iGEM 2025 — Team ynnu-china

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{% block title %}Notebook{% endblock %}
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#### Notebook

[2024](#2024)

* [November](#November2024)
* [December](#December2024)

[2025](#2025)

* [January](#January2025)
* [February](#February2025)
* [March](#March2025)
* [April](#April2025)
* [May](#May2025)
* [June](#June2025)
* [July](#July2025)
* [August](#August2025)
* [September](#September2025)

## 2024

---

### --November

#### --Week 1

**General:**

Project kickoff for Human Practices (HP). The team reviewed global disease reports, biomanufacturing
policies, and company annual reports to understand the societal and industrial needs for high-value plant
natural products.

**Specific Content:**

The team studied major infectious and non-infectious diseases and their treatment methods. Key insights:
plant natural products are crucial for treatment but face high costs, long cycles, and low accessibility.
Policies from different regions supporting biomanufacturing were reviewed to align the project with
regional industry priorities. Initial discussions emphasized the need for enzyme engineering tools to
overcome heterologous synthesis bottlenecks.

#### --Week 2

**General:**

Focused on industry insights and protein design tool needs through corporate annual report surveys.

**Specific Content:**

Analyzed reports from multiple biotechnology and pharmaceutical companies. Identified strong demand for
enzyme engineering and pathway optimization. The team decided to focus on plant enzyme design and
synthetic biology solutions, aiming to create tools that align with industrial pain points and accelerate
product development.

#### --Week 3

**General:**

Initiated expert consultation and academic interviews to refine technical objectives.

**Specific Content:**

Discussed current biomanufacturing challenges, enzyme modification needs, and strategies for
pharmaceutical natural products and pigments. Insights guided the project to target key enzymes in
pathways such as paclitaxel and lycopene biosynthesis.

#### --Week 4

**General:**

Expanded academic consultation to include cell factory and synthetic biology experts.

**Specific Content:**

Learned that missing or low-activity enzymes are the main barriers in heterologous synthesis. Emphasis on
balancing research value with industrial application to maximize societal impact.

### --December

#### --Week 1

**General:**

Conducted market and industry-oriented consultations with agricultural and biomanufacturing enterprises.

**Specific Content:**

Gained practical industry insights on consumer group analysis, market trends, and competition landscape.
Team recognized the importance of developing tools with practical application value and high industrial
adoption potential.

#### --Week 2

**General:**

Analyzed literature and existing protein design platforms to identify limitations.

**Specific Content:**

Compared PROSS, FuncLib, and ProteinMPNN. Found that current platforms are limited in integration with
experiments, complexity, and coverage of active sites. This motivated the development of a tool
specifically addressing heterologous plant enzyme stability and activity.

#### --Week 3

**General:**

Protein design technical consultations with academic experts.

**Specific Content:**

Discussed strategies for enzyme modification, pathway analysis, co-evolutionary analysis,
function-oriented design, multi-dimensional iteration, backbone optimization, and mutation-combination
prediction. Insights informed REvoDesign development, emphasizing stability, activity, and industrial
applicability.

#### --Week 4

**General:**

Continued refinement of tool design and workflow optimization.

**Specific Content:**

Integrated semi-rational modification workflows with co-evolutionary analysis and rational protein
design. Focused on minimal experimental burden, synergistic stability and activity improvement, and
alleviation of epistasis effects. Developed a visualization module to support expert review.

## 2025

---

### --January

#### --Week 1

**General:**

Development of the first version of REvoDesign tool.

**Specific Content:**

Completed initial integration of semi-rational modification workflows, co-evolutionary analysis, and
rational protein design modules.

#### --Week 2

**General:**

Field research in hospitals to understand clinical needs of high-value plant products.

**Specific Content:**

Collected information on limitations in patient access to plant-derived drugs due to high prices and
production cycles. Insights highlighted the societal importance of improving heterologous synthesis of
natural products via REvoDesign.

#### --Week 3

**General:**

Human health and microbiome considerations.

**Specific Content:**

Considered variability in drug metabolism and precision medicine applications. Learned to incorporate
public health awareness alongside technological development.

#### --Week 4

**General:**

Industrial feasibility evaluation.

**Specific Content:**

Analyzed production, supply chain, and regulatory challenges in pharmaceutical manufacturing. Adjusted
REvoDesign goals to align with industrial adoption, R&D cycle reduction, and regulatory standards.

### --February

#### --Week 1

**General:**

Selection and research on high-value plant products for case studies.

**Specific Content:**

Identified potential plant targets with high-value active compounds. Heterologous synthesis proposed as a
solution to resource scarcity.

#### --Week 2

**General:**

Investigation of key enzyme pathways for industrial production.

**Specific Content:**

Observed that precursors are limited, growth cycles are long, and yields are unstable. Identified key
rate-limiting enzymes. REvoDesign set to optimize these enzymes for industrial production efficiency.

#### --Week 3

**General:**

Academic consultations for integration with drug discovery.

**Specific Content:**

Evaluated structure-based approaches and fragmentation methods. REvoDesign identified as a complementary
tool for enzyme modification and large-scale production.

#### --Week 4

**General:**

Traditional medicinal product synthesis analysis.

**Specific Content:**

Analyzed bottlenecks in enzyme activity and multi-gene expression. Confirmed REvoDesign’s potential for
industrial enzyme modification and pathway optimization.

### --March

#### --Week 1

**General:**

Pilot case studies for REvoDesign.

**Specific Content:**

Selected lycopene and paclitaxel pathways as demonstration cases. Focused on increasing catalytic
efficiency and reducing byproducts. Initial computational designs completed; planning wet-lab validation.

#### --Week 2

**General:**

Expert evaluation and tool refinement.

**Specific Content:**

Collected feedback from protein design and industry experts. Adjusted REvoDesign features to improve
interpretability, usability, and industrial applicability.

#### --Week 3

**General:**

Social dissemination and public engagement.

**Specific Content:**

Shared knowledge on heterologous synthesis, plant natural products, and synthetic biology. Engaged public
via webinars and social media to increase awareness of accessibility, cost reduction, and sustainable
production.

#### --Week 4

**General:**

Industrial partnership exploration.

**Specific Content:**

Negotiated strategic cooperation with industry partners. Applied REvoDesign to industrial lycopene
production case. Plan to monitor performance, collect data, and further optimize workflow for real-world
application.

### --April

#### --Week 1

**General:**

Tool validation and data analysis

**Specific Content:**

Analyzed REvoDesign outputs for target enzymes. Evaluated improvements in stability and catalytic
efficiency. Data informed iterative updates to the tool and documentation for industry adoption.

#### --Week 2

**General:**

Expert review and workshop.

**Specific Content:**

Held workshops with scholars and industry experts. Discussed case study results, tool interface
improvements, and future applications. Collected feedback for enhanced workflow and broader applicability.

#### --Week 3

**General:**

Finalization of REvoDesign tool features.

**Specific Content:**

Incorporated all feedback, refined visualization, mutation prediction modules, and DBTL workflow. Ensured
modular outputs and industrial usability.

#### --Week 4

**General:**

Project wrap-up and reporting.

**Specific Content:**

Prepared comprehensive report documenting Human Practices, industry insights, tool development, and case
studies. Ensured alignment with sustainable development goals and iGEM competition requirements.

### - May

#### --Week 1

**General:**

After the conclusion of the school competition selection, students from different departments joined the
YNNU-China team, and everyone is full of energy and enthusiasm! So far, we have successfully identified
the
members in charge of the wet lab section.

We held our first team meeting, where all members introduced themselves and got to know each other. We
also
elected our student team leader and deputy team leader. In addition, we had a preliminary discussion with
our
PI regarding the project direction, aiming to accelerate the development and utilization of the REvoDesign
tool. Meanwhile, our WiKi team is also learning the basic knowledge of Web development and familiarizing
themselves with frameworks to prepare for the subsequent website construction phase.

#### --Week 2

**General:**

A brief introduction was provided to the background of the IGEM (International
Genetically Engineered
Machine) competition project: "using the REvoDesign tool to engineer natural enzymes, enabling the
heterologous efficient synthesis of natural products". Discussions were also held on experimental details
such
as candidate cases and the combination of genes to be used in synthetic pathways.

Task assignments were made for all participants, who were specifically divided
into
the Dry Team and the Wet
Team.

**Wet Team:** Conduct wet experiments, and record and organize wet
experiment data; this data
will be used for the production and distribution of PPTs, animations, and other materials.

**Dry Team responsibilities:** Undertake software development; create
promotional PPTs, design
the team logo, produce animations (with assistance from professionals required in the later stage), and
handle
post-production subtitle dubbing; organize team recruitment presentations and carry out Human Practices
(HP)
work, while being responsible for the team's subsequent communication with potential new members; collate
project-related materials ("Stories") and provide textual materials for subsequent PPTs, animations, etc.;
design and coordinate the procurement of team uniforms and team flags; coordinate personnel and book
meetings;
take meeting minutes, track project progress, and record updates in real time; create promotional handout
cards.

![Overview of YNNU-China team's 2025 comprehensive human practice](https://static.igem.wiki/teams/5915/notebook/fig-1.webp)

Fig. 1 Work schedule and deadlines.

#### --Week 3

**General:**

Discussion on Phased Work and Arrangements for Subsequent Tasks

**Specific Content:**

We held an online meeting with students majoring in media who are interested in
joining the team, introduced the iGEM project, clarified the tasks to be undertaken, and we will follow up
on
the recruitment of new members subsequently.

The team discussed the team logo design, developed the initial version of the
logo,
and discussed its color scheme, implied meaning, and other aspects during the meeting. A consensus was
reached
within the day, and this logo will be used for the production of team uniforms and flags.

Discuss the work arrangements for promotional presentations, including the
following
specific items:

1. Web design (personnel to be determined);

2. Promotional posters (to be produced after the materials are finalized);

3. Promotion Venues and Methods: The initially proposed venues include companies,
hospitals, government agencies, and schools, while the initially proposed methods cover roadshows,
presentations, and more. During the meeting, in-depth discussions were held on how to conduct promotion
efficiently and accurately, with a focus on two key aspects: how to fully introduce the story background
(to
demonstrate its theoretical feasibility, social value, and potential future value) and how to achieve
targeted
delivery of promotional content. All team members expressed their respective opinions; among them, the
person
in charge of this section took Chenguang Biotechnology Co., Ltd. as an example, explaining how to align
corporate pain points with the project's highlights, and planned to conduct a presentation and promotion
at
this company in the subsequent phase.

