iGEM 2024 — Team buct

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

## Inspirations

---

* [2018 Munich](http://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering{% endblock %}
{% block header\_class %}header-engineering{% endblock %}
{% block page\_content %}

* [Cycle1 :Preliminary pathway construction](#section2)
* [Cycle2: Co-expression Plasmid Construction, P450 Enzymes Modification, and Genome Integration](#section3)
* [Cycle3: Fermentation and Yield Verification](#section4)

**Our work adheres to the DBTL principles, undergoing three complete engineering iterations,
ultimately achieving a remarkable yield of sulforaphane.**

![](https://static.igem.wiki/teams/5327/engineering/engineering-figure1-1.png)

# Cycle1 :Preliminary pathway construction

## Discovery and Optimization of Pathways

### Design Goal:

To identify and optimize an efficient pathway for the synthesis of sulforaphane.

### Challenges in Construction:

* We attempted to find the optimal pathway through computer-aided metabolic simulation, facing
  several challenges:
  1. The synthesis pathway from methionine to sulforaphane (SFN) is excessively long, making
     mathematical modeling impractical.
  2. Calculating Gibbs free energy and enzyme kinetic coefficients is complex.
* The natural pathway for sulforaphane is intricate and relatively inefficient, lacking
  detection methods for intermediate products and effective optimization strategies.

### Testing, Learning, and Solutions:

In addressing the calculation of Gibbs free energy, we shifted from quantitative
calculations to qualitative assessments, concluding that ΔG < 0 is sufficient. By simplifying
the calculations, we developed a qualitative approach that narrows the data range, making it
easier to identify feasible pathways through experiential judgment.

![](https://static.igem.wiki/teams/5327/engineering/engineering-figure2.png)

Fig1.Metabolic Simulation Research Plan

Based on the natural pathway, we conducted extensive research on existing studies to screen,
optimize, and replace enzymes in the pathway. Combining these findings with the analysis results
from metabolic simulation, we identified a final feasible pathway.

![](https://static.igem.wiki/teams/5327/engineering/engineering-figure3.png)

Fig2.Optimized Pathway for Sulforaphane Synthesis

## Preliminary Construction of the Pathway

### Design Goal:

To initially construct the genes involved in the pathway onto a suitable vector.

### Challenges Encountered:

* The large number of genes makes conventional plasmid construction methods complex and time-consuming.
* Eukaryotic-derived genes require more sophisticated regulatory systems.

### Testing, Learning, and Solutions:

* A simplified construction strategy was adopted, utilizing a faster in-fusion homologous
  recombination method to build the plasmids, alongside refined team collaboration.
* Extensive literature review was conducted to design a more rational combination of expression regulatory elements.

## Conclusion:

The team conducted a thorough research investigation and successfully developed a metabolic
simulation framework, optimizing existing pathways and creating a universal metabolic simulation
protocol for future iGEM teams. We also constructed a preliminary pathway for sulforaphane synthesis.

However, we discovered that the current number of constructed plasmids is too large and complex
to transform all of them for subsequent experiments or fermentation. Therefore, we decided to
continue learning and improving the project, leading to a second engineering iteration.

# Cycle2: Co-expression Plasmid Construction, P450 Enzymes Modification, and Genome Integration

---

## (1) Co-expression Plasmid Construction

### Design Goal:

To further combine the initially constructed single-frame plasmids for easier transformation and
fermentation processes.

### Challenges in Construction:

* The construction scheme is complex and requires consideration of interactions between genes.
* Literature review indicates that directly transforming plasmids into yeast for fermentation results in unstable yields.

### Testing, Learning, and Solutions:

* A refined design was implemented using overlap PCR to integrate numerous fragments, reducing the
  number of segments introduced during homologous recombination and increasing success rates.
* A genome integration strategy was employed for the final fermentation experiments.

## (2) P450 Enzymes Modification

### Design Goal:

To modify the P450 enzymes in the pathway using computer-aided design, while adjusting subcellular
localization and cofactor supply to enhance P450 enzymes efficiency.

### Challenges in Construction:

* The computer optimization of P450 enzymes is complex and often yields low efficiency.
* The effects of modified enzymes lack direct characterization methods, with conclusions drawn
  only from final fermentation data.

### Testing, Learning, and Solutions:

* Discussions with multiple experts in the field of proteins were conducted to optimize the
  hydrophilicity and hydrophobicity of the enzyme's substrate channel, thereby enhancing enzyme efficiency.

![](https://static.igem.wiki/teams/5327/engineering/engineering-figure4.png)

![](https://static.igem.wiki/teams/5327/engineering/engineering-figure5.png)

Fig3. Computer Representation of Modifications to the CYP79F1，CYP83A1 Protein Sequence.

![](https://static.igem.wiki/teams/5327/header/8888888.png)

Fig4.Mitochondrial Localization Laser scanning Confocal Microscopy Imaging.

## (3) Genome Integration

### Design Goal:

To address the instability of plasmid fermentation in Saccharomyces cerevisiae, we aim to integrate
the pathway into its genome.

### Challenges in Construction:

* **Large Gene Fragment Lengths:** Integration of longer gene fragments is challenging.
* **Long Yeast Cultivation Cycles:** Verification processes are complex and time-consuming.

