Part:BBa_K5217038
pTDH3_AcNES1_ADH1t
BY4741-POT2_pTDH3_AcNES1_ADH1t: A Composite Part for Enhanced Production of Nerolidol in Saccharomyces cerevisiae
Nerolidol Introduction
Nerolidol is a naturally occurring sesquiterpene alcohol renowned for its fresh fruity and woody scent, found widely in essential oils of orange blossoms, ginger, and lavender. As a volatile organic compound, nerolidol plays a significant role in the scent profile of these plants and is extensively used in perfumes, flavorings, and medicinal products.
Chemical Formula: C₁₅H₂₆O
Molecular Weight: 222.37 g/mol
- Figure 1: Nerolidol 3D Conformer.
Biosynthesis
The enzyme responsible for converting precursors to nerolidol is called nerolidol synthase. This enzyme utilizes the terpene precursor farnesyl diphosphate (FPP) to produce nerolidol through a specific enzymatic reaction:
Farnesyl diphosphate → Nerolidol + Diphosphate
In yeast (such as Saccharomyces cerevisiae), the MVA pathway is crucial for the biosynthesis of FPP, which subsequently serves as a substrate for nerolidol synthase. The introduction of nerolidol synthase genes in laboratory conditions has been proven to successfully produce nerolidol, demonstrating the feasibility of this method in both research and commercial applications.
- Figure 2: MVA Pathway.
Applications of Nerolidol
Fragrances and Perfumes
Nerolidol has a fresh, floral, and woody scent, making it widely used in perfumes, soaps, and body washes. Its natural origin and unique aroma make it a common ingredient in high-end perfumes and cosmetic products.
Food and Beverage Additive
Nerolidol is used as a natural flavoring agent in food and beverages, especially in teas, candies, and certain drinks, providing a distinctive aroma and flavor. Its natural safety profile makes it an increasingly preferred option as a food additive.
Medicinal Uses
Nerolidol shows great potential in the pharmaceutical field due to its anti-inflammatory, antioxidant, and antimicrobial properties. It is gaining attention in the health and wellness industry, particularly in natural therapies and supplements.
Introduction to the Components of BY4741-POT-pTDH3-AcNES1-ADH1t
pTDH3 Promoter (pTDH3)
The pTDH3 promoter is derived from the TDH3 gene, which encodes the key enzyme in the glycolytic pathway glyceraldehyde-3-phosphate dehydrogenase. This promoter is commonly used in yeast for metabolic engineering applications due to its ability to drive high levels of protein expression, maximizing production of target metabolic products such as nerolidol.
Nerolidol Synthase (AcNES1)
AcNES1 encodes the nerolidol synthase enzyme, a critical component in the biosynthesis of nerolidol from the precursor farnesyl diphosphate (FPP). This enzyme catalyzes the conversion of FPP into nerolidol through a specific enzymatic reaction.
ADH1 Terminator (ADH1t)
The ADH1 terminator is derived from the gene encoding alcohol dehydrogenase 1, which plays a significant role in the ethanol metabolic pathway. Terminators are crucial for ensuring proper transcriptional termination and polyadenylation signaling in mRNA processing. The ADH1 terminator is known for its stable and efficient transcriptional termination performance, thus enhancing the overall expression stability of the upstream gene.
Rationale for the Construction of BY4741-POT-pTDH3-AcNES1-ADH1t
The design of BY4741-POT-pTDH3-AcNES1-ADH1t is aimed at optimizing nerolidol production in yeast. The goal is to enhance the efficiency of precursor conversion to nerolidol, simplify the biosynthetic process, reduce metabolic burden, and increase yield.
Usage and Biology
Design
Our initiative focuses on establishing a sustainable production process for nerolidol using engineered Saccharomyces cerevisiae BY4741. Nerolidol, a crucial sesquiterpene, is synthesized by a plasmid-encoded nerolidol synthase that transforms farnesyl diphosphate (FPP) into nerolidol. The plasmid selected for this project is HcKan-O.
- Figure 3: HcKan-O Vector.
The three components selected for our project are as shown in the diagram below.
- Figure 4: pTDH3.
- Figure 5: ADH1t.
- Figure 6: HcKan-O-AcNES1.
