Overview

As part of our project Scentinel, we aim to produce seven valuable terpenes: limonene, ocimene, cineole, borneol, myrcene, nerolidol, and nerol. To achieve this goal, we successfully constructed 19 plasmids and transformed them into Saccharomyces cerevisiae chassis strain. This strategic approach enabled us to engineer yeast for terpene biosynthesis. Ultimately, we achieved the successful production of four terpenoids: ocimene, cineole, nerolidol, and nerol. Additionally, we explored the conditional release of these aromatic compounds in yeast.

Synthetic biology applies engineering design principles to modify or recreate organisms for specific functions, typically following an iterative Design-Build-Test-Learn (DBTL) cycle. In accordance with this methodology, we undertook three cycles of genetic editing on Saccharomyces cerevisiae aimed at achieving heterologous expression and conditional release of various terpenoids.

In the initial phase of our study, we investigated the mevalonate (MVA) pathway in Saccharomyces cerevisiae for the production of terpene products, thereby confirming the viability of this metabolic route for biotechnological applications. During this phase, we also identified the critical influence of promoter and terminator selection on fermentation yield, which underscored the importance of regulatory elements in optimizing metabolic processes. This foundational work established a robust basis for further research. In the subsequent phase, we directed our efforts towards synthesizing a variety of functional terpenoid compounds to address product demands, with the objective of enhancing both the yield and diversity of these compounds for industrial use. Finally, in the third phase, we explored conditional expression strategies by employing the GAL1 promoter to tightly regulate the production of target terpenes. Through this iterative process, we aimed to refine our engineering strategies, ultimately leading to a more efficient and controlled biosynthesis of terpenoids in S. cerevisiae.

Engineering Cycle 1: Validating Our System

Proof of Concept with Ocimene

Design

Our goal is to successfully produce terpenes in yeast and to verify the pathway feasibility. 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 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 cloning, and then constructed with promoters (pTHD3, pGPM1, pINO1) and terminators (ADH1t, PGK1t, TEF1t) into shuttle plasmid vector. Heterologous expression of CcOCS with different promoters and terminators in S. cerevisiae.

Snapgene diagrams of ocimene synthase gene: CcOCS.

Build

We have completed the construction and validation of the final plasmid through three main processes.

• Plasmid Extraction.
• The TPS gene was cloned into the HcKan-O vector. （Colony PCR, Enzymatic Digestion, Gene sequencing）
• Assembled the TPS gene with different promoters and terminators into shuttle vectors. （Colony PCR, Enzymatic Digestion, gene sequencing）

Test

The colony PCR Gel Electrophoresis Results of CcOCS

We introduced the constructed plasmids into Saccharomyces cerevisiae through transformation and verify successful transformation by yeast colony PCR. The results showed that we successfully transformed plasmids POT2_pINO1_CcOCS_TEF1t and POT2_pTDH3_CcOCS_ADH1t into the yeast, while the verification for the POT2_pGPM1_CcOCS_PGK1t plasmid failed.

GC-MS analysis of ocimene :

To evaluate ocimene production in S. cerevisiae cells, shake flask fermentation was performed. After 12 hours of fermentation, a dodecane overlay culture was added, and the fermentation continued for a total of 120 hours. The ocimene titer reached 0.83 ± 0.06 mg/L when using the pINO1 promoter and TEF1t terminator, whereas it was 0.33 ± 0.09 mg/L with the pTDH3 promoter and ADH1t terminator.

Learn

The successful production of ocimene demonstrates the feasibility of the MVA pathway in Saccharomyces cerevisiae. The production of ocimene varied significantly under the control of different promoters and terminators, despite no notable differences in OD600 values. In our human practice activities, we focused on synthesizing various terpenes to assist visually impaired women by alerting them to the onset of menstruation and potential leakage. Based on our literature research findings, we planed to utilize synthetic biology to produce a diverse range of terpenes.

Engineering Cycle 2: Expanding Our Terpene Library

Construction for producing Cineole, Nerol, Nerolidol etc.

