1. Introduction
Geraniol reductase catalyzes the conversion of geraniol to citronellol, both of which are the main componants of rose essential oil. Linalool isomerase catalyzes the conversion of geraniol to linalool, which is the main componant of lavender essential oil.
Fig.1 The conversion of geraniol to citronellol and linalool catalyzed by geraniol reductase and linalool isomerase respectively.
The goal of our project is to enable E. coli to produce citronellol and linalool in any ratio using geraniol (synthesized by yeast), so as to meet the needs of different users for different ratios of these two essential oil components.
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2. The First-Generation Part pACYCDuet-1-GER-Lis
2.1 Design of pACYCDuet-1-GER-Lis
To prevent competition among multiple plasmids in E. coli, a high-copy multi-gene co-expression vector pACYCDuet-1 was selected. Two genes, geraniol reductase (GER) and linalool isomerase (Lis), were designed to clone into two distinct multiple cloning sites (MCSs) of this plasmid. Both of them are expressed under the control of the strong T7/lac promoter, enabling the "one-click activation" of the synthesis of multiple essential oil components.
Fig.2 GER and Lis genes were constructed into the plasmid of pACYCDuet-1, followed by transformation into E. coli.
Geraniol Reductase, GER, Linalool isomerase, Lis.
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2.2 Construction of pACYCDuet-1-GER-Lis
Prior to gene synthesis, we optimized the gene sequences based on the codon preference of E. coli. Results from PCR and restriction enzyme digestion confirmed the successful construction of the pACYCDuet-1-GER-Lis plasmid.
Fig.3 Construction and identification of pACYCDuet-1-GER-Lis recombinant vector using EcoR I, Pst I, Nde I and Kpn I restriction enzymes.
M: Marker; 1: pACYCDuet-1 Plasmid; 2: pACYCDuet-1-GER-Lis; 3: Digestion of pACYCDuet-1-GER-Lis with EcoR I and Pst I; 4: PCR product of GER using pACYCDuet-1-GER-Lis as template; 5: Digestion of pACYCDuet-1-GER-Lis with Nde I and Kpn I; 6: PCR product of GER using pACYCDuet-1-GER-Lis as template.
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2.3 Measurement of pACYCDuet-1-GER-Lis
The constructed recombinant plasmid pACYCDuet-1-GER-Lis was then transformed into competent BL21 cells. Under the induction with IPTG, the strain can efficiently express two enzymes (GER and Lis) which are used for the synthesis of citronellol and linalool respectively.
Although the "one-click activation" synthesis can be achieved since both GER and Lis genes are co-controlled by the T7 promoter and IPTG induction, it fails to meet the requirement for regulating the personalized production of different essential oil components, namely citronellol and linalool.
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3. The Second-Generation Part Orthogonal combination plasmid-GER-Lis
To address the problem above, we optimized the previous design using an orthogonal expression system to enable separate control over the synthesis of different essential oil components.
The core of this design lies in the use of two orthogonal promoter-inducer systems. The term "orthogonal" means there is no cross-reactivity between the two systems: inducer A only activates promoter A without affecting promoter B, and vice versa. However, there is currently no "ready-to-use" commercial single plasmid that directly integrates two such promoters on a single plasmid, so we have to construct an orthogonal combinatorial plasmid ourselves.
The T7/lac system consists of the T7 promoter and the repressor protein LacI, which is induced by IPTG to achieve high expression levels and is one of the most commonly used expression systems. The araBAD (pBAD) system comprises the pBAD promoter and the repressor protein AraC, which is induced by L-arabinose. It offers highly precise regulation, with induction levels linearly adjustable according to arabinose concentration, and is completely orthogonal to the T7/lac system. Therefore, we plan to construct an orthogonal combinatorial plasmid containing both T7/lac and pBAD promoters for expression of GER and Lis genes.
