Bioactivities and biosynthesis of monoterpene-based biopesticides: current state and perspectives

Abstract

Biopesticides constitute a category of natural products and organisms effective against pests. Given their advantages, such as easier clearance and degradation compared to chemical pesticides, along with their lack of detrimental effects on ecosystem health, they represent a promising alternative for sustainable pest management and crop protection. The development of natural biopesticides to substitute synthetic pesticides is therefore of paramount importance for securing food safety and advancing agricultural production. Monoterpenes, primarily present in plant volatile oils, are characterized by a strong aroma and a range of biological activities-including insecticidal, antimicrobial, antiviral, anti-inflammatory, antioxidant, antitumor, and anticancer effects. This profile renders them a promising platform for the research and development of new, more effective pesticides. This article reviews the biosynthetic pathways of monoterpenes and contemporary engineering strategies for their microbial synthesis. Additionally, an evolutionary analysis database for monoterpene synthases (MTPS) has been established. Finally, the biological activities of monoterpenes are summarized and analyzed, with particular emphasis on their potential and advantages as biopesticides, thereby providing direction for the future development of monoterpene-based biopesticides.

Graphical Abstract

Supplementary Information

The online version contains supplementary material available at 10.1186/s12934-026-02973-4.

Introduction

Ensuring global food security and boosting crop yields have become pressing challenges amid the continuous growth of the global population. Over the past few decades, pests and diseases have led to substantial losses of major crops worldwide, such as wheat, rice, maize, potatoes and soybeans, with the estimated loss rate ranging from 21% to 30% [1]. Currently, chemical pesticides remain the dominant measure for controlling agricultural pests and diseases. Nevertheless, the application of chemical pesticides not only reduces crop productivity but also raises production costs due to their high expense. Moreover, many chemical pesticides are difficult to decompose into simple and safe substances, and their residues tend to accumulate in soil and water [2], thereby posing severe threats to the ecological environment and human health [3]. In contrast, biopesticides, a category of natural products and organisms with effective pest-suppressive activity, are more easily degraded and removed, and they do not exert adverse effects on ecosystem health when compared with chemical pesticides [4]. These advantages make biopesticides a promising candidate for environmentally friendly pest management and crop protection [5]. Therefore, developing natural biopesticides to replace chemical pesticides is of great significance for safeguarding food safety and promoting sustainable agricultural development.

Monoterpenes constitute a major class of natural products, widely distributed in plants and other organisms. Structurally, they are derived from the condensation of two isoprene units and can be classified into four categories: acyclic, monocyclic, bicyclic, and tricyclic monoterpenes. Beyond their well-established applications in the flavor, fragrance, and cosmetics industries [6], monoterpenes exhibit a broad spectrum of biological activities. For instance, essential oils (EO) containing citronellol have shown notable fumigant and herbicidal properties in laboratory bioassays [7]. Specifically, citronellol has been employed as an effective fumigant against mosquitoes [7] and Aleyrodidae [8]. Limonene, another common monoterpene, has demonstrated fungicidal efficacy against various plant pathogens, including Pseudomonas fluorescens [9]. Additionally, citral has been reported to possess potent insecticidal activity against a wide range of pests and pathogens, such as crop insects, plant-pathogenic fungi, and nematodes [10]. Beyond its nematicidal activity, carvacrol also exerts antifungal effects against diverse microorganisms, including Phytophthora infestans [11] and Mycobacterium avium [12]. Moreover, multiple studies have suggested that certain monoterpenes and their derivatives, such as geraniol, thymol, borneol, camphor, and others, may play crucial roles in the prevention and treatment of various other diseases, including but not limited to cardiovascular diseases and diabetes mellitus. These compounds have been applied in multiple fields of medicine and pharmacy [13]. Overall, monoterpenes have been proven to exhibit beneficial effects in various aspects, including anti-inflammatory [14], anticancer [15], antimicrobial [16], insecticidal and herbicidal [17], and serve as important potential precursors for some clinical drugs and biopesticides. This offers a broad foundation for further research and development of novel, more effective pesticides.

Currently, monoterpenes are mainly extracted from plant biomass through conventional approaches. Although plant genetic engineering can improve monoterpene yield and thereby reduce production costs [18], its efficiency is inferior to other techniques, as geographic location and seasonal plant growth exert significant impacts on production performance [19]. Chemical synthesis is also applicable for monoterpene production, yet it tends to encounter drawbacks due to the complexity and inefficiency of process design, which leads to the generation of large amounts of environmentally harmful organic waste [20]. Compared with plant extraction and chemical synthesis, biosynthesis exhibits remarkable potential in natural terpenoid production [21]. This approach realizes heterologous reconstruction of the monoterpene biosynthetic pathway in microorganisms via metabolic engineering and synthetic biology, enabling high-titer and high-purity production of target compounds. It not only substantially shortens the production cycle [22] but also achieves full reuse of abundant recyclable resources [16]. For example, heterologous expression of high-copy-number genes significantly enhanced limonene production [23], engineered subcellular compartmentalization [24] and overexpression of certain efflux pumps (e.g., Escherichia coli acrAB) [25] also exerted positive effects on microbial synthesis of the target product. Additionally, a cell-free system with modular co-catalysis was constructed by combining an upstream modular E. coli strain (MEVI) and a downstream modular E. coli strain (PINE), which notably promoted pinene production [26]. However, the number of monoterpene species that can be biosynthesized currently remains limited, primarily attributed to the lack of efficient MTPS. Recently, studies have reported the successful achievement of efficient microbial production of nerolidol and borneol: nerolidol production was realized by heterologous expression of Glycine max-derived nerolidol synthase (GmNES) in E. coli, while borneol production was achieved via heterologous expression of truncated bisphosphate borneol synthase (LdtBPPS) from Lippia dulcis, followed by screening of LdtBPPS mutants to optimize production [27]. Therefore, the identification and modification of appropriate MTPS are critical strategies for the efficient biosynthesis of monoterpenes.

In this review, we reviewed the biosynthetic pathways of monoterpenes and the current engineering strategies for microbial biosynthesis of monoterpenes. Additionally, a database of MTPS is constructed with an evolutionary analysis. Finally, the biological activities of monoterpenes were summarized and analyzed, with particular attention paid to the biological activities and potential advantages of monoterpenes as biopesticides. This analysis served to improve the direction for the subsequent development of monoterpene biopesticides (Fig. 1).

Fig. 1.

Biological activities and structures of monoterpenes

The insecticidal and antimicrobial activity of monoterpene-based biopesticides

Monoterpenes, which are abundantly present in plant volatile oils, are known for their intense aroma and diverse biological activities. Their notable insecticidal and antibacterial properties position them as promising candidates for biopesticide development. This section systematically reviews the insecticidal and antibacterial activities of monoterpenes, as summarized in Table 1.

Table 1.