### Testing, Learning, and Solutions:

* **Stepwise Integration:** Integrate all fragments in stages, gradually constructing strains for
  intermediate product synthesis and final sulforaphane production.

![](https://static.igem.wiki/teams/5327/engineering/engineering-figure7.png)

Fig5.Schematic Diagram of Genome Integration Plan

* Rational Design of Verification Plan

![](https://static.igem.wiki/teams/5327/engineering/engineering-figure8.png)

Fig6.Schematic Diagram of Genome Integration Validation Plan

### Conclusion:

We successfully constructed genome-integrated strains for the synthesis of intermediates and the
pathway for producing sulforaphane. However, detailed fermentation validation is necessary to
confirm the final yield and the effectiveness of the optimization, prompting the initiation of the
third engineering iteration.

# Cycle3: Fermentation and Yield Verification

### Design Goal:

Conduct detailed fermentation experiments to validate the production capacity of engineered
strains using HPLC, LCMS, and other methods.

### Challenges in Construction:

* Difficulty in obtaining or the high cost of standard intermediates.
* Instability and low concentration of intermediates complicate detection, with sulforaphane
  requiring GS for analysis.

### Testing, Learning, and Solutions:

* Some key intermediates were analyzed using LC-MS.
* Lyophilization was employed to concentrate the fermentation broth and enhance intermediate detection.
* Research indicated that modifications to myrosinase yielded positive results; therefore, we
  assessed whether GRA, a precursor to SFN, could achieve similar outcomes to validate the pathway.
  GRA can be quantitatively analyzed via HPLC with a UV detector.
* After confirming GRA yield, we proceeded to test sulforaphane, significantly reducing
  experimental costs and time consumption.

### Conclusion:

Through fermentation experiments, we accurately measured the yield of GRA in the fermentation
broth (including samples tested after P450 optimization) and determined the yield of sulforaphane.

![](https://static.igem.wiki/teams/5327/engineering/neengineering-figure9.png)

Fig7.96-Hour Fermentation Yield Detection Results

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](https://parts.igem.org/Main_Page).

## Inspirations

---

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block header\_class %}header-results{% endblock %}
{% block page\_content %}

* [Introduction](#section1)
* [Section1-Plasmid amplification and reconstruction.](#section2)
* [Section2- Construction of Co-expression Plasmids and Genome Integration.](#section3)
* [Section3-P450 enzyme engineering.](#section4)
* [Section4-Fermentation and production testing.](#section5)
* [Summary and Outlook](#section6)

# Introduction

---

This project aims to explore the efficient production of sulforaphane using *Saccharomyces cerevisiae*,
achieving promising results. The project primarily utilizes synthetic biology tools to modify *S. cerevisiae*
through homologous recombination, constructing a pathway in yeast for the de novo synthesis of sulforaphane
from methionine. To achieve these objectives, we constructed 18 single-cassette plasmids: 16 gene cassettes
required for the complete pathway and 2 modified P450 enzyme genes. Additionally, we combined these 18 plasmids
into 6 co-expression plasmids, which were then introduced into *S. cerevisiae* via homologous recombination
to express the desired proteins, enabling the de novo synthesis of sulforaphane from methionine.

# Section1-Plasmid amplification and reconstruction.

---

![](https://static.igem.wiki/teams/5327/result/result1.png)

Fig1. The diagram of constructing the plasmid inserted by in-fusion.

We successfully constructed a single-cassette plasmid encompassing the entire metabolic pathway. The gene
sequences were initially synthesized by the International Gene Synthesis Consortium (IGSC). Using PCR
amplification, we obtained the plasmid backbone, along with gene cassettes, promoters, and terminators
equipped with homologous arms. These components were assembled through seamless cloning via the In-Fusion
method, facilitating homologous recombination.

The resulting recombinant plasmids were transformed into *Escherichia coli* strains Trans-5α and
Trans-T1 for amplification. The engineered strains were cultured in LB medium supplemented with ampicillin
to enable antibiotic selection. Colony PCR was performed using both universal and verification primers to
screen for positive clones. Plasmids from colonies yielding positive PCR results were subsequently sequenced
by IGSC to confirm the accuracy of the plasmid constructs.

![](https://static.igem.wiki/teams/5327/result/result2-1-1.png)

(a)

![](https://static.igem.wiki/teams/5327/result/result2-2.png)

(b)

![](https://static.igem.wiki/teams/5327/result/result2-3.png)

(c)

Fig2. Expression results of *ADH1p-BCAT4-CYC1t.*

a) The PCR result b) The plasmid expression c) The express diagram

BCAT4 catalyzes the transamination step in the methionine chain elongation pathway, serving as a crucial
enzyme in the biosynthesis of methionine-derived glucosinolates. We successfully amplified the
ADH1p-BCAT4-CYC1t construct (approximately 1700 bp) using PCR, and the observed fragment lengths matched
the expected theoretical size of 1751 bp, as calculated based on primer design spanning from the promoter
to the terminator. This result confirms the successful construction and acquisition of the desired plasmids.

