AcNES-Nerolidol coding sequence

AcNES1: A Basic 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.

Part AcNES1 Introduction

Nerolidol Synthase (AcNES1)

The terpene synthase gene AcNES1 from Actinidia chinensis, which 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.

Usage and Biology

Design

Our initiative focuses on establishing a sustainable production process for nerolidol using engineered Saccharomyces cerevisiae strain BY4741. Nerolidol, a crucial sesquiterpene, is synthesized by a plasmid-encoded nerolidol synthase that transforms farnesyl diphosphate (FPP) into nerolidol. We first constructed the terpene synthase gene into the backbone vectors and then constructed shuttle plasmids with various combinations of promoters and terminators using Golden Gate assembly. After transforming Saccharomyces cerevisiae and performing yeast colony PCR, we typically employ a two-phase fermentation system for terpenoid production. We then test terpenoid production using gas chromatography–mass spectrometry.

Figure 3: HcKan-O Vector

Figure 4: HcKan-O-AcNES1

Figure 5: POT vector

Figures 3, 4, and 5 represent our HcKan-O Vector, HcKan-O-AcNES1, and POT Vector, which are commonly used across all three constructed plasmids. The promoters, terminators, and the final POT constructs for each of the three plasmids are shown in Table 1.

Table 1: Constructed Plasmid Systems for AcNES1 Expression

Construct System	Promoter	Terminator	Constructed Plasmid
POT2_pINO1_AcNES1_TEF1t System	Figure 6: pINO1	Figure 7: TEF1t	Figure 8: POT2_pINO1_AcNES1_TEF1t
POT2_pTDH3_AcNES1_ADH1t System	Figure 9: pTDH3	Figure 10: ADH1t	Figure 11: POT2_pTDH3_AcNES1_ADH1t
POT2_pGPM1_AcNES1_PGK1t System	Figure 12: pGPM1	Figure 13: PGK1t	Figure 14: POT2_pGPM1_AcNES1_PGK1t

Build

Method:By using the Golden Gate assembly method, we first cloned these genes into a backbone vector called Kan-O. Next, we selected appropriate promoter and terminator combinations to regulate the expression of these genes and constructed into shuttle vectors. Following this, we validated the constructs through colony PCR and sequencing to ensure accurate assembly. After confirmation, we transformed the vectors into Saccharomyces cerevisiae (yeast) and conducted 120-hour two-phase fermentation in shaking flasks. We then extracted the organic phase and used GC-MS analysis to detect the presence and quantity of the target terpenes.

Figure 15: Experimental operation procedure

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 bacterial liquid was sequencing subsequently to confirme the successful construction of the plasmid.

Colony PCR

We successfully constructed gene AcNES1 into backbone vector HcKan-O, and the shuttle for AcNES1 constructed with four combinations of promoters and terminators: pGPM1 with PGK1t, pINO1 with TEF1t, pTDH3 with ADH1t, and pGAL1 with PGK1t, as confirmed by colony PCR and sequencing results.

Figure 16: Single colonies of plasmid transformants on LB agar plates containing ampicillin or kanamycin

Figure 17: DH5α E.coli colony PCR and enzymatic digestion results of AcNES1 A: HcKan-O-AcNES1. B: enzymatic digestion results of HcKan-AcNES1 and POT2_pGPM1_AcNES1_PGK1t C: POT2_pTDH3_AcNES1_ADH1t D: POT2_pINO1_AcNES1_TEF1t E: POT2_pGPM1_AcNES1_PGK1t (M=DL5000 marker）

Figure 18: DH5α E.coli colony PCR results of POT2_pGAL1_AcNES1_PGK1t. (M=DL5000 marker）

The sequencing result:

Figure 19: Strain 4 and 8 have been confirmed positive by sequencing

Figure 20: Strain 3 and 10 have been confirmed positive by sequencing

Figure 21: Strain 2 and 4 have been confirmed positive by sequencing

Figure 22: Strain 1 has been confirmed positive by sequencing

Figure 23: Strain 2 and 6 have been confirmed positive by sequencing

Transformation and Yeast colony PCR: We utilized a chemical transformation method to introduce the constructed plasmid into Saccharomyces cerevisiae. Following the kit instructions, we prepared competent yeast cells and performed the transformation. The transformed yeast was then plated on SC-URA agar plates for cultivation. We selected single colonies for streaking culture, followed by yeast colony PCR and fermentation. The results of the yeast colony PCR confirmed that two plasmids, POT2_pGPM1_AcNES1_PGK1t and POT2_pTDH3_AcNES1_ADH1t, tested positive in Saccharomyces cerevisiae.

