Microbial Synthesis of Neo-Allo-Ocimene by Celery-Derived Neo-Allo-Ocimene Synthase

Abstract

Neo-allo-ocimene is a monoterpene which could be applied in pesticides, fragrances, and sustainable polymers. In this study, we mined a terpene synthase, AgTPS40, from the transcriptome of celery leaf tissues. Through sequence and phylogenetic analysis, AgTPS40 was characterized as a monoterpene synthase. The AgTPS40 gene was introduced into a heterologous mevalonate pathway hosted in Escherichia coli to enable terpene production. Gas chromatography–mass spectrometry analysis confirmed that AgTPS40 catalyzes the formation of neo-allo-ocimene, marking the first reported identification of a neo-allo-ocimene synthase. Subsequently, we optimized the fermentation conditions and achieved a yield of 933.35 mg/L in a 1 L shake flask, which represents the highest reported titer of neo-allo-ocimene to date. These results reveal the molecular basis of neo-allo-ocimene synthesis in celery and provide a sustainable way to obtain this compound.

1. Introduction

Terpenes are a large and diverse group of organic compounds produced by a wide variety of plants and microbes, as well as some insects [1,2]. The structural diversity structures of terpene is catalyzed by a class of enzymes that convert simple precursors, such as geranyl diphosphate (GPP), farnesyl diphosphate (FPP), and geranylgeranyl diphosphate (GGPP), into a myriad of complex molecules [3]. Monoterpenes, which contain two isoprene units, play important roles in medicine, energy, food, and agriculture [4,5,6]. Based on their structural features, monoterpenes can be categorized into acyclic and cyclic monoterpenes [7]. Representative acyclic monoterpenes include myrcene, limonene, ocimene, and allo-ocimene [8,9,10,11]. Ocimene exists in two isomeric forms: (E)-β-ocimene and (Z)-β-ocimene. Similarly, allo-ocimene has two isomers: (4E,6E)-allo-ocimene and (4E,6Z)-allo-ocimene, the latter also known as neo-allo-ocimene [11].

Allo-ocimene has been widely utilized as a fragrance in the cosmetics industry due to its distinctive aromatic properties and also exhibits multiple biological activities. It can prime neighboring plants to enhance their defense against sweet potato weevils [12]. Volatile allo-ocimene increases the resistance of Arabidopsis thaliana to the pathogenic fungus Botrytis cinerea [13]. It also shows high toxicity to the larvae of four mosquitoes [14]. These characteristics suggest that allo-ocimene holds potential for development as an environmentally friendly pesticide. Furthermore, allo-ocimene can be utilized in the synthesis of bio-based sustainable polymers [15], significantly expanding its application value. Traditionally, allo-ocimene is predominantly produced through chemical synthesis [15]. While this approach can temporarily meet market demands, it suffers from high production costs, low efficiency, excessive natural resource consumption, and potential environmental risks. With the advancement of metabolic engineering and synthetic biology, microbial heterologous biosynthesis has emerged as a promising alternative for monoterpene production. In recent years, myrcene synthase was discovered in Cannabis sativa, where iterative enzyme evolution using the high-throughput biosensor method achieved a remarkable β-myrcene yield of 510.38 mg/L [16]. Similarly, the heterologous expression of (E)-β-ocimene synthase from Cinnamomum camphora, coupled with the fusion co-expression of geranyl pyrophosphate (GPP) synthase and the target enzyme, yielded 34.56 mg/L of (E)-β-ocimene [17]. These successes demonstrated the importance of enzyme activity in terpene biosynthesis [18]. However, allo-ocimene synthase remains uncharacterized [11], representing an unresolved mystery in the field.

Plants are one of the primary sources of terpene synthases. For example, numerous terpene synthases have been identified in species such as Arabidopsis thaliana, Abies grandis, and Mentha arvensis. Celery (Apium graveolens L.), an annual or biennial vegetable belonging to the Angiospermae Apiaceae family, is rich in vitamins, apigenin, cellulose, and other nutrients [19]. Its essential oil is reported to have carminative, stomachic, diuretic, and emmenagogic properties [20]. A previous study identified over 70% of celery’s volatile compounds as terpenes using HS-SPME-GC/MS, with β-myrcene, D-limonene, β-ocimene, and γ-terpinene being the most critical compounds [21]. In addition, allo-ocimene has been detected in the essential oil of celery [20]. Therefore, celery holds significant potential for the discovery of novel monoterpene synthases.

Isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), the universal precursors for terpenoid biosynthesis [6], are generated through two distinct metabolic routes: the methylerythritol-4-phosphate (MEP) pathway and the mevalonate (MVA) pathway. The MEP pathway predominates in prokaryotic systems, whereas the MVA pathway is primarily operational in eukaryotic organisms such as Saccharomyces cerevisiae [22]. However, the native MEP pathway exhibits constrained precursor productivity due to inherent regulatory constraints in its natural hosts [23]. In contrast, heterologous expression of the MVA pathway in Escherichia coli has emerged as a key strategy for enhancing monoterpene biosynthesis [16,24,25].

In this study, the terpene synthase AgTPS40 was successfully mined from celery and identified as a monoterpene synthase by sequence and phylogenetic analysis. We introduced AgTPS40 into a plasmid encoding a heterologous MVA pathway and expressed it in E. coli BS1101. Gas chromatography–mass spectrometry (GC-MS) revealed that AgTPS40 catalyzes the production of neo-allo-ocimene, which is the first report of a neo-allo-ocimene synthase. Through optimization of fermentation conditions, we achieved 933.35 mg/L of neo-allo-ocimene in a 1 L shake flask, which represents the highest titer of neo-allo-ocimene reported to date. This study not only fills the gaps in the field of monoterpene synthases but also provides a robust platform for the production of high-value terpenes using engineered microbial hosts.

2. Materials and Methods

2.1. Strains and Reagents

The strains used in this study are presented in Table S1. E. coli DH5α was used as a routine clone strain. E. coli BS1101 was used as a fermentation chassis [26]. Neo-allo-ocimene ((4E,6Z)-allo-ocimene) (Macklin, N775958, Shanghai, China) was used as a standard. Caryophyllene oxide (Sigma, W509647, St. Louis, MO, USA) was used as an internal reference. LB medium (per liter: 5 g of yeast extract, 10 g of NaCl, 10 g of tryptone) was used for the cultivation of the seed solution. SOC medium (per liter: 5 g of yeast extract, 0.5 g of NaCl, 20 g of tryptone, 10 M KCl, 10 M MgCl₂, 10 M glucose) was used as a recovery medium. M9-MOPS medium [M9 salt (Coolaber, Beijing, China), 75 mM 3-(N-morpholino)propanesulfonic acid (MOPS), 2 mM MgSO₄, 0.01 mM CaCl₂, 1 mg/L thiamine·HCl, and 2.78 mg/L FeSO₄ at pH 7; the micronutrients provided were 3 nM ammonium molybdate, 0.4 μM boric acid, 30 nM cobalt chloride, 23 nM cupric sulfate, 80 nM manganese chloride, 10 nM zinc sulfate, and 2% glucose] [27] was used for fermentation. If necessary, 34 mg/L chloramphenicol (Beyotime, Zhengzhou, China) was added to the medium. Various salts and glucose were purchased from Aladdin (Beijing, China). Yeast extract and tryptone were purchased from Oxoid (Basingstoke, UK).

2.2. Plasmid Construction

The plasmids and primers used in this study are presented in Table S1. DNA sequences were synthesized by BGI (Beijing, China) and Tsingke (Beijing, China). Phanta Max Master Mix polymerase and the ClonExpress^®II one-step cloning kit were purchased from Vazyme (Nanjing, China) for DNA amplification and plasmid construction. The plasmid for monoterpene synthase expression was constructed as follows: pCC24, containing genes coding for the heterologous MVA pathway and the trGPPS gene, was linearized by the BamHI site located after trGPPS as the backbone plasmid to construct the precursor plasmid. The signal peptide sequence of AgTPS40 was predicted by TargetP and then removed. The AgTPS40 gene was amplified by pCC24-AgTPS40-F and pCC24-AgTPS40-R and then ligated into the pCC24 backbone, generating pCC24-AgTPS40. The protein sequence of AgTPS40 and its codon-optimized DNA sequence are listed in the Supplementary Materials.