Structures and biological activities of different monoterpenes

Classification	Name	Structure	Insecticidal activity	Antimicrobial activity	References
Acyclic monoterpenes	Citral		T. cruzi, C. fasciculata	S. aureus, S. anisopliae, S. agalactiae, B. cereus and E. coli, C. sakazakii	[38, 83–85]
	Geraniol		C. maculatus, C. pusillus, R. dominica, S. oryzae, S. zeamais, T. castaneum, S. frugiperda	S. aureus, H. influenzae, S. pneumoniae, S. pyogenes, C. albicans, C. neoformans, H. pylori	[28, 29, 40, 41, 86]
	Linalool		A. aegypti, C. quinquefasciatus (Say) second-instar larvae, M. dirhodum (Walker) adults, S. littoralis (Boisduval) second-instar larvae and T. urticae (Koch) adults	S. putrefaciens, A. flavus, A. tumefaciens and E. carotovora	[43, 44, 87, 88]
	Citronellol		A. aegypti, aphids and mites	S. aureus, E. coli, B. cinerea, Fusarium spp.	[31, 32, 89]
	Myrcene		Myzus persicae, A. aegypti, S. frugiperda	S. aureus, F. graminearum PH-1 and F. culmorum FcUK 99	[34, 90–92]
	Ocimene		T. castaneum and L. serricorne, E. obliqua, A. tristis	S. aureus, E. coli, B. cinerea and Fusarium spp.	[93–98]
	Nerol		Trichoplusia ni	Broad-spectrum antimicrobial activity (e.g., E. coli and S. aureus)	[30, 37]
Monocyclic monoterpenes	Limonene		C. maculatus, A. albopictus, C. quinquefasciatus, M. domestica and C. elegans	P. fluorescens and S. aureus	[49, 66, 99, 100]
	γ-Terpinene		S. littoralis, A. stephensi at al.	E. coli, P. aeruginosa, S. typhimurium, P. mirabilis, L. monocytogenes, B. cereus, M. luteus, S. aureus, A. flavus, A. fumigatus, A. brasiliensis, (A) ochraceus, P. soppii, P. Ochrochloron, T. viride, M. albican, H. armigera (B) cinerea, H. armigera	[101–104]
	Carvacrol		E. kuehniella, P. interpunctella, A. obtectus, M. javanica	E. coli, C. perfringens, Salmonella spp and Lactobacillus spp, A. niger, A. flavus, Candida spp., Streptomyces spp., A. glabrata, P. erythrorhizonticum and T. versicolor, C. acutatum, C. fragariae and C. glabrum	[47, 51, 105, 106]
	Menthol		C. elegans, D. suzukii, Cephalopina spp	Cephalosporium, S. greyi, M. fructicola, B. cinerea, A. niger, P. expansum and R. stolonifer	[54, 107–111]
	1,8-Cineole		T. castaneum	S. aureus, methicillin-resistant S. aureus, E. coli, K. pneumoniae, E. faecalis and C. albicans	[55, 63, 112]
	Phellandrene		storage pests and A. aegypti	L. plantarum	[29, 93, 113]
	α-Terpineol		P. xylostella, P. americana, Camponotus pennsylvanicus De Geer, B. germanica, H. armigera, S. invicta, F. occidentalis, F. insularis	E. coli, S. typhimurium, L. monocytogenes and S. aureus, P. digitatum	[57, 114–120]
	Thymol		P. shantungensis, S. invicta, S. richteri, S. litura, S. exigua, T. cinnabarinus	S. aureus, E. coli, S. pyogenes and S. typhimurium	[52, 53, 56], 121– [123]
	p-Cymene		T. castaneum, L. serricorne, L. bostrychophila	broad-spectrum antimicrobial and anti-biofilm activity	[93, 124–126]
Bicyclic monoterpenes	α-Pinene		H. dromedarii, L. bostrychophila, S. oryzae, C. maculatus, C. quinquefasciatus, D. melanogaster, T. palmi, S. zeamais	methicillin-resistant S. aureus, S. aureus, E. faecalis, E. coli, K. pneumoniae, S. maize	[35, 69, 70], 72– [76, 127]
	β-Pinene		P. interpunctella, mosquitoes, houseflies, and whiteflies, P. palmivora, T. castaneum	B. subtilis, S. aureus, E. coli and P. aeruginosa, B. cereus, C. albicans	[128–132]
	Fenchol		A. aegypti	Gram-positive bacteria, Gram-negative bacteria (e.g., C. albicans)	[133–135]
	Camphor		T. castaneum and L. serricorne, S. littoralis, C. capitata, L. decemlineata, P. interpunctella, S. cerealella and T. putrescentiae	E. coli, Phytophthora nicotianae, Fusarium spp, and Trametes versicolor	[136–143]
	Borneol		stored-product pests, mosquitoes, aphids	E. coli, S. aureus,, Alternaria panax	[144–148]
	Sabinene		T. confusum, S. zeamais, A. pomorum	P. aeruginosa, S. aureus	[77, 78, 149, 150]

Acyclic monoterpenes

Acyclic monoterpene-based biopesticides mainly include citral, geraniol, linalool, citronellol, myrcene, ocimene and nerol. Laboratory bioassays have demonstrated that they all exhibit notable activities against various crop pests, phytopathogenic fungi, and nematodes. The insecticidal mechanisms mainly via disruption of neural function or metabolic pathway. For example, geraniol inhibits acetylcholinesterase in insects such as Callosobruchus maculatus, Cryptolestes pusillus, Rhyzopertha dominica, Sitophilus oryzae, and Sitophilus zeamais [28]. Geraniol also can reduce viability of Spodoptera frugiperda by altering egg morphology and chorion structure even at low concentrations [29]. Nerol have been shown to enhance efficacy against Trichoplusia ni by increasing cell membrane permeability [30]. Citronellol exhibits significant repellent, toxic, and growth-inhibiting properties against insects primarily by disrupting the nervous system, impairing cell membrane integrity, and inhibiting respiratory metabolism [31]. For example, it demonstrates notable repellent activity against dipterans such as mosquitoes and flies by interfering with olfactory reception and host-seeking behavior [31], along with larvicidal activity against A. aegypti [32]. Furthermore, the insecticidal mechanisms of myrcene include acting on transient receptor potential (TRP) channels, inhibiting acetylcholinesterase (AChE) and Na⁺/K⁺-ATPase activity [33, 34]. In the context of A. aegypti larvae, this phenomenon has been observed to induce reactive oxygen species (ROS) accumulation, resulting in tissue damage and a subsequent decrease in trypsin-like activity [35].