![Overview of YNNU-China team's 2025 comprehensive human practice](https://static.igem.wiki/teams/5915/notebook/fig-2.webp)

Fig. 2 We have created the team's LOGO.

#### --Week 4

**General:**

Main Content: The team summarized the work of the previous phase and made
arrangements
for subsequent tasks. The Principal Investigator (PI) attended the meeting online and provided guiding
suggestions. New team members also participated in the online meeting.

**Specific Content:**On the part of the Dry Team, its members
reviewed
the work of the previous phase and assigned personnel for various tasks in the next phase, including the
following details:

1. Public media platforms: These include Bilibili, Xiaohongshu (Little Red Book),
WeChat Official Account, etc. It was initially planned to start with the WeChat Official Account—members
of
the Dry Team will take turns to provide articles on a weekly basis, and Gao Yu will be in charge of
publishing
and management. During this period, the PI suggested that for international competitions, the team should
also
pay attention to the operation and content release of foreign social media platforms, while ensuring the
breadth of the audience and focusing on popular science content.

2. Animation design: Two students from the Academy of Fine Arts of Yunnan Normal
University, introduced by a team member, will be responsible for the production and are required to submit
the
first draft before June 1st.

3. Poster design: Team members will optimize and revise the first draft, and
finalize
the final version before May 15th.

4. PPT production and external promotion: The person in charge of this work
proposed
a
series of practical options for promotion venues, including primary schools, Yunnan Normal University
Provincial Academy of Sciences, Kunming University of Chinese Medicine, Chenguang Biotechnology Co., Ltd.,
Henan Muyuan Laboratory, Zhengzhou Municipal Health Commission, etc. This work will be launched
immediately
after the completion of PPT and poster production, aiming to expand the team's influence and recruit new
team
members at the same time.

5. Website design: Design of the WiKi website, including the webpage framework and
specific content.

On the part of the Wet Team, they reported the current difficulties and problems.
Team
members stated that the Wet Team is facing a tight schedule, heavy tasks, large workload of experiments,
and
a
significant shortage of personnel. After discussion at the meeting, members of the Dry Team will assist in
wet
experiments: Wet Team members will create an experiment schedule and inform Dry Team members in advance
when
assistance is needed to complete the experiments.

During the meeting, the PIs delivered guiding opinions online and informed the
team
that they would invite other teachers to assist in the project implementation later. These teachers will
provide support in areas such as experience in participating in iGEM, social application value of
REvoDesign,
and guidance on enzyme engineering.

![Overview of YNNU-China team's 2025 comprehensive human practice](https://static.igem.wiki/teams/5915/notebook/fig-3.webp)

Fig. 3 Online meeting photos.

### - June

#### --Week 1

**General:**

Main Content of the Meeting: Reviewed the experimental ideas of the Wet Team and
refined the work arrangements for the Dry Team

**Specific Content:** The team analyzed the requirements for various
iGEM
awards and discussed whether objective conditions (such as time) could meet the rigid requirements,
including
the DBTL cycle. Subsequently, the PI (Principal Investigator) explained the wet experiment section from
the
perspectives of experimental ideas and overall planning. He pointed out that currently, plant enzymes
exhibit
poor stability and low activity in microbial hosts, which is a major bottleneck in the production of
high-value natural products in synthetic biology. To address this, we have developed a rational
evolution-guided enzyme reconstruction (REvoDesign) workflow to guide the rapid evolution of enzymes with
target functions and adaptability in microorganisms. The PI emphasized that this iGEM project will only
focus
on the key enzymes (involved in lycopene and taxadien-5α-ol (T5OH) production) modified by REvoDesign, and
the
Wet Team's experiments only need to proceed to verifying that the product yield shows a significant
difference
compared with that before modification. Additionally, the PI suggested that the team create a mind map
based
on the Wet Team's experimental ideas—this will not only facilitate the implementation of subsequent
experiments but also help the team gain a clearer understanding of the tasks to be completed and their
objectives.

Afterwards, the team further subdivided the work for the Dry Team. Due to the
relatively heavy workload of Human Practices (HP) presentations, personnel allocation for other iGEM
projects
was temporarily reduced. Meanwhile, the team coordinated with members from the School of Media and
Communication regarding video and poster production. A work discussion was held with four students from
the
School of Media and Communication, during which the storyboards for the video were further discussed and
the
person in charge of subsequent poster work was clarified. Additionally, these four students decided to
join
the iGEM team and will participate in project-related work as official team members subsequently.

#### --Week 2

**General:**

All-team Meeting: Discussed team task division and next-step plans. We outlined
the
specific technical roadmap for the Wet Team. Our WiKi team also continued to delve deeper into web-related
knowledge. During this busy week, everyone on the team worked tirelessly toward the same goal, hardly
having
any personal rest time.

The addition of new members in areas such as graphic design, video production, and
costume design has made our team officially complete! After the regular meeting on Tuesday, we took a
group
photo in front of the main building of the college. This week, our project design finally began to take
shape.
The team dedicated to enzyme engineering is planning to use REvoDesign to address the issue of
heterologous
synthesis of plant natural products. Meanwhile, work has begun on constructing a fusion expression system
in
yeast cells. We also discussed the feasibility of various technologies such as multi-gene integration. Our
WiKi members are currently collecting materials, and our HP (Human Practices) team member presented their
overall plan—and we are confident that we will definitely succeed!

![Overview of YNNU-China team's 2025 comprehensive human practice](https://static.igem.wiki/teams/5915/notebook/fig-4.webp)

Fig. 4 YNNU-China Team Members.

#### --Week 3

**General:**

Main Content of the Meeting: The PI (Principal Investigator) led the team to
review
the relevant requirements and tasks on the iGEM official website, and made specific arrangements for them.

**Specific Items:**

1.White List Check: Conduct checks on items including materials used in
experiments
to
ensure all materials are included in the iGEM White List (approved materials list).

2.Completion of the Preliminary Safety Form: Submit the preliminary filled content
to
the PI for review and verification before Sunday, followed by the official submission of the form.

![Overview of YNNU-China team's 2025 comprehensive human practice](https://static.igem.wiki/teams/5915/notebook/fig-5.webp)

Fig. 5 YNNU-China team uniform design sketches.

3.Personnel Work Adjustments: Some team members will shift their main work from
the
Dry Team to the Wet Team to assist in completing wet experiments. In addition, with the assistance of
teachers
and after communication, a new student from the School of Information Science and Technology will join the
team, taking charge of the WiKi webpage design.

4.Verification of Experimental Materials: The team will check all expression
vector
sequences to be used by the team to ensure the experiment proceeds without any errors. Additionally, all
chassis strains used have been reported in academic literature. The Wet Team will continue to focus on
vector
construction and natural product expression. First, we will transform unmodified genes (such as T5αH and
CarRP) into yeast cells and conduct PCR identification to confirm whether they have been successfully
transformed.

Students in charge of costume design have started designing team uniforms for the
team.

#### --Week 4

**General:**

Main Content of the Meeting: Divided the work of the Dry Team, conducted in-depth
discussions on the details of video storyboards, and all participants reached a consensus.

Work Allocation for Communication PPTs: The PPT tasks were divided into modules.
The
team focused on collecting materials for the "Problem Statement" and "Current Situation Introduction"
sections, and providing content for presentation—this is to facilitate subsequent discussions and
experience
sharing with gold award winners.

**Specific Content:**

1. Team members learned from previous award-winning teams and proposed additional
tasks to be implemented, including the development of a business plan, modeling, and the design of
questionnaires.

2. The team conducted in-depth analysis and discussion on the content of the video
storyboards, reached a consensus, and confirmed the final version. For the specific storyboard content,
please
refer to the following link: <https://static.igem.wiki/teams/5915/notebook/notebook.pdf>.

![Overview of YNNU-China team's 2025 comprehensive human practice](https://static.igem.wiki/teams/5915/notebook/fig-6.webp)

Fig. 6 Latest task tracker chart.

### - July

#### --Week 1

**General:**

Dry Team Key Content:

1. Arranged the date for the exchange meeting with Nanjing Normal University (NNU)
and
the specific matters to be prepared.

2. Made arrangements for the coordination work for the WiKi video.

3. Discussed the specific implementation strategies for social media operations.

4. Divided the work for promotional presentations.

**Specific Details:**

Confirmed that the exchange meeting with Nanjing Normal University (a two-time
iGEM
gold award winner) will be held this Saturday. The team split the PPT preparation work into tasks and
assigned
them to specific team members.

Social media operations were divided into three segments, corresponding to three
platforms: Xiaohongshu (Little Red Book), WeChat Official Account, and Bilibili Video Platform. In
addition
to
promoting and distributing WeChat Official Account articles across all three platforms, Xiaohongshu and
Bilibili will update video or graphic content regularly. This work is tentatively assigned to members from
the
media team, and a detailed plan will be formulated later.

The work for promotional presentations was subdivided into two parts:
pre-preparation
and the presentation itself. Pre-preparation includes preparing team flags, team uniforms, leaflets,
posters,
and other materials.

![Overview of YNNU-China team's 2025 comprehensive human practice](https://static.igem.wiki/teams/5915/notebook/fig-7.webp)

Fig. 7 iGEM team members at the meeting site.

#### --Week 2

**General:**

iGEM Peer Exchange Meeting

Exchanging Teams: Nanjing Normal University (NNU) – Yunnan Normal University
(YNNU)

Participants: All members of YNNU iGEM Team, representatives of NNU iGEM Team,
etc.

**Main Meeting Content**

Nanjing Normal University and Yunnan Normal University took turns to share their
iGEM
teams' work and progress. The PIs (Principal Investigators) of our team and NNU's PI put forward guiding
suggestions, and suggestions for improvement on the current project progress were also proposed during the
meeting.

**Specific Meeting Content**

Nanjing Normal University reported on its project related to folate metabolism.
Based
on their presentation, we gained a deeper understanding of the specific requirements for iGEM projects and
made corresponding adjustments. As NNU has participated in the iGEM competition many times and won gold
awards
on multiple occasions, it has a profound understanding of the competition and rich experience. Based on
their
report, we made the following adjustments:

1. For the Human Practices (HP) section, we need to intensify efforts and
accelerate
the dual advancement of promotional presentations and research surveys.

