iGEM 2024 — Team scut-china-s

![](https://static.igem.wiki/teams/5409/wet/2023-12-7.jpg)

## 2023.12.7

We established the SCUT-China-S Wet group.

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## 2023.12.23

We read literature, consulted with PI, and conducted brainstorming sessions within the team
to determine the research content of the project.

![](https://static.igem.wiki/teams/5409/wet/2024-2-1.png)

## 2024.2.1

Completion of vector construction:

 Co-introduction of the ndps1 and ls genes to achieve dual-pathway synthesis of limonene
in yeast.

Construct multiple combinations of ndps1 and ls gene promoters and terminators
(hereinafter referred to as the combination of Promoter1, Terminator1, Promoter2, and
Terminator2 as PTPT).

(Experiment 1.1)

![](https://static.igem.wiki/teams/5409/wet/2024-2-9.png)

## 2024.2.9

Fermentation test to select the optimal combination of promoter and terminator:

Construct multiple PTPT-ndps1-ls vectors.

After transformation into yeast, shake-flask fermentation tests were conducted, and from
six biphasic fermentation results, we selected the best promoter and terminator combination.

(Experiment 1.3)

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## 2024.3.1

Using CRISPR/Cas9 genome editing technology, the tHMG1 and IDI1 genes were inserted at the
ROX1 locus in yeast cells, resulting in the ΔROX1::tHMG1, IDI1 strain.

The modified yeast cells were validated in shake-flask experiments, successfully further
increasing limonene production.

(Experiment 2.1)

![](https://static.igem.wiki/teams/5409/wet/2024-3-20.png)

## 2024.3.20

Replacing the constitutive ERG20p element with the glucose-induced HXT1p element.

Decoupled cell growth from product synthesis. The modified yeast used mannitol, which is
more abundant in kelp, as a carbon source when glucose was depleted, and energy and
materials flowed more toward limonene synthesis.

(Experiment 3.1)

![](https://static.igem.wiki/teams/5409/wet/2024-4-5.png)

## 2024.4.5

Completion of the ERG20F96W mutation, significantly altering the substrate preference of
farnesyl pyrophosphate synthase, further reducing the competition between cell growth and
limonene synthesis, promoting limonene production.

(Experiment 3.2)

![](https://static.igem.wiki/teams/5409/wet/2024-4-20.png)

## 2024.4.20

Construction of the yp1062w:

ERG12 gene, accelerating the flux of the MVA pathway.

(Experiment 2.2)

![](https://static.igem.wiki/teams/5409/wet/2024-4-23.jpg)

## 2024.4.23

We recruited members for the art group, publicity group, wiki group, model group, and
business group, officially establishing SCUT-China-S.

[More...](https://2024.igem.wiki/scut-china-s/teammembers)

![](https://static.igem.wiki/teams/5409/wet/2024-4-24.jpg)

## 2024.4.24

We interviewed Guoyin Lai from Guangzhou Flower Flavours & Fragrances Co., Ltd online, and
learned about the application and prospect market of our product limonene.

![](https://static.igem.wiki/teams/5409/wet/2024-4-27.jpeg)

## 2024.4.27

We organized the "Running Yeast" orienteering running activity.

Through this activity, we know how much college students know about synthetic biology,
and we also publicize our project.

[More...](https://2024.igem.wiki/scut-china-s/education)

![](https://static.igem.wiki/teams/5409/wet/2024-5-17.png)

## 2024.5.17

We interviewed Dr. Alexander and asked him to shoot a promotional video for our project.

![](https://static.igem.wiki/teams/5409/wet/2024-5-19.jpg)

## 2024.5.19

We participated in the 8th iGEM Southern China Regional Meeting, exchanging project ideas
with other iGEMers from the South China region.

[More...](https://2024.igem.wiki/scut-china-s/communication)

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## 2024.5.24

The constructed PTPT-ndps1-ls was integrated into the genome from the plasmid to prevent
plasmid loss during cultivation in kelp medium without function.

(Experiment 5.1)

![](https://static.igem.wiki/teams/5409/wet/2024-5-31.png)

## 2024.5.31

We communicated with Biocreatech online and obtained 8,500 RMB sponsorship from the company.

[More...](https://2024.igem.wiki/scut-china-s/sponsor)

![](https://static.igem.wiki/teams/5409/wet/2024-6-2.jpg)

## 2024.5.31-2024.6.2

We visited the production place of kelp - Nanri Island, Fujian, to understand the growth
habits of kelp and the working environment and treatment of kelp workers.

[More...](https://2024.igem.wiki/scut-china-s/communication)

![](https://static.igem.wiki/teams/5409/wet/2024-6-12.jpg)

## 2024.6.12

We went to Guangzhou Experimental Middle School to popularize synthetic biology, and led the
students of the first grade to observe wild yeast cells and learn the related knowledge of
cell factories.

[More...](https://2024.igem.wiki/scut-china-s/education)

![](https://static.igem.wiki/teams/5409/wet/2024-7-11.jpg)

## 2024.7.11-14

We attended the 11th Conference of China iGEMer Community (CCiC), exchanging project ideas
with iGEMer teams from the Chinese region.

