iGEM 2025 — Team scut-china-l

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}This serves as a chronological record of your team's progress throughout the season. It documents your
daily activities, experiments, discussions, and decisions.{% endblock %}
{% block page\_content %}

![Team Banner](https://static.igem.wiki/teams/5843/wiki-template/header/notebook1.webp)

* [February](#february)

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| Timetable | Wetlab | Drylab |
| --- | --- | --- |
| 27 February, 2025 | SCUT-China-L team was established and held the first offline in-person meeting.  ![utili-pic2](https://static.igem.wiki/teams/5843/wiki-notebook/wet1.webp) |  |
| February 28 - March 9, 2025 | Based on the experience from the team's field investigation to the red algae factory and the advisor's suggestions, the project direction was finalized: remodeling the S. cerevisiae to produce rare ginsenoside Rh1 using red algae. |  |
| March 10 - 23, 2025 | We refined the project details through literature review, formulated the experimental design, selected the cosmetics village after analyzing previous iGEM projects and assigned tasks among team members.  ![utili-pic2](https://static.igem.wiki/teams/5843/wiki-notebook/wet2.webp) | By using FBA algorithm, we wrote code for calculations to verify project feasibility and optimal experimental strategy selection.  ![utili-pic2](https://static.igem.wiki/teams/5843/wiki-model/pic03.webp)  ![utili-pic2](https://static.igem.wiki/teams/5843/wiki-model/pic04.webp) |
| March 24 - 30, 2025 | Wet lab members underwent lab safety training, learned experimental techniques for primer design, plasmid construction, strain culture, and the use of tools like CHOPCHOP, SnapGene, and NCBI website searches. Determined the use of wild-type S.cerevisiae CEN.PK2-1D for engineering based on literature review and existing lab research. | Dry lab members further analyzed the FBA algorithm and decided to develop related visualization software. |
| March 31 - April 6, 2025 | Through literature review and database mining, we screened three agarases from different sources and two neoagarobiose hydrolases from different sources.  ![utili-pic2](https://static.igem.wiki/teams/5843/wiki-notebook/wet-table1.webp)  ![utili-pic2](https://static.igem.wiki/teams/5843/wiki-notebook/wet-table2.webp) | We explored platform tools related to protein structure prediction and molecular docking prediction. Based on the five amino acid sequences provided by wet lab members, conducted initial modeling tests, selecting AF2 for structure prediction. Subsequently, we explored the PDB website, used Avogadro and UCSF ChimeraX for computational operations, and learned to use tools like WGLTools, AutoDock Vina, Meeko for molecular docking tests to provide predictive results for wet lab experiments.  ![utili-pic2](https://static.igem.wiki/teams/5843/wiki-notebook/docking1.webp)  ![utili-pic2](https://static.igem.wiki/teams/5843/wiki-notebook/docking2.webp)   | **Enzyme Type** | **Name** | **Binding Energy (kcal/mol)** | | | | | | **Site 1** | **Site 2** | **Site 3** | **Site 4** | **Site 5** | | Agarase | Aga3464 | -6.23 | -7.205 | -7.766 | -6.077 | -7.413 | | AqAga | -8.275 | -10.054 | -7.361 | -6.079 | -7.636 | | PdAgaC | -7.396 | -7.440 | -8.574 | -6.722 | -8.647 | | Neoagarobiose Hydrolase | agaNash | -6.686 | -5.065 | -7.266 | -7.669 | - | | NH852 | 3.725 | -4.691 | -7.297 | -6.736 | - | |
| April 7 - May 4, 2025 | Using Gibson Assembly, we successfully constructed six plasmids. After screening and sequencing verification, the plasmids were transformed into yeast cells using the lithium acetate method. Verified and measured the enzyme activities of agarase and neoagarobiose hydrolase, screening the engineered strain Sq-Ag5 for efficient degradation of red algae polysaccharides.  ![utili-pic2](https://static.igem.wiki/teams/5843/wiki-notebook/wet3.webp) | We continued learning from existing model predictions. While receiving verification from wet lab experiments, we conducted further exploratory analysis on other aspects of the predicted enzyme structures. |
| May 5 - 11, 2025 | To increase squalene production, we designed an experiment to optimize the MVA pathway by overexpressing the tHMG1 and IDI1 genes. | We collaborated with the design team members to discuss the visual aesthetics of the software interface, including theme colors and interactive animations.  ![utili-pic2](https://static.igem.wiki/teams/5843/wiki-notebook/meeting2.webp)  ![utili-pic2](https://static.igem.wiki/teams/5843/wiki-notebook/meeting1.webp) |
| May 12 - June 1, 2025 | We learned experimental operations for CRISPR technology. Selected the genomic GAL80 target site and constructed the Cas9-sgRNA plasmid, simultaneously constructing DNA integration fragments for the tHMG1 and IDI1 genes. After yeast gene editing, primary screening, negative screening, and PCR verification, the obtained strain Sq-0 was used for squalene fermentation experiments. Results confirmed the successful squalene production of strain Sq-0, which significantly outperformed the unmodified strain.  ![utili-pic2](https://static.igem.wiki/teams/5843/wiki-notebook/wet4.webp) | We recruited new dry lab members to assist with modeling for a new direction: calculating the economic and environmental benefits of the ginsenoside manufacturing route.  ![docking-pic1](https://static.igem.wiki/teams/5843/wiki-model/sustain-calculating/pic01.webp)  ![docking-pic1](https://static.igem.wiki/teams/5843/wiki-model/sustain-calculating/pic05.webp) |
| June 2 - June 16, 2025 | Over these two weeks, we integrated the secretory hydrolase module and the MVA modification module, constructing strain Sq-Ag capable of hydrolyzing agar polysaccharides and producing squalene. Simultaneously optimized culture conditions, red algae liquefaction conditions and the optimal of mixed carbon sources. | In software development, through communication and demos with wet lab members, including internal downloads and usage, we obtained user trial feedback. Then we added an AI agent function to enrich software operations. Meanwhile, we began establishing the synthesis pathway for rare ginsenoside Rh1 in the model. We introduced the rare ginsenoside Rh1 synthesis genes into yeast cells model by constructing high and low copy plasmids.  ![utili-pic2](https://static.igem.wiki/teams/5843/wiki-notebook/meeting3.webp) |
| June 17 - July 6, 2025 | After introducing the ginsenoside synthesis genes into yeast cells, neither Rh1 nor any intermediate products were detected upon fermentation. Consequently, we planned to redesign the experiment by integrating the genes into the yeast genome and repeating the experiment.  ![utili-pic2](https://static.igem.wiki/teams/5843/wiki-notebook/wet5.webp) |  |
| July 17 - July 21, 2025 | We further enhanced the yield of the precursor squalene. We screened 3 constitutive promoters and 3 inducible promoters for combination, constructing strains Sq-Ag7 to Sq-Ag23, and compared squalene yields.  Simultaneously, we began redesigning the experimental plan, searching for suitable gene integration sites. Using strain Sq-0 as the starting point, we introduced the five genes in batches, conducted experiments, and detected the yields of corresponding products  ![utili-pic2](https://static.igem.wiki/teams/5843/wiki-notebook/change1.webp) | We recruited new dry lab members to assist in developing the AI agent, conducted online meetings with wet lab members to re-clarify user needs and encountered problems, built a relevant knowledge base, and clarified each member's work.  We analyized promoter characteristics and built a prediction model for optimal promoter combinations. |
| July 22 - July 26, 2025 | Following suggestions from Miss Xie and Miss Wang, we further implemented a chimeric intron, by inserting the RPS25Ai intron near the promoter, which further increased squalene yield.  ![utili-pic2](https://static.igem.wiki/teams/5843/wiki-results/pic002.webp)  We also conducted experiments and finally successfully detected the yields of the corresponding products. |
| August 10 - August 31, 2025 | We used purchased red algae to prepare YPDA medium and cultured Sq-Ag23c, confirming the establishment of the red algae utilization pathway. Meanwhile, constructed strain S.cerevisiae Rh1-Ag for de novo synthesis of Rh1 using red algae polysaccharides as substrate. Verified that introducing the red algae hydrolase genes enabled effective Rh1 production from red algae, and also confirmed that the MVA pathway modifications in yeast cells remained effective after completing the metabolic pathway construction.  ![utili-pic2](https://static.igem.wiki/teams/5843/wiki-notebook/change2.webp)  ![utili-pic2](https://static.igem.wiki/teams/5843/wiki-results/pic45.webp) |  |
| September 1 - September 30, 2025 | We curated data, generated the figures, and wrote the Wiki, while also designed the content for the presentation video. |  |

{% endblock %}

{% extends "layout.html" %}
{% set show\_scroll\_progress = False %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one
iteration of the engineering design cycle.{% endblock %}
{% block page\_content %}

![Team Banner](https://static.igem.wiki/teams/5843/wiki-template/header/engineering.webp)

![coral1](https://static.igem.wiki/teams/5843/wiki-home/coral1.webp)

![coral2](https://static.igem.wiki/teams/5843/wiki-home/coral2.webp)

![coral3](https://static.igem.wiki/teams/5843/wiki-home/coral3.webp)

![coral4](https://static.igem.wiki/teams/5843/wiki-home/coral2.webp)

![coral5](https://static.igem.wiki/teams/5843/wiki-home/coral1.webp)

![coral6](https://static.igem.wiki/teams/5843/wiki-home/coral5.webp)

![coral7](https://static.igem.wiki/teams/5843/wiki-home/coral3.webp)

![coral8](https://static.igem.wiki/teams/5843/wiki-home/coral6.webp)

Design

Engineering Principles: To circumvent the drawbacks of glucose
metabolism (such
as the Crabtree effect leading to by-product ethanol production), utilize
non-conventional carbon source red algae hydrolysate to improve the theoretical
yield and process sustainability of Rh1 synthesis.

Model-Assisted Design: Adopt the latest consensus metabolic model
GEM-Yeast9 as
the computational foundation, which comprehensively covers the metabolic network
of Saccharomyces cerevisiae. Then introduce heterologous synthesis pathways: Add
the complete synthesis pathway reactions and corresponding metabolites from
2,3-oxidosqualene to ginsenoside Rh1 into the model. Then introduce the AHG
metabolic pathway: Add the AHG absorption and degradation pathways, enabling it
to finally enter central metabolism. Objective function definition: To simulate
the resource trade-off between cell "growth" and "production", construct a
weighted dual objective function Z = w · vbiomass + (1-w) · vRh1, where w is the
weight factor.

Build

In silico Construction: In the expanded metabolic model, use the
COBRApy toolbox
to set reaction boundary conditions, simulating the implementation effect of
genetic engineering. For example, close the glucose exchange reaction, open the
input of galactose and AHG, and allow the output of Rh1.

Test

In silico Testing and Functional Analysis:

Step 1: Determine the optimal weight (w): Vary w from 0 to 1,
maximizing the objective function Z. By analyzing the curves of biomass
synthesis flux vbiomass and Rh1 synthesis flux vRh1 against w, find the balanced
point w=0.55. This point avoids excessive dominance by a single objective,
better aligning with the metabolic phenotype of actual engineered strains.

Step 2: Compare different carbon source strategies: Under the
optimal weight w=0.55, fix the total carbon source input concentration, use
glucose and galactose+AHG mixture as carbon sources respectively, and calculate
the Rh1 synthesis rate vRh1 when Z is maximized.

Step 3: Analyze metabolic flux distributions: At a specific carbon
source concentration (e.g., 1 mmol/gDW/h), analyze the flux distribution
differences in the central metabolic pathways.