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3.1 Design of the orthogonal combination plasmid-GER-Lis
The procedure was performed in two steps: first, two recombinant plasmids, pET28a(+)-GER and pBAD-Lis, were constructed. pET28a(+) contains the T7/lac promoter and is induced for expression by IPTG; pBAD contains the araBAD promoter and is induced for expression by L-arabinose. Subsequently, the fusion of the two plasmids, namely the orthogonal combination plasmid-GER-Lis, was constructed using homologous recombination PCR. This plasmid contains KanR, GER, T7/lac promoter and LacI genes from pET28a(+)-GER, as well as AraC, pBAD promoter, Lis，ApR genes，and Ori site from pBAD-Lis, with a total length of 9926 base pairs.
Fig.4 Construction of Orthogonal combination plasmid-GER-Lis using recombinant PCR method.
Geraniol Reductase, GER, Linalool isomerase, Lis.
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3.2 Construction and identification of orthogonal combination plasmid-GER-Lis
The construction and identification of the orthogonal combination plasmid-GER-Lis were showed as follows:
Fig.5 Construction and identification of Orthogonal combination plasmid-GER-Lis using homologous recombination PCR method.
M: Marker; 1: PCR product of pET28a(+)-GER containing KanR, GER, T7/lac promoter and LacI genes, etc (4.4kb) ; 2: PCR product of pBAD-Lis containing AraC, pBAD promoter, Lis, ApR genes and Ori site (5.5kb) ; 3: Orthogonal combination plasmid-GER-Lis; 4: PCR product of GER using Orthogonal combination plasmid-GER-Lis as template; 5: PCR product of Lis using Orthogonal combination plasmid-GER-Lis as template.
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3.3 Expression of GER and Lis in E. coli tranformed with orthogonal combination plasmid-GER-Lis
To verify the transformation of the orthogonal combination plasmid-GER-Lis in E. coli, protein purification using His-tag and SDS-PAGE electrophoresis were performed on the overexpressed GER and Lis proteins. The results showed that the plasmid was well-expressed in E. coli, and the amounts of GER (46.8 kDa) and Lis (76.1 kDa) proteins were significantly higher than those in the control group.
Fig.6 SDS-PAGE indicates the expression and purification of GER and Lis expressed in E. coli transformed with Orthogonal combination plasmid-GER-Lis.
M: protein marker; 1: The supernatant obtained by centrifuging the total lysate of transformed cells; 2. The supernatant obtained by centrifuging the lysate of untransformed cells; 3. The residual liquid remaining after purifying the overexpressed proteins from the lysate of transformed cells using a His-tag column; 4. The purified overexpressed GER (46.8kD) and Lis (76.1kD) proteins.
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3.4 Measurement of orthogonal combination plasmid-GER-Lis in E. coli
Although we verified that the GER and Lis genes can be overexpressed in BL21 cells, further validation is required to confirm whether the expressed enzymes are active and capable of converting geraniol into citronellol and linalool. We used gas chromatography (GC) for analysis.
We are aware that overexpression of exogenous genes imposes a burden on cells, so we selected inducible plasmids. When E. coli grew to an OD₆₀₀ of 1.0, 100 µM IPTG and 500 µM L-arabinose were used to simultaneously induce the expression of GER and Lis. Additionally, 200 mM geraniol standard was added every two hours to a final concentration of 50 mg/L, followed by continued fermentation for 24 h. The culture was overlaid with n-dodecane to extract terpenoids, which were analyzed by GC using geraniol, citronellol, and linalool standards as controls.
Fig.7 GC analysis for geraniol, citronellol and linalool production by E. coli. The retention times of geraniol, citronellol, and linalool standards were 5.64 min, 5.16 min, and 3.26 min, respectively. The retention times of these components in the samples are essentially consistent with those of the standard substances..
From the GC results, we observed that in addition to the expected main peaks of geraniol, citronellol, and linalool, other miscellaneous peaks appeared. However, we are unaware of the identity of these substances, which requires further in-depth investigation.