![](https://static.igem.wiki/teams/5327/result/result3-1.png)

(a)

![](https://static.igem.wiki/teams/5327/result/result3-2.png)

(b)

![](https://static.igem.wiki/teams/5327/result/result3-3.png)

(c)

Fig3. Expression results of *CDC19p-MAM1-ADH1.*

a) The PCR result b) The plasmid expression c) The express diagram

MAM1 specifically catalyzes the first and second condensation reactions in the methionine carbon chain
elongation pathway, thereby determining the side chain length of aliphatic glucosinolates. The PCR-amplified
fragment CDC19p-MAM1-ADH1t (approximately 2500 bp) was found to be consistent with the theoretical length
of 2853 bp, as predicted from the primer locations spanning from the promoter to the terminator. This
result confirms the successful construction and acquisition of the plasmid containing the MAM1 gene.

![](https://static.igem.wiki/teams/5327/result/result4-1.png)

(a)

![](https://static.igem.wiki/teams/5327/result/result4-2.png)

(b)

![](https://static.igem.wiki/teams/5327/result/result4-3.png)

(c)

Fig4. Expression results of *TEF1p-IPMI-LSU1-ADH1t.*TEF1p-IPMI-LSU1-ADH1t.

a) The PCR result b) The plasmid expression c) The express diagram

LSU1 plays a crucial role in catalyzing the isomerization between 2-isopropylmalate and 3-isopropylmalate.
The PCR-amplified fragment TEF1p-IPMI-LSU1-ADH1t (approximately 2000 bp) was found to match the theoretical
length of 2265 bp, as estimated from the designed primer locations spanning from the promoter to the
terminator. This result confirms the successful construction and acquisition of the LSU1 plasmid.

![](https://static.igem.wiki/teams/5327/result/result5-1.png)

(a)

![](https://static.igem.wiki/teams/5327/result/result5-2.png)

(b)

![](https://static.igem.wiki/teams/5327/result/result5-3.png)

(c)

Fig5. Expression results of *ADH1p-IPMI-SSU2-PYK1t.*

a) The PCR result b) The plasmid expression c) The express diagram

SSU2 catalyzes the isomerization between 2-isopropylmalate and 3-isopropylmalate, potentially via the
formation of 2-isopropylmaleate, and functions redundantly with LEUD2 in the methionine chain elongation
pathway for aliphatic glucosinolate formation. The PCR-amplified fragment ADH1p-IPMI-SSU2-PYK1t
(approximately 1500 bp) was found to be consistent with the theoretical length of 1697 bp, as estimated
from the designed primer locations spanning from the promoter to the terminator. This result confirms the
successful construction and acquisition of the SSU2 plasmid.

![](https://static.igem.wiki/teams/5327/result/result6-1.png)

(a)

![](https://static.igem.wiki/teams/5327/result/result6-2.png)

(b)

![](https://static.igem.wiki/teams/5327/result/result6-3.png)

(c)

Fig6. Expression results of *GPDp-IPMDH1-CYC1t.*

a) The PCR result b) The plasmid expression c) The express diagram

IPMDH1 catalyzes the conversion of alkylmalate into homoketo acid. The PCR-amplified fragment GPDp-IPMDH1-CYC1t
(approximately 2000 bp) was found to match the theoretical length of 2164 bp, as estimated from the designed
primer locations spanning from the promoter to the terminator. This result confirms the successful construction
and acquisition of the IPMDH1 plasmid.

![](https://static.igem.wiki/teams/5327/result/result7-1.png)

(a)

![](https://static.igem.wiki/teams/5327/result/result7-2.png)

(b)

![](https://static.igem.wiki/teams/5327/result/result7-3.png)

(c)

Fig7. Expression results of *ENO2p-BCAT3-HXT7t.*

a) The PCR result b) The plasmid expression c) The express diagram

BCAT3 catalyzes the transamination of homoketo acid to produce dihomomethionine. The PCR-amplified fragment
ENO2p-BCAT3-HXT7t (approximately 2000 bp) was found to be consistent with the theoretical length of 2259
bp, as estimated from the designed primer locations spanning from the promoter to the terminator. This result
confirms the successful construction and acquisition of the BCAT3 plasmid.

![](https://static.igem.wiki/teams/5327/result/result8-1.png)

(a)

![](https://static.igem.wiki/teams/5327/result/result8-2.png)

(b)

![](https://static.igem.wiki/teams/5327/result/result8-3.png)

(c)

Fig8. Expression results of *CDC19p-CYP79F1-PYK1t.*

a) The PCR result b) The plasmid expression c) The express diagram

In this experiment, the enzyme specifically catalyzes the conversion of dihomomethionine (DHM). The
PCR-amplified fragment CDC19p-CYP79F1-PYK1t (approximately 3000 bp) was found to match the theoretical
length of 3144 bp, as estimated from the designed primer locations spanning from the promoter to the
terminator. This result confirms the successful construction and acquisition of the CYP79F1 plasmid.

![](https://static.igem.wiki/teams/5327/result/result9-1.png)

(a)

![](https://static.igem.wiki/teams/5327/result/result9-2.png)

(b)

![](https://static.igem.wiki/teams/5327/result/result9-3.png)

(c)

Fig9. Expression results of *PGI1p- CYP83A1-HXT7t.*

a) The PCR result b) The plasmid expression c) The express diagram

CYP83A1 metabolizes (E)-aldoximes derived from chain-elongated methionine, such as dihomomethionine.
The PCR-amplified fragment PGI1p- CYP83A1-HXT7t (approximately 2000 bp) was found to match the theoretical
length of 2464 bp, as estimated from the designed primer locations spanning from the promoter to the
terminator. This result confirms the successful construction and acquisition of the CYP83A1 plasmid.