2. Accelerate project progress: provide timely feedback when problems arise,
establish
additional collaboration mechanisms and a "responsibility assignment" mechanism (ensuring each task is
assigned to a specific person), to promote project progress more efficiently.

3. Combine the core of the project with traditional culture: promote the green
production of traditional Chinese medicines using synthetic biology, elaborate on the project's
contributions
of synthetic biology to society at a deeper level, and embody the entrepreneurial spirit.

![Overview of YNNU-China team's 2025 comprehensive human practice](https://static.igem.wiki/teams/5915/notebook/fig-8.webp)

Fig.8 Photos of the online exchange meeting.

#### --Week 3

**General:**

**Meeting Content:** Reviewed the exchange meeting with Nanjing
Normal
University held last Saturday. Based on the improvement suggestions proposed earlier, a task tracker was
created: tasks were broken down among each individual, and deadlines were clearly marked. The team
communicated with the art team to redesign the posters, and planned to have a further discussion with the
art
team tomorrow evening to provide a detailed description of the project background, so as to facilitate the
drawing of video storyboards.

The meeting focused on discussing the content of the WiKi and video. Team members
made
new arrangements for work schedules; all members participated in the discussion and clarified the specific
details of tasks as well as their completion dates. The WiKi tasks were mainly divided into two sections:
the
text section and the webpage design section. These two sections were assigned to different team members
respectively, and the work content under each section was further broken down. After discussion, it was
decided that the art team would submit the first draft of the video in early August, and the revised final
version would be submitted in late August. Meanwhile, the meeting also discussed the phased team work
summary
meeting to be attended by the PI, and summarized the work that has been completed so far and the tasks
planned
to be finished in the future.

After modifying the CarRP protein, the Wet Team successfully turned the final
product
into lycopene, with almost no β-carotene produced. This case demonstrates the feasibility of modifying
enzymes
using the REvoDesign tool. In subsequent experiments, the team will also continue to explore the optimal
conditions for its fermentation production, aiming to achieve the goal of efficient heterologous synthesis
of
lycopene.

#### --Week 4

**General:**

**Human Practices and Wet Team Progress**

In terms of Human Practices (HP), the team organized a science outreach activity
titled "Sowing the Seeds of Science, Illuminating the Light of the Future" at Jinning Primary School. The
activity included sessions such as educational lectures, DIY iGEM logo painting, knowledge quizzes, and
rolling lucky draws. The children were highly enthusiastic, and everyone developed a great interest in
synthetic biology and iGEM!

As always, the Wet Team remained busy in the laboratory—both protein modification
and
product extraction experiments have entered a critical stage.

After modification using the REvoDesign tool, the CarRP protein successfully
enabled
the synthesis of lycopene, which confirmed the tool's reliability. This marks a major success for the
enzyme
modification project!

However, the success of a single case is not sufficient to convince more people of
the
REvoDesign tool's effectiveness. Therefore, our team will start working on protein modification for the
second
case next week, and we believe that the team's full collaboration will help overcome these challenges.

### - August

#### --Week 1

**General:**

As we enter August, the experiments are entering the final stage.

The Wet Team made significant progress this week—after adjusting the experimental
conditions, they successfully obtained recombinant plasmids based on the modified T5αH gene. Following
this
method, we will construct the remaining mutants in one batch and introduce the constructed plasmids into
Saccharomyces cerevisiae (baker’s yeast).

The team conducted an objective analysis of the experimental data from the
lycopene
case, discussed the performance of product data under different modification conditions, and is now
working
to
find the optimal solution.

The HP Team attended the CCiC (China Conference for iGEMers) meeting, conducted
in-depth exchanges with Chinese teams participating in the iGEM competition, and held profound discussions
after listening to each other’s presentations.

![Overview of YNNU-China team's 2025 comprehensive human practice](https://static.igem.wiki/teams/5915/notebook/fig-9.webp)

Fig. 9 Photos of CCiC exchange meeting participation.

#### --Week 2

**General:**

This week, the experiments achieved another significant breakthrough. Members of
the
Wet Team successfully expressed and purified taxadien-5α-ol (T5OH)—the catalytic product of T5αH—in yeast.
They also continued to adjust enzyme modification sites and test more optimization schemes.

After multiple experiments, the team finally identified some enzyme modification
sites
and successfully completed the double-site mutations that enable the efficient synthesis of taxadien-5α-ol
(T5OH). This marks another important milestone in the successful enzyme modification using the REvoDesign
tool.

The WiKi team made solid progress by improving the basic framework of the website.
When we showcase it later, it will definitely be impressive.

Additionally, this week we held a collaborative exchange meeting with students
from
Sichuan University (SCU). We learned about their innovative design concepts and strategies, which led to
an
exciting exchange of ideas and insights, leaving valuable practical experience for both teams.

![Overview of YNNU-China team's 2025 comprehensive human practice](https://static.igem.wiki/teams/5915/notebook/fig-10.webp)

Fig. 10 Live footage of online exchanges with SCU.

#### --Week 3

**General:**

Our experimental progress is going more smoothly day by day! The Wet Team is
approaching the final stage of data collection, and all experiments are advancing steadily—this makes us
more
and more confident in the stability of the experimental data.

What is exciting is that after conducting instrumental detection and analyzing the
calculated data of the product, we found that the T5OH yield of multiple groups of mutants has
significantly
increased compared with that of the unmodified protein.

The Modeling Team continues to test various mutants—this is a complex process, but
progress is steady.

Although the pace in the laboratory is busy, the preparation work for the WiKi is
still proceeding in parallel, with continuous efforts to enhance the website's functions.

#### --Week 4

**General:**

The Laboratory Team (consistent with the "Wet Team" in previous contexts,
referring
to
the team responsible for experimental operations) continued to adjust the conditions for lycopene
fermentation
experiments. Repeated experiments require patience, but each attempt seems to boost everyone's confidence
even
more.

The team had a relatively smooth week with revisions and discussions on HP (Human
Practices) and WiKi. Notably, the team has started drafting some basic documents in the WiKi, marking
steady
progress in the team's tasks.

### - September

#### --Week 1

**General:**

In September, the team focused its efforts on the writing and organizing of WiKi
documents. Members of the Wet Team fully compiled and carefully reviewed their experimental results, and
now
everyone is focused on systematically integrating these results and data into the WiKi. Additionally, the
team
has begun drafting documents for its models to ensure that the integration of experiments and theory is
well
reflected in the WiKi. The entire team is working together to integrate the data in the WiKi, ensuring
that
our team's achievements are clearly and accurately presented.

#### --Week 2

**General:**

This week, the WiKi writing process has entered a critical stage. Most of the
experimental content from the team responsible for enzyme modification using REvoDesign has been
incorporated
into the WiKi, and the documentation from the Modeling Team has also begun to take shape. The team is now
focused on refining the language and logical structure to ensure that the content is not only
scientifically
rigorous but also easily understandable. The team is working hard to integrate the various modules in the
WiKi, and overall, progress is going smoothly.

![Overview of YNNU-China team's 2025 comprehensive human practice](https://static.igem.wiki/teams/5915/notebook/fig-11.webp)

Fig. 11 Photos of the on-site discussion.

#### --Week 3

**General:**

The WiKi document is nearly complete. All experimental data, model results, and
relevant discussions have been fully integrated into the document. We have conducted multiple rounds of
discussions with the PI (Principal Investigator) to ensure the content is both comprehensive and
scientifically accurate. We have conducted multiple careful reviews of the results obtained from enzyme
modification using REvoDesign, to ensure there are no omissions or misrepresentations. The Modeling Team
has
also completed their model documentation, successfully linking the theoretical and experimental aspects of
the
project. The team has now entered the final stage of adjusting the format and layout.

#### --Week 4

**General:**

The final version of the WiKi document has been completed! All content from each
experimental module has been fully integrated, and the theoretical content developed by the Modeling Team
is
rigorous and detailed. The team has done an excellent job with the document formatting and proofreading in
the
WiKi, making the overall presentation of the project highly professional and polished.

Thank you all for your hard work—we have finally showcased our team's efforts in a
clear and impressive manner, highlighting all the work we put in to achieve our goals.

{% endblock %}

{% extends "layout\_no\_header.html" %}
{% block title %}Engineering{% endblock %}
{% block page\_content %}

#### Engineering

[Dry lab](#DryLab)

* [Software Design](#Software)
* [Protein Design](#Protein)

[Wet lab](#WetLab)

* [Engineering of T5αH plant enzyme modification](#modification)
* [CarRP Enzyme Engineering](#CarRP)

[Integrated Human Practices](#Integrated)

## Dry Lab

## Software Design

---

### Software Development and Platform Construction

### Design：

The initial design phase adopted modular principles from industrial software engineering. Core
frameworks include a plugin-based PyMOL interface, the MAGIC scheduling protocol, a YAML-based
function–window mapping mechanism, and the Hydra+OmegaConf configuration bus (Fig. 1). Optional component
management was implemented through pip extras, separating tools such as RFdiffusion, DLPacker, and
OpenMM into modular packages. REvoDesign leverages the Qt environment embedded within PyMOLto avoide
potential commercial licensing issues (Fig. 2).

![Primary school science popularization activity](https://static.igem.wiki/teams/5915/engineering/fig-1.webp)

Fig. 1 Architectural Design of the Evolutionary Data Computation Service for
RevoDesign.

![Primary school science popularization activity](https://static.igem.wiki/teams/5915/engineering/fig-2.webp)

Fig. 2 MAGIC protocol architecture.

### Build：

The development environment is based on VS Code, Miniconda for environment management, Git for version
control, and GitHub private repositories for secure hosting. A standardized workflow—branch creation,
incremental commits, pull requests, CI checks, squash merging, and branch removal—ensures traceability
and compliance with conventional commit practices.

### Test：

Continuous integration and delivery were realized through GitHub Actions, automatically triggering
tests across operating systems, processor architectures, Python versions, and PyMOL distributions.
Testing employed PyTest to organize 585 unit and integration tests, achieving ~70% coverage. Graphical
interface testing combined PyTest-qt with an internally developed TestWorker utility to simulate real
interactions. Lightweight cases were parallelized, whereas more demanding tasks were executed
sequentially to prevent resource conflicts.