Beyond insecticidal effects, they all possesses broad-spectrum antibacterial activity against both Gram-positive (e.g., S. aureus) and Gram-negative bacteria (e.g., E. coli) through disruption of microbial membrane integrity or interference with essential enzymatic processes [36, 37]. For example, citral could impair cellular activity of Cronobacter sakazakii by disrupting its cell membrane, including decreasing intracellular ATP, reducing internal pH, and membrane hyperpolarization [38]. Notably, citral combined with other natural compounds or antibiotics could enhance its efficacy [39], which suggests a promising strategy for combating antibiotic-resistant bacteria and developing biopesticides with reduced resistance risk. As a key antifungal component in palm oil, geraniol is active against Candida albicans by inhibiting its biofilm formation, and suppresses growth of Cryptococcus neoformans [40, 41]. Combined with conventional antibiotics, geraniol may enhance therapeutic potential against infections caused by S. aureus, E. coli, and Helicobacter pylori [42]. In addition, antimicrobial studies indicate that linalool is effective against Streptococcus mutans (MIC = 1.5 µL/mL), causing membrane depolarization, leakage of alkaline phosphatase, and release of cellular components such as DNA, RNA, and proteins [43]. Furthermore, EO of Pimenta dioica, in which linalool is a major constituent, inhibits mycelial growth of Aspergillus flavus [44]. Currently, some monoterpene-based biopesticides performed better, for example, the larvicidal activity against mosquito larvae of myrcene is surpassing some inorganic phosphate insecticides [45]. Some ocimene-based formulations show higher efficacy (LC₅₀ < 4.5 µg/mL) than commercial insecticides like pymetrozine and flonicamid [46]. Additionally, citronellol suppresses weed seed germination and seedling growth, indicating herbicidal potential [47, 48]. These multifunctional properties support its application as an effective biopesticide in organic and ecological agricultural systems.

Monocyclic monoterpenes

Monocyclic monoterpenes includes limonene, γ-terpinene, carvacrol, menthol, 1,8-cineole, phellandrene, α-terpineol, thymol and p-cymene, exhibits insecticidal, antimicrobial, and antiseptic properties, highlighting its potential for agricultural applications. For example, essential oils rich in D-limonene, such as that from Protium heptaphyllum, demonstrate toxicity, strong repellency, and ovicidal activity against pests like C. maculatus [49]. Under laboratory conditions, carvacrol exhibits pronounced nematicidal activity (250–1000 ppm) against different life stages of Meloidogyne javanica [47]. It also acts as an effective mosquito repellent, outperforming DEET, while reducing egg hatchability and inducing sterility [50]. Owing to its favorable antimicrobial properties, carvacrol is widely used as a natural food preservative [51]. The mixture of some monoterpenes can exert a synergistic insecticidal effect. Synergistic insecticidal effects against Spodoptera exigua are observed when thymol is combined with extracts from Piper ribesioides [52], and the acaricidal activity against Tetranychus cinnabarinus is enhanced in mixtures with carvacrol or linalool [53].

Similar to the insecticidal mechanisms of acyclic monoterpenes, menthol acts as an insecticide by rapidly penetrating the cuticular wax layer, disrupting the body surface membrane, and interfering with the nervous system, ultimately leading to pest mortality [54]. 1,8-Cineole inhibits stored-product pests through multiple mechanisms, such as disrupting neurotransmitter transmission, altering neural membrane permeability, and inhibiting detoxification enzymes such as cytochrome P450 [55]. Against Spodoptera litura, thymol exhibits high toxicity (24-h LC₅₀ = 5.61 µg/larva) and significantly suppresses key detoxification enzymes, including carboxylesterase and cytochrome P450 [56]. α-Terpineol acts as a repellent and growth inhibitor against pests such as Periplaneta americana, Camponotus pennsylvanicus and Blattella germanica, likely through modulation of the insect octopaminergic system [57]. Phellandrene insecticidal potency is notable, reaching up to 90% lethality against certain pests at 1.0 mg/L, significantly exceeding that of evodiamine (10%), matrine (45%), and rotenone (30%) [58]. This activity may be mediated via activation of insect ryanodine receptors (RyRs), leading to elevated intracellular Ca²⁺ levels [58, 59].

Beyond insecticidal activity, 1,8-cineole shows broad-spectrum antimicrobial effects by increasing bacterial surface charge, enhancing membrane permeability, inducing leakage of cellular contents, and promoting ROS production [60]. Activity spans Gram-positive and Gram-negative bacteria as well as yeasts [61], with Gram-negative bacteria often showing greater susceptibility, likely due to enhanced lipopolysaccharide efflux and cytoplasmic membrane disruption [62]. Synergy with chlorhexidine gluconate has been observed against pathogens including S. aureus, methicillin-resistant S. aureus, E. coli, Klebsiella pneumoniae, Enterococcus faecalis and C. albicans [63]. Although generally regarded as safe and hypoallergenic [64], most evidence derives from in vitro and animal studies; further clinical research is needed to translate its antimicrobial potential into practical applications. Thymol inhibits bacterial (e.g., E. coli) growth by disrupting cytoplasmic membrane permeability and inducing depolarization [65]. Limonene exerts bactericidal effects against pathogens such as P. fluorescens and S. aureus by disrupting the morphology of cell and the integrity of cell wall, and by interfering with the TCA cycle and EMP pathway [66]. These broad and potent activities position limonene as a promising active ingredient for eco-friendly insecticides and fungicides. Moreover, carvacrol inhibits the replication of several viruses, such as human rotavirus, acyclovir‑resistant herpes simplex virus type 1, human respiratory syncytial virus, and pandemic H1N1 influenza virus [67, 68].

Bicyclic monoterpenes

Bicyclic monoterpenes includes α-pinene, β-pinene, fenchol, camphor, borneol and sabinene. They have extensive insecticidal activities. In fumigation assays, α-pinene demonstrates toxicity against a range of arthropods, including ticks (e.g., Hyalomma dromedarii) [69], stored-product insects (e.g., L. bostrychophila; LC₅₀ = 1.4 mg/L air) [70], S. oryzae and C. maculatus [35], as well as vectors like C. quinquefasciatus, Drosophila melanogaster, and the crop pest Thrips palmi [71]. Additionally, the antibacterial activities contain gram-positive and gram-negative bacteria, including methicillin-resistant S. aureus (MRSA) [72], and common pathogens such as E. coli, K. pneumoniae, S. aureus, and E. faecalis [73–76]. The insecticidal and antibacterial mechanisms of this type of compound are consistent with those of above monoterpene compounds. For example, α-Pinene exerts its bioactivity primarily by inhibiting key enzymes in pests, such as AChE and Na⁺/K⁺-ATPase [34]. Studies indicate that sabinene can inhibit the growth of pathogens such as P. aeruginosa [77] and S. aureus [78], of which mechanisms include disruption of bacterial cell wall synthesis, damage to cell membrane integrity, inhibition of protein synthesis, and interference with nucleic acid synthesis, collectively impairing normal bacterial growth and reproduction [77, 78].

Tricyclic monoterpenes

Tricyclic monoterpenes constitute a structurally distinct and relatively rare class of monoterpenoid compounds [79]. The fundamental skeleton of these compounds consists of ten carbon atoms, meeting the definition of a monoterpene and arranged into three fused rings. The formation of three rings from a fixed number of carbon atoms typically results in a structure characterized by significant strain [79], leading to a much lower prevalence of naturally occurring species compared to monocyclic and bicyclic monoterpenes [6, 19]. A review of the extant literature reveals an absence of explicit descriptions of typical tricyclic monoterpene structures. Tricyclic arrangements are more commonly observed in higher-carbon terpenoids, such as diterpenes and triterpenes [80, 81]. In the event that tricyclic monoterpenes are present, they may be indicative of rare derivatives formed through specialized cyclization reactions. However, specific examples remain unreported in the extant literature. The artificial construction of tricyclic monoterpene analogues is a potential outcome of synthetic biology or chemical modification [19, 82].