Learn

Computational test results indicate that under
the same carbon molar concentration, the predicted Rh1 synthesis rate using the
galactose+AHG mixed carbon source is significantly higher than using the glucose
single carbon source. This theoretically validates the initial design principle,
indicating the advantage of red algae hydrolysate as a carbon source.

Flux distribution analysis revealed the differences in the central
metabolic network under the two carbon source strategies, explaining the
potential reasons for the different Rh1 synthesis efficiencies (e.g., carbon
flow direction, cofactor regeneration efficiency).

Model improvement and iterative design inspiration: The success of
this cycle proves the reliability of the expanded model. The key parameters and
metabolic bottleneck information learned provide precise guidance for the next
DBTL cycle. For example, the rate-limiting enzymes in the red algae
decomposition process can be further adjusted to seek higher yield.

CYCLE 1: Expressing Agarase and Neoagarobiose Hydrolase

Design

Through literature research, it was found that the agar in
red algae needs to be hydrolyzed into monosaccharides in two steps. First,
agarase hydrolyzes it into neoagarobiose, and then neoagarobiose hydrolase
hydrolyzes it into galactose and 3,6-anhydro-L-galactose[1][2]. We decided to
introduce heterologous hydrolases into Saccharomyces cerevisiae to achieve
extracellular secretion of hydrolases.

Build

We obtained the signal peptide gene from the α-factor mutant[3]([BBa\_258RYIFY](https://registry.igem.org/parts/bba-258ryify))
of Saccharomyces
cerevisiae, which contains the Kozak sequence and helps to enhance
the efficiency of translation initiation. Three types of agarases[4][5][6][7][31] and two types
of neoagarobiose hydrolases[5][8][32] from different sources were screened, and 6 types of
plasmids were constructed. We introduced the 6 types of plasmids into the Saccharomyces
cerevisiae CEN.PK2-1D by lithium acetate transformation method, and
obtained 6 engineered strains (namely Sq-Ag1, Sq-Ag2, Sq-Ag3, Sq-Ag4, Sq-Ag5,
Sq-Ag6).

Table 1 Gene Sources of 5 Hydrolases

![table5](https://static.igem.wiki/teams/5843/dbtl/table7.webp)

![table6](https://static.igem.wiki/teams/5843/dbtl/table8.webp)

Table 2 Gene Sources of 5 Hydrolases

![table4](https://static.igem.wiki/teams/5843/dbtl/table4.webp)

A

![build2-image1](https://static.igem.wiki/teams/5843/dbtl/build2-image2.svg)

B

![build2-image1](https://static.igem.wiki/teams/5843/dbtl/build2-image3.svg)

C

![build2-image1](https://static.igem.wiki/teams/5843/dbtl/build2-image4.svg)

D

![build2-image1](https://static.igem.wiki/teams/5843/dbtl/build2-image5.svg)

E

![build2-image1](https://static.igem.wiki/teams/5843/dbtl/build2-image6.svg)

F

![build2-image1](https://static.igem.wiki/teams/5843/dbtl/build2-image1.svg)

Figure Plasmids of 6 Hydrolases

Test

We cultured the 6 strains in defective medium and conducted
qualitative detection using the Lugol's iodine plate method. As shown in the
figure, large transparent hydrolysis circles were formed around the colonies,
indicating that all 6 strains had the ability to secrete agarase
extracellularly.

![test2-image4](https://static.igem.wiki/teams/5843/dbtl/test2-image4.webp)

Lugol's iodine staining result
diagram

Learn

This cycle initially verified the ability of the
reconstructed yeast to decompose agar, but we need to further conduct
quantitative research on the activity of hydrolases to obtain the combination
with the best activity.

CYCLE 2: Expressing Agarase and Neoagarobiose Hydrolase

Design

In Cycle 2, we confirmed the activity of two enzymes. To improve the dual-enzyme catalytic system, we plan to use a computational model to predict how well enzymes bind to substrates, find the best enzyme combination, and test it with wet experiments. The main goal is to build an efficient process for predicting and verifying enzyme function, to support future metabolic pathway construction.

Build

We made a prediction model (see [Model Part 3](https://2025.igem.wiki/scut-china-l/model#docking) for details) based on protein structure modeling and molecular docking. It uses enzyme sequences to predict enzyme-substrate binding，which shows enzyme activity level. The model found that the AqAga + agaNash combination scored highest—meaning it should work best.

To check the model, we designed 7 different enzyme combinations for wet experiments, to prepare for later comparison.

![build3-image1](https://static.igem.wiki/teams/5843/dbtl/build3-image1.webp)

Test

First, we made a standard curve for substrate concentration (to measure enzyme activity accurately).

![test3-image1](https://static.igem.wiki/teams/5843/dbtl/test3-image1.webp)

Then we tested the 7 enzyme combinations.

![test3-image2](https://static.igem.wiki/teams/5843/dbtl/test3-image2.webp)

Results showed AqAga + agaNash had the highest activity—matching the model. This proves the model works.

Learn

We analyzed the model’s docking results and experimental data (see [Model Part 3](https://2025.igem.wiki/scut-china-l/model#docking)).

AqAga and agaNash act on different agar substrates, but both use their active pockets to recognize and catalyze substrates. They work because of matching structures, polar or non-polar interactions, and suitable substrate channels.

1. How AqAga binds and catalyzes tetramers efficiently?

AqAga uses 9 hydrogen bonds (most < 3.5 Å) to hold tetramers in its active pocket. Key residues (e.g., TRP133, ASP137) form strong polar interactions (for tight binding). Hydrophobic pockets (e.g., VAL631, PHE663) also help (via van der Waals forces), but some conflicts (e.g., with GLU677) mean the binding could be better.

Tetramers can pass through the whole active channel. The binding area has both hydrophilic and hydrophobic parts—this matches polysaccharide-degrading enzymes: hydrophilic parts help substrate binding and catalysis (e.g., GLU/ASP donates protons), while hydrophobic parts help position and stabilize.

2. How agaNash binds and catalyzes dimers?

agaNash and the dimer form a moderately strong polar interaction network via 8 hydrogen bonds, among which the key hydrogen bonds formed by ASN 149 and ARG 234 provide crucial anchoring for the binding. Meanwhile, the hydrophobic interface composed of aromatic residues such as TRP 88 and PHE 76 serves as the main driving force for binding through dense negative overlapping contacts.

This binding mode exhibits local steric clashes, most prominently in the regions of PHE 125 and THR 126; these clashes may affect conformational stability. Some hydrogen bond sites also show steric strain, indicating the need for coordination between polar interactions and stereomatching.

The binding region presents a typical feature of alternating hydrophilic-hydrophobic distribution, which meets the substrate recognition requirements of glycoside hydrolases. The polar network is responsible for substrate-specific recognition, while the hydrophobic region provides positional stabilization. The synergistic effect of the two endows the system with moderate binding affinity, which can support the binding and catalytic functions towards the dimer substrate.

CYCLE 3: Expressing Agarase and Neoagarobiose Hydrolase

Design

After obtaining the Sq-Ag5 strain with the highest enzyme
activity, we hoped that Sq-Ag5 could integrate the exogenous enzymatic
hydrolysis pathway and the endogenous fermentation pathway, and successfully
produce the rare ginsenoside precursor squalene using agar. We first attempted
to conduct fermentation experiments using 20 g/L glucose and 20 g/L agar.

Build

We inoculated the Sq-Ag5 strain on a nutrient-deficient
plate, then carried out shake flask fermentation in YPD medium and YPA liquid
medium respectively, and finally detected the squalene content in the
fermentation broth using a HPLC.

Test

Since the fermentation temperature of yeast is 30°C, the
medium with only agar added solidified, making it impossible to detect the
product. Strangely, the group with only glucose added also had a very low
squalene content, only 5.89 mg/L, which could not provide sufficient precursor
substances for the subsequent conversion of ginsenosides.

![test4-image1](https://static.igem.wiki/teams/5843/dbtl/test4-image1.svg)

Figure HPLC Detection Chromatogram of Sq-Ag5
Squalene Fermentation Broth

Learn

After communication with Dr. Xie, it was initially
determined that the MVA pathway for squalene production in Saccharomyces
cerevisiae was limited. Literature research showed that up-regulating
the expression of genes encoding key enzymes in the MVA pathway can effectively
increase the overall flux of this metabolic pathway.

CYCLE 4: Optimization of the MVA Pathway

CYCLE 4.1: Try to Produce Squalene Using Sq-Ag5

Design

To enhance the MVA pathway for improved squalene synthesis capacity, we inserted the tHMG1 and IDI1 genes[9] into the GAL80 locus of the genome of wild-type Saccharomyces cerevisiae CEN PK2-1D. tHMG1[10][11] is a
rate-limiting enzyme in the MVA pathway, catalyzing the conversion of HMG-CoA to
mevalonate (MVA) and determining the metabolic flux of the MVA pathway. IDI1
catalyzes the reversible conversion between isopentenyl pyrophosphate (IPP) and
dimethylallyl pyrophosphate (DMAPP). Meanwhile, knockout of the GAL80 gene[12] not
only helps to relieve the inhibition of galactose utilization in subsequent
experiments but also enhances the expression intensity of galactose-regulated
promoters.

Build

We constructed a Cas9-sgRNA plasmid targeting the GAL80 locus, integrated the tHMG1 gene, IDI1 gene, and corresponding promoters and
terminators as donor DNA, and finally performed gene editing on yeast. After
primary screening and negative screening, the engineered strain S. cerevisiae
Sq-0 was obtained, in which the genes were successfully integrated into the
genome and the tags were removed.

![figure1](https://static.igem.wiki/teams/5843/dbtl/figure1.webp)

Figure: IDI1 and tHMG1 Gene Modules

Test

The experimental procedure was consistent with CYCLE 4.1-Test. Under the culture condition of 20 g/L glucose, the squalene yield of
strain Sq-0 was detected to reach 806 mg/L.

![test4-image1](https://static.igem.wiki/teams/5843/dbtl/figure4-2-2.webp)

Figure: HP-LC Detection Spectrum of Squalene
Fermentation Broth from Strain Sq-0

Learn

The detection results showed that the optimization of the MVA pathway by
overexpressing the tHMG1 and IDI1 genes was highly effective, and the engineered
strain Sq-0 could be used as the starting strain for subsequent engineering
development

CYCLE 4: Optimization of the MVA Pathway

CYCLE 4.2: Construction of Engineered Strain Sq-0 with Overexpressed tHMG1 and IDI1

Design

According to the conclusion of CYCLE 4.1, we found that the
excessively high concentration of red algae polysaccharides would increase the
viscosity of the medium, which is unfavorable for the growth of strains.
Therefore, it is important to explore the specific conditions for the
liquefaction under the action of low-concentration hydrochloric acid: it is
necessary to ensure the fluidity of the fermentation medium to facilitate the
increase of microbial movement; it is also necessary to ensure that no
monosaccharides are hydrolyzed during the liquefaction pretreatment.

Build

We used hydrochloric acid with a final concentration
gradient of 0.001 to 0.01 M in YPA medium and carried out high-temperature
treatment. To verify whether the polysaccharides were hydrolyzed during the
liquefaction process, we planned to use the engineered strain Sq-Ag5 for
fermentation experiments after finding the optimal liquefaction conditions to
detect whether neoagarobiose and galactose were produced.

Test

The results showed that an extremely low acid concentration
(0.005 M hydrochloric acid) could open the structure of agar to be in a fluid
state, and the medium did not solidify during the entire shake flask culture of
yeast. After liquefying the yeast medium with 0.005 M hydrochloric acid, no
neoagarobiose or monosaccharides were detected by HPLC, and the medium could be
used for yeast fermentation experiments after adjusting the pH to 6.0.