![](https://static.igem.wiki/teams/5327/result/result10-1.png)

(a)

![](https://static.igem.wiki/teams/5327/result/result10-2.png)

(b)

![](https://static.igem.wiki/teams/5327/result/result10-3.png)

(c)

Fig10. Expression results of *ADH1p-GGP1-PYK1t.*

a) The PCR result b) The plasmid expression c) The express diagram

GGP1 participates in the biosynthesis of glucosinolates by hydrolyzing the γ-glutamyl bond in glutathione
(GSH) conjugates, producing Cys-Gly conjugates. The PCR-amplified fragment ADH1p-GGP1-PYK1t (approximately
1500 bp) was found to match the theoretical length of 1550 bp, as estimated from the designed primer locations
spanning from the promoter to the terminator. This result confirms the successful construction and
acquisition of the GGP1 plasmid.

![](https://static.igem.wiki/teams/5327/result/result11-1.png)

(a)

![](https://static.igem.wiki/teams/5327/result/result11-2.png)

(b)

![](https://static.igem.wiki/teams/5327/result/result11-3.png)

(c)

Fig11. Expression results of *HXT7p-SUR1-HXT7t.*

a) The PCR result b) The plasmid expression c) The express diagram

SUR1 plays a crucial role in glucosinolate biosynthesis by catalyzing the conversion of
S-(alkylacetohydroximoyl)-L-cysteine to thiohydroximate. The PCR-amplified fragment HXT7p-SUR1-HXT7t
(approximately 2000 bp) was found to match the theoretical length of 2185 bp, as estimated from the designed
primer locations spanning from the promoter to the terminator. This result confirms the successful
construction and acquisition of the SUR1 plasmid.

![](https://static.igem.wiki/teams/5327/result/result12-1.png)

(a)

![](https://static.igem.wiki/teams/5327/result/result12-2.png)

(b)

![](https://static.igem.wiki/teams/5327/result/result12-3.png)

(c)

Fig12. Expression results of *PGK1p-UGT74B1-ADH1t.*

a) The PCR result b) The plasmid expression c) The express diagram

UGT74B1 plays a crucial role in glucosinolate biosynthesis by participating specifically in the glycosylation
step. It is specific for the thiohydroximate functional group and does not glycosylate the carboxylate or
hydroxyl groups. The PCR-amplified fragment PGK1p-UGT74B1-ADH1t (approximately 2500 bp) was found to match
the theoretical length of 2532 bp, as estimated from the designed primer locations spanning from the promoter
to the terminator. This result confirms the successful construction and acquisition of the UGT74B1 plasmid.

![](https://static.igem.wiki/teams/5327/result/result13-1.png)

(a)

![](https://static.igem.wiki/teams/5327/result/result13-2.png)

(b)

![](https://static.igem.wiki/teams/5327/result/result13-3.png)

(c)

Fig13. Expression results of *PGI1p-SOT18-HXT7t.*

a) The PCR result b) The plasmid expression c) The express diagram

SOT18 exhibits a preference for long-chain desulfo-glucosinolates derived from methionine. The PCR-amplified
fragment PGI1p-SOT18-HXT7t (approximately 1800 bp) was found to be consistent with the theoretical length
of 1954 bp, as estimated from the designed primer locations spanning from the promoter to the terminator.
This result confirms the successful construction and acquisition of the SOT18 plasmid.

![](https://static.igem.wiki/teams/5327/result/result14-1.png)

(a)

![](https://static.igem.wiki/teams/5327/result/result14-2.png)

(b)

![](https://static.igem.wiki/teams/5327/result/result14-3.png)

(c)

Fig14. Expression results of *GPDp-FMO-PYK1t.*

a) The PCR result b) The plasmid expression c) The express diagram

FMO GS-OX1 encodes a flavin monooxygenase that catalyzes the conversion of methylthioalkyl glucosinolates
into methylsulfinylalkyl glucosinolates. The PCR-amplified fragment GPDp-FMO-PYK1t (approximately 2000 bp)
was found to match the theoretical length of 2464 bp, as estimated from the designed primer locations
spanning from the promoter to the terminator. This result confirms the successful construction and acquisition
of the FMO GS-OX1 plasmid.

![](https://static.igem.wiki/teams/5327/result/result15-1.png)

(a)

![](https://static.igem.wiki/teams/5327/result/result15-2.png)

(b)

![](https://static.igem.wiki/teams/5327/result/result15-3.png)

(c)

Fig15. Expression results of *PGI1p-Myrosinase(TGG)-PYK1t.*

a) The PCR result b) The plasmid expression c) The express diagram

Myrosinase 1 is a key enzyme involved in the degradation of glucosinolates. In this experiment, it primarily
catalyzes the hydrolysis of Glucoraphanin to produce Sulforaphane, a compound known for its anticancer,
antibacterial, and antioxidant properties. The PCR-amplified fragment PGI1p-Myrosinase(TGG)-PYK1t
(approximately 2000 bp) was found to match the theoretical length of 2499 bp, as estimated from the designed
primer locations spanning from the promoter to the terminator. This result confirms the successful
construction and acquisition of the Myrosinase 1 plasmid.