Biosynthesis of monoterpene-based biopesticides

Biosynthetic pathways

As with other terpenes, the precursors of monoterpenes are isopentenyl pyrophosphate (IPP) and diallyl pyrophosphate (DMAPP), which are biosynthesized primarily through the mevalonate (MVA) pathway, the methylenepentylate (MEP) pathway [151] and engineered synthetic route IPA/IUP pathway (Fig. 2).

Fig. 2.

Biosynthetic pathway of monoterpene biosynthesis. Abbreviation of metabolites: HMG-CoA,3-hydroxy-3-methylglutaryl-CoA; MVA, mevalonate; MVA-P, 5-phosphomevalonate; MVA-PP, 5-diphosphomevalonate; MG-CoA, 3-methyl-glutaryl-CoA; DMA-CoA, 3-methyl-2-butenoyl-CoA; DMAP, dimethylaminopyridine; IPP, isoprene diphosphate; DMAPP, dimethylallyl diphosphate; FPP, farnesyl diphosphate; G3P, glyceraldehyde-3-phosphate; DXP, 1-deoxy-D-xylulose 5-phosphate; MEP, 2-C-Methyl-d-erythritol-4-phosphate; CDP-ME, 4-(cytidine-5′-diphospho)-2-C-methyl-D-erythritol; MEC, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate; ERG10, acetoacetyl-CoA thiolase; ERG13, 3-hydroxy-3-methylglutaryl-CoA synthase; HMGR, 3-hydroxy-3-methylglutaryl-CoA reductase; ERG12, mevalonate kinase; ERG8, phosphomevalonate kinase; ERG19, mevalonate diphosphatedecarboxylase; IDI, isopentenyl di-phosphate isomerase; DXS: 1-Deoxy-D-xylulose-5-phosphate synthase; DXR: 1-deoxy-D-xylulose-5-phosphate reductoisomerase; CMS: 4-(cytidine 5′-diphospho)-2-C-methyl-D-erythritol synthase; CMK: cytidine monophosphate kinase; MECS: 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase; HDS: 4-hydroxy-3-methylbut-2-enyldiphosphate synthase; HDR: 4-hydroxy-3-methylbut-2-enyl diphosphate reductase; GPPS, geranyl diphosphate synthase; EDH, Enoyl-CoA hydratase; GCD, membrane-bound glucose dehydrogenase; AD, acyl-CoA reductase; ADH, alcohol dehydrogenase; PK, phosphoketolase; IP, isopentenyl phosphate; IPK, isopentenyl phosphate kinase; MTPS, monoterpene synthase

MVA pathway

The MVA pathway serves as a primary route for monoterpene biosynthesis, widely distributed across plants, fungi, and animals, while absent in some prokaryotes such as cyanobacteria [152]. In addition, the MVA pathway is also found in lepidoptera and some archaea. In eukaryotes, the MVA pathway is primarily localized in the cytoplasm. It initiates with acetyl‑CoA and proceeds through a series of enzymatic steps to yield IPP and DMAPP, the universal five‑carbon precursors of monoterpenes [153–155]. The expression of key enzymes (e.g., 3-hydroxy-3-methylglutaryl-coenzyme A reductase, HMGR) can be regulated to enhance the titer of mevalonic acid, which in turn provides precursors for monoterpene synthesis [156, 157]. For example, metabolic engineering of the MVA pathway in Corynebacterium glutamicum improved mevalonic acid titer to 4.16 g/L [156]. In lepidoptera, the LMVA pathway can facilitate the synthesis of non-natural monoterpene derivatives (e.g., sodorifen and 9-methoxycamptothecin) by introducing non-classical building blocks (e.g., C-methyltransferases) or optimising enzyme combinations, thereby expanding the structural diversity of monoterpenes [158, 159]. It has been demonstrated that metabolic flux optimization of LMVA pathway can significantly enhance the production of farnesyl diphosphate (FPP) analogues, thereby enabling the synthesis of non-natural terpenoids [158, 159]. In certain microbial hosts, such as Saccharomyces cerevisiae, the introduction of the LMVA pathway has been observed to substitute for traditional MVA or MEP pathways, thereby simplifying metabolic networks [160]. Additionally, the introduction of archaeal MVA pathway has been shown to circumvent regulatory bottlenecks present within the host’s native pathway. For example, the combination of archaeal enzymes (e.g., methoxypyruvate kinase MmMK from Methanosarcina octaedrica) has the potential to optimize rate-limiting steps and enhance the supply of monoterpene precursors, resulting in a substantial increase in limonene titer in S. cerevisiae [161]. It is notable that archaeal MVA pathway enzymes frequently demonstrate distinctive cofactor preferences (e.g., NADH-dependent), thereby complementing the host’s endogenous pathways [162]. For example, the screening for archaeal NADH-dependent MVA synthases has been demonstrated to allow for a more flexible utilization of intracellular reducing power, thereby improving carbon flux efficiency [162]. This property is of particular importance in engineered bacteria, where the requirement for redox balance is paramount. It is noteworthy that archaeal protein activity may be subject to limitations in bacterial or eukaryotic systems. As indicated by extant literature, the hierarchical optimisation of pathway assembly (e.g., Kronecker product combinations) is critical for maintaining activity. In contrast, heterologous expression of archaeal enzymes often requires additional optimization [163, 164]. Moreover, the introduction of archaeal pathways necessitates precise regulation of upstream and downstream metabolic flows. Research has demonstrated that the synthesis of monoterpenes necessitates the orchestrated regulation of flux at both the “top” (acetyl-CoA to MVA) and “bottom” (MVA to IPP/DMAPP) of the MVA pathway. The kinetic properties of archaeal enzymes have been observed to disrupt existing equilibria [164, 165]. For example, in C. glutamicum, an excessive boost in upstream flux driven by an engineered pathway led to intermediate accumulation and subsequent growth inhibition [166].

Several strategies have been employed to enhance monoterpene production, primarily focusing on increasing MVA pathway flux, optimizing precursor supply, and mitigating the toxicity of pathway intermediates. For example, the optimisation of the expression of MVA pathway enzymes such as HMG-CoA reductase has been demonstrated to significantly increase mevalonic acid production, with a titer improvement of 7660% to ultimately reach 4.16 g/L [156]. Since acetyl‑CoA serves as the primary substrate for the MVA pathway, its supply can be enhanced by engineering related metabolic nodes, including the glycolytic, citrate, and TCA cycles, thereby further improving MVA flux [156]. Furthermore, phosphorylated intermediates in the MVA pathway (e.g., M5P and M5DP) have been observed to exhibit cellular toxicity, particularly when localized in mitochondria [167]. Localizing part of the MVA pathway (from acetyl-CoA to MVA) to mitochondria and enhancing cytosolic mevalonic acid synthesis has been shown to mitigate toxicity and boost production [167]. Co-production of squalene (a downstream MVA pathway product) has been shown to alleviate cellular stress from monoterpenes while concomitantly increasing monoterpene production (e.g., α-pinene) [168]. Research indicates that regulating competing pathways can reduce substrate diversion and enhance monoterpene synthesis efficiency, such as the knockout of soluble methanomonoxygenase (sMMO) via CRISPR base editing [169].