![test5-image3](https://static.igem.wiki/teams/5843/dbtl/test5-image3.webp)

Figure Medium State During Fermentation

Learn

During the fermentation process, we monitored the state of
the medium at different stages (0 h, 48 h, 120 h) and observed the effect of
hydrochloric acid concentration on the fluidity of the medium. The above
phenomena confirmed the effectiveness of this experimental system.

CYCLE 5: Fermentation Production of Squalene by the Engineered Strain Sq-Ag Using
Red Algae Polysaccharides as Substrate

CYCLE 5.1: Determine Liquefaction Conditions

Design

There is a distribution limit of metabolic resources in
Saccharomyces
cerevisiae, so it is necessary to establish a dynamic balance between
cell growth and target product synthesis[13]. In the early stage of the experiment,
it was predicted by the Flux Balance Analysis (FBA) model that the flux of the
Rh1 synthesis reaction is greater under the condition of mixed carbon sources.
To optimize the carbon source utilization efficiency, we designed an orthogonal
fermentation experiment to explore the optimal ratio of glucose to red algae
polysaccharides. The strategy was to use low-concentration glucose to initiate
microbial growth, while maximizing the efficiency of galactose as a carbon
source.

Build

We introduced the plasmid p426-AqAga-agaNash into the
engineered strain Sq-0 by lithium acetate transformation method to obtain the
engineered strain Sq-Ag, and carried out orthogonal fermentation experiments in
a series of media (liquefied with 0.005 M hydrochloric acid).

Test

Through detection, it was found that yeast could achieve the
step-by-step degradation and saccharification of agar from scratch. The
experimental results showed that 10 g/L glucose and 25 g/L agar could achieve
the highest squalene yield of 667.02 mg/L.

![test5-image5](https://static.igem.wiki/teams/5843/dbtl/test5-image5.webp)

Figure Orthogonal Experimental Results of Squalene
Synthesis by Glucose and Agar in Shake Flasks

Learn

When the concentration of red algae polysaccharides was ≤25
g/L, the squalene yield increased with the increase of red algae polysaccharide
concentration. However, high-concentration polysaccharides (exceeding 25 g/L)
began to inhibit the increase of squalene. The possible reason is that
high-concentration red algae polysaccharides lead to excessively high medium
viscosity, which affects oxygen transfer or cell metabolism. This reminds us
that in the development of new carbon source fermentation processes, the
selection of carbon sources requires multi-dimensional systematic evaluation.

CYCLE 5: Fermentation Production of Squalene by the Engineered Strain Sq-Ag Using
Red Algae Polysaccharides as Substrate

CYCLE 5.2: Orthogonal Experiment

Design

Since the squalene yield under mixed carbon source conditions was still not
ideal, we aimed to further improve the squalene yield through promoter
engineering. Considering the large number of promoter combinations, we planned
to calculate the optimal promoter combination based on wet lab experiments
combined with biokinetic system analysis, so as to provide support and reference
for the conclusions of the wet lab.

Build

Through literature research, 3 constitutive promoters
(PTEF, PGPD, PTDH) and 3 inducible promoters (PGAL1, PGAL7, PGAL10) were
identified, and 18 plasmids with different promoter combinations were
constructed using the Gibson assembly method( the same as Cycle2-Build).

![](https://static.igem.wiki/teams/5843/dbtl/figure6-1.webp)

Figure: Schematic Diagram of Promoter Engineering

Test

We cultured the strains for 96 hours under the conditions of 5 g/L glucose and
25 g/L agar, and detected the squalene yield. The reason for choosing this
carbon source combination was that, according to Cycle5.2-Test, when the glucose
concentration increased from 2.5 g/L to 5 g/L, the squalene yield showed a jump;
before the agar concentration reached 25 g/L, the squalene yield increased
rapidly, and after exceeding 25 g/L, the increase rate of the yield became
gentle and even showed a downward trend. To meet the growth needs of cells while
maximizing the utilization rate of agar polysaccharides, 5 g/L glucose and 25
g/L agar were finally determined as the carbon source conditions for the
promoter engineering experiment.

The detection results showed that the inducible promoter combination PGAL10
(regulating agarase expression) + PGAL7 (regulating neoagarobiose hydrolase
expression) had the best effect, and the corresponding squalene yield reached
594.68 mg/L.

![test5-image3](https://static.igem.wiki/teams/5843/dbtl/figure6-2-6.webp)

Figure: Experimental Verification Diagram of Different Promoter Combinations

Learn

The experiment found that inducible promoters had
significant advantages in the presence of galactose. Inducible promoters can be
induced and activated by galactose, thereby accurately regulating the expression
of agarase and neoagarobiose hydrolase. Different from constitutive promoters
that continuously express enzymes, inducible promoters can start enzyme
synthesis at the appropriate time according to the presence of galactose,
avoiding the problems of excessive enzyme expression or improper expression
timing that may be caused by constitutive promoters, thus utilizing substrates
more efficiently and reducing the metabolic burden of cells at the same time. To
further verify the accuracy of the wet lab results, we constructed a promoter
prediction model.

CYCLE 6: Promoter Engineering

CYCLE 6.1: Promoter Selection (Wet lab)

Design

This cycle aimed to construct a promoter prediction model to provide systematic
guidance for the screening of optimal promoter combinations in product
synthesis. During the design phase, the core goal of the model was clarified: to
quantify the dynamic regulatory mechanism of different promoter combinations on
product yield. A complete set of ordinary differential equation (ODE) model
framework was constructed by integrating the Monod kinetic equation, carbon
source allocation mechanism, cell growth inhibition term, and promoter
expression intensity quantification method. At the same time, the types of wet
lab data required for the model were planned, including mRNA expression level,
green fluorescent protein (GFP) intensity, and product yield time series, which
were used for parameter calibration and verification of the model in the
subsequent steps. In terms of algorithm selection, the Runge-Kutta method
(ode45) was used for numerical solution, and constrained nonlinear optimization
(Sequential Quadratic Programming, SQP) was combined to realize parameter
fitting, so as to ensure the accuracy and reliability of the model.

For detailed process, please refer to [Model Part 4](https://2025.igem.wiki/scut-china-l/model#biody).

Build

During the construction phase, the mathematical expression and preliminary
implementation of the model were completed. Firstly, based on the wet lab data,
the relative expression intensities of the three promoters PTEF, PGPD, and PTDH
were normalized, and their weights were obtained as 0.265, 0.418, and 0.317
respectively. Furthermore, the synergy coefficients α and β for homologous and
heterologous promoter combinations were defined. On this basis, an ODE system
including glucose and agar consumption kinetics, cell growth equation, and
product synthesis flux model was established, and a time-dependent carbon source
allocation function r(t) was introduced to dynamically reflect the resource
allocation strategy. To ensure the biological rationality of the model, a number
of constraint conditions were set, including carbon source residual amount,
lower limit of cell density, and lower limit of synthesis rate. The ODE solution
and optimization algorithm code were written using MATLAB, and parameter
training was conducted using data from 6 groups of known promoter combinations.
To further expand the application scope of the model, inducible promoters such
as PGAL1, PGAL7, and PGAL10 were also introduced, the Analytic Hierarchy Process
(AHP) was used to evaluate their comprehensive scores, and the model was
modified to incorporate galactose metabolism and induction delay effects.

Test

The performance of the model was comprehensively evaluated by comparing the
model prediction results with independent wet lab data. For promoter
combinations not involved in training (such as PTEF+PTDH, PGAL10+PGAL1), the
model successfully predicted their squalene yields, and the predicted values
showed good consistency with the experimental values. Statistical evaluation
showed that the coefficient of determination R² of the model increased from the
initial 0.7987 to 0.8384 after expansion; the K-S test result (p > 0.05)
indicated that the predicted data and experimental data had the same
distribution characteristics. It is particularly worth noting that the yield
ranking predicted by the model was basically consistent with the experimental
ranking, which proved its guiding value in practical screening. Through the
visual analysis of scatter plots and confidence ellipses, it was further
confirmed that the model had no significant systematic deviation, and the
prediction results were reliable.

Learn

Through in-depth analysis in the learning phase, the effectiveness and
improvement directions of the model were summarized. This ODE model could well
capture the dynamic impact of promoter combinations on product synthesis and
showed strong explanatory and predictive capabilities. However, the model had a
slight underestimation phenomenon in some combinations, which might be related
to the excessive setting of constraint conditions or incomplete parameter
optimization. In the future, the generalization ability of the model will be
improved by increasing experimental data of more promoter combinations, and
machine learning methods will be introduced to assist parameter optimization, so
as to further improve the fitting efficiency and prediction accuracy of the
model.

CYCLE 6: Promoter Engineering

CYCLE 6.2: Promoter Selection (Model)

Design

Through literature research, it was found that inserting introns into promoters could further improve gene expression levels, thereby increasing squalene yield. Studies have shown that the RPS25Ai[27] intron in Saccharomyces cerevisiae is widely used in yeast genetic engineering and metabolic pathway modification, and has a significant effect. Therefore, we planned to chimerize the RPS25Ai intron with the PGAL10 and PGAL7.

Build

The RPS25Ai intron was inserted into the proximal regions of the PGAL10 and PGAL7. Both the PGAL10 and PGAL7 were connected to the RPS25Ai intron at the -1 position, and 4 promoter combinations containing chimeric introns were constructed.

![figure6-3-3](https://static.igem.wiki/teams/5843/dbtl/figure6-3-3.webp)

Figure: Schematic Diagram of Chimeric Intron Construction

Test

The strains were cultured for 96 hours under the conditions of 5 g/L glucose and 25 g/L agar, and the squalene yield was detected. The experimental results showed that when the RPS25Ai intron was inserted into the proximal end of the PGAL10 and no intron was inserted into the PGAL7, the squalene yield was the highest, reaching 682 mg/L.

![figure6-3-5](https://static.igem.wiki/teams/5843/dbtl/figure6-3-5.webp)

Figure: Squalene Yield of Four Promoter Combinations with Chimeric Introns

Learn

Inserting introns into promoters could enhance the expression of agarase and neoagarobiose hydrolase genes in Saccharomyces cerevisiae to varying degrees, thereby increasing squalene yield. After promoter engineering modification, the squalene yield was significantly improved, laying a foundation for obtaining higher yields of rare ginsenoside Rh1 in subsequent experiments.

CYCLE 6: Promoter Engineering

CYCLE6.3: Engineered Promoter (Chimeric Intron)

Design

The synthesis of the rare ginsenoside Rh1 from squalene requires five exogenous
genes[28] - PgDDS (Dammarenediol-II synthase gene，[BBa\_255LOGT6](https://registry.igem.org/parts/bba-255logt6)), CYP716A47
(Protopanaxadiol synthase gene，[BBa\_25RY2RMX](https://registry.igem.org/parts/bba-25ry2rmx)), PgCPR1 (Cytochrome P450 reductase
gene), CYP716A53v2 (Protopanaxatriol synthase gene，[BBa\_25NSM6TW](https://registry.igem.org/parts/bba-25nsm6tw)) and UGTPg100
(Glycosyltransferase gene，[BBa\_255ROLW2](https://registry.igem.org/parts/bba-255r0lw2)). The vector was too large when five
genes were placed on one plasmid. Therefore, we assigned the five genes to
low-copy and high-copy plasmids, and then simultaneously transformed the
high-copy and low-copy plasmids into the engineered strain S.cerevisiae Sq-0.

Build

The genes PgDDS, CYP716A47, PgCPR1 were placed on high-copy
plasmids, the genes CYP716A53v2 and UGTPg100 was placed on low-copy plasmids.
After the sequencing was correct, the plasmids were transformed into the
engineered strain S.cerevisiae Sq-0 by lithium acetate conversion method to
obtain the engineered strain S.cerevisiae Rh1.

