibited efficient crocin formation, with an expanded product profile that included cleavage of β-carotene, lycopene, and β-apo-8′-carotenal, yielding diverse C17–C20 dialdehyde apocarotenoids (Zheng et al., 2022). A recent CCD-independent route in E. coli using an engineered CrtMLIKE, CrtN, and CrtP enzymes to produce crocetin dialdehyde, where CrtM catalyzes head-to-head condensation of geranylpyrophosphate (GPP) to C20-phytoene; CrtN mediates sequential desaturation to form 8,8′-diapo-carotenoids; and CrtP performs further oxidation leading to crocetin dialdehyde. This strategy directly converted GPP to a C20 intermediate, thereby bypassing the multi-step β-carotene- and CCD-dependent route, reducing enzyme load and yielding 1.13 mg/L of crocetin dialdehyde. (Lee et al., 2025).
Beyond these metabolites, carotenoid cleavage also drives the biosynthesis of strigolactone (SL), a class of apocarotenoid-derived phytohormones with crucial developmental and ecological functions. SLs originate from the sequential cleavage of 9-cis-β-carotene by CCD7 and CCD8, yielding carlactone, which is further oxidized by cytochrome P450s of the MAX1 (CYP711) family into carlactonoic acid and various canonical and non-canonical SLs (Yoneyama et al., 2009; Alder et al., 2012; Jia et al., 2018). These compounds regulate shoot branching and root architecture and mediate rhizosphere signaling, promoting arbuscular mycorrhizal symbiosis but also triggering germination of parasitic weeds such as Striga and Orobanche (Ito et al., 2017; López-Ráez et al., 2017). Recent work has advanced microbial reconstruction of SL biosynthesis. A peach (Prunus) CYP711A homolog was shown to catalyze multi-step oxidation to strigol, the first identified SL, and the entire pathway from xylose feedstock to strigol was built in an E. coli–yeast consortium, proving the feasibility of industrial-scale SL production (Wu et al., 2023).
Collectively, cleavage chemistry extends the pathway beyond pigments into hormones, aromas, and specialty ingredients, as shown by the side of the modules in Figures 1c–f, marking the transition from carotenoids to apocarotenoids.
A consolidated overview of all engineering strategies, associated host systems, yields, and fold improvements across Section 2 is presented in Table 1.
Table 1. Summary of metabolic engineering strategies for carotenoid and apocarotenoid biosynthesis across hosts, core-pathways, and tailoring modules.
3 Discussion
The engineering advances outlined above and summarized in Table 1 reveal how each biosynthetic module contributes to the overall efficiency and diversity of carotenoid and apocarotenoid production. The discussion that follows synthesizes these developments to highlight common design principles, challenges, and opportunities.
3.1 Current challenges in microbial carotenoid and apocarotenoid biosynthesis
The challenges limiting predictable, scalable carotenoid and apocarotenoid production stem from several interconnected biological constraints. Precursor limitations and imbalanced fluxes remain central issues, as even strengthened MVA and MEP pathways exhibit fluctuations tightly coupled to growth rate and cellular redox balance (Kang et al., 2016; Zhao et al., 2022; Raghavan et al., 2024). Redox imbalance caused by demanding enzymes, such as CrtI, can deplete reducing equivalents (NADPH/NADH) during sequential desaturation steps, introducing flux bottlenecks and metabolic stress. Regulatory limitations further constrain pathway performance because most systems rely on constitutive promoters; however, carotenoid synthesis requires dynamic control to prevent metabolic burden. Product specificity is strongly influenced by the balance of cyclases and tailoring enzymes LCYB/LCYE, CrtZ, BKT/CrtW, and ZEP, whose optimization is complicated by cofactor requirements and substrate competition. Spatial factors add another challenge, as unstable intermediates would diffuse or degrade without appropriate compartmentalization. Additionally, the field remains heavily centered on S. cerevisiae and E. coli, leaving algae, fungi, and other bacterial hosts underexplored despite their unique advantages. Finally, industrial implementation demands solutions for scale-up, especially low-cost feedstocks, robust high-stress sustaining strains, and efficient downstream processing tailored to highly hydrophobic carotenoids.
3.2 Strategies emerging from recent engineering advances
The engineering concepts discussed in Section 2 address the interconnected challenges above. Precursor balancing and redox management remain foundational. Strengthened MVA and MEP pathways increase IPP/DMAPP availability. However, optimal flux requires redox-balanced pathway design, the use of feedback-resistant enzyme variants, and machine-learning-guided promoter tuning. AlphaFold-enabled structural models now support rational redesign of PSY, a longstanding catalytic bottleneck.