MEP pathway

The MEP pathway is primarily localized in plastids and serves as another essential route for terpenoid biosynthesis in most bacteria, algae, and plants [152–154]. This pathway starts with pyruvate and glyceraldehyde 3-phosphate as initial substrates and ultimately yields the universal five‑carbon building blocks IPP and DMAPP [153, 170]. Particularly prominent in photosynthetic microorganisms such as cyanobacteria, the MEP pathway can directly utilize light and CO₂ to generate terpenoid precursors [152]. Studies have shown that enhancing the expression of key MEP pathway enzymes, including DXS and DXR, can significantly increase the production of monoterpenes [152, 171]. Furthermore, the activity of this pathway is modulated by various environmental stimuli, such as UV‑A radiation and plant hormones [170, 172]. Although compartmentalized differently within the cell, the MVA and MEP pathways engage in metabolic crosstalk and coordinated regulation [173, 174]. For example, monoterpenes derived from the MEP pathway, including many volatile terpenes, play important roles in plant responses to abiotic stress, a process that may rely on certain precursors supplied by the MVA pathway [172]. The carbon flux between these two pathways is finely tuned through the integrated actions of transcription factors, feedback inhibition, and metabolite‑sensing mechanisms, thereby maintaining the homeostasis of terpenoid biosynthesis [172–174].

The toxicity of MEP pathway intermediates [41], competition with host endogenous compounds [175] are identified as the rate-limiting steps. The enhancement of monoterpene titer can be achieved through the over-expression of rate-limiting enzymes in the MEP pathway, such as DXS and DXR [152]. For example, the overexpression of MEP pathway genes in cyanobacteria has been demonstrated to significantly increase terpenoid precursor supply [152], while the functional characterization of the McDXS gene in lemon balm has provided new targets for monoterpene synthesis [176]. Furthermore, studies have indicated that the accumulation of MEP pathway metabolites can feedback-inhibit pathway activity [165], dynamic regulation or feedback inhibition can relief to optimise flux [175]. Research on Plasmodium falciparum revealed that NADPH supply (regulated by GAPDH2) directly impacts MEP pathway flux [177], suggesting the need to balance reducing power availability. The localization of synthetic pathways to specific compartments, such as peroxisomes, has been shown to circumvent metabolic interference. For example, compartmentalization strategies in yeast have been shown to set a record for monoterpene titer [178]. Alternatively, the introduction of exogenous MVA pathways or synthetic pathways has been shown to achieve monoterpene precursor yields of 1504.6 mg/L, such as the construction of a MVA bypass pathway in Bacillus glutamicus [153].

IPA/IUP pathway

The IPA/IUP is an engineered synthetic route that directly phosphorylates isopentenol and prenol in two steps to yield the universal terpenoid precursors IPP and DMAPP, respectively [179]. This pathway has been demonstrated to significantly enhance IPP/DMAPP pool levels, with experimental evidence indicating a 147-fold increase in efficiency compared to the MVA pathway [179]. The engineered IUP pathways become decoupled from the host’s native metabolic network. The design of additional orthogonal synthetic pathways (e.g., non-natural biosynthetic routes) has been shown to reduce competition with metabolites that are essential for cell growth [163, 179]. For example, the co-overexpression of MEP pathway genes and engineered MVA pathway genes in cyanobacteria has been demonstrated to amplify the supply of terpenoid precursors [152]. In S. cerevisiae, compartmentalization strategies, such as targeting IUP enzymes to mitochondria or peroxisomes, combined with multifunctional synthetic enzymes have elevated diterpene (e.g., miltiradiene) production to 146.1 mg/L [180]. Furthermore, IUP pathway has been shown to synergise with the canonical MVA pathway through the co-feeding of isopentenol and prenol, thereby enhancing the supply of the monoterpene precursor geranyl diphosphate (GPP) [179]. It has been demonstrated that IUP outperforms the canonical MVA pathway in synthesising complex terpenes, particularly under conditions of high ATP demand. Further efficiency gains have been achieved through the optimisation of enzyme activity via high-throughput screening [160]. In summary, through strategic engineering, compartmentalization, and orthogonal operation, the IUP pathway provides an efficient and flexible platform for enhancing terpenoid precursor supply and target compound yields, offering a novel and powerful optimization avenue for terpenoid biosynthesis [160, 179, 180].

Heterologous synthesis in microbial cell factories

In recent years, the employment of model and non-model microorganisms for monoterpene synthesis has emerged as a pivotal strategy to replace traditional plant extraction and chemical synthesis. Optimizing endogenous pathways (MVA or MEP) and introducing plant-derived MTPS has enabled the microbial synthesis of various monoterpenes [19, 181, 182]. Table 2 compiled the highest reported titers of monoterpenes achieved through microbial heterologous synthesis to date. According to the investigation, the direct heterologous synthesis of several monoterpenes, including carvacrol, phellandrene, camphor, and fenchol, has not been reported. Nevertheless, studies have provided insights into potential conversion routes and pathway optimization strategies for related compounds [95, 183, 184]. For example, Krause demonstrated that thymol could be hydroxylated to thymohydroquinone by expressing relevant enzymes in yeast, offering a potential route for the microbial production of thymol derivatives [185]. Additionally, when direct synthesis remains challenging, accessing target monoterpenes through the conversion of other monoterpenes presents a feasible alternative. Citronellol, for instance, serves as a key intermediate in the biosynthetic pathway toward citral [186]. Nerol has been observed to undergo spontaneous conversion into d‑limonene, terpinene, linalool, and α‑pinene, with the conversion rate to terpinene reaching up to 5.94% [187]. Enzyme‑catalyzed routes also offer promising avenues: the LcTPS28 enzyme can produce multiple monoterpenes such as geraniol, nerol, and d‑limonene [182], while LcCYP94A1 oxidizes nerol to nerol aldehyde [182]. Furthermore, Zhao et al. [188] improved geraniol production by first converting geraniol into less‑toxic geraniol acetate, followed by hydrolysis, which significantly enhanced overall yield. Collectively, these studies lay an important theoretical foundation for advancing the heterologous synthesis and scalable microbial production of monoterpenes.

Table 2.