![test5-image3](https://static.igem.wiki/teams/5843/dbtl/figure7-1-1.webp)

High-copy plasmids

![test5-image3](https://static.igem.wiki/teams/5843/dbtl/figure7-1-9.webp)

Low-copy plasmids

![test5-image3](https://static.igem.wiki/teams/5843/dbtl/figure7-1-3.webp)

High-copy sequencing is correct

![test5-image3](https://static.igem.wiki/teams/5843/dbtl/figure7-1-4.webp)

Low-copy plasmids

Test

The medium containing 20g/L glucose was used for fermentation for 144 hours.
With the LC-16 high performance liquid chromatograph equipped with SPD-16
dual-wavelength ultraviolet detector, no rare ginsenoside Rh1 and any
intermediate products were detected at multiple retention times.

![figure7-1-10](https://static.igem.wiki/teams/5843/dbtl/figure7-1-10.webp)

Liquid phase detection graph

Learn

We conducted numerous repeated experiments. Despite
verifying the correctness of the gene sequence and the copy plasmid sequencing,
no rare ginsenoside products or any intermediate products were detected. We
consulted relevant professors. Although the cause was not identified, after
communication, the professor suggested that we integrate the gene into the
genome of yeast cells for the experiment.

Cycle 7: Using red algae polysaccharides as the substrate to produce the rare
ginsenoside Rh1

Cycle 7.1: Construction of the biosynthetic module of rare ginsenoside Rh1 and
its compatibility with Saccharomyces cerevisiae cells

Design

In order to enable Saccharomyces cerevisiae to produce the rare ginsenoside Rh1
using squalene, we redesigned the experimental protocol and integrated five
exogenous genes into the corresponding genomic sites of the engineered strain
S.cerevisiae Sq-0[29] using CRISPR-Cas9 technology. An engineered strain
S.cerevisiae Rh1-con capable of synthesizing the rare ginsenoside Rh1 was
constructed. Integrate the gene PgDDS at the X-3 site, the gene CYP716A47 and
the gene PgCPR1 at the XI-3 site, the gene CYP716A53v2 and the gene UGTPg100 is
integrated at the LPP1 site. Among them, since the expression of the LPP1 gene
will promote the flow of the precursor substance FPP of squalene towards the
synthesis of farnyl alcohol, inserting the gene at the LPP1 site can increase
the yield of squalene and its downstream products[30].

Build

We constructed the Cas9-sgRNA plasmid with sgRNA sequences targeting X-3, XI-3,
and LPP1 sites. Then, after gene editing of each gene, high concentrations of
TADH1-PgDDS-PGAL1,10-linear donor DNA,
PGAL1,10-CYP716A47-TALT1-PGAL7-PgCPR1-TCYC1 linear donor DNA, and
TADH1-CYP716A53v2-PGAL1,10-UGTPg100-TALT1 linear donor DNA were obtained
respectively. These linear donor DNAs were successively integrated into the
genome of yeast cells through gene editing. After primary screening and negative
screening, the engineering strain that successfully integrated the gene into the
genome and discarded the label was obtained. After all the gene editing was
completed, the engineered strain S.cerevisiae Rh1-con was obtained.

![figure7-2-8](https://static.igem.wiki/teams/5843/dbtl/figure7-2-8.webp)

The engineered strain S.cerevisiae Rh1-con

Test

The medium containing 20g/L glucose was used for fermentation for 144 hours. The
amount of the corresponding product after each gene introduction was determined
using an LC-16 high performance liquid chromatograph equipped with a SPD-16
dual-wavelength ultraviolet detector. The test results showed that the yield of
Dammarenediol-II was 1 g/L, that of Protopanaxadiol was 273.8 mg/L, and that of
Rare ginsenoside Rh1 was 170.8 mg/L.

![test5-image3](https://static.igem.wiki/teams/5843/dbtl/figure7-2-3.webp)

The yield of Dammarenediol-II

![figure7-2-11](https://static.igem.wiki/teams/5843/dbtl/figure7-2-11.webp)

The yield of Protopanaxadiol

![test5-image3](https://static.igem.wiki/teams/5843/dbtl/figure7-2-5.webp)

The yield of Rare ginsenoside Rh1

Learn

Based on the experimental results, after integrating the Rh1 synthesis gene into
the genome of yeast cells, a yeast strain capable of producing Rh1 was
successfully obtained. We plan to further explore whether the introduction of
the red algae hydrolase gene into yeast cells can utilize red algae to produce
rare ginsenosides, and further investigate whether the modification of the MVA
pathway remains effective after constructing a complete metabolic pathway from
red algae to Rh1 in yeast cells.

Cycle 7: Using red algae polysaccharides as the substrate to produce the rare ginsenoside Rh1

Cycle 7.2: A recombinant Saccharomyces cerevisiae strain S.cerevisiae Rh1-con
with stable accumulation of rare ginsenoside Rh1

Design

We used the lithium acetate conversion method to introduce the plasmid
p426-AqAga-agaNash into the engineered strain S.cerevisiae Rh1-con to construct
the engineered strain S.cerevisiae Rh1-Ag. Under the optimal mixed carbon source
conditions, By comparing the Rh1 yield with that of the engineered strain
S.cerevisiae Rh1-Con, it was verified whether the introduction of the red algae
hydrolase gene into yeast cells could produce rare ginsenosides from red algae.
We designed and constructed the strain S.cerevisiae SC0Rh1-Ag, which contains
red algae hydrolase and integrates the Rh1 synthesis pathway but has not been
modified by the MVA pathway. Under the optimal mixed carbon source conditions,
the Rh1 yield was compared with that of the engineered strain S.cerevisiae
Rh1-Ag To verify whether the modification of the MVA pathway remains effective
after a complete metabolic pathway from red algae to Rh1 is constructed in yeast
cells.

Build

Firstly, using the same method, five exogenous genes of the Rh1 synthesis
pathway were integrated into the corresponding genome of wild-type Saccharomyces cerevisiae CEN PK2-1D by CRISPR-Cas9 technology to obtain the engineered strain
S.cerevisiae SC0Rh1. Then, the plasmid p426-AqAga-agaNash was introduced into
the engineered strains S.cerevisiae SC0Rh1 and S.cerevisiae Rh1-con by lithium
acetate conversion method. The engineered strains S.cerevisiae SC0Rh1-Ag and
S.cerevisiae Rh1-Ag were obtained.

![figure7-3-5](https://static.igem.wiki/teams/5843/dbtl/figure7-3-5.webp)

The engineered strains S.cerevisiae SC0Rh1-Ag

The engineered strains S.cerevisiae Rh1-Ag

Test

The YPDA medium containing 10g/L glucose and 25g/L agar was shaken and fermented
for 144 hours at 30℃ and 220 rpm. Samples were taken at regular intervals of 0,
12, 24, 48, 72, 96, 120 and 144 hours after inoculation to detect sugar
consumption, bacterial growth and the yield of rare ginsenoside Rh1. The test
results showed that compared with the control strain Rh1-con, the Rh1 yield of
Rh1-Ag significantly increased in the later stage of fermentation (72-144
hours), reaching the maximum yield of 141.78 mg/L at 144 hours. The engineered
strain S.cerevisiae SC0Rh1-Ag completely failed to detect the rare ginsenoside
Rh1.

![figure7-3-4](https://static.igem.wiki/teams/5843/dbtl/figure7-3-4.webp)

Liquid phase detection image of rare ginsenoside Rh1

![test5-image3](https://static.igem.wiki/teams/5843/dbtl/figure7-3-3.webp)

A comparison chart of Rh1 yield between strains Rh1-con and Rh1-Ag

Learn

The test results show that the engineered strain can continuously utilize red
algae polysaccharides to synthesize the rare ginsenoside Rh1. The synergistic
utilization of red algae polysaccharides and glucose in the culture medium
optimizes carbon flow distribution: glucose preferentially supports the growth
and primary metabolism of the bacteria, while the degradation products of red
algae polysaccharides drive the synthesis of Rh1. It has also been demonstrated
that tHMG1 and IDI1 enzymes can enhance the MVA pathway in the yeast substrate
and balance IPP/DMAPP, thereby increasing the yield of products such as
cycloalene and downstream terpene rare ginsenoside Rh1.

Cycle 7: Using red algae polysaccharides as the substrate to produce the rare ginsenoside Rh1

Cycle 7.3: The recombinant strain was used to synthesize the rare ginsenoside Rh1
with red algae polysaccharides as the substrate

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References:

{% 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 %}

![Team Banner](https://static.igem.wiki/teams/5843/wiki-template/header/experiments.webp)

* [1 Materials and Methods](#material)
  + [Strains](#mat-1)
  + [Methods](#mat-2)
    - [1 Utilization of Red Algal
      Polysaccharides](#mat-2-1)
    - [2 Determination of Fermentation
      Conditions](#mat-2-2)
    - [3 Promoter Engineering](#mat-2-3)
    - [4 Squalene Production from Red Algal
      Polysaccharides](#mat-2-4)
    - [5 Rh1 Synthesis](#mat-2-5)
* [2 Protocols](#protocols)
  + [2.1 Culture Media Formulations](#protocols-cul)
  + [2.2 Plasmid Construction](#protocols-pla)
  + [2.3 Gene Expression](#protocols-gen)
  + [2.4 Chemical Experiments & Process
    Optimization](#protocols-che)
* [3 Sequences](#sequence)

# 1 Materials and Methods

## Strains

| **Strains** | **Related Description** |
| WT | Refers to Saccharomyces cerevisiae CEN.PK2 - 1D |
| Sq-Ag1 | WT with plasmid p426-Aga3463-NH852 |
| Sq-Ag2 | WT with plasmid p426-AqAga-NH852 |
| Sq-Ag3 | WT with plasmid p426-PdAgaC-NH852 |
| Sq-Ag4 | WT with plasmid p426-Aga3463-agaNash |
| Sq-Ag5 | WT with plasmid p426-AqAga-agaNash |
| Sq-Ag6 | WT with plasmid p426-PdAgaC-agaNash |
| Sq-0 | WT: ΔGAL80 :: tHMG1 + IDI1 |
| Sq-Ag | Sq-0 with p426-AqAga-agaNash |
| SCORh1 | WT: ΔX-3:: PgDDS, ΔXI-3:: CYP716A47 + PgCPR1, ΔLPP1:: CYP716A53v2 + UGTPg100 |
| SCORh1-Ag | SCORh1 with p426-AqAga-agaNash |
| Rh1-con | Sq-0:ΔX-3:: PgDDS, ΔXI-3:: CYP716A47 + PgCPR1, ΔLPP1:: CYP716A53v2 + UGTPg100 |
| Rh1-Ag | Rh1-con with p426-AqAga-agaNash |

## Methods

### 1 Utilization of Red Algal Polysaccharides

**1.1 Hydrolase Plasmid Construction**

A series of Saccharomyces cerevisiae expression plasmids were constructed
using
Gibson assembly based on
the p426 Gal backbone vector (containing 2μ ori, pBR322 ori, F1 ori, selection marker URA3, ampicillin
(Amp)
resistance tag, and strong promoters PGAL7 and PGAL1). Specific primers with
homologous arms were
designed,
and target fragments and vector backbone fragments were amplified by PCR using PrimeSTAR® Max DNA
polymerase
with specific reaction systems and procedures. The amplified products were detected by agarose gel
electrophoresis, purified and recovered using SanPrep Column PCR Product Purification Kit (Cat. No.
B518141-0100), and their concentrations were determined using a K5600C micro-spectrophotometer. For
example,
p426-AqAga-agaNash was constructed by mixing α-factor mutant-AqAga (amplified from S. cerevisiae CEN PK2-1D
genome), AqAga (amplified from PUC57-AqAga plasmid), α-factor
mutant-agaNash
(amplified from S.cerevisiae
CEN PK2-1D genome), agaNash (amplified from PUC57-agaNash plasmid), backbone
fragment A (amplified from p426
Gal plasmid), and backbone fragment B (amplified from p426 Gal plasmid) at a molar ratio of 1:1:1:1:1:1.
The
mixture was added to Gibson assembly reaction solution, ligated at 50°C for 45 min, and transformed into
E.coli DH5α. Positive clones were selected using Amp resistance,
verified
by
colony PCR and sequencing to
obtain the corresponding expression plasmids.