### Learn：

Automated reviews and coverage reports provided immediate feedback on design flaws, security risks, and
performance bottlenecks. Development efficiency was further enhanced through exploratory “Vibe Coding,”
where large language models (e.g., ChatGPT, Claude) were used to generate code templates, refine logical
structures, and accelerate prototyping. Insights from these practices informed subsequent iterations of
architectural refinement, testing strategy upgrades, and feature development.

### Rational Protein Design Workflow

### Design：

Within the graphical PyMOL plugin interface, the workflow was modularized into distinct functional
units: loading of protein structures and evolutionary information; prioritization of candidate hotspots
using evolutionary data and structure; rational design of mutants based on PSSM, ddG, and ESM-1v DMS
predictions; integration of external design tools (Cartesian ddG, ColabDesign, RFdiffusion) for
cross-method validation; Clustering and co-evolutionary analysis, selection of final mutation
combinations.

### Build：

Mutant modeling was conducted using side-chain prediction engines such as DLPacker, PIPPack, and
Rosetta-MutateRelax.Declarative YAML-based window configuration enables rapid conversion of Python
functions into interactive modules. Input parameters can be archived and reloaded through configuration
recipes, ensuring reproducibility across experiments

### Test：

Rational validation involved superimposing modeled side chains onto original structures, combining
these with PSSM and ddG calculations to distinguish conservative from high-risk substitutions. Critical
residues underwent both visual inspection and computational confirmation to maintain consistency with
evolutionary constraints.For computational scalability, evolutionary data processing was implemented as
a standalone Flask+Celery+Docker service, supporting parallel task submission, monitoring, and result
packaging under high-concurrency conditions.

### Learn：

Feedback from computational predictions (e.g., PSSM distributions, ddG changes) was cross-validated
with experimental functional data, enabling refinement of hotspot prioritization and mutation
strategies. The Hydra configuration bus and recipe files facilitate complete reconstruction of prior
experimental environments, supporting dynamic adjustment of design strategies.

Cross-platform benchmarking of components such as DLPacker, RFdiffusion, and RosettaPy provided
insights into performance and stability under different hardware conditions, guiding optimal resource
allocation for future experiments.

## Protein Design

---

### Design：

The use of microbial cell factories enabled by synthetic biology to produce natural products has become
a major focus of current research. However, the key enzymes required for natural product biosynthesis
often exhibit poor stability and low catalytic efficiency when expressed in heterologous systems,
thereby reducing overall biosynthetic efficiency. Current protein design platforms are primarily
tailored for industrial enzyme applications and face limitations in addressing challenges such as
epistatic interactions across multiple sites, the intrinsic trade-off between activity and stability,
and reliance on high-throughput approaches.

### Build：

REvoDesign integrates state-of-the-art protein design technologies and tools, leveraging high-precision
complex structures and evolutionary information to guide rational protein engineering. By applying
cross-regional combinatorial optimization between catalytic sites and protein surface regions, and
accounting for long-range structural interactions, the platform alleviates challenges associated with
multi-site epistasis and the trade-off between activity and stability. Furthermore, by employing
cross-validation and screening of variants through multiple algorithms, REvoDesign enables functional
enzyme improvement using only a compact and focused mutant library, thereby overcoming the reliance on
high-throughput approaches.

### Test：

To evaluate the reliability of our tool, we reconstructed a carotenoid biosynthetic pathway in
Saccharomyces cerevisiae and engineered its key gene, CarRP. CarRP comprises two functional domains: the
R domain, which exhibits lycopene cyclase activity, and the P domain, which possesses phytoene synthase
activity but depends on the proper conformation of the R domain for functionality. In designing
loss-of-function mutations in the R domain, it was critical to ensure that its overall folding remained
intact. Accordingly, we employed DiffDock and AlphaFold3 to perform enzyme–substrate complex modeling
and molecular docking. We identified the region within approximately 6 Å of the substrate lycopene in
the R domain as the putative active site. To avoid disrupting the phytoene synthase function of the P
domain, we targeted only residues distant from this domain for mutagenesis and subsequent experimental
testing. Using the “visualization” function of REvoDesign, we conducted rational design-based evaluation
and selected two candidate mutation sites (F81A and Y145F). Experimental validation confirmed that the
Y145F mutant completely lost lycopene cyclase activity and produced the highest lycopene yield, whereas
the F81A mutant still accumulated a small amount of β-carotene (Fig. 3).

![Primary school science popularization activity](https://static.igem.wiki/teams/5915/engineering/fig-3.webp)

Fig. 3 CarRP transformation results.

a HPLC detection results of yeast fermentation products. b Yeast transformation results and product
ratios.

Based on the structure of T5αH, we applied REvoDesign for active-site pocket analysis and rational
mutagenesis design. Using the visualization tools of REvoDesign, we assessed substrate binding energy
and protein folding free energy, and identified 23 candidate mutants. Experimental validation revealed
that mutations in the active-site region (L72M, V226E) and in the stability region (Q122A, Q266A)
significantly enhanced the yield of the target product. Building upon the high-yield single mutants, we
combined beneficial mutations from the active and stability regions, ensuring that the functional
regions did not overlap, to construct double mutants. The results demonstrated that the double mutants
produced significantly higher levels of the target product compared with all single mutants and the wild
type (Fig. 4).

![Primary school science popularization activity](https://static.igem.wiki/teams/5915/engineering/fig-4.webp)

Fig. 4 T5αH transformation results.

### Learn：

Molecular dynamics simulations are employed to analyze combinations of beneficial mutations and to
elucidate the mechanisms by which these variants enhance yield. The resulting insights serve to guide
the further optimization of enzyme function.

## Wet Lab

## Engineering of T5αH plant enzyme modification

---

### Construction of yeast chassis

### Design：

Heterologous production of paclitaxel by reconstituting the biosynthetic pathway in microorganisms is a
highly promising route. However, it is still highly challenging to achieve de novo and efficient
synthesis of paclitaxel in microbial hosts due to bottlenecks constituted along the pathway. T5αH
represents one such enzyme-level bottleneck due to its poor activity and selectivity when expressed in
microbial hosts (Fig. 5). Therefore, it is imperative to enhance the conversion of taxadiene into T5OH,
which, as such, would be advantageous for improved pathway control and performance. To address this, we
constructed a yeast chassis to support downstream engineering.

![Primary school science popularization activity](https://static.igem.wiki/teams/5915/engineering/figure-5-schematic-diagram-of-the-t5oh-biosynthesis-pathway.webp)

Fig. 5 Schematic diagram of the T5OH biosynthesis pathway。

### Build：

* Recombinant plasmids were designed and assembled using SnapGene, and the target genes were amplified
  by PCR with gene-specific primers. The BTS1 and TS genes were cloned into the yeast expression vector
  pMASC02-Ura3, and the resulting recombinant plasmids were transformed into chemically competent
  Escherichia coli cells for verification.
* The recombinant plasmids were linearized with NotI (NEB) and subsequently transformed into
  Saccharomyces cerevisiae to construct the engineered chassis strain.

### Test：

Recombinant vectors carrying BST1 and TS were successfully introduced into S. cerevisiae, generating
the engineered chassis strain. Sequencing confirmed correct assembly(Fig. 6).

![Primary school science popularization activity](https://static.igem.wiki/teams/5915/engineering/figure-6-vector-backbone-and-sequencing-results.webp)

Fig. 6 Vector backbone and sequencing results.

### Learn：

The engineered chassis strain was capable of producing T5OH. When cultured in glucose medium, it
proliferated normally, and upon transfer to galactose medium, it generated precursor metabolites
required for T5OH biosynthesis.

### Generation and screening of mutants

### Design：

We set out to design and engineer T5αH variants using our REvoDesign approach.The three-dimensional
complex structure of T5αH, the cofactor heme, and the substrate taxadiene was predicted using AlphaFold2
and DiffDock.In parallel, evolutionary information for T5αH was obtained through position-specific
scoring matrix (PSSM) analysis and Gremlin coevolutionary modeling.Based on these structural and
evolutionary insights, we carried out rational engineering of T5αH to guide the design of improved
enzyme variants.

### Build：

We identified two functional regions of T5αH for targeted mutagenesis: activity-related sites located
within the substrate pocket and stability-related sites outside the substrate and heme pockets. Mutants
were designed using REvoDesign, excluding proline and cysteine substitutions, and screened based on
predicted substrate binding energy (activity) or protein folding free energy (stability). This strategy
yielded a focused set of variants with enhanced catalytic efficiency or improved structural stability
for subsequent experimental validation.

### Test：

Finally, 23 mutants were selected—including 15 targeting activity-related sites(Tab. 1) and 8 targeting
stability-related sites(Tab. 2)—with the goal of separately verifying how mutations in the two types of
sites influence T5OH production.

![Primary school science popularization activity](https://static.igem.wiki/teams/5915/engineering/table-1-screening-of-activity-related-mutants.webp)

Tab. 1 Screening of Activity-Related Mutants

![Primary school science popularization activity](https://static.igem.wiki/teams/5915/engineering/table2.webp)

Tab. 2 Screening of Stability-Related Mutants

### Learn：

This design phase yielded 15 variants targeting catalytic activity and 8 targeting structural
stability. These will undergo functional validation in subsequent experiments.

### Validation of the mutant

Iteration1

### Design:

The constructed T5αH mutants were cloned into a yeast expression vector and transformed into the
engineered chassis strain. The resulting transformants were subjected to small-scale fermentation,
followed by extraction of the target product T5OH and analysis using GC–MS.

### Build:

In the initial fermentation trials, the wild-type (WT) strain was used to preliminarily optimize
culture conditions. Fermentations were carried out in 100-, 150-, 250-, and 500-mL Erlenmeyer flasks
containing either 10 mL or 20 mL of medium, incubated at 30 °C and 220 rpm for 5 days. The fermentation
products were then extracted for analysis.

### Test:

Unfortunately, no detectable T5OH was observed in any of the fermentation broths.

### Learn:

Comprehensive verification of both the recombinant expression vectors and the chassis strain revealed
no construction errors, indicating that the failure to detect T5OH was not due to upstream genetic
assembly. We hypothesized that the absence of product was likely caused by insufficient precursor supply
within the chassis strain, thereby limiting T5OH biosynthesis. Given time constraints, reconstructing a
chassis with an enhanced precursor pathway was not feasible. Literature reports suggested that lowering
the fermentation temperature can increase the accumulation of the T5OH precursor taxadiene (Fig. 7).
Therefore, we planned to perform subsequent fermentations at 20 °C to enhance T5OH production（1）.