Summary of strategies for the heterologous synthesis of monoterpenes

MTP	MTPS	Sources	Microbial hosts	Engineering strategies	Titer (mg/L)	Culture scale	Reference
Geraniol	CrGES	Catharanthus roseus	P. pastoris	CRISPR-mediated ribonucleic acid (rDNA) integration and fluorescence screening are employed to optimize pathways, and identifying the most efficient carbon source (glycerol or glucose).	6270	5 L Fermenter	[206]
Linalool	t67OMcLISM	Mentha citrata	S. cerevisiae	The semi-rational design of t67OMcLISM combines cytoplasmic and peroxisomal engineering to target the localization of t67OMcLISM expression.	2600	5 L Fermenter	[178]
Citronellol	CrGES	Catharanthus roseus	S. cerevisiae	ATF1, mutation of endogenous ERG20 to ERG20F96W, protein scaffold SF1 (SH31PDZ1GBD1) was used for fusion protein, CrIS enzyme and IDI1.	8300	5 L Fermenter	[207]
Myrcene	GES	Ocimum basilicum	E. coli	The utilization of truncated linalool dehydratase isomerase in a one-step biotransformation system facilitates the synthesis of myrcene from geraniol. This approach employs an aerobic-anaerobic two-stage fermentation process, integrating induction temperature regulation and site-directed mutation.	1250	1 L Fermenter	[208]
Nerol	GmNES	Glycine max	E. coli	Truncation of N-terminal of BPPS, sitedirected mutation of BPPS, condition optimization (induction cell density, inducer concentration, glucose concentration).	966.6	Shake flask	[209]
γ-Terpinene	TvTPS	T. vulgaris	E. coli	Condition optimization (carbon source, Mg2+ and IPTG).	275.4	5 L Fermenter	[210]
Limonene	tLimS	Mentha spicata	S. cerevisiae	Dynamic suppression of ERG20 regulation of competitive bypass and optimization of tLimS copy number, enhanced supply of acetyl-CoA and NADPH, reestablished the citrulline synthesis pathway in mitochondria.	2630	3 L Fermenter	[211]
Menthol	LimS	Mentha spicata	S. cerevisiae	Enhancing the MVA pathway and dynamically regulating ERG20 expression.	6.28	Shake flask	[212]
1,8-cineole	HYP3	Hypoxylon sp. E74060B	R. toruloides	Optimization of the culture medium for wood cellulose hydrolysate, overexpression of the MVA pathway genes (HMGR, MK and PMK).	1400	2 L Fermenter	[213]
α-Terpineol	VvTS	Vitis vinifera	S. cerevisiae	Truncation of N-terminal of VvTS, ERG20-VvTS fusion protein by a GSGSGSGSGS linker, overexpression of tHMG1, ERG20 and IDI.	21.9	5 L Fermenter	[214]
α-Pinene	Pt30	Pinus taeda	S. cerevisiae	Dynamic regulation of design production and cell growth and enhancement of the transport protein Sge1.	1800	3 LFermenter	[215]
β-Pinene	AgPS	Abies grandis	D. radiodurans	Codon optimization of PS, promoter engineering for PS, condition optimization (glycerol concentration).	3.2	Shake flask	[216]
Camphor	PsBDH	Pseudomonas sp	S. cerevisiae	Express PsBDH, a borneol dehydrogenase from Pseudomonas and perform two-phase fermentation.	23.5	Shake flask	[217]
Borneol	LdBPPS	L. dulcis	E. coli	Truncation of N-terminal of BPPS, sitedirected mutation of BPPS, condition optimization (induction cell density, inducer concentration, glucose concentration).	87.2	Shake flask	[209]
Sabinene	SpSabS	Salvia pomifera	E. coli	Culture medium and condition optimization (carbon source, organic nitrogen source, induction temperature and inducer concentration).	2650	5 L Fermenter	[218]

Monoterpene synthesis in model microorganisms

S. cerevisiae and E. coli are the most commonly used microbial platforms for monoterpene synthesis [189]. These model organisms possess well-defined genetic backgrounds, mature genetic manipulation tools, and efficient heterologous expression systems [166]. Furthermore, the selection of microbial chassis necessitates a comprehensive consideration of the compatibility between their endogenous metabolic networks and the monoterpene synthesis pathway. For example, the canonical MVA pathway in yeast is more conducive to supplying terpenoid precursors [189, 190]. The key to constructing monoterpene synthesis pathways in model microorganisms lies in the heterologous reconstruction of MTPS catalytic templates [181, 191]. Enzyme engineering strategies, such as directed evolution or rational design, are employed to enhance the functional expression and catalytic efficiency of MTPS. One strategy involves modifying the dimensions of the active site pocket to regulate product specificity [19, 192]. The localization of MTPS to specific compartments, such as mitochondria or plasma membranes, has been demonstrated to enhance substrate accessibility and reduce toxicity [193]. For example, VvPDS (initially misannotated as a geranyl diphosphate synthase, GPPS) was found to participate in mitochondrial ubiquinone synthesis, with its localization study correcting earlier misconceptions about the monoterpene pathway [193]. Furthermore, metabolic engineering optimizations, such as enhancing carbon flux in the MEP or MVA pathways, have been shown to significantly boost monoterpene production [181, 194]. To circumvent the metabolic burden resulting from intermediate accumulation, inducible promoters or RNA-based switches can orchestrate the concurrent expression of GPPS and MTPS [19, 181]. Recent studies highlight the role of rhizosphere microbes in promoting monoterpene accumulation, either by activating host immune systems or by directly participating in terpenoid metabolic pathways [195]. For example, certain endophytes found in citrus plants have been observed to enhance monoterpene synthesis by regulating host genes, such as LcTPS42 and LcGPPS.SSU1 [196]. Experiments with synthetic microbial communities (SynComs) further validate the positive regulatory impact of microbe-plant interactions on monoterpene production [195]. The synthesis of monoterpenes in model microorganisms has evolved from a reconstruction of a single pathway to a multidimensional optimization of systems. The integration of tools from metabolic engineering, synthetic biology, and microbiomics will continue to enhance its production efficiency and expand its application scope.

Monoterpene synthesis in non-model microorganisms

While research has predominantly focused on model microorganisms like E. coli and S. cerevisiae, non-model microorganisms offer distinct advantages and considerable potential. This category encompasses microbes that are less studied or lack established genetic toolkits, yet often possess unique metabolic traits or environmental resilience that make them attractive hosts for monoterpene production [197]. In contrast to traditional model organisms, numerous non-model microorganisms possess diverse secondary metabolic pathways, which provide additional precursors for monoterpene synthesis [189, 197]. For example, certain actinomycetes and cyanobacteria possess unique enzyme systems for modifying terpenoid skeletons [198]. Extremophiles (e.g., thermophiles, halophiles) have been observed to exhibit high levels of viability under harsh conditions, while photosynthetic microorganisms such as cyanobacteria have demonstrated the capacity to directly utilize CO₂ as a carbon source [199], thereby reducing cultivation costs [198]. For example, engineering the MEP pathway in Chlamydomonas reinhardtii significantly improved monoterpene titers [200]. Furthermore, non-model microorganisms such as Streptomyces naturally produce terpenoid compounds, possessing complete synthetic and modification systems [201, 202]. Genomic analysis has identified key genes involved in monoterpene synthesis, including MTPS and GPPS, in various non-model microorganisms [19, 203]. However, the reconstruction of plant monoterpene synthesis pathways in non-model microorganisms is currently encumbered by numerous challenges, including the absence of efficient transformation systems and gene editing tools [6, 197], the potential toxicity of monoterpenes and their precursors to microbial hosts [6], and limitations in heterologous pathway expression due to translation efficiency or protein folding [181, 204]. In order to address these challenges, several effective solutions have emerged, which includes the development of CRISPR-Cas9 genome editing systems or transposon-mediated random insertion [204, 205], the overexpression of efflux pumps or membrane modification genes to engineer toxicity tolerance in host cells [189, 198], the optimization of MTPS gene codons based on host preferences [19, 181], and the co-expression of chaperones (e.g., the GroEL/ES system) to enhance enzyme stability [181, 204]. Non-model microorganisms present a novel platform for monoterpene synthesis; however, their application is hindered by challenges in genetic manipulation, metabolic regulation, and product toxicity. The integration of synthetic biology, metabolic engineering, and systems biology approaches holds promise for the efficient and sustainable production of monoterpenes in these hosts in the future [6, 181, 204].