**1.2 Screening Engineered Strains**

Six plasmids were introduced into wild-type S.cerevisiae using the lithium
acetate transformation method
to obtain engineered strains Sq-Ag1 to Sq-Ag6. Lugol's plate assay was used for qualitative
determination
of
enzyme activity. The DNS method was employed to determine agarase activity, and a galactose detection
kit
was used to measure neoagarobiose hydrolase activity.

**1.3 Optimization of the MVA Pathway**

To optimize the MVA pathway in S. cerevisiae, tHMG1
and IDI1 were integrated into the GAL80 locus of
wild-type S. cerevisiae CEN PK2-1D using the CRISPR-Cas9 system to construct
engineered strain Sq-0. The
construction process was as follows: For Cas9-sgRNA plasmid construction, the ORF of the GAL80 gene was
localized, and a sgRNA target sequence (ACGATAGTTGCAGTATGGCG) was screened using the CHOPCHOP tool.
Using
this sequence as homologous arms, PCR linear amplification of
p426-PTEF1-SpCas9-TCYC1-PSNR52-sgRNA-TSUP4
(constructed by Gibson assembly of p426-SNR52p-gRNA.csr-1.Y-SUP4t and SpCas9 fragments) was performed
with
primers GAL80-sgRNA-F/R. The recovered fragment was transformed into E. coli
DH5α, and sequencing
verification was performed to obtain the Cas9-sgRNA plasmid targeting GAL80.
For
donor DNA integration
fragment construction, tHMG1 and IDI1 gene
fragments
were amplified by PCR using S. cerevisiae CEN PK2-1D
genome as the template; the bidirectional promoter PGAL1, PGAL10 was amplified
using
PGAL1,10-MCS-His-MCS-Flag-URA plasmid as the template; terminators TADH1 and
TCYC1 were also amplified. The
above fragments were mixed at a molar ratio of 1:1:1:1:1, subjected to Gibson assembly, transformed into
E.
coli DH5α, and positive clones were selected using Amp resistance. After verification by
colony PCR
and
sequencing, the donor DNA was obtained by PCR amplification and purification. For yeast gene editing,
competent S.cerevisiae cells were prepared using the ZYMO Frozen-EZ Yeast
Transformation II Kit, and 500 ng
of Cas9-sgRNA plasmid and 1 μg of linear donor DNA were co-transformed. The transformation system was
spread
on uracil-deficient YNB plates and incubated at 30°C for 4-5 days. After verifying the integration of
target
genes by colony PCR, positive transformants were inoculated into YPD liquid medium for culture, spread
on
YPD plates containing 1 mg/mL 5-fluoroorotic acid for negative screening to remove the URA3 tag, and
verified again by PCR to obtain engineered strain Sq-0 (△GAL80::tHMG1+IDI1).

### 2 Determination of Fermentation Conditions

Gradient concentrations of hydrochloric acid (0.001-0.01 M) were used to treat the medium at 121°C for
20
min. The optimal conditions were screened by observing medium fluidity and detecting the presence of
monosaccharides and neoagarobiose via HPLC. The liquefied medium was prepared under the optimized
conditions
(25 mL medium with 0.005 M hydrochloric acid, treated at 121°C for 20 min, pH adjusted to 6.0 after
cooling). The engineered strain Sq-Ag was inoculated at 1% (v/v) into the liquefied medium and cultured
in
a
shaker at 30°C and 220 rpm, with changes in medium status during fermentation observed.

### 3 Promoter Engineering

Plasmids were constructed using Gibson assembly as described in 1.1, containing different promoter
combinations (constitutive promoters TEF, GPD, TDH and inducible promoters GAL1, GAL7, GAL10) to
regulate
the expression of agarase and neoagarobiose hydrolase. The constructed plasmids were introduced into
S.
cerevisiae engineered strain Sq-0, respectively. The inoculated strains were cultured in a
mixed
carbon
source medium containing 5 g/L glucose and 25 g/L agar in a shaker at 30°C and 220 rpm for 96 h. After
cultivation, fermentation broth was collected, and squalene content was determined by high-performance
liquid chromatography. Furthermore, the intron RPS25Ai was inserted into the proximal end of the
promoters
to construct four engineered strains: [PGAL10-RPS25Ai]-[PGAL7-RPS25Ai],
PGAL10-[PGAL7-RPS25Ai],
[PGAL10-RPS25Ai]-PGAL7, and PGAL10-PGAL7. Squalene yields
were
compared under the same culture and detection
conditions.

### 4 Squalene Production from Red Algal Polysaccharides

Red algae were pretreated, and the engineered strain was inoculated into a medium with red algal
biomass
hydrolysate as the sole carbon source, followed by shake flask culture at 30°C and 220 rpm. During
cultivation, the contents of reducing sugars, ethanol, acetate, glycerol, and OD600nm (reflecting cell
growth) were detected at different incubation times (0-120 h), and squalene yield was determined
simultaneously.

### 5 Rh1 Synthesis

Wild-type S. cerevisiae CEN PK2-1D and engineered strain Sq-0 (containing
tHMG1 and IDI1) were used as
starting strains to screen and integrate enzyme genes required for the heterologous synthesis pathway of
rare ginsenoside Rh1 (PgDDS, CYP716A47, PgCPR1, CYP716A53v2, UGTPg100, all codon-optimized for S. cerevisiae).
Using
the CRISPR-Cas9 system, sgRNAs for X-3, XI-3,
and
LPP1 loci were designed via CHOPCHOP to
construct corresponding Cas9-sgRNA plasmids. Each gene fragment, promoter, and terminator were
amplified,
mixed at a molar ratio of 1:1:1, and subjected to Gibson assembly to construct donor DNA for each locus,
which was then transformed into E. coli DH5α for screening,
verification,
and purification. Competent cells
were prepared using the ZYMO yeast transformation kit, and 500 ng of Cas9-sgRNA plasmid and 1 μg of
donor
DNA were co-transformed. The transformation system was spread on uracil-deficient YNB plates and
incubated
at 30°C for 5 days. After initial screening by colony PCR, positive transformants were cultured in YPD
liquid medium, spread on YPD plates containing 5-fluoroorotic acid for negative screening to remove the
URA3
tag, and verified again by PCR to obtain strains SC0Rh1 and Rh1-con. Plasmid p426-AqAga-agaNash was
introduced into SC0Rh1 and Rh1-con using the lithium acetate transformation method to obtain strains
SC0Rh1-Ag and Rh1-Ag. The primary seed liquid was inoculated at 1% into YPDA medium containing 25 g/L
red
algal polysaccharides and 10 g/L glucose, cultured at 30°C and 220 rpm for 144 h, and samples were taken
at
0, 12, 24, 48, 72, 96, 120, and 144 h. Rh1 yield was detected using an LC-16 high-performance liquid
chromatograph, and sugar consumption and cell growth were monitored simultaneously.

# 2 Protocols

## 2.1 Culture Media Formulations

1 Uracil-Deficient YNB Plate

![Toggle](https://static.igem.wiki/teams/5843/wiki-template/chevron-up-solid-full.svg)

| Yeast Nitrogen Base (without amino acids) | 6.7 g/L |
| Glucose | 20 g/L |
| Histidine | 50 mg/L |
| Tryptophan | 50 mg/L |
| Leucine | 50 mg/L |
| Agar | 15 g/L |

2 YPD Liquid Medium

![Toggle](https://static.igem.wiki/teams/5843/wiki-template/chevron-up-solid-full.svg)

| Yeast Extract | 10 g/L |
| Peptone | 20 g/L |
| Glucose | 20 g/L |
| Deionized Water | 1 L |

3 YPA Liquid Medium

![Toggle](https://static.igem.wiki/teams/5843/wiki-template/chevron-up-solid-full.svg)

| Yeast Extract | 10 g/L |
| Peptone | 20 g/L |
| Agar | 20 g/L |
| Deionized Water | 1 L |

4 Solid YPD Plate with 5-Fluoroorotic Acid (5-FOA)

![Toggle](https://static.igem.wiki/teams/5843/wiki-template/chevron-up-solid-full.svg)

| Yeast Extract | 10 g/L |
| Peptone | 20 g/L |
| Glucose | 20 g/L |
| Agar | 15 g/L |
| 5-Fluoroorotic Acid (5-FOA) | 1 g/L |

5 Lugol's Iodine Plate

![Toggle](https://static.igem.wiki/teams/5843/wiki-template/chevron-up-solid-full.svg)

| Yeast Nitrogen Base (without amino acids) | 6.7 g/L |
| Glucose | 20 g/L |
| Histidine | 50 mg/L |
| Tryptophan | 50 mg/L |
| Leucine | 50 mg/L |
| Agar | 15 g/L |
| Lugol's Iodine Solution | 5 mL/L |

6 LB Medium

![Toggle](https://static.igem.wiki/teams/5843/wiki-template/chevron-up-solid-full.svg)

| Yeast Extract | 5 g/L |
| Peptone | 10 g/L |
| NaCl | 10 g/L |

Sterilize at 121 °C for 20 minutes, and then add
the corresponding antibiotics as needed.

7 YPDA Medium

![Toggle](https://static.igem.wiki/teams/5843/wiki-template/chevron-up-solid-full.svg)

| Yeast Extract | 10 g/L |
| Peptone | 20 g/L |
| Red Algal Polysaccharides | 25 g/L |
| Glucose | 10 g/L |
| Deionized Water | 1 L |

## 2.2 Plasmid Construction

1 PCR (Polymerase Chain Reaction)

![Toggle](https://static.igem.wiki/teams/5843/wiki-template/chevron-up-solid-full.svg)

**Purpose:**

Amplify target gene fragments using specific primers.

**Steps:**

1. In a sterile PCR tube, add the following reagents in order:

   | DNA Template (50-200 ng) | 0.5 μL |
   | Forward Primer (10 μM) | 0.4 μL |
   | Reverse Primer (10 μM) | 0.4 μL |
   | High-Fidelity PCR Buffer (2X) | 10 μL |
   | ddH2O | 9.1 μL |
2. Place the tube in the PCR instrument for PCR amplification.

   PCR thermal cycling conditions:

   | Step | Time(s) | Temperature(℃) | Cycle |
   | Initial Denaturation | 180 | 98 | 1 |
   | Denaturation | 10 | 98 | 30 |
   | Annealing | 5 | 58 | 30 |
   | Extension | 30-180 | 72 | 30 |
   | Final Extension | 600 | 72 | 1 |

2 Agarose Gel Electrophoresis

![Toggle](https://static.igem.wiki/teams/5843/wiki-template/chevron-up-solid-full.svg)

**Purpose:**

Separate and visualize DNA fragments by size; excise target bands.

**Steps:**

Agarose Gel Preparation

1. Weigh 0.4g agarose powder in a flask; add 50mL 1×TAE buffer.
2. Heat in microwave with intermittent shaking until fully dissolved and clear.
3. Cool solution to 50-60°C.
4. Add 5μL Ultra GelRed dye; mix gently.
5. Seal gel tray ends; insert comb.
6. Pour solution into tray avoiding bubbles. Solidify at RT for 20-30 min.