![Primary school science popularization activity](https://static.igem.wiki/teams/5915/engineering/figure-7-taxadiene-production.webp)

Fig. 7 Taxadiene production.

Iteration2

### Design:

Based on the preceding analysis, we adopted a strategy reported in the literature to re-evaluate T5OH
production by lowering the fermentation temperature.

### Build:

The wild-type (WT) strain was again used to optimize the culture conditions. Fermentations were
conducted at 20 °C and 220 rpm for 5 days, and the products were subsequently extracted for
analysis，after which the products were extracted with n-hexane for GC–MS analysis.

### Test:

Under these modified conditions, T5OH was successfully detected in the 20mL fermentation cultures,
demonstrating that lowering the fermentation temperature facilitated product formation.

### Learn:

This improvement in fermentation conditions enabled the detection of T5OH, confirming the feasibility
of the revised approach. The optimized parameters (20 °C, 220 rpm, 5 days, 20 mL culture volume) were
subsequently applied to mutant strains to evaluate their effects on T5OH biosynthesis.

Iteration3

### Design:

Following preliminary optimization of the fermentation parameters, a total of 23 rationally designed
T5αH mutants—targeting both activity-related and stability-related sites—were selected for functional
validation.

### Build:

Wild-type (WT) strain served as the control. For each mutant, three independent single-colony isolates
were cultured in 100-mL Erlenmeyer flasks containing 20 mL fermentation medium at 20 °C and 220 rpm for
5 days. The fermentation products were extracted with n-hexane, and β-caryophyllene was added as an
internal standard for quantitative analysis.

### Test:

Among the 23 mutants tested, several variants exhibited significantly enhanced T5OH production relative
to the WT control. Notably, activity-related mutants L72M and V226E demonstrated markedly improved
catalytic activity (Fig. 8a), while stability-related mutants Q122A and Q266A also showed substantial
increases in T5OH yield (Fig. 8b).

![Primary school science popularization activity](https://static.igem.wiki/teams/5915/engineering/figure-8-effects-of-t5-h-activity-related-and-stability-related-mutations-on-t5oh-production.webp)

Fig. 8 Effects of T5αH activity-related and stability-related mutations on
T5OH production.

a T5OH production efficiency of activity-related mutants vs WT T5αH. The vertical axis represents
relative T5OH yield. Compared to WT, mutants targeting activity-related sites showed distinct yield
changes: L72M and V226E exhibited highly significant increases in T5OH yield, with yields ~2.3-fold
and ~2.1-fold that of WT, respectively—this is attributed to enhanced substrate binding affinity and
catalytic turnover rate. b Residual activity of stability-related mutants and their correlation with
T5OH yield. Mutants targeting stability-related sites showed clear stability-yield correlations:
Q122A, Q266A exhibited highly significant yield improvement. Statistical significance is marked by
asterisks: \*\*\*p < 0.001.

### Learn:

These results validated the effectiveness of our structure-guided, evolution-informed design strategy,
identifying key mutations that enhance either catalytic efficiency or structural stability of T5αH. The
best-performing mutants will serve as the basis for further combinatorial mutagenesis to evaluate
potential additive or synergistic effects on T5OH biosynthesis.

### Generation and Screening of Combined Mutants

### Design:

To further enhance T5OH (taxadien-5α-ol) production efficiency, we leveraged the results of single-site
mutant verification and adopted a rational combination strategy for activity-related and
stability-related mutation sites—selecting high-performance single mutants that target non-overlapping
functional regions (to avoid mutual interference between mutations) for site-directed combination. This
design was guided by REvoDesign’s predictive analysis: the tool confirmed that the selected
activity-related sites (located in the substrate pocket) and stability-related sites (located in the
structural maintenance region) have independent spatial distributions and functional roles, ensuring
that their combination would not disrupt the enzyme’s overall structure or catalytic core.

### Build:

We constructed recombinant plasmids carrying different double mutant sequences via molecular cloning.
We performed various types of arrangement and combination on the aforementioned high-yield single
mutants to further obtain double mutants, and observed whether they possess a higher yield.

### Test:

Fermentation assays revealed that the L72M+Q122A double mutant achieved the highest improvement in T5OH
yield compared to single mutants and WT(Fig. 9).

![Primary school science popularization activity](https://static.igem.wiki/teams/5915/engineering/figure-9-comparison-of-t5oh-yields-among-t5-h-wild-type-single-mutants-and-double-mutants.webp)

Fig. 9 Comparison of T5OH yields among T5αH Wild-Type, single mutants, and
double mutants.

### Learn:

In summary, the yield data of the double mutants not only confirms that multi-dimensional optimization
of T5αH can drastically improve T5OH production but also further validates REvoDesign’s effectiveness as
a powerful tool for rational enzyme engineering.

## CarRP Enzyme Engineering

---

### Construction of the Yeast Chassis

### Period 1

### Design：

Saccharomyces cerevisiae possesses an endogenous mevalonate (MVA) pathway that provides precursors for
terpenoid biosynthesis; however, its native metabolic network lacks the complete enzymatic machinery
required for lycopene production(Fig. 10). To overcome this limitation, we previously integrated the
CarG and CarB genes into a genomic integration vector, thereby creating an engineered yeast strain
capable of synthesizing the precursor metabolites necessary for lycopene biosynthesis（2-5）.

![Primary school science popularization activity](https://static.igem.wiki/teams/5915/engineering/figure-10-lycopene-synthesis-pathway.webp)

Fig. 10 Lycopene synthesis Pathway.

### Build:

* Recombinant plasmids were designed and assembled using SnapGene (Fig. 11), and the target genes
  were amplified by PCR with specific primers. The CarG and CarB genes were cloned into the
  dual-expression vector pMASC02-Ura3.
* The recombinant plasmids were then transformed into chemically competent Escherichia coli cells to
  complete vector construction.

![Primary school science popularization activity](https://static.igem.wiki/teams/5915/engineering/figure-11-construction-of-gene-expression-vector.webp)

Fig. 11 Construction of gene expression vector.

### Test:

To identify positive clones, colony PCR was performed using primer pairs flanking the CarG and CarB
genes, generating expected amplicons of 912 bp and 1740 bp (Fig. 12). respectively. Colonies exhibiting
the correct PCR profiles were subsequently selected for Sanger sequencing to confirm construct integrity
(Fig. 13).

![Primary school science popularization activity](https://static.igem.wiki/teams/5915/engineering/figure-12-colony-pcr-identification.webp)

Fig. 12 Colony PCR Identification.

![Primary school science popularization activity](https://static.igem.wiki/teams/5915/engineering/figure-13-sequencing-results.webp)

Fig. 13 Sequencing results.

### Learn:

Through this round of experimentation, we successfully constructed the recombinant plasmid
pMASC02-TTPI1-CarB-PGAL10-PGAL1-CarG-TPGI- Ura3. Initially, plasmid assembly using a conventional
single-fragment recombinase failed to yield the desired construct. Subsequently, NEBuilder HiFi DNA
Assembly Master Mix was employed for multi-fragment assembly, which resulted in successful plasmid
construction. As the next step, we plan to integrate this plasmid into the Saccharomyces cerevisiae
BY4742 strain to further reprogram its metabolic pathway for the production of lycopene precursors.

### Period 2

### Design:

Based on literature reports, *Saccharomyces cerevisiae* **BY4742** was selected as
the host strain for chassis construction because of its well-characterized genetic background, ease of
genetic manipulation, and suitability for heterologous protein expression. Moreover, the endogenous
**mevalonate (MVA) pathway** in S. cerevisiae provides precursor metabolites for lycopene
biosynthesis, and
the strain offers the advantages of simple cultivation and low production cost.

### Build:

* The recombinant plasmid pMASC02-TTPI1-CarB-PGAL10-PGAL1-CarG-TPGI- Ura3 was digested with
  **NotI** (Fig. 14).
* Competent cells of S. cerevisiae BY4742 were prepared, and the digested vector fragments were
  transformed into the yeast competent cells for integration.
* Integration of CarG and CarB into the yeast genome was verified to confirm successful chassis
  construction.

![Primary school science popularization activity](https://static.igem.wiki/teams/5915/engineering/figure-14-schematic-diagram-of-enzyme-digestion.webp)

Fig. 14 Schematic diagram of enzyme digestion.

### Test:

PCR screening identified positive transformants with the expected 912 bp and 1740 bp bands (Fig. 15a).
The verified clone, designated BY01 (Fig. 15b), was stored in 20% glycerol at –80 °C for long-term
preservation.

![Primary school science popularization activity](https://static.igem.wiki/teams/5915/engineering/figure-15-construction-of-lycopene-engineering-strain.webp)

Fig. 15 Construction of lycopene engineering strain.

a The electrophoresis profile of PCR detection for transformed yeast strains. b The colony morphology of recombinant yeast inoculated onto Ura-deficient (Uracil-deficient) medium plates after 2 days of cultivation.

### Learn：

Through this round of experimentation, we successfully constructed a *Saccharomyces cerevisiae*
chassis
strain capable of supporting **lycopene biosynthesis.** During the initial yeast
transformation, no colonies
were observed on **uracil-deficient (Ura⁻)** selection medium. This failure was attributed
to an
insufficient concentration of the NotI-digested plasmid. After repeating the digestion and increasing
the plasmid concentration, transformation yielded normal single colonies. The next phase of this work
will focus on **functional engineering of CarRP**, aiming to convert the native
bifunctional enzyme into a
**monofunctional variant.**

### Generation and screening of mutants

### Design：

CarRP is a bifunctional enzyme consisting of an N-terminal R domain (lycopene cyclase) and a C-terminal
P domain (phytoene synthase). The R domain can function independently, whereas the P domain requires
proper folding of the R domain. Using REvoDesign, we targeted the R domain to suppress lycopene cyclase
activity while retaining phytoene synthase activity. Two variants, F81A and Y145F, were obtained (Fig.
16).