Our project won the most popular poster award.

[More...](https://2024.igem.wiki/scut-china-s/communication)

![](https://static.igem.wiki/teams/5409/wet/2024-7-24.jpg)

## 2024.7.24

We held an exchange meeting with the shanghai-city team.

This exchange provides an opportunity for us to ask questions and seek solutions to
challenges together.

[More...](https://2024.igem.wiki/scut-china-s/communication)

![](https://static.igem.wiki/teams/5409/wet/2024-7-31.png)

## 2024.7.31

Completion of long-term cultivation and evolution to select yeast cells that use mannitol as
the sole carbon source, enhancing the yeast's ability to utilize kelp hydrolysate.

(Experiment 4.1)

![](https://static.igem.wiki/teams/5409/wet/2024-8-6.jpg)

## 2024.8.6-8.10

We participated in SynBio Challenges.

Our project won a silver award.

[More...](https://2024.igem.wiki/scut-china-s/communication)

![](https://static.igem.wiki/teams/5409/wet/2024-8-8.png)

## 2024.8.8

We visited Biocreatech (Shenzhen).

[More...](https://2024.igem.wiki/scut-china-s/sponsor)

![](https://static.igem.wiki/teams/5409/wet/2024-9-7.png)

## 2024.9.7

We participated in the iSDG (sustainable development) online conference to share the SDG of
our project and learn from the SDG of other iGEM teams.

[More...](https://2024.igem.wiki/scut-china-s/sustainable)

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## 2024.9.13

After drying and pulverizing kelp into powder, it was hydrolyzed and enzymatically treated,
followed by filtration to prepare kelp medium for Saccharomyces cerevisiae fermentation.

(Experiment 6.1)

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## 2024.9.20

We achieved a 144-hour fed-batch fermentation in 200 ml scale, with a significant increase
compared to the 10 ml 48-hour shake-flask fermentation.

(Experiment 7.2)

![](https://static.igem.wiki/teams/5409/wet/2024-9-22.png)

## 2024.9.22

We performed vacuum rotary evaporation for the limonene and IPM mixture, followed by phase
separation after evaporation, and successfully purified the limonene product.

(Experiment 8.2)

![](https://static.igem.wiki/teams/5409/wet/2024-9-22.jpg)

## 2024.9.22

We held a debate on the theme of environmental protection. In the heat debate, people understand the meaning of environmental protection and what should be done to make it.

![](https://static.igem.wiki/teams/5409/engineering/engineering.png)

[Overview](#intro1)[Strategy 1—Dual Expression
Cassette Screening](#intro2)[Strategy 2—Synthetic Pathway
Modification](#intro3)

[·ΔROX1::tHMG1, IDI1](#intro31)
[·ERG20p::HXT1p & ERG20F96W](#intro32)
[·ypl062w::ERG12](#intro33)

[Strategy 3—Optimization of Kelp
Component Utilization](#intro4)[Strategy 4—Product
Purifications](#intro5)[References](#intro6)

![](https://static.igem.wiki/teams/5409/engineering/engi11.png)

Overview

Utilizing kelp as a raw material for limonene fermentation is an effective way
to
expand the use of blue carbon
resources, promote the development of third-generation biomass resources, and provide an environmentally
friendly and
efficient method for producing high-value terpenoids, such as limonene.

To better achieve this goal, we designed our experiments around four key
processes: dual expression cassette plasmid
screening, synthetic pathway modification, optimization of kelp component utilization, and product
purification. This
page outlines the design principles and related achievements of each section.

For detailed steps and methods, please refer to the [experiment](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiment%27%29%20%7D%7D) and [protocol](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocol%27%29%20%7D%7D) page.

Strategy 1—Dual Expression Cassette Screening

(1) Promoter and Terminator Screening

The *ndps1* gene and *limonene* synthase gene are integrated into
a
plasmid, and
various combinations of promoters and
terminators are tested to find the optimal dual expression cassette that maximizes the efficiency of
limonene synthase
production.

![](https://static.igem.wiki/teams/5409/engineering/engi2.png)

(2) YJL064w::*ndps1-(R)-ls*

The YJL064w gene does not encode a functional protein in yeast, but studies
have
shown that its knockout can effectively
enhance the synthesis of downstream terpenoids. After screening the optimal promoter and terminator
combination, we
insert the YJL064w locus into this expression cassette to shift the expression of limonene synthase from
the
plasmid to
the genome level, preventing plasmid loss and enabling the strain to permanently synthesize limonene.

![](https://static.igem.wiki/teams/5409/engineering/engi33.png)

Strategy 2—Synthetic Pathway Modification

![](https://static.igem.wiki/teams/5409/engineering/engi44.png)

ΔROX1::tHMG1, IDI1

**(1)** The *ROX1* gene is a transcriptional repressor in yeast that primarily regulates genes
related to
anaerobic growth. Knockout of this gene can alter yeast metabolism under anaerobic conditions. Studies
have
shown that
*rox1* gene deletion significantly increases the expression of intermediates mevalonic acid in the
MVA
pathway,
and other research has found that *rox1* acts as a transcriptional regulator that represses gene
expression in
the MVA pathway and ergosterol biosynthesis. Its deletion enhances MVA pathway flux.