Sample Loading Electrophoresis

1. Remove comb and end seals after solidification.
2. Place gel in tank with wells facing cathode.
3. Add 1×TAE buffer to cover gel by 1-2mm.
4. Load 5-10μL DNA samples mixed with loading buffer into wells. Include DNA ladder in one well.
5. Run gel at 100V for 30 min.
6. Stop when bromophenol blue reaches 2/3-3/4 gel length.
7. Transfer gel to UV transilluminator; visualize and photograph bands.
8. Excise target DNA fragments based on marker reference for purification.

3 DNA Purification and Recovery

![Toggle](https://static.igem.wiki/teams/5843/wiki-template/chevron-up-solid-full.svg)

### Using SanPrep Column PCR Product Purification Kit (Cat. B518141-0100)

**Purpose:**

Remove enzymes, primers, and other impurities while concentrating target DNA to obtain
high-purity templates for downstream experiments.

**Steps:**

1. Preparation: Ensure ethanol has been added to Wash Solution, and isopropanol to Buffer B3.
   Check for precipitation in Buffer B3.
2. Add 5 volumes of Buffer B3 to the PCR reaction mixture and mix thoroughly.
3. Centrifuge at 8,000 × g for 30 seconds. Discard the flow-through.
4. Add 500 μL of Wash Solution, centrifuge at 9,000 × g for 30 seconds, and discard the
   flow-through.
5. Repeat step 4 once.
6. Centrifuge the empty column at 9,000 × g for 1 minute.
7. Transfer the column to a clean 1.5 mL tube. Add 15–40 μL of Elution Buffer to the center of
   the membrane. Incubate at room temperature for 1 minute, then centrifuge for 1 minute. Collect
   and store the DNA solution.

4 Gibson Assembly

![Toggle](https://static.igem.wiki/teams/5843/wiki-template/chevron-up-solid-full.svg)

**Purpose:**

Efficiently and accurately join multiple linear DNA fragments (e.g., target gene and linearized
vector) into a complete circular recombinant plasmid in vitro.

**Steps:**

1. Prior to step 2, perform PCR and DNA purification for the target gene fragment.
2. Measure concentrations of purified linearized vector and insert using a K5600C
   microspectrophotometer.
3. Calculate molar concentrations based on measured concentrations and fragment lengths (bp).
4. Precisely pipette calculated volumes of linearized vector and insert into a new sterile PCR
   tube or microcentrifuge tube; mix gently.
5. Add an equal volume of Gibson Assembly Master Mix to the DNA mixture.
6. Incubate at 50°C in a PCR instrument or water bath for 45 minutes.

5 Vector Transformation into E. coli DH5α
Strain

![Toggle](https://static.igem.wiki/teams/5843/wiki-template/chevron-up-solid-full.svg)

**Purpose:**

Amplify plasmids efficiently using E. coli DH5α transformation
and rapidly obtain positive clones via antibiotic selection.

**Steps:**

1. Thaw 50 μL of DH5α competent cells slowly on ice.
2. Add 1–5 μL of ligation product or plasmid DNA to the cells; mix gently and incubate on ice for
   5 minutes.
3. Heat-shock at 42°C for 1 minute, then return to ice for 3 minutes.
4. Under sterile conditions, spread the mixture directly onto an LB agar plate containing 150
   μg/mL ampicillin.
5. Incubate at 37°C for 12–15 hours (no satellite colonies observed). Pick colonies for
   verification by colony PCR.

6 Colony PCR

![Toggle](https://static.igem.wiki/teams/5843/wiki-template/chevron-up-solid-full.svg)

**Purpose:**

Rapidly and efficiently screen for positive E. coli clones containing
the target agarase gene insert in the plasmid, rather than empty vector clones.

**Steps:**

1. In a sterile PCR tube, add the following reagents in order:

   | DNA Template (50-200 ng) | 5 μL |
   | Forward Primer (10 μM) | 0.2 μL |
   | Reverse Primer (10 μM) | 0.2 μL |
   | High-Fidelity PCR Buffer (2X) | 0.3 μL |
   | ddH2O | 4.3 μL |
2. Place the tube in the PCR instrument for PCR amplification.

   PCR thermal cycling conditions:

   | Step | Time(s) | Temperature(℃) | Cycle |
   | Initial Denaturation | 180 | 98 | 1 |
   | Denaturation | 10 | 98 | 30 |
   | Annealing | 5 | 58 | 30 |
   | Extension | 30-180 | 72 | 30 |
   | Final Extension | 600 | 72 | 1 |

## 2.3 Gene Expression

1 Episomal Plasmid Expression

![Toggle](https://static.igem.wiki/teams/5843/wiki-template/chevron-up-solid-full.svg)

1. Lithium Acetate Transformation Method

   **Purpose:**

   Efficiently introduce in vitro constructed recombinant plasmid DNA into engineered Saccharomyces cerevisiae cells.

   **Steps:**

   1. Pick a single colony from a solid plate and inoculate into YPD liquid medium; incubate at
      30°C, 220 rpm for 16 h.
   2. Take 500 μL culture, centrifuge at 8000 rpm, and discard supernatant.
   3. Add sequentially: 3 μL of 10 mg/mL salmon sperm ssDNA (denatured at 95°C for 5 min and
      chilled on ice), 100 μL transformation mix (800 μL 50% PEG3350, 200 μL 2 mol/L LiAc, 7.5 μL
      β-mercaptoethanol, balance ddH₂O per 1 mL), and 0.1–1 μg plasmid; mix well.
   4. Incubate at 37°C for 30 min, centrifuge at 8000 rpm for 3 min, discard supernatant.
   5. Resuspend cells in 500 μL sterile water, plate 80 μL on uracil-deficient YNB plates,
      incubate at 30°C for 3–5 days.
2. Preliminary Verification by Lugol’s Iodine Plate Assay

   **Purpose:**

   Qualitatively and rapidly determine whether engineered yeast successfully secretes active
   extracellular enzymes for degrading red algal polysaccharides, based on clear hydrolysis zones
   around colonies.

   **Steps:**

   1. Inoculate a single colony of engineered strain into 5 mL uracil-deficient YNB liquid
      medium; culture at 30°C, 220 rpm for 16–24 h.
   2. Spot 2 μL culture onto Lugol’s iodine plates, incubate for 48 h, and observe clear zones.
      Undegraded areas appear dark blue.
3. Pre-treatment for Enzyme Activity Assay

   **Steps:**

   1. From a fresh plate (≤1 month old), pick a single S. cerevisiae
      colony and inoculate into 3 mL uracil-deficient YNB liquid medium; culture overnight at
      30°C, 220 rpm to obtain primary seed culture in log phase.
   2. Inoculate 1% activated seed culture of 6 engineered strains into YPD medium; after 96 h
      fermentation, collect broth for agarase and neoagarobiose hydrolase activity assays.
4. Agarase Activity Assay

   **Purpose:**

   Quantitatively evaluate the overall ability of extracellular agarase secreted by engineered
   strains to degrade agar polysaccharides into reducing sugars, for screening the most efficient
   degraders.

   **Steps:**

   1. Precisely weigh dried glucose standard and prepare a 1.0 mg/mL stock solution with
      deionized water.
   2. Prepare standard series in test tubes as follows:

      | Tube No. | Glucose Stock (mL) | Deionized Water (mL) | Glucose Content (μg) |
      | 0 | 0.0 | 1.0 | 0 |
      | 1 | 0.2 | 0.8 | 200 |
      | 2 | 0.4 | 0.6 | 400 |
      | 3 | 0.6 | 0.4 | 600 |
      | 4 | 0.8 | 0.2 | 800 |
      | 5 | 1.0 | 0.0 | 1000 |
   3. Add 1.5 mL DNS reagent to each tube, heat in boiling water bath for exactly 5 min, then
      immediately cool.
   4. Add 8.5 mL deionized water to each tube, mix well.
   5. Measure absorbance at 540 nm using Tube 0 as blank.
   6. Plot standard curve with glucose content (μg) as X and OD540 as Y. Obtain regression
      equation: Y = 1.391X - 0.07451 (R² = 0.9974).

   **Enzyme Activity Assay:**

   1. Add 200 μL fermentation broth to 800 μL Tris-HCl buffer (pH 8.0) containing 0.3% w/v agar
      in a colorimetric tube. React with shaking at 40°C for 20 min.
   2. Add 1 mL DNS reagent, heat in boiling water bath for 5 min, cool, and dilute to 10 mL with
      distilled water. Mix well.
   3. Measure absorbance at 540 nm using inactivated enzyme as control. Calculate reducing sugar
      content using standard curve (Y=1.391X-0.07451, R²=0.9974).

   Unit definition (U): Enzyme amount required to produce 1 μg reducing sugar per minute under
   above conditions.
5. Neoagarobiose Hydrolase Activity Assay

   **Purpose:**

   Specifically detect the efficiency of engineered strains hydrolyzing neoagarobiose (agar
   degradation product) into galactose, ensuring complete degradation of algal polysaccharides
   into utilizable monosaccharides.

   **Steps:**

   Galactose Standard Curve (Using Abcam Galactose Assay Kit):

   1. Dilute kit-provided galactose standard (100 mmol/μL) to 1 mmol/μL working solution using
      Assay Buffer.
   2. Take 10 μL working solution, add 990 μL Assay Buffer, mix thoroughly to obtain 10 nmol/μL
      diluted standard.
   3. Add reagents to wells as per table:

      | Tube No. | Glucose Stock (mL) | Deionized Water (mL) | Glucose Content (μg) |
      | 0 | 0.0 | 1.0 | 0 |
      | 1 | 0.2 | 0.8 | 200 |
      | 2 | 0.4 | 0.6 | 400 |
      | 3 | 0.6 | 0.4 | 600 |
      | 4 | 0.8 | 0.2 | 800 |
      | 5 | 1.0 | 0.0 | 1000 |
   4. Then add sequentially to each well: 2 μL Galactose Probe, 2 μL Galactose Enzyme Mix, and 2
      μL HRP.
   5. Incubate the reaction system at 37°C in the dark for 40 min.
   6. Measure the absorbance (OD₅₇₀) of each well at 570 nm.
   7. Plot the galactose standard curve with galactose content (nmol) as X and OD₅₇₀ as Y,
      obtaining the regression equation: Y = 0.07252X + 0.03044 (R² = 0.9945).

   **Enzyme Activity Assay:**

   1. Dilute the galactose standard (Abcam kit) to 1 nmol/μL: Add 10 μL of 100 nmol/μL galactose
      standard to 990 μL galactose assay buffer and mix thoroughly.
   2. Add 0, 2, 4, 6, 8, and 10 μL of the diluted standard to a series of wells. Adjust each
      well to 50 μL with galactose assay buffer, resulting in 0, 2, 4, 6, 8, and 10 nmol galactose
      standards per well.
   3. Plot the standard curve with galactose concentration (nmol) as X and OD570 as Y.
   4. Calculate galactose content in the fermentation broth using the standard curve
      (Y=0.07252X+0.03044, R²=0.9945).
   5. To 25 μL fermentation broth (using inactivated enzyme as control), add 25 μL of 2 mM
      neoagarobiose, 44 μL galactose assay buffer, 2 μL galactose probe, 2 μL galactose enzyme
      mix, and 2 μL HRP. Total reaction volume is 100 μL. Incubate at 37°C for 40 min.
   6. Measure absorbance at 570 nm.

   Unit definition (U):Enzyme amount required to convert 1 μmol galactose per minute.