2.3. Bioinformatics Analysis of Putative Terpene Synthase

Leaf tissue from celery (Apium graveolens L.) was subjected to full-length transcriptome sequencing using the PacBio Sequel platform (Novogene, Beijing, China) to obtain high-quality complete transcripts. TPS genes were subsequently identified through HMMER search (v3.3.2) software targeting the conserved TPS domains (PF01397 and PF03936) within the transcriptome assembly.

The phylogenetic tree was inferred by using the Maximum Likelihood method based on the JTT matrix-based model [28]. The tree with the highest log likelihood (−14,360.28) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. The initial tree(s) for the heuristic search were obtained automatically by applying the Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using a JTT model and then selecting the topology with a superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 34 amino acid sequences. All positions containing gaps and missing data were eliminated. There were 277 positions in the final dataset. Evolutionary analyses were conducted in MEGA 7 [29]. Sequence comparison analysis was performed using ESPript 3.0 (https://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi, accessed on 27 December 2024). All sequences can be retrieved from Table S2.

2.4. Shake Flask Fermentation

E. coli BS1101 containing a single monoterpene-producing plasmid was cultured in M9-MOPS medium supplemented with glucose and appropriate antibiotics for fermentation as follows: The strain was pre-cultured for 16 h at 37 °C in LB medium (chloramphenicol, 35 mg/mL) and then inoculated to 10 mL M9-MOPS at a ratio of 2% in 50 mL shake flasks. The cells were incubated at 37 °C for 5 h, and when OD₆₀₀ reached 0.6–0.8, it was induced by the addition of isopropyl-β-d-1-thiogalactopyranoside (IPTG) at a final concentration of 50 μM, and an in situ extraction was performed by the addition of a 20% (v/v) isopropyl cinnamate (IPM) overlay. The cells were incubated at 30 °C and 220 rpm to produce monoterpenes, and the samples were collected after 72 h of fermentation. The expansion of the system was achieved by proportionally scaling up each component.

2.5. Characterization and Quantification of Neo-Allo-Ocimene

After 72 h of fermentation, the upper layer of IPM was collected following centrifugation at 12,000 rpm for 1 min. Subsequently, the collected IPM was diluted with ethyl acetate containing an internal standard of caryophyllene oxide. The resulting sample was then filtered using a nylon membrane with a pore size of 0.22 μM. The samples were analyzed by gas chromatography–mass spectrometry (7890A GC and 5975C MS detectors, Thermofisher, Waltham, MA, USA). The inlet temperature was set at 250 °C with a flow rate of 1 mL/min. The oven temperature was initially set at 50 °C for 0.5 min, rising to 150 °C at 25 °C/min and then to 260 °C at 40 °C/min, and the column was TG-5SLMS [30]. GC-MS analysis was performed in SCAN mode. Qualitative identification of the primary product was achieved by comparative analysis of retention times and mass spectral profiles with certified reference standards. Secondary metabolites were tentatively identified through spectral matching against the National Institute of Standards and Technology (NIST) Mass Spectral Database. Quantitative analysis of neo-allo-ocimene was performed using a calibration curve method. A standard series (7771, 3108, 1243, 497.2, and 198.9 mg/L) was prepared by serial dilution of the stock standard solution. Caryophyllene oxide (100 mg/mL) was added as an internal standard to the sample solution at 0.005% (v/v). The calibration curve was established through linear regression of the peak area ratios (neo-allo-ocimene to the internal standard) against the corresponding standard concentrations, enabling quantitative analysis of samples.

2.6. Optimization of Fermentation Conditions

To evaluate the impact of different fermentation conditions on monoterpene production biosynthesis in E. coli BS1101, fermentation variables, including inducer concentration gradients (25–200 μM) and carbon source supplementation [1–6% (w/v) glucose], were selected. Samples were taken and analyzed after 72 h of fermentation, and the strain’s OD₆₀₀ was determined on a Mutiskan FC (Thermofisher, USA).

