Coding

Part:BBa_K5217009

Designed by: CHUQIAO LIN   Group: iGEM24_BGI-MammothEdu-East   (2024-09-25)

CcOCS-Ocimene coding sequence

Ocimene Synthase: Enzymatic Production of Ocimene in Yeast

Background and Principles

Ocimene is a naturally occurring monocyclic terpene known for its sweet, herbal, and woody aroma, prevalent in the essential oils of various plants such as basil, mint, and parsley. As a volatile organic compound, ocimene contributes significantly to the scent profile of these plants and is used extensively in the fragrance and flavor industries.

Chemical Formula: C₁₀H₁₆

Molecular Mass: 136.24 g/mol

Figure 1: Ocimene Structural Formula.

In natural environments, ocimene serves as a key aromatic compound, primarily obtained through extraction from plant essential oils. However, we utilize engineered Saccharomyces cerevisiae for its production, which offers several distinct advantages: it reduces dependence on plant resources and minimizes ecological impacts. Additionally, by optimizing production conditions, this method enhances yield and reduces costs, providing a more sustainable and economically efficient production alternative for industrial applications.

Biosynthesis

The enzyme responsible for catalyzing the conversion of precursors to ocimene is known as ocimene synthase. This enzyme leverages geranyl pyrophosphate (GPP), a universal precursor for monoterpenoids, to produce ocimene through a specific enzymatic reaction:

Geranyl pyrophosphate → Ocimene + diphosphate

In yeast (e.g. Saccharomyces cerevisiae), the mevalonate pathway is crucial for the biosynthesis of GPP, which subsequently serves as a substrate for ocimene synthase. Under laboratory conditions and with the introduction of the ocimene synthase gene, engineered yeast strains have been shown to successfully produce ocimene, demonstrating the feasibility of this approach for both research and commercial applications.

Figure 2: MVA Pathway.

Application

The (E)-β-ocimene isomer is particularly noted for its pleasant aroma, resembling that of wood and fresh floral notes, making it a valuable component in the formulation of various commercial products ranging from perfumes to flavored beverages. The integration of ocimene synthase into yeast provides a bio-based method to produce this terpene, potentially reducing reliance on plant extraction methods and enhancing the sustainability of its production.

Usage and Biology

Design

Our project aims to develop a sustainable production method for ocimene, a key aromatic terpene, using engineered Saccharomyces cerevisiae BY4741. To accomplish this, we are focusing on the construction of a plasmid that expresses the ocimene synthase enzyme, which catalyzes the conversion of geranyl diphosphate (GPP) to ocimene. To ensure the proper expression of the ocimene synthase, we have designed two different sets of promoter and terminator combinations for the construction of our engineering elements. These include the pINO1-TEF1t,pTDH3-ADH1t and pGPM1-PGK1t systems. Additionally, we have compared the wild-type strain with our engineered Saccharomyces cerevisiae strains to determine whether the introduction of ocimene synthase can significantly enhance ocimene production.

Figure 3: HcKan-O Vector

Figure 4: HcKan-O-CcOCS

Figure 5: POT vector

Figures 3, 4, and 5 represent our HcKan-O Vector, HcKan-O-CcOCS, 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 CcOCS Expression

Construct System Promoter Terminator Constructed Plasmid
POT2_pINO1_CcOCS_TEF1t System
Figure 6: pINO1
Figure 7: TEF1t
Figure 8: POT2_pINO1_CcOCS_TEF1t
POT2_pTDH3_CcOCS_ADH1t System
Figure 9: pTDH3
Figure 10: ADH1t
Figure 11: POT2_pTDH3_CcOCS_ADH1t
POT2_pGPM1_CcOCS_PGK1t System
Figure 12: pGPM1
Figure 13: PGK1t
Figure 14: POT2_pGPM1_CcOCS_PGK1t



Build

Gene Source: After reviewing relevant literature, we selected the CcOCS gene as the template for the ocimene synthase.

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 fast growth rate of E.coli and the ease of molecular cloning, we decide to construct and verify plasmids in E.coli. The gene CcOCS encoding ocimene synthase from C.camphora and were synthesized by Gene Synthesis Platform according to the codon bias of S. cerevisiae and ligated into the plasmid, we then assembled it into vector HcKan-O by golden gate assembly, and then constructed with promoters (pTHD3, pGPM1, pINO1) and terminators (ADH1t, PGK1t, TEF1t) into shuttle plasmid vector.


