Preface
As shown in the figure above, the design of our project is divided into two parts: the production of geraniol in S. cerevisiae, and the production of citronellol and linalool in E. coli. Accordingly, the project results will also be presented in these two aspects, plus a third part focusing on metabolic engineering optimization, with each part elaborated separately.
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1.Production of Geraniol by S. cerevisiae
1.1 Construction and expression of pYES2-GPPS-GES
The native MVA pathway in yeast cells is active. When the key enzyme GPPS is overexpressed, a relatively large amount of GPP can be synthesized. Subsequently, a substantial quantity of geraniol is produced under the catalysis of exogenous GES, and accumulates inside the cells and is later excreted into the extracellular medium. Therefore, the construction of overexpression vectors for GPPS and GES constitutes the foundation and prerequisite of this project.
We selected pYES2 (5.9 kb), the most commonly used expression vector in S. cerevisiae, which carries the URA3 selection marker but has only one multiple cloning site (MCS). To reduce the metabolic burden on yeast cells and enable the co-expression of GPPS and GES in cells, we linked these two genes via a ribosomal binding site (RBS) and constructed them together under the control of the galactose-inducible promoter PGAL1 (Fig. 1). This design allows the two genes to be transcribed simultaneously while being translated into two independent proteins.
Fig. 1 Construction and identification of pYES2-GPPS-GES recombinant vector using BamH I and EcoR I restriction enzymes.
M: Marker; 1: pYES2 plasmid; 2: pYES2-GPPS-GES recombinant plasmid; 3: Digestion of pYES2-GPPS-GES using BamH I and EcoR I; 4: PCR product of GPPS using pYES2-GPPS-GES as template; 5: PCR product of GES using pYES2-GPPS-GES as template.
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1.2 Knock down the ERG20 gene in the genome of S. cerevisiae
In yeast cells, endogenous geranyl pyrophosphate (GPP) can not only undergo pyrophosphate elimination to form geraniol, but also be converted into farnesyl pyrophosphate (FPP) under the catalysis of FPP synthase (encoded by the ERG20 gene) for the subsequent synthesis of other terpenoids. Notably, the endogenous metabolic flux of S. cerevisiae naturally favors the synthesis of FPP and sterols. To reduce this metabolic flux and enhance the conversion of GPP to geraniol, we used pUG6 plasmid to introduce a mutant FPP synthase (ERG20-F96W-N127W) into the S. cerevisiae genome, which replaced the wild-type ERG20 gene in situ while introducing the KanMX selection marker. The activity of the mutant FPP synthase is significantly reduced, retaining only the enzymatic activity necessary for maintaining cell viability (complete knockout of this gene is lethal).
After PCR amplification, the following fragments were ligated using the Gibson assembly method: vector backbone + upstream homology arm + KanMX expression cassette (containing the TEF1 promoter and terminator) + mutant ERG20 gene + downstream homology arm. The ligated product was transformed into E. coli; the extracted plasmid was verified by restriction enzyme digestion with EcoR I and Xho I (Fig. 2). Positive yeast clones were screened on plates containing G418. Additionally, the ERG20 genes from the wild-type and mutant strains were amplified separately via PCR using the same pair of homology arm primers for detection: the PCR product of the wild-type strain was approximately 2 kb (containing some homology arm sequence), while that of the mutant strain was approximately 3.7 kb (with the additional KanMX gene). The electrophoresis results are shown below:
Fig. 2 ERG20 gene was replaced by the mutant ERG20-F96W-N127W in the genome of S. Cerevisiae.
M: Marker; 1: Gene knockdown plasmid digested with EcoR I and Xho I (5.0 kb and 3.0 kb) ; 2: PCR product of wild-type ERG20 gene (2kb) using homology arm primers; 3: PCR product of mutant ERG20 gene (ERG20-F96W-N127W) (3.7 kb, containing KanMX gene) using homology arm primers.
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1. 3 Detection geraniol production using gas chromatography
To investigate whether yeast cells can synthesize geraniol after overexpressing GPPS and GES, gas chromatography (GC) was employed for analysis (Fig. 3). When the yeast cells grew to a certain concentration (OD₆₀₀ = 0.8), 2% galactose was used to induce the expression of GPPS and GES, followed by continued cultivation for 24 h. A two-phase extraction method was adopted: n-dodecane was used to extract geraniol from the culture, and geraniol standard was used as a control for analysis via gas chromatography. Two samples were included in the experiment: Sample 1 was yeast (without ERG20 gene knockdown) overexpressing GPPS and GES; Sample 2 was yeast (with ERG20 gene knockdown) overexpressing GPPS and GES.
Fig. 3 Gas chromatography analysis for geraniol production by S. cerevisiae.
The retention time of geraniol standard was 5.65 min; the retention time of geraniol produced by S. cerevisiae was 5.63 min.
The experimental results showed that the retention time of the geraniol standard was 5.65 min. Sample 1 contained little to no geraniol. Sample 2 exhibited a peak with a retention time (5.63 min) essentially consistent with that of the geraniol standard, indicating geraniol synthesis. The concentration of geraniol in Sample 2 was determined to be 8.58 mg/L. However, this concentration is relatively low, necessitating further optimization of the synthesis conditions.
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2. Production of citronellol and linalool by E. coli
2.1 Construction of pACYCDuet-1-GER-Lis plasmid
To prevent competition among multiple plasmids in E. coli, a high-copy multi-gene co-expression vector pACYCDuet-1 was selected. Two genes, GER and Lis, were cloned into two distinct multiple cloning sites (MCSs) of this plasmid. Both genes, however, are expressed under the control of the strong T7/lac promoter, enabling the "one-click activation" of the synthesis of multiple essential oil components. Results from PCR and restriction enzyme digestion confirmed the successful construction of the pACYCDuet-1-GER-Lis plasmid (Fig. 4).