2.7. Scanning Electron Microscopy (SEM) Observation

Strain morphology was imaged using a Scanning Electron Microscope (S-4800, Hitachi, Tokyo, Japan). Bacterial cells from the fermentation broth were harvested by centrifugation (5000 rpm, 5 min) and fixed in 4% paraformaldehyde overnight. After removing the supernatant via centrifugation, the cells were sequentially treated in an ethanol gradient (30%, 50%, 80%, and 100%, 10 min per concentration). Subsequently, the samples were washed three times with water (2 min each time), pelleted by centrifugation, flash-frozen at −20 °C, and subjected to freeze-drying for 24 h. After drying, the samples were prepared for observation of strain morphology. For microscopy, low magnification was initially employed to locate target regions, followed by high-magnification imaging. The accelerating voltage was set to 5 kV.

2.8. Statistical Analysis

Graphs were plotted using Origin 2018 SR2 software (OriginLab Inc., Hampton, MA, USA) and PPT 2021 software. All experimental data (including fermentation yields and OD₆₀₀ measurements) represent mean values ± standard deviation derived from triplicate biological replicates.

3. Results

3.1. Sequence and Phylogenetic Analysis of Putative Terpene Synthase

Based on the transcriptome of celery, a putative terpene synthase (TPS) gene was mined. By aligning this gene with the 39 previously reported TPSs in celery [21], it was determined to be a novel TPS that had not been discovered before, and thus, it was designated as AgTPS40. To elucidate its characteristics, a phylogenetic analysis was performed on AgTPS40. The phylogenetic tree revealed that AgTPS40 belongs to the TPS-b subfamily (Figure 1). The TPS-b subfamily is recognized as an angiosperm-specific group that predominantly contains monoterpene synthases [31].

Sequence alignment analysis showed that AgTPS40 has a conserved RRx₈W motif (Figure 2), a hallmark of monoterpene synthases in the TPS-b subfamily and a crucial element for their catalytic function. Additionally, AgTPS40 possesses the conserved DDxxD motif and the NSE/DTE motif, which are essential for divalent metal ion coordination [32,33]. Given these facts, it is strongly indicative that AgTPS40 from Apium graveolens L. functions as a monoterpene synthase.

3.2. Function Characterization of AgTPS40 In Vivo

To characterize the function of AgTPS40, we cloned the gene into the linearized pCC24 vector and transformed it into E. coli BS1101 (Figure 3A). The compounds produced after 72 h of fermentation were analyzed by GC-MS (Figure 3B and Figure S1). The major compound, representing 76.58% of the total terpenoid fraction, was identified by comparing its retention time (RT: 4.62 min) and mass spectrometry data (parent ion at 136 m/z; major daughter ions at 121 and 105 m/z) with those of the standard neo-allo-ocimene (Figure 3B,C). This confirmed that the primary product was neo-allo-ocimene. In addition to neo-allo-ocimene, other compounds were tentatively identified by comparing their mass spectra with the National Institute of Standards and Technology (NIST) Database. Specifically, (Z)-β-ocimene (19.2%) at RT = 4.01 and (E)-β-ocimene (1.8%) at RT = 4.09 were produced (Figure 3D). However, the compound detected at RT = 3.95 (2.42%) could not be confidently assigned based on retention time and mass spectra alone (Figure S2). Control experiments showed that E. coli BS1101 without any plasmid or with only the pCC24 plasmid did not produce any terpenoid compounds (Figure 3B). These results indicate that AgTPS40 functions as a neo-allo-ocimene synthase. Specifically, the fermentation yield was determined to be 426.70 mg/L of neo-allo-ocimene, corresponding to an OD₆₀₀ of 4.76 for the cell culture (Figure 3E).