![](https://static.igem.wiki/teams/5327/result/result16-1.png)

(a)

![](https://static.igem.wiki/teams/5327/result/result16-2.png)

(b)

![](https://static.igem.wiki/teams/5327/result/result16-3.png)

(c)

Fig16. Expression results of *GPDp-CYB5-PYK1t.*

a) The PCR result b) The plasmid expression c) The express diagram

Cytochrome b5 primarily functions as an auxiliary electron transfer protein, facilitating the targeted
metabolic reactions. The PCR-amplified fragment GPDp-CYB5-PYK1t (approximately 1500 bp) was found to match
the theoretical length of 1518 bp, as estimated from the designed primer locations spanning from the
promoter to the terminator. This result confirms the successful construction and acquisition of the
cytochrome b5 plasmid.

![](https://static.igem.wiki/teams/5327/result/result17-1.png)

(a)

![](https://static.igem.wiki/teams/5327/result/result17-2.png)

(b)

![](https://static.igem.wiki/teams/5327/result/result17-3.png)

(c)

Fig17. Expression results of *ADH1p-ATR2-TDH2t.*

a) The PCR result b) The plasmid expression c) The express diagram

The enzyme ATR2 facilitates electron transfer not only to cytochrome P450 but also interacts with cytochrome
b5, thereby assisting its function. Additionally, ATR2 can reduce a range of substrates, including cytochrome
c, ferricyanide, and dichlorophenol indophenol, demonstrating its broad substrate reduction capability.
The PCR-amplified fragment ADH1p-ATR2-TDH2t (approximately 3000 bp) was found to match the theoretical
length of 3066 bp, as estimated from the designed primer locations spanning from the promoter to the
terminator. This result confirms the successful construction and acquisition of the ATR2 plasmid.

![](https://static.igem.wiki/teams/5327/result/result18-1-1.png)

(a)

![](https://static.igem.wiki/teams/5327/result/result18-2.png)

(b)

![](https://static.igem.wiki/teams/5327/result/result18-3.png)

(c)

Fig18. Expression results of *CDC19p- CYP79F1(truncated)-PYK1t.*

a) The PCR result b) The plasmid expression c) The express diagram

In this experiment, the enzyme specifically catalyzes the conversion of dihomomethionine (DHM).
CDC19p- CYP79F1(truncated)-PYK1t (3000bp+) from PCR are identical to the theoretical lengths of 3028bp
estimated by the designed primer locations (promoter to terminator), which could demonstrate that these
plasmids had successfully been obtained.

![](https://static.igem.wiki/teams/5327/result/result19-1.png)

(a)

![](https://static.igem.wiki/teams/5327/result/result19-2.png)

(b)

![](https://static.igem.wiki/teams/5327/result/result19-3.png)

(c)

Fig19. Expression results of *PGI1p- CYP83A1(truncated)-HXT7t.*

a) The PCR result b) The plasmid expression c) The express diagram

CYP83A1 metabolizes (E)-aldoximes derived from chain-elongated methionine, such as dihomomethionine.
PGI1p- CYP83A1(truncated)-HXT7t (2000bp+) from PCR are identical to the theoretical lengths of 2322bp
estimated by the designed primer locations (promoter to terminator), which could demonstrate that these
plasmids had successfully been obtained.

The results above could demonstrate that these plasmids are correctly constructed, allowing the combination
of these single-cassette plasmids into co-expression plasmids.

![](https://static.igem.wiki/teams/5327/result/result20-1.png)

(a)

![](https://static.igem.wiki/teams/5327/result/result20-2.png)

(b)

![](https://static.igem.wiki/teams/5327/result/result20-3.png)

(c)

Fig20. Expression results of *GPDp- GSTF9-ADH1t.*

a) The PCR result b) The plasmid expression c) The express diagram

Possesses glutathione S-transferase activity toward 1-chloro-2,4-dinitrobenzene (CDNB) and benzyl
isothiocyanate (BITC), and glutathione peroxidase activity toward cumene hydroperoxide and linoleic
acid-13-hydroperoxide. GPDp-GSTF9-ADH1t（1500+）from PCR are identical to the theoretical lengths of 1627bp
estimated by the designed primer locations (promoter to terminator), which could demonstrate that these
plasmids had successfully been obtained.

The results above could demonstrate that these plasmids are correctly constructed, allowing the combination
of these single-cassette plasmids into co-expression plasmids.

![](https://static.igem.wiki/teams/5327/result/result21-1.png)

(a)

![](https://static.igem.wiki/teams/5327/result/result21-2.png)

(b)

![](https://static.igem.wiki/teams/5327/result/result21-3.png)

(c)

Fig21. Expression results of ADH1p-pntA-TDH2t.

a) The PCR result b) The plasmid expression c) The express diagram

The pntA gene encodes a component of the membrane-bound transhydrogenase complex, which facilitates the
transhydrogenation between NADH and NADP. This reaction is coupled to respiration and ATP hydrolysis,
functioning as a proton pump across the membrane. The PCR-amplified fragment ADH1p-pntA-TDH2t
(approximately 2400 bp) was found to match the theoretical length of 2430 bp, as estimated from the
designed primer locations spanning from the promoter to the terminator. This result confirms the successful
construction and acquisition of the pntA plasmid.