The final construction results are shown in Figure 7
- Figure 7: POT2_pTDH3_AcNES1_ADH1t.
Build
Plasmid Construction: Due to the rapid growth rate of E. coli and the convenience of molecular cloning, we conducted the plasmid construction and verification in E. coli. Initially, we cloned the AcNES1 gene into the HcKan-O vector using the Golden Gate assembly method. Once constructed, the resulting plasmid was transformed into E. coli DH5α and spread onto antibiotic-selective agar plates for cultivation. Colony PCR was utilized to screen the transformed bacteria initially, and gel electrophoresis analyzed the PCR products to confirm the construction's success. Bands of the expected size were excised, and the DNA was subsequently purified. This purified DNA was sent for sequencing to verify the accuracy of the gene sequence. The sequencing results confirmed the successful construction of the plasmid.
- Figure 8: POT2_pTDH3_AcNES1_ADH1t Colony PCR result.
Transformation: We employed a chemical transformation method to introduce the constructed plasmid into Saccharomyces cerevisiae. First, we purified the constructed plasmid to ensure its quality was suitable for transformation. Next, the yeast cells were treated with calcium chloride (CaCl₂) to temporarily increase the permeability of the cell membranes, facilitating the plasmid's entry into the cells. The purified plasmid was then mixed with the treated yeast cells and incubated under optimal conditions to promote plasmid uptake. Finally, the treated yeast cells were plated onto selective agar plates containing antibiotics to cultivate and select successfully transformed cells. Through this process, we successfully introduced the target plasmid into Saccharomyces cerevisiae, setting the stage for subsequent functional testing and expression analysis.
Test
In test part, we utilized GC-MS (Gas Chromatography-Mass Spectrometry) to analyze the products of engineered Saccharomyces cerevisiae after 120 hours of fermentation. GC-MS is an analytical technique that separates different components of a mixture using gas chromatography, then identifies and quantifies these components using mass spectrometry. Additionally, we monitored the growth of the yeast, recording OD600 values from 0 to 48 hours to plot the growth curve.
- Figure 9: BY4741-POT2_pTDH3_AcNES1_ADH1t GC-MS result.
We conducted three parallel experiments for this part, and the OD values and nerolidol production data are in Table 1.
Table 1: OD600 and Nerolidol Production Results
| Experiment Number | 120h OD600 | Nerolidol Production (μg/L) |
|---|---|---|
| 1 | 4.301 | 44481.37 |
| 2 | 4.444 | 43750.43 |
| 3 | 4.467 | 46956.02 |
These results indicate consistent growth and production across the experiments, demonstrating the stability and efficiency of the engineered yeast strain in producing nerolidol under controlled conditions.
- Figure 10: The OD600 and nerolidol yield by AcNES1 under the control of different promoters and terminators. The data represent the mean ± standard deviation of three independent experiments.
Learn
The experimental results from this project indicate that the engineered Saccharomyces cerevisiae BY4741 successfully expressed the nerolidol synthase and stably produced nerolidol during fermentation. These findings validate the effectiveness of using specific promoter and terminator combinations and demonstrate the potential of this approach in producing high-value terpenes. With the rapid advancements in synthetic biology and metabolic engineering, this composite part has the potential to be further optimized for industrial-scale biosynthesis of nerolidol, not just at laboratory scale. Additionally, this technology platform can be expanded to the production of other terpenes
Reference
[1]Li W, Yan X, Zhang Y, Liang D, Caiyin Q, Qiao J. Characterization of trans-Nerolidol Synthase from Celastrus angulatus Maxim and Production of trans-Nerolidol in Engineered Saccharomyces cerevisiae. J Agric Food Chem. 2021 Feb 24;69(7):2236-2244. doi: 10.1021/acs.jafc.0c06084. Epub 2021 Feb 15. PMID: 33586967.
[2]De Carvalho RBF, De Almeida AAC, Campelo NB, Lellis DROD, Nunes LCC. Nerolidol and its Pharmacological Application in Treating Neurodegenerative Diseases: A Review. Recent Pat Biotechnol. 2018;12(3):158-168. doi: 10.2174/1872208312666171206123805. PMID: 29210667.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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