Figure 16: Single colonies of plasmid transformants on LB Ampicillin plate

Figure 17: Colony PCR results performed to screen strain HcKan-O-CcOCS.（M=DL5000 marker；P=Positive; N=Negative）

Figure 18: Colony PCR results performed to screen strain. A: POT2_pGPM1_CcOCS_PGK1t, B: POT2_pINO1_CcOCS_TEF1t, C: POT2_pTDH3_CcOCS_ADH1t.（M=DL5000 marker；P=Positive; N=Negative）

Figure 19: Strain 2 and 16 have been confirmed positive by sequencing

Figure 20: Strain 4 and 7 have been confirmed positive by sequencing

Figure 21: Strain 6 and 8 have been confirmed positive by sequencing

Figure 22: Strain 1 and 5 have been confirmed positive by sequencing

Transformation and yeast colony PCR

We employed a chemical transformation method to introduce the constructed plasmid into Saccharomyces cerevisiae. We prepared competent yeast cells according to the kit instructions and performed transformation. We spreaded the transformed yeast on SC-URA agar plates for cultivation, then selected single colonies for streaking culture, followed by yeast colony PCR and fermentation. The results showed that we successfully transformed the plasmids POT2_pINO1_CcOCS_TEF1t and POT2_pTDH3_CcOCS_ADH1t into the yeast, while the verification for the plasmid POT2_pGPM1_CcOCS_PGK1t failed. We then conducted heterologous expression of CcOCS using different promoters and terminators in Saccharomyces cerevisiae, with fermentation lasting 120 hours.

Figure 23: Yeast single colonies of plasmid transformants on SC-URA plate

Figure 24: Yeast colony PCR result performed to screen strain A: BY4741-POT2_pGPM1_CcOCS_PGK1t, B: BY4741-POT2_pINO1_CcOCS_TEF1t, C: BY4741-POT2_pTDH3_CcOCS_ADH1t.（M=DL5000 marker；P=Positive; N=Negative）

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 no ocimene. In contrast, when the shuttle plasmid containing the ocimene synthase gene was expressed in yeast, the titer reached 0.83 ± 0.06 mg/L using the pINO1 promoter and TEF1t terminator, while it was 0.33 ± 0.09 mg/L with the pTDH3 promoter and ADH1t terminator. We conducted three parallel experiments for this part, and the OD600 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 ocimene under controlled conditions.

Table 2: Test results for CcOCS Expression

ID 12h OD600 120h OD600 Ocimene Yield (µg/L)
BY4741-POT 4.14 5.49 0
BY4741-POT 4.14 5.44 0
BY4741-POT 4.14 4.88 0
BY4741-POT-pINO1-CcOCS-TEF1t 3.85 5.67 901.02
BY4741-POT-pINO1-CcOCS-TEF1t 4.81 5.58 780.50
BY4741-POT-pINO1-CcOCS-TEF1t 4.46 5.22 807.78
BY4741-POT-pTDH3-CcOCS-ADH1t 5.33 5.59 242.19
BY4741-POT-pTDH3-CcOCS-ADH1t 4.35 4.61 409.94
BY4741-POT-pTDH3-CcOCS-ADH1t 4.86 5.17 265.27
BY4741-POT-pTDH3-CcOCS-ADH1t 4.76 4.65 398.95


Figure 25: Heterologous expression of CcOCS in S. cerevisiae. A: GC-MS analyses of ocimene extracts of S. cerevisiae expressing CcOCS with pTDH3 promoter and ADH1t terminator. B: GC-MS analyses of ocimene extracts of S. cerevisiae expressing CcOCS with the pINO1 promoter and TEF1t terminator.

Figure 26: Comparison of different promoter and terminator combinations in yeast production and growth.

Learn

In this study, our primary objective was to produce ocimene using engineered Saccharomyces cerevisiae BY4741 strains. We constructed three different promoter and terminator combinations in shuttle plasmids: pINO1-TEF1t, pTDH3-ADH1t and pGPM1-PGK1t, and successfully transformed the plasmids POT2_pINO1_CcOCS_TEF1t and POT2_pTDH3_CcOCS_ADH1t into the yeast to monitor their growth and product expression. Through systematic testing and analysis, including GC-MS and growth curve monitoring, we found that these engineered strains not only grew robustly but also successfully produced ocimene. Our experimental results clearly demonstrated that different promoter and terminator combinations significantly impact ocimene yield. Among these combinations, the pINO1-TEF1t expression system showed higher ocimene production and superior cell growth performance. This discovery provides critical ocimene information for future optimization of other metabolic products' production. Given the feasibility of the current system, our next steps involve expanding this platform to produce a variety of metabolic products. By further adjusting and optimizing promoter and terminator combinations, we aim to enhance the production of other valuable compounds, thereby exploring and developing the potential of more biosynthetic pathways to meet industrial and research needs. This will not only broaden our research scope but also further validate the role of different genetic combinations in regulating complex biological pathways. At the same time, we utilized AlphaFold3 to predict the protein structure expressed by the CcOCS gene. This provided us with a detailed protein model, laying a solid foundation for potential optimization work in the future.

Figure 27: Protein Structure


References

Zeng W, Jiang Y, Shan X, Zhou J. Engineering Saccharomyces cerevisiae for synthesis of β-myrcene and (E)-β-ocimene. 3 Biotech. 2023 Dec;13(12):384. doi: 10.1007/s13205-023-03818-2. Epub 2023 Nov 1. PMID: 37928439; PMCID: PMC10620350.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 157
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 217
    Illegal BglII site found at 889
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 157
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 157
  • 1000
    COMPATIBLE WITH RFC[1000]
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Categories
Parameters
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