![Primary school science popularization activity](https://static.igem.wiki/teams/5915/engineering/figure-16-model-of-interactions-of-the-amino-acids-in-the-active-site-of-the-protein-with-lycopene.webp)

Fig. 16 Model of interactions of the amino acids in the active site of the
protein with lycopene.
Green: Wild-type；yellow: lycopene；magenta: acid mutant.

### Build：

* Construction of fusion expression vectors harboring **CarRP** and its mutant
  variants.
* Preparation of competent cells of *Saccharomyces cerevisiae* **BY01**,
  followed by transformation with
  the **digested vector fragments** and cultivation under selective conditions.
* For each construct (**CarRP** and its mutants), **three single
  colonies** were selected for shake-flask
  fermentation. Metabolites were extracted from the cultures and analyzed by **liquid
  chromatography–mass
  spectrometry (LC–MS)** to identify and quantify the products.

### Test：

Analysis results showed that the fermentation products of CarRP\_F81A and CarRP\_Y145F mutants both
exhibited a high lycopene peak, with yields of 1.42 g/L and 1.46 g/L, respectively. CarRP\_F81A produced
a very small amount of β-carotene, while CarRP\_Y145F produced no β-carotene at all. Furthermore, the
lycopene yields of each mutant strain were comparable to the β-carotene yield of the strain containing
wild-type CarRP, indicating that the two CarRP mutants designed in this study had almost lost their
lycopene cyclase activity (Fig. 17). As a result, lycopene no longer cyclized to form β-carotene but
accumulated as the final product in Saccharomyces cerevisiae.

![Primary school science popularization activity](https://static.igem.wiki/teams/5915/engineering/figure-17-functional-validation-of-carrp-mutants.webp)

Fig. 17 Functional validation of CarRP mutants.

### Learn：

In this round of experiments, the functionality of the two engineered CarRP mutants was successfully
validated. The introduced mutations effectively blocked β-carotene biosynthesis, resulting in enhanced
**lycopene accumulation** in *Saccharomyces cerevisiae*. As the next step, we plan
to perform scaled-up
fermentations to evaluate lycopene production yields.

### Pilot-Scale Fed-Batch Fermentation

### Design：

The above experiments confirmed that the engineered mutants enabled greater *lycopene
accumulation* in
 *Saccharomyces cerevisiae*. The CarRP\_Y145F strain was selected for pilot-scale fermentation in
a
bioreactor to evaluate lycopene production.

### Build：

* Strains were streaked onto **YPD agar plates** for activation. Deep red, well-grown
  *Saccharomyces
  cerevisiae* colonies were selected and inoculated into **50 mL YPD broth**,
  followed by incubation at 30 °C
  with shaking in a constant-temperature orbital shaker.
* Inoculate the fermentation broth into a 3 L fermenter containing 950 mL of medium for fed-batch
  fermentation.
* Low-speed feeding was initiated after 48 hours of fermentation, Samples were taken every 24 hours
  to determine the OD600 and lycopene yield.

### Test：

During the initial 48 h, carbon flux supported rapid biomass accumulation, with low lycopene
productivity (0.033 g/L/h). As glucose was depleted, metabolism shifted toward secondary metabolism,
including lycopene biosynthesis (Fig. 18a). After 216 h of fermentation, the strain achieved a titer of
~9.0 g/L lycopene, corresponding to a productivity of 0.049 g/L/h (Fig. 18b).

![Primary school science popularization activity](https://static.igem.wiki/teams/5915/engineering/figure-18-growth-curve-and-lycopene-production-of-the-strain.webp)

Fig. 18 Growth curve and lycopene production of the strain.

a High-density fermentation in a 3L fermenter. b Comparison of cell growth rate and lycopene productivity at different stages of fermentation.

### Learn：

Pilot-scale fermentation confirmed that the engineered CarRP mutant achieved high-level lycopene
production (9.0 g/L). These results demonstrate both the robustness of the engineered strain and the
effectiveness of REvoDesign-guided enzyme engineering.

### References

* Nowrouzi, B., Li, R. A., Walls, L. E., d'Espaux, L., Malcı, K., Liang, L., Jonguitud-Borrego, N.,
  Lerma-Escalera, A. I., Morones-Ramirez, J. R., Keasling, J. D., & Rios-Solis, L. (2020). Enhanced
  production of taxadiene in Saccharomyces cerevisiae. Microbial cell factories, 19(1), 200.
* Zhou, K., Yu, C., Liang, N., Xiao, W., Wang, Y., Yao, M., & Yuan, Y. (2023). Adaptive Evolution and
  Metabolic Engineering Boost Lycopene Production in Saccharomyces cerevisiae via Enhanced Precursors
  Supply and Utilization. Journal of agricultural and food chemistry, 71(8), 3821–3831.
* Velayos, A., Blasco, J. L., Alvarez, M. I., Iturriaga, E. A., & Eslava, A. P. (2000). Blue-light
  regulation of phytoene dehydrogenase (carB) gene expression in Mucor circinelloides. Planta, 210(6),
  938–946.
* Velayos, A., Eslava, A. P., & Iturriaga, E. A. (2000). A bifunctional enzyme with lycopene cyclase
  and phytoene synthase activities is encoded by the carRP gene of Mucor circinelloides. European
  journal of biochemistry, 267(17), 5509–5519.
* Ma, Y., Liu, N., Greisen, P., Li, J., Qiao, K., Huang, S., & Stephanopoulos, G. (2022). Removal of
  lycopene substrate inhibition enables high carotenoid productivity in Yarrowia lipolytica. Nature
  communications, 13(1), 572.

## Integrated Human Practices

---

### Design:

pWe first systematically reviewed disease reports, global health policies, and annual reports from
biomanufacturing companies to elucidate the necessity of heterologous synthesis of plant natural products.

* **Expert & Industry Input:**
  Interviews with clinicians, researchers, and executives revealed the key bottleneck:
  **low activity and poor stability of plant-derived enzymes in heterologous hosts.**
* **Surveys:**
  We collected 204 valid responses from researchers and the public. Over 60% of researchers reported “low
  catalytic efficiency” as a major pain point. Public surveys showed 77% would accept biosynthetic
  products if efficacy was maintained and cost was lower.
* **Goal Definition:**
  Based on these insights, we designed **REvoDesign** — a co-evolution-driven, semi-rational
  protein design tool to improve enzyme activity and stability for heterologous synthesis.

![Primary school science popularization activity](https://static.igem.wiki/teams/5915/engineering/fig-19.webp)

Fig. 19 Research reports, expert consultations, and questionnaire surveys.

a Logos of organizations involved in disease reports and global health policy references, including the World Health Organization (WHO), International Agency for Research on Cancer, National Center for Cardiovascular Diseases, and World Heart Federation. b Logos of biomanufacturing companies whose annual reports were reviewed to elucidate industry insights, namely Angel Yeast Co, Ltd, Shanghai Pharmaceuticals Holding Co.,ltd, Chenguang Biotech Group Co.,Ltd, and Anhui Huaheng Biotechnology Co., Ltd. c Representative individuals interviewed for expert and industry input, including clinicians, researchers, and executives from relevant fields. d Promotional material for the synthetic biology - based survey carried out by the YNNU - China team, which contains a QR code for respondents to participate in the survey.

### Build:

We built REvoDesign by integrating:

* **Co-evolutionary analysis**
  to identify key sites with evolutionary constraints.
* **Semi-rational mutation libraries**
  to reduce blind screening.
* **User-friendly workflow**
  with modular outputs and traceable records to meet industry R&D needs.

During this stage, we consulted protein design experts (Yuan Zhou, Haiyan Liu, Bin Huang) to refine the
algorithm:

* Added **evolutionary conservation** filters to protect key active sites.
* Optimized workflow by removing unnecessary preprocessing steps.
* Strengthened interpretability and visualization for quick expert assessment.

![Primary school science popularization activity](https://static.igem.wiki/teams/5915/engineering/fig-20.webp)

Fig. 20 Interview with Protein Design Consultant.

a Associate Professor Yuan Zhou. b Professor Haiyan Liu. c Professor Bin Huang.

### Test:

We selected two case studies representing different industries:

* **T5αH (Paclitaxel P450 Enzyme, Pharmaceutical):**  Improved catalytic activity in yeast
  for higher paclitaxel precursor yield.
* **Lycopene Cyclase (Nutritional Health):**  Deactivated cyclase function of CarRP to
  boost lycopene production in yeast.

### Results:

* Achieved significant improvement in catalytic efficiency for both enzymes.
* Reduced experimental screening workload.
* Received high praise from corporate partners (Chenguang Biotech Group Co.,Ltd, Hubei Meiqi Health
  Technology Co., Ltd) for addressing real production bottlenecks.

![Primary school science popularization activity](https://static.igem.wiki/teams/5915/engineering/fig-21.webp)

Fig. 21 REvoDesign tool case test.

a bifunctional phytoene synthase/lycopene cyclase(CarRP). b taxadiene-5α-hydroxylase(T5αH)

### Learn:

From feedback by users, scholars, and companies:

* **Usability:**  Added layered versions (easy/advanced), video tutorials, and improved error
  feedback.
* **Scalability:**  Plan multi-enzyme optimization and multi-objective design (activity,
  stability, solubility); introduce QM/MM calculations to explain mutation mechanisms; integrate machine
  learning with physical energy functions for speed and interpretability; support active learning; and
  provide natural language interfaces and visual documentation (Feng Jiang, Yuan Liu, Xiaolin Shen, Long
  Liu).
* **Industry Integration:**  Signed a three-party collaboration agreement with Hubei Meiqi
  Health Technology Co., Ltd for pilot-scale validation, and engaged with Shanghai Pharma for future
  paclitaxel pathway optimization.

![Primary school science popularization activity](https://static.igem.wiki/teams/5915/engineering/fig-22.webp)

Fig. 22 Evaluations and feedback from businesses and experts on REvoDesign help
to iterate the tool.

### Future Plans:

We will enhance REvoDesign with AI-guided mutation combination prediction, build a “question-guided”
design assistant, and expand to other high-value natural products, turning REvoDesign into an
**intelligent,
interpretable, and scalable protein design platform.**

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your project. It is designed to provide sufficient information for other teams to replicate our work.{% endblock %}
{% block page\_content %}

## What Should this Page Contain?

---

Provide comprehensive, step-by-step protocols for all experiments conducted. Include a list of materials, reagents, and equipment used. Specify concentrations, volumes, incubation times, temperatures, and other critical parameters. Document any modifications or optimizations made to standard protocols.