![](https://static.igem.wiki/teams/5327/result/result22-1.png)

(a)

![](https://static.igem.wiki/teams/5327/result/result22-2.png)

(b)

![](https://static.igem.wiki/teams/5327/result/result22-3.png)

(c)

Fig22. Expression results of ADH1p-pntB-TDH2t.

a) The PCR result b) The plasmid expression c) The express diagram

The pntB gene encodes a component of the membrane-bound transhydrogenase complex, which facilitates the
transhydrogenation between NADH and NADP. This reaction is coupled to respiration and ATP hydrolysis,
functioning as a proton pump across the membrane. The PCR-amplified fragment ADH1p-pntB-TDH2t
(approximately 2200 bp) was found to match the theoretical length of 2286 bp, as estimated from the designed
primer locations spanning from the promoter to the terminator. This result confirms the successful
construction and acquisition of the pntB plasmid.

# Section2- Construction of Co-expression Plasmids and Genome Integration

---

After constructing the single-cassette plasmid, to facilitate the integration of gene cassettes into the
Saccharomyces cerevisiae genome, we first combined each gene into five co-expression plasmids.

![](https://static.igem.wiki/teams/5327/result/result23-1.png)

(a)

![](https://static.igem.wiki/teams/5327/result/result23-2.png)

(b)

![](https://static.igem.wiki/teams/5327/result/result23-3.png)

(c)

Fig23. Expression results of *ADH1p-BCAT4-CYC1t-CDC19p-MAM1-ADH1t.*

a) The PCR result b) The plasmid expression c) The express diagram

BCAT4 encodes an aminotransferase and MAM1 encodes a monooxygenase. The process involves the transamination
of methionine by BCAT4 and subsequent coupling with acetyl-CoA to produce 2-Alkylmalate, thus facilitating
the effective synthesis of intermediates in sulforaphane biosynthesis. ADH1p-BCAT4-CYC1t-CDC19p-MAM1-ADH1t
(4000+) from PCR are identical to the theoretical lengths of 4512bp estimated by the designed primer
locations (promoter to terminator), which could demonstrate that these plasmids had successfully been
obtained.

![](https://static.igem.wiki/teams/5327/result/result23-1.png)

(a)

![](https://static.igem.wiki/teams/5327/result/result24-2.png)

(b)

![](https://static.igem.wiki/teams/5327/result/result24-3.png)

(c)

Fig24. Expression results of *TEF1p-LSU-ADH1t-ADH1p-IPMI2-PYK1t-ENO2p-BCAT3-HXT7t-GPDp-IPMDH1-CYC1t.*

a) The PCR result b) The plasmid expression c) The express diagram

We constructed a catalytic element utilizing the IPMI, IPMDH1, and BCAT3 genes, capable of catalyzing the
isomerization of 2-alkylmalate, oxidative decarboxylation, and transamination, synthesizing the intermediate
dihomomethionine within this pathway. TEF1p-LSU-ADH1t-ADH1p-IPMI2-PYK1t-ENO2p-BCAT3-HXT7t-GPDp-IPMDH1-CYC1t
(8000+) from PCR are identical to the theoretical lengths of 8294bp estimated by the designed primer
locations (promoter to terminator), which could demonstrate that these plasmids had successfully been
obtained.

![](https://static.igem.wiki/teams/5327/result/result23-1.png)

(a)

![](https://static.igem.wiki/teams/5327/result/result25-2.png)

(b)

![](https://static.igem.wiki/teams/5327/result/result25-3.png)

(c)

Fig25. Expression results of *CDC19p-CYP79F1-PYK1t-PGI1p-CYP83A1-HXT7t.*

a) The PCR result b) The plasmid expression ; c) The express diagram

We constructed a catalytic element utilizing the CYP79F1 and CYP83A1 genes, catalyzing the conversion of
dihomomethionine (DHM) to aldoxime, followed by the oxidation of aldoxime to an aci-nitro compound, thereby
achieving the oxidation of DHM.CDC19p-CYP79F1-PYK1t-PGI1p-CYP83A1-HXT7t (5000+) from PCR are identical to
the theoretical lengths of 5491bp estimated by the designed primer locations (promoter to terminator),
which could demonstrate that these plasmids had successfully been obtained.

![](https://static.igem.wiki/teams/5327/result/result23-1.png)

(a)

![](https://static.igem.wiki/teams/5327/result/result26-2.png)

(b)

![](https://static.igem.wiki/teams/5327/result/result26-3-3.png)

(c)

Fig26. Expression results of *ADH1p-GGP1-PYK1t-GPDp-GSTF9-ADH1t-HXT7p-SUR1-HXT7t-ADH1t-UGT-PGK1p.*

a) The PCR result b) The plasmid expression; c) The express diagram

We constructed a catalytic element using the GGP1, GSTF9, SUR1, and UGT genes. The GGP1 enzyme hydrolyzes
the γ-glutamyl peptide bond of glutathione (GSH) conjugates, producing S-alkyl thiohydroxime. SUR1, acting
as a CS lyase, converts S-alkyl thiohydroxime to thiohydroxime, while UGT is involved in glycosylation
reactions to synthesize desulfo-glucosinolate. ADH1p-GGP1-PYK1t-GPDp-GSTF9-ADH1t-HXT7p-SUR1-HXT7t-ADH1t-UGT-PGK1p
(7000+) from PCR are identical to the theoretical lengths of 7894bp estimated by the designed primer
locations (promoter to terminator), which could demonstrate that these plasmids had successfully been
obtained.