Design

Based on product characteristics and literature research, we synthesized terpene synthase genes for three compounds: cineole, nerolidol, and nerolidol. The genes AcNES1 (nerolidol synthase from Actinidia chinensis), PgfB (nerol synthase from Penicillium griseofulvum), and HpyCins, StrCins, and SfCins (cineol synthases from Hypoxylon, Streptomyces clavuligerus, and Salvia fruticosa) were synthesized by Gene Synthesis Platform to match the codon bias of S. cerevisiae and then ligated into the plasmid.

Building on the first cycle, we constructed shuttle plasmids with various combinations of promoters and terminators using Golden Gate assembly. After transformed Saccharomyces cerevisiae and performed yeast colony PCR, we typically employed two-phase fermentation system for terpenoid production. We then test terpenoid production using gas chromatography–mass spectrometry.

Snapgene diagrams of three Cineole synthase genes: HpyCins, StrCins, SfCins.

Snapgene diagrams of nerol synthase gene: PgfB.

Snapgene diagrams of nerolidol synthase gene: AcNES1.

Build

New primers were ordered, and then we have completed the construction and validation of the final plasmid through the same method as cycle 1. We have successfully constructed 9 shuttle plasmids, which are

POT2_pTDH3_HpyCins_ADH1t, POT2_pINO1_HpyCins_TEF1t, POT2_pGPM1_StrCins_PGK1t,

POT2_pINO1_PgfB_TEF1t, POT2_pGPM1_PgfB_PGK1t, POT2_pTDH3_PgfB_ADH1t,

POT2_pINO1_AcNES1_TEF1t, POT2_pGPM1_AcNES1_PGK1t, POT2_pTDH3_AcNES1_ADH1t.

The colony PCR Gel Electrophoresis Results of HpyCins, StrCins and SfCins:

The sequencing result:

The colony PCR Gel Electrophoresis Results of PgfB

The sequencing result:

The colony PCR Gel Electrophoresis Results of AcNES1

The sequencing result:

Test

The yeast colony PCR Gel Electrophoresis Results of HpyCins, PgfB and AcNES1:

Yeast colony PCR results confirmed that seven plasmids—POT2_pTDH3_HpyCins_ADH1t,

POT2_pINO1_HpyCins_TEF1t, POT2_pINO1_PgfB_TEF1t, POT2_pGPM1_PgfB_PGK1t,

POT2_pTDH3_PgfB_ADH1t, POT2_pGPM1_AcNES1_PGK1t, and POT2_pTDH3_AcNES1_ADH1t—were positive in Saccharomyces cerevisiae.

GC-MS analysis of cineole:

Flask fermentation of HpyCins yielded 11.88 ± 2.03 mg/l of ocimene with the pINO1 promoter and TEF1t terminator, compared to 17.58 ± 3.20 mg/l with the pTDH3 promoter and ADH1t terminator.

GC-MS analysis of nerol:

Flask fermentation of PgfB yielded 6.07 ± 0.23 mg/l of nerol with the pTDH3 promoter and ADH1t terminator, but no terpenes are produced with pINO1 and GPM1 promoter.

GC-MS analysis of nerolidol:

Flask fermentation of AcNES1 yielded 45.06 ± 1.68 mg/l of nerolidol with the pTDH3 promoter and ADH1t terminator, compared to 12.92 ± 1.37 mg/l with the pGPM1 promoter and TEF1t terminator.

Learn

The production of cineole varied significantly between different promoters and terminators. Flask fermentation using the pINO1 promoter and TEF1t terminator yielded 11.88 ± 2.03 mg/l of ocimene, while the pTDH3 promoter and ADH1t terminator produced 17.58 ± 3.20 mg/l, despite both constructs having similar OD600 values around 4.4. Similarly, flask fermentation with the pTDH3 promoter and ADH1t terminator resulted in 45.06 ± 1.68 mg/l of nerolidol, compared to 12.92 ± 1.37 mg/l with the pGPM1 promoter and TEF1t terminator, even though OD600 values for both constructs were similar at approximately 5. The results highlight the importance of the promoter in heterologous expression.