2 Genomic Integration Expression

![Toggle](https://static.igem.wiki/teams/5843/wiki-template/chevron-up-solid-full.svg)

1. CRISPR-Cas9 Plasmid Construction for Targeted Genomic Loci

   **Purpose:**

   Construct Cas9 plasmids expressing specific sgRNAs to create DNA double-strand breaks at
   specific yeast genomic loci, providing targets for subsequent homologous recombination and
   foreign gene integration.

   **Steps:**

   1. Use online tool CHOPCHOP (https://chopchop.cbu.uib.no/) to design efficient and
      low-off-target 20 nt sgRNA sequences for target genomic loci (e.g., GAL80, X-3).
   2. Amplify sgRNA fragment using p426-SNR52p-gRNA.csr-1.Y-SUP4t as template with primers sg-F
      and sg-R; amplify Cas9 fragment using Addgene SpCas9 plasmid as template with primers Cas9-F
      and Cas9-R. Mix sgRNA and Cas9 fragments at 1:1 molar ratio to assemble
      p426-PTEF1-SpCas9-TCYC1-PSNR52-sgRNA-TSUP4
      plasmid.
   3. Use Cas9 empty vector
      (p426-PTEF1-SpCas9-TCYC1-PSNR52-sgRNA-TSUP4) as
      template and designed sgRNA sequence as 5' homology arm in primers for PCR linearization to
      obtain linearized vector backbone.
   4. Transform 100 ng linearized plasmid into E. coli DH5α
      competent cells after purifying the target gene fragment.
   5. Pick 3 positive transformants for culture, extract plasmids, and send for sequencing
      verification (e.g., Sangon Biotech) to obtain validated Cas9-sgRNA plasmids targeting the
      gene locus.
2. Donor DNA Preparation

   **Purpose:**

   Prepare linear DNA fragments containing the target gene and homology arms as repair templates
   for homologous recombination, guiding precise integration of foreign genes into specific
   genomic loci.

   **Steps:**

   1. Amplify target gene, promoter, terminator, etc., by PCR using yeast genomic DNA or plasmid
      as template.
   2. Assemble all elements using Gibson Assembly.
   3. Transform the assembled product into E. coli DH5α,
      screen positive clones using ampicillin resistance, and verify by colony PCR and sequencing.
   4. Amplify the correct construct using specific primers and purify the gene fragment with
      SanPrep Column PCR Product Purification Kit to obtain donor DNA.
3. Preparation of S. cerevisiae CEN.PK2-1D Competent Cells

   **Using ZYMO Frozen-EZ Yeast Transformation II Kit**

   **Purpose:**

   Disrupt the cell wall barrier to enable efficient uptake of foreign DNA by S. cerevisiae for genetic modification.

   **Steps:**

   1. Inoculate CEN.PK2-1D strain in 10 mL YPD liquid medium. Incubate at 30°C with vigorous
      shaking (200-250 rpm) until mid-log phase (OD600 ≈ 0.8-1.0).
   2. Centrifuge at 500 × g for 4 min, discard supernatant. Resuspend cell pellet in 10 mL
      Solution 1, centrifuge again at 500 × g for 4 min, and discard supernatant to thoroughly
      wash cells.
   3. Add 1 mL Solution 2 to the washed cell pellet, resuspend completely by pipetting or
      vortexing. The resulting cell suspension is competent cells, ready for immediate
      transformation or freezing.
4. Primary Screening

   **Purpose:**

   Rapidly screen for positive clones with potential correct homologous recombination from a
   large number of transformants.

   **Steps:**

   1. Verify by colony PCR: Pick single colonies from uracil-deficient YNB plates, transfer to
      tubes containing PCR mix, and use GAL80 locus verification
      primers F and R to check target gene integration in the yeast genome.
5. Counter-Selection

   **Purpose:**

   Eliminate the URA3 selection marker gene from positive clones to obtain engineered strains
   containing only the target gene without exogenous markers.

   **Steps:**

   1. Inoculate transformants with correct primary screening bands into YPD liquid medium;
      incubate at 30°C, 220 rpm for 16 h.
   2. Spread onto solid YPD plates containing 1 mg/mL 5-fluoroorotic acid (5-FOA) for
      counter-selection. Perform PCR verification again on colonies growing on the plates; strains
      verified correctly indicate successful marker loss.

## 2.4Chemical Experiments & Process Optimization

1 Red Algae Hydrolysate Preparation

![Toggle](https://static.igem.wiki/teams/5843/wiki-template/chevron-up-solid-full.svg)

**Purpose:**

Mild hydrolysis of red algal powder using dilute HCl to convert hard-to-utilize polysaccharides
into fermentable reducing sugars, providing a carbon source for yeast.

**Steps:**

1. Mix 5g algal powder with 0.1 mM HCl solution at 1:10 ratio (total volume 50 mL); react at
   115°C for 15 min.
2. After reaction, adjust hydrolysate pH to 7.0 using NaOH solution to suit yeast growth.
3. Dilute the neutralized hydrolysate 1:1 with deionized water, use as YP fermentation medium in
   shake flasks.
4. Measure initial reducing sugar concentration of diluted medium to assess carbon source
   content.

2 HPLC Analysis

![Toggle](https://static.igem.wiki/teams/5843/wiki-template/chevron-up-solid-full.svg)

1. Quantification of Squalene Accumulation

   **Steps:**

   **Squalene Standard Curve Preparation:**

   1. Add 0.5 mL grinding beads to a disruption tube, then add 0.5 mL fermentation broth and 1
      mL ethyl acetate.
   2. Disrupt cells using a Bioprep-24R instrument, then centrifuge at 12,000 × g for 1 min.
   3. Use a needle to aspirate the supernatant (ethyl acetate layer), attach a 0.22 μm organic
      solvent filter, discard the first few drops (~3 drops) to prime the filter and remove dead
      volume. Collect subsequent filtrate into clear HPLC vials for analysis.
   4. Precisely weigh 10.0 mg squalene standard (purity ≥95%), dissolve in ethyl acetate, and
      dilute to 10 mL in a volumetric flask to prepare 1.0 mg/mL stock solution.
   5. Perform gradient dilution of the stock solution with ethyl acetate to prepare a series of
      standard working solutions:

      | Tube No. | 100 μg/mL Stock (μL) | Ethyl Acetate (μL) | Squalene Content (μg) | Final Concentration (μg/mL) |
      | 0 | 0 | 1000 | 0 | 0 |
      | 1 | 50 | 950 | 50 | 50 |
      | 2 | 100 | 900 | 100 | 100 |
      | 3 | 200 | 800 | 200 | 200 |
      | 4 | 300 | 700 | 300 | 300 |
      | 5 | 400 | 600 | 400 | 400 |
      | 6 | 500 | 500 | 500 | 500 |
      | 7 | 1000 | 0 | 1000 | 1000 |
   6. Filter the upper ethyl acetate layer through a 0.22 μm membrane for HPLC analysis. Record
      the squalene chromatographic peak area for each concentration.
   7. Plot squalene concentration (μg/mL) as X and the measured average peak area as Y. Perform
      linear regression to obtain the equation: Y = 14498.99X + 387171.62, R² = 0.9992.

   **Squalene Quantification:**

   1. Add 0.5 mL fermentation broth to a disruption tube containing 0.5 g of 0.5 mm glass
      beads and 1 mL ethyl acetate.
   2. Disrupt cells using a Bioprep-24R instrument, then centrifuge at 10,000 × g for 1 min.
   3. Filter the upper ethyl acetate layer through a 0.22 μm membrane for HPLC analysis.

   **HPLC Conditions:**

   System: Shimadzu LC-16 with SPD-16 UV detector

   Column: Agilent Poroshell 120 EC-C18 (2.1 × 100 mm)

   Eluent: 100% acetonitrile

   Flow rate: 0.5 mL/min

   Injection volume: 2 μL

   Detection wavelength: 210 nm
2. Detection of Rare Ginsenoside Rh1 Production

   **Steps:**

   **Rh1 Standard Curve Preparation:**

   1. Precisely weigh 3.0 mg of 20(S)-Rh1 standard, dissolve in 1000 μL methanol to prepare a 3
      mg/mL stock solution.
   2. Label 7 clean HPLC vials as 1-7.
   3. Pipette corresponding volumes of 3 mg/mL stock into each vial and add HPLC-grade methanol
      to adjust the total volume to 400 μL:

      | Tube No. | Stock (mL) | Methanol (μL) | Concentration (mg/mL) |
      | 1 | 4 | 396 | 0.01 |
      | 2 | 10 | 390 | 0.025 |
      | 3 | 20 | 380 | 0.05 |
      | 4 | 40 | 360 | 0.10 |
      | 5 | 100 | 300 | 0.25 |
      | 6 | 200 | 200 | 0.50 |
      | 7 | 400 | 0 | 1.00 |
   4. Filter the methanol layer through a 0.22 μm membrane for HPLC analysis. Record the Rh1
      peak area for each concentration.
   5. Plot Rh1 concentration (g/L) as X and average peak area as Y. Perform linear regression to
      obtain the equation: Y = 3878X + 38658, R² = 0.9990.

   **Rh1 Quantification:**

   1. Add 0.5 mL fermentation broth to a disruption tube containing 0.5 g of 0.5 mm glass beads
      and 1 mL n-butanol.
   2. Disrupt cells using a Bioprep-24R instrument, then centrifuge at 10,000 × g for 1 min.
   3. Filter the upper n-butanol layer through a 0.22 μm membrane for HPLC analysis.

   **HPLC Conditions:**

   System: Shimadzu LC-16 with SPD-16 dual-wavelength UV detector

   Column: Neptune 5u C18 (250 × 4.6 mm)

   Mobile phase: Water/acetonitrile

   Detection wavelength: 203 nm

   Column temperature: 35°C

   Gradient elution: 0-6 min (40-100% B), 6-18 min (100% B), 18-25 min (100-40% B), 25-35 min
   (40%
   B)

3 Determination of Liquefaction Conditions

![Toggle](https://static.igem.wiki/teams/5843/wiki-template/chevron-up-solid-full.svg)

**Purpose:**

High concentrations of red algal polysaccharides increase medium viscosity, inhibiting strain
growth. Determine the specific conditions for liquefaction using low HCl concentrations.

**Steps:**

1. Treat 25 mL YPDA medium with HCl gradients (0.001-0.01 M) at 121°C for 20 min.
2. Analyze by HPLC to detect neoagarobiose and monosaccharides.

4 Orthogonal Experiment: Red Algal Polysaccharide & Glucose Concentrations

![Toggle](https://static.igem.wiki/teams/5843/wiki-template/chevron-up-solid-full.svg)

**Purpose:**

Determine the optimal ratio of red algal polysaccharides to glucose in the medium for squalene
production by engineered strain Sq-Ag5.

**Steps:**

1. Prepare YPDA liquid media with glucose concentrations (0, 2.5, 5, 7.5, 10 g/L) and red algal
   polysaccharide concentrations (10, 15, 20, 25, 30, 40 g/L).
2. Inoculate 1% (v/v) primary seed culture into 250 mL flasks containing 25 mL YPDA medium.
   Ferment at 30°C, 220 rpm for 96 h.
3. Measure squalene content in fermentation broth by HPLC to determine the optimal carbon source
   composition.