![](https://static.igem.wiki/teams/5327/result/result27-1.png)

(a)

![](https://static.igem.wiki/teams/5327/result/result27-3.png)

(b)

![](https://static.igem.wiki/teams/5327/result/result27-2.png)

(c)

Fig27. Expression results of *PGI1p-SOT18-HXT7t-ADH1p-ATR2-TDH2t-GPDp-FMO-PYK1t.*

a) The PCR result b) The plasmid expression c) The express diagram

We constructed a catalytic element using the SOT18, ATR2, and FMO genes, which accomplished the sulfation
and sulfoxidation of desulfo-glucosinolate, leading to the synthesis of glucoraphanin, the precursor
compound for sulforaphane. PGI1p-SOT18-HXT7t-ADH1p-ATR2-TDH2t-GPDp-FMO-PYK1t (7000+) from PCR are identical
to the theoretical lengths of 7400bp estimated by the designed primer locations (promoter to terminator),
which could demonstrate that these plasmids had successfully been obtained.

After constructing the co-expression plasmids, we need to electroporate the target gene cassettes into
Saccharomyces cerevisiae. To prevent plasmid loss, we utilized the yeast's native homologous recombination
system to integrate the genes at the corresponding loci in the genome.

![](https://static.igem.wiki/teams/5327/result/result28.png)

Fig28. Recombinant gene table view.

First, we used PCR technology to design and amplify the gene cassettes of all co-expression plasmids,
obtaining the corresponding gene cassettes with integration sites. These cassettes were then electroporated
into the Saccharomyces cerevisiae genome via homologous recombination for expression validation. Below
is an introduction to the cassettes:

* **G1: BCAT4 -MAM1.**
* **G2: LSU -IPMI2 -BCAT3 -IPMDH1.**
* **G3: CYP79F1 -CYP83A1 -SOT18 -ATR2.**
* **G4: GGP1 -GSTF9 -SUR1 -UGT - FMO**
* **G5:Myrosinase.**

The following are the PCR amplification results for each fragment：

![](https://static.igem.wiki/teams/5327/result/result29-29.png)

Fig29. The PCR result for each gene sagment.

The design strategy and validation results for the above complex segments are as follows (using Ⅴ as a
validation reference example:

![](https://static.igem.wiki/teams/5327/result/result30-1.png)
![](https://static.igem.wiki/teams/5327/result/result29.png)

Fig30. The design strategy of genome Ⅴ.

All gene fragment bands were consistent with the theoretical sizes, indicating successful amplification of these cassettes.

Next, we designed a unique electroporation protocol: first, we prepared competent Saccharomyces cerevisiae
cells, and then used electroporation to introduce the cassette with genomic homologous arms into the yeast
cells. The yeast utilized homologous recombination to assemble the gene cassettes, successfully constructing
five strains, defined as I, II, III, IV, and V.

Below are the PCR verification results for the genomes of these strains:

All bands were consistent with the theoretical sizes, indicating that the gene cassettes with genomic
homologous arms have been successfully transformed.

# Section3-P450 enzyme engineering

---

What work we have done:

* Further validation of the expression and function of the fusion protein.
* Specific in vitro characterization of the fusion protein and pntAB

Work has not finished yet:

* Further validation of the expression and function of the fusion protein.
* Specific in vitro characterization of the fusion protein and pntAB

We first used computational modification methods to predict the improvement of the stability and expression
efficiency of the two P450 enzymes. This was achieved by modifying the hydrophobicity and hydrophilicity
of the substrate channel and evaluating through molecular dynamics simulations.

## 1.Results of computer-aided enzyme structure optimization

The specific semi-rational sequence modifications are as follows:

![](https://static.igem.wiki/teams/5327/result/result31.png)

Fig31. Computer representation of modifications to the CYP79F1 protein sequence.

![](https://static.igem.wiki/teams/5327/result/result32.png)

Fig32. Computer representation of modifications to the cyp83a1 protein sequence.

Next, we constructed the fusion protein. To enhance reaction efficiency, we introduced a flexible linker
peptide between the CYP79F1 and CYP83A1 genes, reducing the spatial distance between them to form a fusion
protein with improved functionality, aiming to increase catalytic efficiency. The introduction of the
linker peptide optimized the interaction between the proteins, thereby improving enzymatic reaction efficiency.

![](https://static.igem.wiki/teams/5327/result/result33.png)

Fig33. Comparison of computer-simulated homology docking results.

![](https://static.igem.wiki/teams/5327/result/result34.png)

Fig34. Computer-Assessed RMSD Data Representation of CYP79F1(truncated).

![](https://static.igem.wiki/teams/5327/result/result35.png)

Fig35. Computer-assessed RMSD data representation of Cyp83a1(Truncated).