Explain the purpose of each experiment and its relevance to your project goals. Describe the experimental design, including controls and replicates. Provide a clear rationale for the chosen methods and approaches.

Document any troubleshooting steps taken and optimizations made during the experimental process. Share any lessons learned that could benefit future teams.

## Inspirations

---

* [2024 Heidelberg](https://2024.igem.wiki/heidelberg/experiments)
* [2024 Marburg](https://2024.igem.wiki/marburg/experiments)
* [2024 EPFL](https://2024.igem.wiki/epfl/experiments)
* [2023 Estonia-TUIT](https://2023.igem.wiki/estonia-tuit/experiments)

{% endblock %}

{% extends "layout\_no\_header.html" %}
{% block title %}Results{% endblock %}
{% block page\_content %}

#### Results

- [Taxadiene-5α-hydroxylase(T5αH)](#Taxadiene)
- [Bifunctional phytoene synthase/lycopene
  cyclase(CarRP)](#Bifunctional)

## Taxadiene-5α-hydroxylase(T5αH)

---

### Construction of Heterologous T5OH Synthesis Pathway

To validate the generality and robustness of REvoDesign, we first
verified its performance and then applied this protein design approach to engineer
taxadiene-5α-hydroxylase (T5αH) which is involved in the biosynthesis of paclitaxel (Fig.1).
Heterologous production of paclitaxel by reconstituting the biosynthetic pathway in
microorganisms is a highly promising route. However, it is still highly challenging to
achieve de novo and efficient synthesis of paclitaxel in microbial hosts due to bottlenecks
constituted along the pathway. T5αH represents one such enzyme-level bottleneck due to its
poor activity and selectivity when expressed in microbial hosts. Therefore, it is imperative
to enhance the conversion of taxadiene into T5OH and reduce the accumulation of byproducts,
which, as such, would be advantageous for improved pathway control and performance.

![Primary school science popularization activity](https://static.igem.wiki/teams/5915/result/fig-1.webp)

Fig.1 Schematic diagram of the T5OH biosynthesis pathway.

### Vector Construction of Genes Related to the T5OH Biosynthesis Pathway

Through molecular cloning, the taxadiene synthesis genes GGPPs and TS
derived was cloned into vectors pMASC02-Ura3, resulting in the recombinant plasmids
pMASC02-TTPI1-TS-Linker-GGPPs-PGAL10-Ura3 (Fig.2a). Meanwhile, T5αH and CRP was cloned
into vector pMASC04-HIS3, yielding the recombinant plasmid
pMASC04-TFBA1-CPR-PGAL10-PGAL1-T5αH-TPDC1-His3 (Fig.2b). The plasmid map and results of E.
coli colony PCR identification are shown in Figure 2. The E. coli colony PCR identification
results were consistent with the expected outcomes, and the sequencing results were correct.

![Primary school science popularization activity](https://static.igem.wiki/teams/5915/result/fig-2.webp)

Fig.2 Construction of gene expression vector.

a The plasmid has integrated the GGPPs and TS genes. Meanwhile, key functional elements
are labeled: promoter GAL10, terminator TPI1, and the selectable marker gene Ura3. b The
T5αH and CRP genes were inserted into the plasmid, with key elements including the GAL1
and GAL10 promoter, PDC1 and FBA1 terminator, and His3 selectable marker gene. The
figure below shows the PCR amplification bands of the recombinant plasmid.

### - Construction of T5OH-Engineering Strains

The linearized recombinant plasmid
pMASC02-TTPI1-TS-Linker-GGPPs-PGAL10- Ura3 was integrated into the genome of BY4742, and the
strain TY01 was obtained. Subsequently, the linearized plasmid
pMASC04-TFBA1-CPR-PGAL10-PGAL1-T5αH-TPDC1 -His3 was transformed into TY01 (used as the
chassis cell), resulting in the T5OH-producing strain TY02. After verification by genomic
PCR (Fig.3a), the correct integration of the plasmid was confirmed. The recombinant yeast
was plated on Leu-deficient plates, and white single colonies were observed on the surface
of the medium after 2 days (Fig.3b).

![Primary school science popularization activity](https://static.igem.wiki/teams/5915/result/fig-3.webp)

Fig.3 Construction of T5OH engineering strain.

a The electrophoresis profile of PCR detection for transformed yeast strains. b The
colony morphology of recombinant yeast inoculated onto Leu-deficient (leucine-deficient)
medium plates after 2 days of cultivation.

### Rational Design of T5αH Mutant Proteins Using REvoDesign

We set out to design and engineer T5αH variants using our REvoDesign
approach. An apo structural model of T5αH was created using AlphaFold2, and the heme group
referring to its top hit crystal structure (PDB: 6A15) was transplanted via the pair-fit
wizard tool of PyMOL, which was followed by a FastRelax using Rosetta to remove spatial
crashes. A DiffDock blind docking inference was next performed against the top hit complex
model of relaxed T5αH-heme model and taxadiene molecule. By aligning the diffusion
trajectories, the top model as the initial model was then selected for further model
refinement using RosettaLigand (Fig.4). The final optimized model was used for further
design and evaluation processes.

![Primary school science popularization activity](https://static.igem.wiki/teams/5915/result/fig-4.webp)

Fig.4 presents the optimized T5αH-heme complex model.

### Functional Verification of T5αH Mutant

To systematically explore how mutations targeting activity-related
sites and stability-related sites affect T5OH (taxadien-5α-ol) production, we first defined
two functional regions of T5αH to guide site-specific mutation design:

1. Activity-related region (substrate pocket): Residues within a
6-angstrom radius of T5OH (used as a substrate analog) were designated as the substrate
pocket—these residues directly interact with the substrate taxadiene and participate in
catalytic reactions, so mutations here primarily regulate T5αH’s catalytic activity (Tab.1).

![Primary school science popularization activity](https://static.igem.wiki/teams/5915/result/table-1.webp)

Tab. 1 Screening of activity-related mutants.

2. Stability-related region: Residues that are non-overlapping with
the substrate pocket and cofactor pocket (heme-binding region) but critical for maintaining
T5αH’s three-dimensional structure were defined as stability-related sites—mutations here
aim to improve the enzyme’s thermal stability or structural robustness without interfering
with catalytic function (Tab. 2).

![Primary school science popularization activity](https://static.igem.wiki/teams/5915/result/table-2.webp)

Tab. 2 Screening of Mutants Based on Thermal Stability

During mutant design, substitutions involving proline (Pro) or cysteine (Cys) were excluded:
Pro has a rigid side chain that is essential for maintaining the protein backbone’s
stability (disrupting it may cause structural collapse), while Cys can form unwanted
disulfide bonds or interfere with heme binding, both of which are detrimental to T5αH’s
function. Using the "Visualize" function of REvoDesign, we further screened mutants by
evaluating two key metrics: (1) for activity-related mutants, changes in substrate binding
energy (to predict catalytic efficiency); (2) for stability-related mutants, changes in
protein folding free energy (to predict structural stability). Finally, 23 mutants were
selected—including 15 targeting activity-related sites and 8 targeting stability-related
sites—with the goal of separately verifying how mutations in the two types of sites
influence T5OH production. The catalytic activity and stability of these mutants were
measured, with the results shown in Fig.5.

![Primary school science popularization activity](https://static.igem.wiki/teams/5915/result/fig-5.webp)

Fig.5 Effects of T5αH activity-related and stability-related
mutations on T5OH production.

a. T5OH production efficiency of activity-related mutants vs WT T5αH. The vertical axis
represents relative T5OH yield. Compared to WT, mutants targeting activity-related sites
showed distinct yield changes: L72M and V226E exhibited highly significant increases in
T5OH yield, with yields ~2.3-fold and ~2.1-fold that of WT, respectively—this is
attributed to enhanced substrate binding affinity and catalytic turnover rate. b.
Residual activity of stability-related mutants and their correlation with T5OH yield.
Mutants targeting stability-related sites showed clear stability-yield correlations:
Q122A, Q266A exhibited highly significant yield improvement. Statistical significance is
marked by asterisks: \*\*\*p < 0.001.

### T5OH Yield Verification of T5αH Double Mutants

To further enhance T5OH (taxadien-5α-ol) production efficiency, we
leveraged the results of single-site mutant verification (Fig.5) and adopted a rational
combination strategy for activity-related and stability-related mutation sites—selecting
high-performance single mutants that target non-overlapping functional regions (to avoid
mutual interference between mutations) for site-directed combination. This design was guided
by REvoDesign’s predictive analysis: the tool confirmed that the selected activity-related
sites (located in the substrate pocket) and stability-related sites (located in the
structural maintenance region) have independent spatial distributions and functional roles,
ensuring that their combination would not disrupt the enzyme’s overall structure or
catalytic core.

We constructed recombinant plasmids carrying different double mutant
sequences via molecular cloning. We performed various types of arrangement and combination
on the aforementioned high-yield single mutants to further obtain double mutants, and
observed whether they possess a higher yield.

All strains were subjected to shake-flask fermentation under the same
conditions, and T5OH yields were detected by HPLC. The results are shown in the T5OH yield
diagram below (Fig.6).

![Primary school science popularization activity](https://static.igem.wiki/teams/5915/result/fig-6.webp)

Fig.6 Comparison of T5OH yields among T5αH Wild-Type, single
mutants, and double mutants.

In summary, the yield data of the double mutants not only confirms that multi-dimensional
optimization of T5αH can drastically improve T5OH production but also further validates
REvoDesign’s effectiveness as a powerful tool for rational enzyme engineering.

## Bifunctional phytoene synthase/lycopene cyclase(CarRP)

---

### Construction of yeast chassis

Saccharomyces cerevisiae can utilize its intrinsic MVA pathway to
provide precursors for terpenoid synthesis; however, its natural metabolic network lacks the
complete enzyme system required for lycopene biosynthesis. Therefore, in this study, the
CarRP and CarB genes — which encode phytoene synthase and phytoene desaturase, respectively—
were constructed into a gene integration vector. These genes were randomly integrated into
the genome of Saccharomyces cerevisiae, so as to achieve stable lycopene synthesis.