3.3. Optimization of the Production Process

In the MVA pathway, glucose serves as a crucial source of metabolic precursors and energy, primarily through the glycolytic pathway and the pentose phosphate pathway [34,35]. To optimize glucose supply, we tested concentrations of 1%, 2%, 3%, 4%, 5%, and 6% while expanding the fermentation system to 1 L. After 72 h of fermentation, we observed that at a glucose concentration of 2%, the production of neo-allo-ocimene reached up to 933.35 mg/L, corresponding to an OD₆₀₀ of 7.95 (Figure 4A), which is the highest yield titer reported. Scanning electron microscopy revealed that at a glucose concentration of 2%, the cells exhibited a more robust and larger-diameter morphology compared to those at 1% (Figure 4C).

Additionally, we varied the concentration of IPTG inducer, testing final concentrations of 25 μM, 50 μM, 100 μM, 150 μM, and 200 μM, while maintaining the glucose concentration at 2%. The highest titer of 933.35 mg/L was achieved at 50 μM (Figure 4B), whereas titers at other induction concentrations were significantly lower than that observed at 50 μM. This indicates that the optimal final concentration of IPTG for neo-allo-ocimene production in this study was 50 μM.

4. Discussion

Neo-allo-ocimene is an acyclic monoterpene with high commercial value, widely used in food, cosmetics, and pharmaceuticals. It has been detected in celery for a long time [20], but its biosynthetic pathway has not been fully characterized. Previous studies proposed a putative pathway for neo-allo-ocimene biosynthesis through the dehydration of geraniol in Hedychium coronarium [11], but this hypothesis lacks direct evidence. We mined the transcriptome of celery and successfully identified a novel monoterpene synthase, AgTPS40, which catalyzes the direct synthesis of neo-allo-ocimene. This finding demonstrates that neo-allo-ocimene in plants is synthesized directly by monoterpene synthases, rather than through the proposed geraniol pathway. This is the first time that neo-allo-ocimene synthase has been discovered, a breakthrough that has filled a gap in understanding neo-allo-ocimene biosynthesis.

Monoterpene synthases, in addition to catalyzing the formation of primary products, often produce multiple by-products. For example, the (E)-β-ocimene synthase from Arabidopsis thaliana synthesizes not only (E)-β-ocimene but also small amounts of (Z)-β-ocimene and myrcene [36]. Similarly, AgTPS40 primarily produces neo-allo-ocimene, but it also generates three by-products. Two of these by-products were identified as (Z)-β-ocimene and (E)-β-ocimene. The third product is likely allo-ocimene [(4E,6E)-allo-ocimene] based on mass spectral predictions, but its retention time pattern could not be matched with known standards [37]. Consequently, we were unable to make a definitive inference about this product. Due to its low yield, alternative identification methods could not be performed, and thus, the product remained unidentified. It could only be tentatively classified as a monoterpene based on its mass spectrum.

Currently, neo-allo-ocimene is primarily obtained through extraction from plant essential oils or chemical synthesis. However, these methods face significant natural resources constraints and may cause harm to the environment [11,38]. In this study, we selected E. coli BS1101 as the host and successfully achieved microbial synthesis of neo-allo-ocimene, reaching a titer as high as 933.35 mg/L. This represents the highest reported microbial production of this compound to date. Although the purity of the product has not yet reached a single component and further optimization is required, this work lays the foundation for the sustainable production of neo-allo-ocimene. By constructing a biosynthetic pathway using microorganisms as chassis, high yields of neo-allo-ocimene can be achieved without causing environmental harm.

5. Conclusions

In this study, a monoterpene synthase was identified from celery and confirmed as a neo-allo-ocimene synthase through GC-MS analysis combined with standards. A biosynthetic pathway for neo-allo-ocimene production was constructed by integrating the identified synthase with a vector encoding the heterologous MVA pathway. Using E. coli as the host, the fermentation process was optimized to achieve a final neo-allo-ocimene titer of 933.35 mg/L, which is currently the highest reported yield. In conclusion, this study not only fills the gap in the understanding of neo-allo-ocimene biosynthesis but also provides a new perspective for the production of high-value neo-allo-ocimene in engineered microbial systems.