Mechanism and evolutionary analysis of MTPS

MTPS involved in monoterpenes

It has become a convenient and effective means in synthetic biology research to mine enzymes with similar functions from different plants and to find key sites to improve the reaction characteristics of synthetases by simulation calculation. However, at present, the synthetic biology research on plant terpenoids is usually based on the established components, and the chassis suitability improvement and metabolic pathway optimization are carried out. There are few reports on the systematic mining and transformation of components for target products. As more and more plant genomes have been analyzed, a large amount of plant MTPSs information has been obtained. Systematically combing the information and using bioinformatics methods to explore the intrinsic correlation of their functions will provide theoretical support for rational design of component functions and then obtain ideal synthetic components through directional evolution. Currently, 253 MTPSs have been identified to the above- monoterpenes with biological pesticides (Table S1). They were from bryophytes (2), ferns (4), gymnosperms (61), angiosperms (183), microorganisms (1, Hypoxylon sp.) and protists (2, Physarum polycephalum). The phylogenetic tree shown in Fig. 3 shows the evolutionary relationship of these MTPSs. The amino acid sequences of MTPSs from different species were well clustered in the subregions of the phylogenetic tree. The terpenoid synthases of gymnosperms and angiosperms are differentiated from a common ancestor, and the MTPSs of gymnosperms represented by Abies grandis and Picea abies form a separate branch (Fig. 3A). MTPSs of gymnosperms contain synthetic acyclic, monocyclic and bicyclic monoterpenes, and their sequence similarity is higher than that of MTPSs of the same type of products of angiosperms. The amino acid sequences of MTPSs encoding the same monoterpene product type have certain aggregation in the sub-regions of the phylogenetic tree (Fig. 3B), and there are also higher amino acid sequences of MTPSs that synthesize different types of products. The MTPS sequence in angiosperms is similar. It is worth noting that MTPSs that synthesize acyclic products in angiosperms have good aggregation and high sequence similarity.

Fig. 3.

Phylogenetic tree of monoterpene synthases. A Phylogenetic tree of monoterpene synthases in different species. B Phylogenetic tree of acyclic/monocyclic/bicyclic monoterpene synthases in gymnosperms and angiosperms

Mechanism of MTPS reaction

MTPS, which typically utilize GPP or neryl diphosphate (NPP) as substrates, catalyze the formation of diverse monoterpene structures through mechanisms including cyclization, methylation, and rearrangement [219]. Based on the structural classification of monoterpenes into cyclic and acyclic types, the catalytic pathways of these enzymes are broadly divided into cyclization and non-cyclization routes (Fig. 4).

Fig. 4.

Mechanistic scheme of the early steps of MTS catalysis

Initially, GPP or NPP undergoes ionization with the assistance of divalent metal ions (e.g., Mg²⁺ or Mn²⁺) to remove the pyrophosphate group from GPP and NPP. This process can further result in the conversion of GPP or NPP into the cationic intermediate geranyl cation. During the process of non-cyclized monoterpene product formation, the geranyl cation has been observed to form laurene [220] and (E)-β-ocimene [221] through direct proton loss. Alternatively, the geranyl cation can undergo hydration to yield linalool [222, 223] or geraniol [224, 225]. In the formation of cyclic monoterpene products, the geranyl cation combines with a free diphosphate anion to form a key universal intermediate, linalyl diphosphate (LPP) [226]. Subsequently, the resulting allylic cation-diphosphate anion pair undergoes a rearrangement, resulting in the formation of enzyme-bound tertiary allylic isomers, namely 3R- or 3 S-linalyl diphosphate. Following the rotation of LPP into a cis-conformation, ionization and cyclization in an anti-endo manner occur, resulting in the formation of the corresponding 4R- or 4 S-α-terpinyl cation. This cation serves as a critical branching point for the formation of all cyclic monoterpenes. From the α-terpinyl cation, deprotonation can yield limonene [227], which can be further oxidized by cytochrome P450 enzymes to produce perillyl alcohol. This reaction can be catalyzed, resulting in the formation of perillaldehyde. Subsequent conversion of perillaldehyde to perillic acid occurs via dehydrogenase activity [228]. Alternatively, the α-terpinyl cation can undergo a 2,7-cyclization to generate a pinyl cation, which can lose a proton to form α-pinene or β-pinene [220]. The α-terpinyl cation can also undergo a 3,7-cyclization to produce a Borneol cation, a precursor for borneol and camphor [229]. Another cyclization pathway of the α-terpinyl cation involves a 5,7-closure to form a cyclopropyl ring, leading to the generation of 3-carene [230]. Water capture by the α-terpinyl cation yields α-terpineol, which can undergo heterocyclization to form 1,8-cineole [231]. Additionally, under the action of terpene synthases, bicyclic monoterpenes with cyclopropyl rings can be formed. For instance, a 1,2-hydride shift in the α-terpinyl cation followed by a 2,6-closure yields a sabinyl cation, a precursor for sabinene and related monoterpenes [232]. These cyclization reactions demonstrate the versatility of the α-terpinyl cation in generating diverse monoterpene structures, which are further modified through enzymatic reactions to yield the final monoterpene products. The specific reaction pathways and products depend on the participating enzymes (terpene synthases) and reaction conditions.

MTPSs exhibit remarkable flexibility in regulating the reaction pathways of carbocation intermediates (e.g., cyclization, hydride shifts, rearrangements) and terminating reactions (e.g., deprotonation, water capture), enabling the generation of a wide array of monoterpenes. This diversity not only reflects the catalytic capabilities of these enzymes but also provides critical insights into their reaction mechanisms. Whether forming primary or secondary products, their generation underscores the complexity and multifunctionality of MTPSs in the biosynthesis of natural products.