In the MVA pathway, acetyl-CoA generates intermediate products through
a series of enzymatic reactions, which provide the basis for lycopene synthesis.
Nevertheless, limited by the catalytic capacity of enzymes involved in each step of the
pathway, the rate of product synthesis remains rather low. Hence, while constructing the
lycopene synthesis pathway, this study also introduced CarG, CarRP, and CarB into the
synthetic pathway (as shown in Fig.7) to enhance lycopene production.

![Primary school science popularization activity](https://static.igem.wiki/teams/5915/result/fig-7.webp)

Fig. 7 Lycopene synthesis Pathway.

### Vector Construction of Genes Related to the Lycopene Biosynthesis Pathway

Through molecular cloning, the lycopene synthesis genes CarB and CarG
derived from Mucor circinelloides were cloned into vectors pMASC02-Ura3, resulting in the
recombinant plasmids pMASC02-TTPI1-CarB-PGAL10-PGAL1-CarG-TPGI-Ura3 (Fig.8a). Meanwhile,
CarRP was cloned into vector pMASC04-HIS3, yielding the recombinant plasmid
pMASC04-PGAL1-CarRP-TPDC1-His3 (Fig.8b). The plasmid map and results of E. coli colony PCR
identification are shown in Fig.8. The E. coli colony PCR identification results were
consistent with the expected outcomes, and the sequencing results were correct.

![Primary school science popularization activity](https://static.igem.wiki/teams/5915/result/fig-8.webp)

Fig. 8 Construction of gene expression vector.

a. The plasmid has integrated the CarB and CarG genes. Meanwhile, key functional elements
are labeled: promoter GAL10, promoter GAL1, terminator PGI , terminator TPI1, and the
selectable marker gene Ura3. b. The CarRP gene is inserted into the plasmid, with key
elements including the GAL1 promoter, PDC1 terminator, and His3 selectable marker gene.
The figure below shows the PCR amplification bands of the recombinant plasmid.

### Construction of Lycopene Engineering Strains

The linearized recombinant plasmid
pMASC02-TTPI1-CarB-PGAL10-PGAL1-CarG-TPGI- Ura3 was integrated into the genome of BY4742.
After verification by genomic PCR (Fig.9a), the strain BY01 was obtained. Subsequently, the
linearized plasmid pMASC04-PGAL1-CarRP-TPDC1-His3 was transformed into BY01 (used as the
chassis cell), resulting in the lycopene-producing strain BY02. Lycopene is a natural
pigment with a vivid red color, allowing positive strains to be screened based on color. The
recombinant yeast was plated on His-deficient plates, and orange single colonies were
observed on the surface of the medium after 2 days (Fig.9b).

![Primary school science popularization activity](https://static.igem.wiki/teams/5915/result/fig-9.webp)

Fig. 9 Construction of lycopene engineering strain.

a The electrophoresis profile of PCR detection for transformed yeast strains. b The
colony morphology of recombinant yeast inoculated onto His-deficient (Histidine-deficient)
medium plates after 2 days of cultivation.

The integration of CarG, CarB, and CarRP into the genome of Saccharomyces cerevisiae aimed
to construct the lycopene biosynthesis pathway. However, CarRP possesses dual functions as
phytoene synthase and lycopene cyclase, with its cyclization activity dominating in the
heterologous expression system of Saccharomyces cerevisiae. This causes lycopene, a key
intermediate in the biosynthesis pathway, to be rapidly cyclized into β-carotene, making it
difficult to achieve substantial accumulation of lycopene. Shake flask fermentation followed
by HPLC detection revealed that the products of BY02 ( <https://doi.org/10.1038/s41467-022-28277-w> ) contained a large
amount of β-carotene (1.4 g/L), while the lycopene yield was only 0.024 g/L (Fig.10a,b).

![Primary school science popularization activity](https://static.igem.wiki/teams/5915/result/fig-10.webp)

Fig. 10 HPLC Determination of β-Carotene and Lycopene Contents.

a. There are no characteristic peaks of β-carotene and lycopene in the chromatogram of the
wild-type strain; two key peaks appear in the chromatogram of the BY02 strain: one
strong peak corresponding to β-carotene, and one weak peak corresponding to lycopene. b.
The β-carotene yield of the BY02 strain reaches 1.4 g/L, while the lycopene yield is
only 0.024 g/L.

### CarRP: Inactivation of the Lycopene Cyclase Function

### Rational Design of CarRP Mutant Proteins Using REvoDesign

Therefore, functional modification of the key enzyme CarRP through
protein engineering approaches—attenuating or even eliminating its cyclization activity
while enhancing the ability to convert phytoene to lycopene—will be a crucial breakthrough
for efficient synthesis and accumulation of lycopene. CarRP contains two functional domains:
the R domain exhibits lycopene cyclase activity, and the P domain possesses phytoene
synthase activity, though the P domain requires the correct conformation of the R domain to
exert its function. In designing loss-of-function mutations for the R domain of CarRP, it is
essential to ensure that the overall folding structure of the R domain is not disrupted /
remains undisrupted. To this end, modeling of the enzyme-substrate complex and molecular
docking were first carried out by DiffDock and AlphaFold3. The area around 6Å of substrate
lycopene in R domain was selected as the potential active center. Furthermore, to avoid
interrupting the function of phytoene synthase in P domain in CarRP, we opted to only target
the residues that are far from the P domain for mutagenesis and experiments. We finally
chose two mutant sites (F81A and Y145F) through rational inspection using the
"visualization" function of REvoDesign, as shown in Fig. 11 and Tab. 3.

![Primary school science popularization activity](https://static.igem.wiki/teams/5915/result/fig-11.webp)

Fig. 11 Complex model of CarRP and lycopene and the active center
sites.

Finally, 2 sites were used for mutant verification, which may alter the electric field
distribution in this region, thereby affecting interactions with other charged molecules
or alter the channel size. This change could modify the binding properties of CarRP's
active site and weaken the interaction between CarRP and lycopene.

![Primary school science popularization activity](https://static.igem.wiki/teams/5915/result/table-3.webp)

Tab. 3 Design strategies for protein modification

### Functional Verification of CarRP Mutant

The mutated CarRP sequences were cloned into the vector pMASC04-HIs3,
yielding the recombinant plasmids pMASC04-PGAL1-CarRP\_F81A-TPDC1-His3 and pMASC04-PGAL1
-CarRP\_Y145F-TPDC1-His3. The mutant plasmids were linearized and separately transformed into
strain BY01, resulting in strains BY03 and BY04. These two strains, together with strain
BY02, were subjected to shake flask fermentation simultaneously, and the products were
detected by HPLC.

HPLC analysis showed that the fermentation products of CarRP\_F81A and
CarRP\_Y145F mutants both exhibited a high lycopene peak, with yields of 1.42 g/L and 1.46
g/L, respectively. CarRP\_F81A produced a very small amount of β-carotene, while CarRP\_Y145F
produced no β-carotene at all. Furthermore, the lycopene yields of each mutant strain were
comparable to the β-carotene yield of the strain containing wild-type CarRP, indicating that
the two CarRP mutants designed in this study had almost lost their lycopene cyclase
activity. As a result, lycopene no longer cyclized to form β-carotene but accumulated as the
final product in Saccharomyces cerevisiae.

The cellular appearance further confirmed that the mutations
significantly impaired cyclase activity, leading to massive accumulation of lycopene and
giving the cells a bright red color. All these experimental results are presented in Fig.
12.

![Primary school science popularization activity](https://static.igem.wiki/teams/5915/result/fig-12.webp)

Fig. 12 Functional validation of CarRP mutants.

a Show the chromatograms of fermentation products from three strains (BY02: wild-type
CarRP; BY03: CarRP\_F81A; BY04: CarRP\_Y145F). b Clarify the product content of each
strain: the lycopene yield of BY03 is 1.42 g/L, and that of BY04 is 1.46 g/L; the
lycopene yields of both strains are comparable to the β-carotene yield of BY02.

The above experimental results indicate that the RvoDesign tool
exhibits excellent efficiency and practicality, as it can quickly identify the key sites
that play a decisive role in mediating the binding of CarRP to lycopene and the cyclization
of lycopene to β-carotene. Mutations at these sites can significantly reduce or even
completely abrogate cyclization activity, thereby promoting lycopene accumulation. Among
these mutants, Y145F has completely lost its lycopene cyclase function and exhibited the
highest lycopene yield; therefore, strain BY04 was selected for subsequent fermentation use.

### High-Cell-Density Fermentation (HCDF) of Recombinant Strains in Fermenters

Recombinant strain BY04 containing the pMASC04-PGAL1
-CarRP\_Y145F-TPDC1-His3 gene expression cassette was activated and then inoculated into a 3
L fermenter for high-cell-density fermentation. During the initial phase, a higher aeration
rate was set, and the stirring speed was adjusted in real-time to maintain the dissolved
oxygen level at 20% saturation, so as to promote cell proliferation and rapidly increase
cell biomass. 0.2 M PBS was added to maintain the pH stability of the medium, while
regulating ion balance to optimize the cell growth environment. After 48 hours of cell
growth, fed-batch feeding at a low rate was initiated to maintain a low glucose
concentration and optimize the carbon-nitrogen ratio, thereby regulating the dynamic balance
of carbon flux between the MVA pathway and the lipid synthesis pathway, and ultimately
promoting the efficient synthesis and accumulation of lycopene Fig.13a.

![Primary school science popularization activity](https://static.igem.wiki/teams/5915/result/fig-13.webp)

Fig. 13 Growth and lycopene yield of BY05 fermented in fermenter.

a High-density fermentation in a 3L fermenter. b Comparison of cell growth rate and
lycopene productivity at different stages of fermentation.

As shown in Fig.13b, during the early stage of fermentation (0 - 48
h), cells grew rapidly, and carbon flux was more inclined to support the primary metabolic
activities of cells to promote rapid cell proliferation. At this time, the biomass was low,
with a low lycopene productivity (0.033 g/L/h). With the depletion of glucose in the medium,
the preferential utilization of carbon source shifted from cell proliferation and lipid
synthesis to lycopene synthesis, and the rate of biomass growth gradually slowed down.
Finally, after 216 h of continuous fed-batch fermentation, ~9.0 g/L of lycopene was
obtained, with a productivity of 0.042 g/L/h. These data demonstrate the robustness of our
engineered strains guided by REvoDesign in scale-up and high-cell-density fermentation.

{% endblock %}