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3.5 Optimization of inducer concentration and induction times
Our target final products are mixed essential oil components containing citronellol and linalool in different proportions, which can be produced in a single fermation process. The achievement of this goal mainly depends on the activity ratio of the key enzymes responsible for synthesis of these two substances, as they share the same substrate, geraniol. These two key enzymes (GER and Lis) are constructed under the control of two distinct promoters (pT7/lac and pBAD) on the same plasmid, with their expression induced by IPTG and L-arabinose respectively. These two promoters form a fully orthogonal regulatory system (non-interfering regulation).
However, the induction efficiencies of these two fully orthogonal regulatory systems differ, leading to variations in the expression levels of the enzymes and the catalytic efficiency of product synthesis. Additionally, there exists competition between the two enzymes for substrate utilization and carbon source consumption.
Therefore, to determine the working efficiency of the two promoters, we conducted experiments on the combined induction of expression using different concentrations of IPTG and L-arabinose. The efficiency was evaluated based on the ratio of the product concentrations of citronellol (synthesized under IPTG induction) to linalool (synthesized under L-arabinose induction).
Fig.8 The measured product ratio of citronellol to linalool after 12 h of combined induction with different concentrations of IPTG and L-arabinose.
The thermodynamic diagram (heatmap) shows the ratio of two products induced by IPTG and Arabinose at different concentrations. Different colors are used in the figure to represent different product ratios, with darker colors indicating lower ratios and lighter colors indicating higher ratios. A: Induced production ratio with higher arabinose concentration; B: Induced production ratio with lower arabinose concentration.
Comparing the heatmap of A and B in Fig.8, we figure out that after 12 h of co-induction with a relatively high concentration of arabinose (1 mM – 10 mM, Fig. 8 A) and a relatively low concentration of IPTG (0.1 mM – 1.0 mM), the product ratio (citronellol:linalool) fluctuates within the range of 1 to 10. In contrast, after 12 h of co-induction with a relatively low concentration of arabinose (0.1 mM–2.5 mM, Fig. 8 B) and a relatively low concentration of IPTG (0.1 mM – 1.0 mM), the product ratio (citronellol:linalool) fluctuates within the range of 0.1 to 3. The latter is more likely to meet the needs of most users for different proportions of these two components.
Next, we designed two additional experiments involving sequential induction with the two inducers: Experiment A: First, induce expression with 0.4 mM IPTG for 6 h then co-induce with different concentrations of arabinose for 12 h, during which the ratio of the two products was measured. Experiment B: First, induce expression with 1 mM arabinose for 6 h, then co-induce with different concentrations of IPTG for 12 h, during which the ratio of the two products was measured.
Fig.9 The Effect of Different Induction Orders of IPTG and Arabinose on the Ratio of the Two Products.
A: The thermodynamic plot (heatmap) illustrates the effects of different arabinose concentrations and induction durations on the product ratio after 6h of IPTG (0.4 mM) induction. B: The thermodynamic plot (heatmap) shows the effects of different IPTG concentrations and induction durations on the product ratio after 6 h of Arabinose (1 mM) induction. In the plot, different colors represent different product ratios: darker colors indicate lower ratios, while lighter colors indicate higher ratios.
As can be seen from Fig. 9 above, even if the two inducers have different induction timings (i.e., induced sequentially), the final desired product ratio can still be achieved by adjusting the concentrations of the inducers.
From the above figures, the optimal configuration can be identified according to the requirements for different product ratios. It is expected that these data will provide support for the personalized production of essential oil components with different proportions.
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4. Summary
In conclusion, through iterative design and optimization, we have successfully constructed an orthogonal expression plasmid. This plasmid contains two non-interfering promoter control systems, which can respectively induce the expression of two different genes (GER and Lis) under the regulation of two different inducers. After these two genes are expressed, they can catalyze the synthesis of citronellol and linalool with their respective efficiencies, so as to meet the needs of different users for the ratio of the two essential oil components.
In the future, we plan to integrate machine learning algorithms with real-time metabolic flux analysis to establish a dynamic mapping model between user preferences and fermentation parameters. Users only need to input their preference for the aroma ratio of rose to lavender (e.g., 7:3 or 5:5), and the system will automatically optimize culture parameters and induction parameters to precisely allocate carbon flow to target products.
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