Modular desaturation and isomerization strategies combine the robustness of bacterial CrtI with the regulatory precision of plant desaturation complexes (Schaub et al., 2012; Li et al., 2020). Dynamic control using inducible, quorum-sensing, or metabolite-responsive promoters (Wan et al., 2019; Stephens and Bentley, 2020) enables tunable desaturation while preventing redox collapse.
Cyclization precision is strongly governed by the LCYB/LCYE ratio, which determines the ratio of α-to β-carotene formation (Kang et al., 2018; Liang et al., 2019). Directed evolution and CRISPR-based genome editing provide powerful means for modulating cyclase specificity, especially in hosts with existing carotenoid machinery.
Hydroxylation, ketolation, and epoxidation efficiencies depend heavily on tailoring enzymes such as CrtZ, BKT/CrtW, and CYP97 homologs, whose activities remain constrained by cofactor requirements and kinetic limitations (Zhang et al., 2022; Lautier et al., 2023). ZEP engineering is still in its early stages, which requires improving catalytic efficiency and electron-transfer support (Liu et al., 2023; He et al., 2024).
Spatial engineering strategies address the instability of intermediates by leveraging organelle targeting, peroxisome engineering (Choi et al., 2022), lipid droplet remodeling (Lin et al., 2024), scaffold-based assemblies (Wang Y. et al., 2017; Kang et al., 2019), and emerging synthetic bacterial microcompartments (Yung, 2015; Moon et al., 2024) that enhance colocalization.
Tailoring apocarotenoid enzymes such as CCD1 and NCED unlocks higher-value apocarotenoids, including β-ionone and ABA. Specialized CCDs, such as CCD2, catalyze the formation of crocetin dialdehyde, a key intermediate in saffron crocins (López et al., 2020; Chen et al., 2025). Future opportunities include extremophile enzyme mining, intermediate stabilization, and engineered microbial consortia to distribute pathway burden.
3.3 Future perspectives
Drawing from these challenges and strategies, several future directions emerge. First, comprehensive kinetic and structural enzyme characterization remains essential, particularly for PSY, CRTISO, ε-hydroxylases, ZEP, and NCEDs, which represent key catalytic nodes that currently constrain carotenoid bioengineering. Second, system-level flux design integrating genome-scale metabolic models, proteomics, and AI-guided optimization will improve our ability to predict pathway performance and design balanced flux distributions that respond dynamically to metabolic demands (Blazeck and Alper, 2010; Mukherjee et al., 2022; Zhang and Wang, 2025). Third, diversification of host chassis, including algae, cyanobacteria, and non-model yeasts, offers untapped advantages in compartmentation, redox environment, and tolerance to metabolic stress (Santos-Merino et al., 2019). Fourth, dynamic pathway regulation using CRISPRi/a, optogenetics, and synthetic feedback loops presents a frontier for controlling flux, stress responses, and metabolite toxicity with temporal precision (Han et al., 2023). Finally, industrial translation depends on innovations in low-cost feedstocks, co-product strategies, strain robustness, and scalable downstream processing tailored to carotenoids and apocarotenoids (Su et al., 2023). Collectively, carotenoid and apocarotenoid engineering are moving towards precision synthetic biology, bridging enzyme discovery with rational design, expanding into novel hosts, and transforming proof-of-concept pathways into robust platforms for nutrition, pharmaceuticals, and sustainable agriculture.
Author contributions
BR: Conceptualization, Data curation, Formal Analysis, Investigation, Visualization, Writing – original draft, Writing – review and editing. ZW: Data curation, Formal Analysis, Funding acquisition, Investigation, Project administration, Resources, Validation, Writing – review and editing.
Funding
The author(s) declared that financial support was received for this work and/or its publication. Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R35GM150552 to Z.Q.W.
Conflict of interest
The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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The author(s) declared that generative AI was used in the creation of this manuscript. Generative AI was used to compress some sections of the initial draft.
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Keywords: apocarotenoids, biosynthesis, carotenoids, metabolic engineering, synthetic biology
Citation: Ravi Gopal B and Wang ZQ (2026) Beyond pigments and perfumes: engineering in the carotenoid and apocarotenoid spectrum, novel enzymes, and synthetic biology strategies. Front. Bioeng. Biotechnol. 13:1716709. doi: 10.3389/fbioe.2025.1716709
Received: 30 September 2025; Accepted: 25 December 2025;
Published: 15 January 2026.
Edited by:
Akaraphol Watcharawipas, Mahidol University, ThailandReviewed by:
Alice Stra, King Abdullah University of Science and Technology, Saudi ArabiaCopyright © 2026 Ravi Gopal and Wang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Zhen Q. Wang, emhlbndAYnVmZmFsby5lZHU=