Fig. 4 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 withNde I and Kpn I; 6: PCR product of GER using pACYCDuet-1-GER-Lis as template.
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2.2 Construction and identification of orthogonal combination plasmid-GER-Lis
After the successful construction of the recombinant plasmid pACYCDuet-1-GER-Lis, it was noted that although the two genes, under the control of the same strong T7/lac promoter, enable the "one-click activation" of the synthesis of two essential oil components, namely citronellol and linalool, separate regulation cannot be achieved.
This issue can be addressed using an orthogonal expression system, which allows the expression of two promoters to be induced by two different inducers on the same plasmid. A literature search revealed no such ready-to-use commercial plasmids, necessitating de novo construction.
We first cloned the GER gene into plasmid pET28a(+) (Fig. 5), which contains a LacI-IPTG regulated promoter, and then cloned the Lis gene into plasmid pBAD (Fig. 6), which contains an AraC-arabinose regulated promoter. These two regulatory systems are fully orthogonal and do not interfere with each other. Subsequently, they were fused using homologous recombination PCR (Fig. 7).
Fig. 5 Construction and identification of pET28a (+)-GER recombinant vector using Hind III and EcoR I restriction enzymes.
M: Marker; 1: pET28a (+) Plasmid; 2: pET28a (+) – GER plasmid; 3: Digestion pET28a (+) – GER with Hind III and EcoR I; 4: PCR product of GER using pET28a (+) – GER as template.
Fig. 6 Construction and identification of pBAD-Lis recombinant vector using Pst I and EcoR I restriction enzymes.
M: Marker; 1: pBAD Plasmid; 2: pBAD-Lis plasmid; 3: Digestion of pBAD-Lis plasmid with Pst I and EcoR I; 4: PCR product of Lis using pBAD-Lis as template.
Fig.7 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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2.3 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 purificationu 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. 8).
Fig.8 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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2.4 Detection the production of linalool and citronellol after transformation of Orthogonal combination plasmid-GER-Lis into 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 also used gas chromatography 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 gas chromatography using geraniol, citronellol, and linalool standards as controls (Fig. 9).
Fig.9 Gas chromatography 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.
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3. Optimization for Metablic Engineering
3.1 Reducing Product Cytotoxicity
The core challenge in microbial synthesis of monoterpenes is product toxicity. A common solution to this issue is the use of biphasic extraction fermentation (in-situ product removal, ISPR), which involves introducing a water-immiscible organic solvent as the second phase into the fermentation system. This allows real-time extraction of the produced terpenoids from the aqueous phase (culture medium), thereby maintaining the product concentration in the aqueous phase at a low-toxicity level and ensuring cell growth and metabolic activity.
Literature search revealed that researchers have used different organic phases for extraction, some of which are inherently toxic to microorganisms. To identify the most suitable solvent for extracting citronellol and linalool, we compared three substances: isopropyl myristate (IPM), n-dodecane, and n-hexane, each used as an organic overlay, to determine which performs best.
Induction of GER and Lis expression was initiated when the OD₆₀₀ of E. coli reached 0.8. Four hours after induction, 10% (v/v) of each of the three organic extraction solvents mentioned above was added for fermentation. Samples were taken every 12 h, totaling five samplings. The concentrations of citronellol and linalool in the organic phase, as well as the OD₆₀₀ and glucose concentration in the aqueous phase, were measured for each sample. The results are shown in the figure below (Fig. 10).
Fig.10 Comparative analysis for the citronellol and linalool production, OD600, and glucose in the medium of E. coli, using different extraction solvent.
(A): Concentration of extracted citronellol; (B): Concentration of extracted linalool; (C): OD600 value of E. coli; (D): Concentration of glucose in the medium.
By comparing the growth curves (OD₆₀₀) of the IPM group, n-dodecane group, n-hexane group, and the control group, it was found that the growth curves of the IPM group and n-dodecane group were similar to that of the control group, indicating good biocompatibility. In contrast, the growth rate of the n-hexane group was significantly reduced, suggesting that this solvent exerted a distinct toxic inhibitory effect on E. coli. Analysis revealed that n-hexane, with its small molecular size and strong hydrophobicity, tends to damage cell membranes, making it unsuitable as an extractant during fermentation. The IPM group and n-dodecane group showed similar product extraction efficiencies and good biocompatibility, making them suitable as in-situ extraction solvents for citronellol and linalool.
The characteristics of the three extractants were further analyzed by evaluating the biomass-specific productivity (= total yield/final OD₆₀₀ value) and product yield (= total mass of product formed/mass of glucose consumed) (Fig. 11).
Fig.11 Comparative Analysis of Biomass-Specific Productivity and Product Yield When Using Three Extractants.
(A): Biomass-specific productivity of citronellol; （B): Biomass-specific productivity of linalool; （C): Product yield of citronellol; （D): Product yield of linalool.
Comparative analysis revealed that n-hexane exhibited the highest biomass-specific productivity; however, further analysis showed that the product yields of the three extractants were nearly consistent. Therefore, the conclusion was drawn: IPM and n-dodecane are suitable for extracting products during fermentation, while n-hexane can be used as an extractant after fermentation.
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3.2 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 (Fif. 12). 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.12 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.12, we figure out that after 12 h of co-induction with a relatively high concentration of arabinose (1 mM – 10 mM, Fig. 12 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. 12 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 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 induce with different concentrations of IPTG for 12 h, during which the ratio of the two products was measured (Fig. 13).
Fig.13 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 6 h 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. 13 , 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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