Yeast colony PCR results:

Figure 24: Yeast colony PCR result of nerolidol performed to screen strain. A: BY4741-POT2_pGPM1_AcNES1_PGK1t. B: BY4741-POT2_pTDH3_AcNES1_ADH1t. C: BY4741-POT2_pINO1_AcNES1_TEF1t.（M=DL5000 marker；P=Positive; N=Negative）

Figure 25: Yeast colony PCR result of BY4741-POT2_pGAL1_AcNES1_PGK1t performed to screen strain.（M=DL5000 marker；P=Positive; N=Negative）

Figure 26: Yeast single colonies of plasmid transformants on SC-URA plate

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. The results showed that Saccharomyces cerevisiae carrying the empty shuttle plasmid produced 1.18 ± 0.14 mg/l of nerolidol, flask fermentation of AcNES1 yielded 45.06 ± 1.68 mg/l of nerolidol with the pTDH3 promoter and ADH1t terminator, 12.92 ± 1.37 mg/l with the pGPM1 promoter and TEF1t terminator, and 4.78 ± 0.88 mg/l of nerolidol with the pGAL1 promoter and PGK1t terminator. We conducted three parallel experiments for this part, and the OD values and nerolidol production data are in table 1. 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.

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

ID	12h OD600	120h OD600	Yield (µg/L)
BY4741-POT	4.14	5.49	1027.64
BY4741-POT	4.14	5.44	1301.40
BY4741-POT	4.14	4.88	1209.89
BY4741-POT-pTDH3-AcNES1-ADH1t	3.96	4.301	44481.37
BY4741-POT-pTDH3-AcNES1-ADH1t	3.98	4.444	43750.43
BY4741-POT-pTDH3-AcNES1-ADH1t	3.89	4.464	46956.02
BY4741-POT-pGal1-AcNES1-PGK1t(Glu)	3.66	4.33	478.95
BY4741-POT-pGal1-AcNES1-PGK1t(Glu)	3.63	4.41	384.99
BY4741-POT-pGal1-AcNES1-PGK1t(Glu)	3.62	4.57	484.32
BY4741-POT-pGal1-AcNES1-PGK1t(Gal)	4.49	4.99	4756.69
BY4741-POT-pGal1-AcNES1-PGK1t(Gal)	3.88	4.83	3907.79
BY4741-POT-pGal1-AcNES1-PGK1t(Gal)	4.15	4.81	5666.31
BY4741-POT-pGPM1-AcNES1-PGK1t	4.08	4.53	12222.56
BY4741-POT-pGPM1-AcNES1-PGK1t	4.07	4.28	14501.07
BY4741-POT-pGPM1-AcNES1-PGK1t	4.05	4.38	12043.18

Figure 27: Heterologous expression of AcNES1 in S. cerevisiae. A: GC-MS analyses of nerolidol extracts of S. cerevisiae expressing AcNES1 with pTDH3 promoter and ADH1t terminator. B: GC-MS analyses of nerolidol extracts of S. cerevisiae expressing AcNES1 with the pGPM1 promoter and PGK1t terminator.

Figure 28: Heterologous expression of AcNES1 in S. cerevisiae A: GC results of AcNES1 with glucose under the control of the pGAL1 promoter and PGK1t terminator. B: GC results of AcNES1 with galactose under the control of the pGAL1 promoter and PGK1t terminator.

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 29: 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. By employing various promoter-terminator combinations, we observed a significant impact on nerolidol production. The AcNES1 gene with the pTDH3 promoter and ADH1t terminator achieved the highest yield of 45.06 ± 1.68 mg/L, compared to 12.92 ± 1.37 mg/L using the pGPM1 promoter and TEF1t terminator. The GAL1 promoter is recognized for its strength in driving high levels of enzyme expression, facilitating the conditional release of terpenes from Saccharomyces cerevisiae. Under induction conditions with galactose as the carbon source, the yield reached 4.78 mg/L, while using glucose resulted in a much lower yield of 0.45 mg/L, which was still below the 1.18 ± 0.14 mg/L produced by S. cerevisiae carrying the empty shuttle plasmid. Despite the galactose-inducible expression, the yield remained significantly lower than that achieved with the constitutive promoter (45.06 mg/L).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.

At the same time, we utilized AlphaFold3 to predict the protein structure expressed by the AcNES1 gene. This provided us with a detailed protein model, laying a solid foundation for potential optimization work in the future.</p>

Figure 30: Protein Structure

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

Assembly Compatibility:

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COMPATIBLE WITH RFC[10]
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COMPATIBLE WITH RFC[12]
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COMPATIBLE WITH RFC[21]
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COMPATIBLE WITH RFC[23]
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COMPATIBLE WITH RFC[25]
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COMPATIBLE WITH RFC[1000]