Although we are able to produce the scent molecules, we have not yet fully achieved the primary goal of our project: to provide reminders through the conditional release of sanitary napkins or laundry detergent and to make our product accessible to visually impaired women. Part of this limitation arises from safety restrictions imposed by the competition, but more significantly, we currently lack a viable implementation method. Our research confirms that scented sanitary napkin products are available on the market; however, there are no studies on the conditional release of fragrance in sanitary napkin products. Therefore, we have decided to focus on the conditional release of terpenes to explore the feasibility of this approach.

Engineering Cycle 3: Conditional Release test

Regulated synthesis of aromatic compounds by inducible promoters

Design

From our previous two experimental cycles, we have successfully engineered yeast to produce scents. Our next main focus will be to build a conditional release test for terpenes aimed at assisting visually impaired women. To achieve this, we will explore the use of regulatory promoters for the conditional release of terpenoids through synthetic biology. We have preliminarily selected two genes for investigation: AcNES1 (nerolidol synthase from Actinidia chinensis) and PgfB (nerol synthase from Penicillium griseofulvum). We will examine strategies for achieving conditional release from a synthetic biology perspective. The pGAL1 promoter is known for its strength in driving high levels of enzyme expression, which facilitates efficient production of the desired product. Therefore, we plan to utilize the pGAL1 promoter for plasmid construction, enabling the conditional release of terpenes from Saccharomyces cerevisiae.

Snapgene diagrams of three Cineole synthase genes: PgfB and AcNES1.

Build

We have completed the construction and validation of the final plasmid through the same method as cycle 1.

We have successfully constructed two shuttle plasmids, which were POT2_pGAL1_PgfB_PGK1t, POT2_pGAL1_AcNES1_PGK1t.

The colony PCR Gel Electrophoresis Results of PgfB and AcNES1:

The sequencing result:

Test

The colony PCR gel electrophoresis Results of PgfB and AcNES1:

Yeast colony PCR results confirmed that two plasmids—BY4741-POT2_pGAL1_AcNES1_PGK1t and BY4741-POT2_pGAL1_PgfB_PGK1t—were positive in Saccharomyces cerevisiae. We then selected single colonies for fermentation. In contrast to the previous two cycles, we employed a galactose-inducible promoter and chose SC-URA medium with both glucose and galactose as carbon sources for the control fermentation culture.

GC-MS analysis of nerol and nerolidol:

Flask fermentation of AcNES1 with the pGAL1 promoter and PGK1t terminator yielded 4.78 ± 0.88 mg/l of nerolidol, while no nerol production was detected.

Learn

In detecting nerol from the PgfB gene with various combinations, only the pTDH3 and ADH1t combination yielded 6.07 ± 0.23 mg/l, while no terpenes were produced with the other combinations. GC-MS analysis revealed that the impurity peak around 8.2 minutes was identified as a solvent peak (dodecane), whereas the retention time for nerol was 8.4 minutes. Thus, the fermentation results from the three plasmids are likely influenced by the solvent peak. Future experiments will consider using n-hexane or ethyl acetate as extraction agents for re-evaluation.

In our comparison of nerolidol yields from the AcNES1 gene using various promoter and terminator combinations, we observed that galactose induction increased the yield 12-fold compared to glucose. However, this yield remained lower than the yield achieved by the constitutive promoter (45.1 mg/l). These results indicate that our approach to conditional release has shown initial success.

Reference

Yakun Guo and others, ‘YeastFab: The Design and Construction of Standard Biological Parts for Metabolic Engineering in Saccharomyces Cerevisiae’, Nucleic Acids Research, 43.13 (2015), pp. e88–e88, doi:10.1093/nar/gkv464.

Hongting Tang and others, ‘Promoter Architecture and Promoter Engineering in Saccharomyces Cerevisiae’, Metabolites, 10.8 (2020), p. 320, doi:10.3390/metabo10080320.