**(2)** *tHMG1* is a homolog of the human HMG-CoA reductase gene, which catalyzes the reduction of
HMG-CoA
(3-hydroxy-3-methyl glutaryl coenzyme A) to mevalonate. Overexpression of this gene can increase metabolic
flux,
enhancing terpene production.

**(3)** *IDI1* (isopentenyl-diphosphate delta-isomerase 1) catalyzes the interconversion of
isopentenyl
diphosphate (IPP) and its electrophilic isomer dimethylallyl diphosphate (DMAPP) in cells. Overexpression
of
this gene
increases the synthesis of limonene precursors, thus boosting limonene production.

![](https://static.igem.wiki/teams/5409/engineering/engi55.png)

ERG20p::HXT1p & ERG20F96W

**(1)** The *ERG20* gene encodes farnesyl diphosphate (FPP) synthase, which synthesizes FPP from
GPP
and
IPP.
FPP synthesis is essential for yeast growth, but this metabolic pathway competes with limonene synthesis
for
substrates.

**(2)** *HXT1p* encodes a glucose transporter protein responsible for glucose transport in yeast
cells.
Replacing the native promoter of *ERG20* with the *HXT1* promoter allows regulation of
*ERG20*
expression, thereby controlling carbon source utilization of yeast—favoring rapid growth in the early
stages
and
directing more carbon to limonene synthesis later.

**(3)** Substituting phenylalanine (Phe) at position 96 of the *ERG20* gene with tryptophan (Trp)
affects the
activity or properties of FPP synthase. This mutation shifts carbon flux from FPP to GPP, significantly
influencing
terpenoid production in yeast.

![](https://static.igem.wiki/teams/5409/engineering/engi66.png)

ypl062w::ERG12

(1) The function of YPL062W in yeast is not well characterized, but studies have shown that its deletion
upregulates
the pyruvate dehydrogenase bypass and the mevalonate pathway, increasing the production and accumulation
of
acetyl-CoA, thereby promoting terpenoid production.

(2) ERG12 encodes mevalonate kinase, which is involved in the mevalonate pathway. This enzyme is
responsible
for
the
biosynthesis of IPP. Overexpression of this enzyme in yeast promotes IPP synthesis.

![](https://static.igem.wiki/teams/5409/engineering/engi7.png)

Strategy 3—Optimization of Kelp Component Utilization

**(1) Mannitol Adaptive Laboratory Evolution**

Analysis shows that mannitol accounts for about 15%-30% of the dry weight of kelp. Mannitol, as a
non-fermentable
carbon source, has many favorable characteristics. To enhance yeast's ability to utilize mannitol in kelp,
we
subjected the strain to long-term adaptive laboratory evolution (ALE) using SM△T medium.

**(2) Optimization of Kelp Hydrolysate Medium**

Analysis shows that mannitol accounts for about 15%-30% of the dry weight of kelp. Mannitol, as a
non-fermentable
carbon source, has many favorable characteristics. To enhance yeast's ability to utilize mannitol in kelp,
we
subjected the strain to long-term adaptive laboratory evolution (ALE) using SM△T medium.engi

![](https://static.igem.wiki/teams/5409/engineering/engi88.png)

Strategy 4—Product Purification

After performing small-scale (10 ml) biphasic fermentation, we scaled up the fermentation. To obtain
high-purity
limonene for industrial production, we used vacuum rotary evaporation based on the boiling point
characteristics
of
limonene and the organic phase (IPM) to separate and purify the organic phase obengitained after fermentation.

![](https://static.igem.wiki/teams/5409/engineering/engi9.png)
![](https://static.igem.wiki/teams/5409/engineering/engi10.png)

References

- [1] ÖZAYDıN B, BURD H, LEE T S, et al. Carotenoid-based phenotypic screen of the yeast deletion
  collection reveals new
  genes with roles in isoprenoid production.[J]. Metabolic Engineering, 2012.
- [2] IGNEA C, PONTINI M, MAFFEI M E, et al. Engineering Monoterpene Production in Yeast Using a
  Synthetic
  Dominant
  Negative Geranyl Diphosphate Synthase[J]. ACS Synthetic Biology, 2014.
- [3] CHEN Y, WANG Y, LIU M, et al. Primary and Secondary Metabolic Effects of a Key Gene Deletion
  (ΔYPL062W) in
  Metabolically Engineered Terpenoid-Producing Saccharomyces cerevisiae.[J]. Applied and Environmental
  Microbiology, 2019.
- [4] LIU Y, JIANG X, CUI Z, et al. Engineering the oleaginous yeast Yarrowia lipolytica for production
  of
  α-farnesene.[J]. Biotechnology for Biofuels, 2019.
- [5] CHEN Y, XIAO W, WANG Y, et al. Lycopene overproduction in Saccharomyces cerevisiae through
  combining
  pathway
  engineering with host engineering.[J]. Microbial Cell Factories, 2016.