**Computational analysis revealed that the stability of the modified CYP79F1 increased by 31.22%,
while the stability of the modified CYP83A1 increased by 29.46%.**

[**For detailed results, please visit the model👈**](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D)

## 2.Fusion protein construction and subcellular localization modification results

We added a mitochondrial targeting sequence (MLS) at the N-terminus. To better modify its electron transfer
environment, we targeted the two P450 enzymes involved in the pathway to the mitochondrial inner membrane.

![](https://static.igem.wiki/teams/5327/result/result36.png)

Fig36. The express diagram of CDC19p-MLS-CYP79F1(truncated,Δter)-Linker-CYP83A1(truncated)-HXT7t.

After constructing the fusion protein, we integrated it into the Saccharomyces cerevisiae genome through
homologous recombination and added EGFP to verify its successful targeting to the mitochondrial inner membrane. And we validated the fusion protein through SDS-PAGE gel electrophoresis.

![](https://static.igem.wiki/teams/5327/result/result37.png)

Fig37. SDS-PAGE image of the fusion protein

![](https://static.igem.wiki/teams/5327/result/result38.png)

Fig38. The express diagram of CDC19p-MLS-CYP79F1(truncated,Δter)-Linker-CYP83A1(truncated)-EGFP-HXT7t.

![](https://static.igem.wiki/teams/5327/result/result39.png)

Fig39. Laser confocal scanning images of mitochondria before and after semi-rational modification.

## 3. Cofactor supply

However, since one of the cofactors for P450 enzymes is NADPH, which lacks the necessary cofactors and
electron transfer-associated proteins in the mitochondria, we plan to further modify the enzyme system:
introducing the cyb5 and pntAB proteins into the mitochondrial inner membrane using the same system,
allowing the fusion protein to be localized and expressed on the mitochondrial inner membrane.

![](https://static.igem.wiki/teams/5327/result/result40-1.png)

(a)

![](https://static.igem.wiki/teams/5327/result/result40-2.png)

(b)

![](https://static.igem.wiki/teams/5327/result/result40-3.png)

(c)

Fig38. Expression results of MLS-CYP79F1(truncated,Δter)-CYP83A1(truncated)-pntAB-CYB5. a) The PCR result; b) The plasmid; c) The express diagram.

a) The PCR result; b) The plasmid; c) The express diagram.

MLS-CYP79F1(truncated,Δter)-CYP83A1(truncated)-pntAB-CYB5（8000+）from PCR are identical to the theoretical
lengths of 9978bp estimated by the designed primer locations (promoter to terminator), which could
demonstrate that these plasmids had successfully been obtained.

We constructed the aforementioned P450s modification system into the yeast plasmid vector pRS425 and
transformed it into the engineered strains producing GRA and SFN for fermentation experiments.

# Section4-Fermentation and production testing

---

The detection of final and intermediate products during the fermentation process for sulforaphane production
is relatively complex and involves numerous challenges. According to our investigation, the fermentation
process for sulforaphane faces issues such as the instability of intermediate products, a lack of
established quantitative detection methods, low product concentrations, and making detection difficult.
To address this, we contacted relevant companies to analyze the lyophilized and concentrated samples using
LC-MS or HPLC techniques.

After the construction of the strains, we proceeded with fermentation experiments in YPD medium at a
constant temperature of 30°C,220 rpm for 96 h. Samples were taken, and cells were disrupted using ultrasonic
treatment. The supernatant was collected, freeze-dried, and concentrated before being sent to a testing
company for analysis. Using techniques such as LC-MS and HPLC, we achieved groundbreaking high yields,
marking the first successful de novo synthesis of sulforaphane in Saccharomyces cerevisiae, reaching the
highest yields reported to date.

We present the LC-MS raw data spectrum for GRA, one of the most important precursors and final products of
the pathway, along with the final yield data from the 96 h fermentation.

![](https://static.igem.wiki/teams/5327/result/result41.png)

Fig41. 96-hour fermentation results.

![](https://static.igem.wiki/teams/5327/result/result42.png)

Fig42. LC-MS data from GRA.

We conducted large-scale fermentation of sulforaphane using a 3L bioreactor. With the assistance of our
partner company, we processed the product through freeze-drying, concentration, ethyl acetate extraction,
and purification steps, achieving a purity of over 95%. We also performed cellular experiments (approved
by the safety committee) to validate the inhibitory effect of our produced sulforaphane on PANC-1 cells.

![](https://static.igem.wiki/teams/5327/result/result44.png)

Fig 43.Purified sulforaphane.

![](https://static.igem.wiki/teams/5327/result/result43.png)

Fig 44.Sulforaphane exhibits high-efficiency inhibition of PANC-1 cells.

# Summary and Outlook

---

**In fact, the construction, optimization, fermentation, and eventual industrialization of such a complex
metabolic pathway is an extremely challenging process. We are thrilled to have completed this "impossible
mission" in less than five months, a feat made possible through everyone's collective effort. Although
our results have not yet reached the anticipated industrial standards, we have successfully completed
the most critical first step!**

**Looking ahead, we will continue to explore, optimize, and increase yields in pursuit of our target
goals. It's worth noting that many naturally bioactive isothiocyanates share similar synthesis pathways,
meaning that our engineered strains have the potential to evolve into a platform capable of efficiently
synthesizing natural isothiocyanate compounds in the future.**

{% endblock %}