Research progress on enhancing MTPS function through protein engineering

MTPS are pivotal enzymes in plant secondary metabolic pathways, demonstrating remarkable functional plasticity. The majority of plant MTPSs are characterized by broad substrate or product characteristics [19]. This property facilitates the modulation of substrate selectivity, product distribution, or enzyme activity through active site mutations and domain swaps [19]. For example, certain MTPs primarily produce linalool [233], while others may yield multiple monoterpene products. This inherent diversity provides a foundation for the implementation of engineering modifications. Rational design has been demonstrated to be an effective approach to enhance MTP functionality [234]. The employment of tools such as AlphaFold2 in the modeling of enzyme structures facilitates the comparison of variations in active site residues across enzymes, thereby providing a framework for the guidance of protein engineering [235]. For example, by comparing active site residues of AsDMS and the fungal albicanol synthase, researchers successfully converted AsDMS activity from 2-synthase to 4-synthase [235]. This approach is equally applicable to MTP modification. A combined molecular dynamics simulation and experimental strategy has also been employed to explore the structure-function relationships of terpenoid synthases [236]. Despite the fact that terpenoid synthases (including MTPS) generate diverse terpenoid compounds through complex cyclization pathways, significant untapped potential remains due to limited understanding of their structure-function relationships [236]. This integrated approach facilitates a more precise prediction of which amino acid residues affect enzyme activity and specificity. The mutation of active site residues has been demonstrated to be an effective strategy for modifying the function of MTP. For example, studies on the terpenoid synthase from Sacred Lotus revealed that N314 is a key amino acid controlling γ-eugenol synthesis [237]. A similar approach can be applied to the systematic analysis of active sites across different MTPS. This analysis enables the identification of key residues that affect product specificity for targeted modification. In the domain of citral synthase engineering, researchers have achieved substantial gains in yield enhancement through the strategic augmentation of monoterpene precursor (GPP) supply [186]. The wild-type farnesyl diphosphate synthase (Erg20) was substituted with a mutant form, resulting in citral titers reaching 406.01 mg/L without compromising cell growth [186]. This finding underscores the necessity of a multifaceted approach to optimize precursor supply and enzyme activity in tandem.

Perspective and outlook

As key constituents of plant-derived natural products, monoterpenes have demonstrated considerable potential in the field of biopesticides. Their core advantages include renewable sourcing, favorable environmental compatibility, and diverse mechanisms of action. Specifically, monoterpenes can serve not only as effective insecticides and repellents, disrupting the nervous system and behavior of pests through contact or fumigation, but also as broad-spectrum antibacterial and antifungal agents that damage the cell structures of pathogens, contributing significantly to green pest control and post-harvest preservation. Additionally, they can act as plant immunity inducers to activate crop self-defense mechanisms, and as effective synergists to enhance the performance of other pesticides, thereby reducing reliance on conventional chemical products. Despite the persistent challenges associated with the synthesis and application of monoterpenes, particularly concerning stability, persistence, and cost, the potential for future technological advancements to transform monoterpene compounds into essential components of next-generation biopesticides is promising. These advancements may include formulation innovations, synthetic biology-driven yield enhancements, and molecular structure optimizations. This advancement will support greener, more sustainable agricultural development.

Microbial synthesis of monoterpenes, as a sustainable manufacturing paradigm to replace traditional plant extraction and chemical synthesis, has shown great application potential in the fields of spices, medicine and biofuels due to its advantages of environmental friendliness and controllable process. At present, the technology has established a mature chassis cell system represented by S. cerevisiae and E. coli, and has made significant progress through a series of metabolic engineering strategies. Specifically, it includes strengthening the supply of endogenous precursors to solve the fundamental problem of insufficient supply of precursors such as GPP; the second is the mining and optimization of key enzymes, that is, heterologous expression of MTPs derived from plants, and directed evolution or rational design of them by means of protein engineering to improve their catalytic efficiency and stability in microbial hosts. The third is the process innovation to deal with the toxicity of the product. For example, the introduction of organic phases such as dodecane into the fermentation system to form a two-phase culture system for in-situ product removal, or the use of output proteins to promote intracellular product efflux to alleviate the damage of monoterpenes to cell membrane structure. Despite the deepening of research, the road of microbial synthesis of monoterpenes to large-scale industries still faces several severe challenges. For example, monoterpenoids are highly volatile and exhibit toxicity toward microbial hosts, resulting in low yields and difficulties in scaling up production. In addition, plant-derived MTPS exhibit low activity or produce impurities when expressed in microorganisms. Terpenoid synthesis involves multi-step reactions, with metabolic flux potentially diverted by competing pathways. Furthermore, there exists a gap between laboratory-scale and industrial production, such as high substrate costs and low yields, insufficient structural diversity of natural products, and the influence of transcription factors (e.g., MYB, ERF) and epigenetic regulation on monoterpene biosynthesis, with limited research on these mechanisms in non-model plants. Consequently, future solution research will primarily focus on the following areas. First, the optimization of host strains (e.g., E. coli, yeast) through metabolic engineering is necessary. This can be achieved by modifying membrane structures or secretion systems to reduce intracellular accumulation. Then, tolerant hosts can be selected via adaptive laboratory evolution (ALE). Secondly, the enhancement of MTPS catalytic efficiency and product specificity through directed evolution or rational design, while concurrently augmenting precursor supply by integrating MVA or MEP pathways, is imperative. Thirdly, the development of strains capable of utilizing non-grain feedstocks, such as lignocellulosic hydrolysates, in conjunction with high-density culture or continuous fermentation technologies, is imperative. Fourthly, the prediction and elimination of metabolic bottlenecks through network analysis, as well as the precise control of pathway gene expression via CRISPRi/a or promoter libraries, are crucial. The fifth objective is to identify novel MTPS genes from plants or microbes and to synthesize new terpenoid skeletons through hybrid pathways or non-natural enzyme design. Breaking through these bottlenecks requires the cross-integration of multidisciplinary technologies. The potential directions include: using synthetic biology tools to develop intelligent dynamic regulation systems that can sense the physiological state of cells, so that product synthesis is only initiated after the cells grow to a high density; through the combination of adaptive laboratory evolution and global metabolic engineering, a new generation of chassis microorganisms with intrinsic high tolerance to monoterpenes was selected. To further explore the natural MTP from microbial sources to obtain catalytic elements with better compatibility with the host; by optimizing the integrated fermentation process, such as the coupling of in-situ extraction and adsorption technology, the continuous removal and purification of the product can be realized, thus systematically promoting the industrialization process of microbial synthesis of monoterpene technology.

Supplementary Information

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Author contributions

Analyzing and Writing- review & editing, Ruiqi Chen; Investigation and writing-original draft, weilin Liu; Provide guidance for constructing the framework of the entire review paper in the early stages, Daoguang Tian & Shengli Wang; Revising it critically for intellectual content and final approval of the version to be published, Jianjun Qiao, Weiguo Li, Qinggele Caiyin. All authors agree to be accountable for all aspects of the manuscript.

Funding

This work was supported by “Pioneer” and “Leading Goose” R&D Program of Zhejiang (Grant 2025C01094) and Zhejiang Provincial Natural Science Foundation of China (No. LQN25C010006).

Data availability

Data sharing is not applicable to this article as no new data were created or analyzed in this study.

Declarations

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.