# 3 Sequences

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{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}Present the results of your project, along with a detailed analysis and discussion of their
significance. Also outline future plans and reflections on the impact of your project.{% endblock %}
{% block page\_content %}

![Team Banner](https://static.igem.wiki/teams/5843/wiki-template/header/results1.webp)

* [1 Overview](#overview)
* [2 Utilization of Red Algal Polysaccharides](#utili)
  + [2.1 Hydrolase Plasmid Construction](#utili-pla)
  + [2.2 Screening Engineered Strains](#utili-s-1)
  + [2.3 Optimize the MVA pathway](#utili-cons-of-g)
* [3 Determination of Fermentation Conditions](#deter)
  + [3.1 Liquefaction Condition
    Optimization](#deter-liq)
  + [3.2 Carbon Source Condition
    Optimization](#deter-car)
* [4 Promoter Engineering](#prom)
  + [4.1 Promoter Screening](#prom-pro)
  + [4.2 Insertion of Chimeric Intron](#prom-ins)
* [5 Squalene Production from Red Algal Polysaccharides](#squa)
* [6 Rh1 Synthesis](#rh1)
  + [6.1 Insert the PgDDS
    Gene at the X-3 Locus](#rh1-01)
  + [6.2 Insert the CYP716A47 and PgCPR1 Genes at
    the XI-3 Locus](#rh1-02)
  + [6.3 Insert the CYP716A53v2, UGTPg100 Genes
    at the LPP1 Locus](#rh1-03)
  + [6.4 Rh1 Synthesis Using Red Algal
    Polysaccharides](#rh1-04)
* [7 References](#reference)

# 1 Overview

Our project primarily consists of the red algal polysaccharide utilization module and the Rh1 synthesis
module. This page presents the experimental results of our project and our interpretation of the data. It
mainly includes plasmid construction maps for each module, screening results of engineered strains,
determination of fermentation conditions and product yields.

# 2 Utilization of Red Algal Polysaccharides

## 2.1 Hydrolase Plasmid Construction

We selected three agarases and two neoagarobiose hydrolases from different sources. After codon
optimization, six plasmids were constructed through combinatorial assembly. The p426 Gal backbone vector
was used to construct Saccharomyces cerevisiae expression plasmids [1],
featuring URA3 as the selectable marker, ampicillin (Amp) resistance, and the strong promoters
PGAL7 and
PGAL1. Secretion was directed using the α-factor signal peptide ([BBa\_258RYIFY](https://registry.igem.org/parts/bba-258ryify)).

![Plasmid Map](https://static.igem.wiki/teams/5843/wiki-results/pic-pla-2.webp)

#### Figure 1 Maps of the 6 constructed plasmids.

#### (A) Aga3463 - NH852, (B) AgAga - NH852, (C) PdAgac - NH852,

#### (D) Aga3463 - agaNash, (E) AgAga - agaNash, (F) PdAgac - agaNash.

Target fragments were successfully amplified and assembled into the vector using Gibson Assembly.
Constructed plasmids were transformed into E. coli DH5α. Successful
ligation was confirmed by colony PCR and sequencing, yielding agarase system expression vectors.

## 2.2 Screening Engineered Strains

The six plasmids were transformed into wild-type S. cerevisiae CEN.PK2-1D
using the lithium acetate method, yielding six engineered strains (Sq-Ag1, Sq-Ag2, Sq-Ag3, Sq-Ag4, Sq-Ag5,
Sq-Ag6).

#### Table 1 Genotypes of engineered strains.

| **Strain** | **Genotype** | |
| **Agarase** | **Neoagarobiose Hydrolase** |
| **Sq-Ag1** | Aga3463 | NH852 |
| **Sq-Ag2** | AqAga | NH852 |
| **Sq-Ag3** | PdAgaC | NH852 |
| **Sq-Ag4** | Aga3463 | agaNash |
| **Sq-Ag5** | AqAga | agaNash |
| **Sq-Ag6** | PdAgaC | agaNash |
| **Sq-pls** | p426 empty plasmid | |
| **WT** | The control group without any treatment | |

All six strains were cultured in uracil-deficient YPD medium. Qualitative detection using Lugol's iodine
plate assay confirmed extracellular agarase secretion capability for all strains.

As can be seen from the size of the transparent zones, Sq-Ag4, Sq-Ag5, and Sq-Ag6 exhibit strong
secretion
abilities.

![utili-pic2](https://static.igem.wiki/teams/5843/wiki-results/pic02-5.webp)

#### Figure 2 Results of Lugol's iodine plate assay.

Activated seed cultures of the six engineered strains were transferred to YPD medium for agarase and
neoagarobiose hydrolase activity assays.

By constructing a standard curve of enzyme activity and comparing it with the samples, we obtained the
enzyme activity data of the engineering strain [2].

![utili-pic5](https://static.igem.wiki/teams/5843/wiki-results/change2.webp)

#### Figure 3 (A) Calibration curves of galactose for the quantitative determination of neoagarobiose hydrolase activity,

#### (B) agarase activity, (C) enzyme activity assays for combinations of agarase and neoagarobiose hydrolase.

By combining the results of the Lugol's iodine solution plate assay and the enzyme activity assay, the
optimal enzyme combination was the agarase gene AqAga ([BBa\_258028F8](https://registry.igem.org/parts/bba-258028f8))
from
Aquimarina agarilytica ZC1 and the neoagarobiose hydrolase gene agaNash ([BBa\_257L75AN](https://registry.igem.org/parts/bba-257l75an)) from Cellvibrio sp. OA-2007. This
combination (strain Sq-Ag5) was selected for subsequent engineering.

## 2.3 Optimize the MVA pathway

### Construction of Cas9-sgRNA Plasmid

The sgRNA 20 nt sequence was selected using the CHOPCHOP website. The sgRNA sequence was cloned into
a Cas9 expression
plasmid, constructing individual GAL80-targeting plasmids
(p426-PTEF1-SpCas9-TCYC1-PSNR52-sgRNA-TSUP4). Each plasmid
expressed Cas9 protein and a specific sgRNA in yeast to form a complex for precise genomic cutting.

![utili-pic6](https://static.igem.wiki/teams/5843/wiki-results/pic06.webp)
![utili-pic7](https://static.igem.wiki/teams/5843/wiki-results/change-right01.webp)

#### Figure 4 (Left Panel) Plasmid map.

#### (Right Panel) Gel electrophoresis of the PCR-linearized p426-PTEF1-SpCas9-TCYC1-PSNR52-sgRNA-TSUP4 plasmid (10251 bp fragment).

### Construction of Donor DNA

Homology arms for the GAL80 locus were introduced via primers for tHMG1 and
IDI1. Fragments were amplified by PCR and assembled.

![utili-pic8](https://static.igem.wiki/teams/5843/wiki-results/pic-ld.webp)

#### Figure 5 Linear donor DNA map.

The assembled donor was transformed into E. coli DH5α. Successful
ligation was confirmed by colony PCR and sequencing. The correct construct was PCR-amplified, and the
donor DNA fragment was purified using a SanPrep PCR Product Purification Kit.

![utili-pic9](https://static.igem.wiki/teams/5843/wiki-results/pic09.webp)
![utili-pic10](https://static.igem.wiki/teams/5843/wiki-results/change-right2.webp)

#### Figure 6 (Left Panel) Successfully transformed E. coli DH5α colonies.

#### (Right Panel) Gel electrophoresis confirming successful vector ligation.

### Screening Engineered Strains

Plasmids were transformed into prepared competent S. cerevisiae CEN.PK2-1D.
Primary and counter-selection were performed to obtain strains with successful genomic integration.
Primary screening was performed using colony PCR.

![utili-pic11](https://static.igem.wiki/teams/5843/wiki-results/pic11.webp)

![utili-pic12](https://static.igem.wiki/teams/5843/wiki-results/change-right3.webp)

#### Figure 7 (Left Panel) Primary selection plate results.

#### (Right Panel) Gel electrophoresis results showing bands of expected length, indicating successful genomic integration of the target gene.

Counter-selection was used to remove the URA3 selectable marker from positive clones.

![utili-pic14](https://static.igem.wiki/teams/5843/wiki-results/pic14.webp)

![utili-pic15](https://static.igem.wiki/teams/5843/wiki-results/change-right4.webp)

#### Figure 8 (Left Panel) Counter-selection plate results.

#### (Right Panel) Gel electrophoresis results confirming successful excision of the URA3 marker.

The final engineered strain, S. cerevisiae Sq-0, with successful genomic
integration and marker excision, was obtained.

Squalene content detection showed that overexpressing tHMG1 and IDI1 to optimize the MVA pathway significantly increased squalene production.
Strain Sq-0 was used as the starting strain for subsequent engineering.

By preparing a squalene standard curve and comparing it with the samples, we obtained the squalene yield
data [3].

![utili-pic14](https://static.igem.wiki/teams/5843/wiki-results/pic004.webp)

#### Figure 9 (Left Panel) Squalene HPLC standard curve.

#### (Right Panel) HPLC chromatogram of squalene from Sq-0 fermentation broth.

# 3 Determination of Fermentation Conditions

## 3.1 Liquefaction Condition Optimization

YPA medium (25 mL) was treated with HCl at final concentrations ranging from 0.001 M to 0.01 M at 121°C
for 20 min.

The results showed that an extremely low acid concentration (0.005 M hydrochloric acid) was able to break
down
the structure of agar, rendering it fluid. Moreover, it remained non-coagulated throughout the yeast shake
flask culture and gradually became clear, indicating that the agar had been completely utilized.

![deter-pic16](https://static.igem.wiki/teams/5843/wiki-results/pic005.webp)

#### Figure 10 State of the medium during fermentation.

HPLC detection confirmed that 0.005 M HCl liquefaction did not degrade agar into neoagarobiose or
monosaccharides. The medium pH was adjusted to 6.0 for yeast fermentation experiments.

![deter-pic17](https://static.igem.wiki/teams/5843/wiki-results/pic006.webp)

#### Figure 11 HPLC chromatogram of yeast medium liquefied with 0.005 M HCl.

## 3.2 Carbon Source Condition Optimization

Plasmid p426-AqAga-agaNash was transformed into engineered strain Sq-0 using the lithium acetate method,
yielding engineered strain Sq-Ag. Orthogonal fermentation experiments were conducted in media liquefied
with 0.005 M HCl.

Strain Sq-Ag successfully degraded and saccharified red algal polysaccharides stepwise. Squalene yield
increased with red algal polysaccharide concentration up to ≤ 25 g/L, and decreased with higher
concentrations (>25 g/L) of the substrate. The highest squalene yield of 667.02 mg/L was
achieved with 10 g/L glucose and 25 g/L red algal polysaccharides.

![deter-pic18](https://static.igem.wiki/teams/5843/wiki-results/pic008.webp)

#### Figure 12 Orthogonal experimental results of squalene synthesis with glucose and agar in flasks.

# 4 Promoter Engineering

## 4.1 Promoter Screening

We screened 3 constitutive promoters and 3 inducible promoters, constructed plasmids with 18 promoter
combinations, and transformed them into strain Sq-0 [4].

Yeast cells were cultured for 96 hours under the conditions of 5 g glucose and 25 g agar, and the
squalene yield was detected. The inducible combination of PGAL10 (regulating agarase expression) + PGAL7
(regulating neoagarobiose hydrolase expression) led to the highes squalene yield of 594.68 mg/L.

![prom-pic1](https://static.igem.wiki/teams/5843/wiki-results/013-2.webp)

#### Figure 13 Results of Squalene Fermentation with 18 Promoter Combinations.

## 4.2 Insertion of Chimeric Intron

In the strain Sq-Ag23, the RPS25Ai intron was inserted into the proximal regions of the PGAL10 and PGAL7 promoters, strains Sq-Ag23a, Sq-Ag23b and Sq-Ag23c were constructed.

![prom-pic1](https://static.igem.wiki/teams/5843/wiki-results/pic002.webp)

#### Figure 14 Construct the PGAL10-RPS25Ai chimeric promoter.

Yeast cells were cultured for 96 hours under the conditions of 5 g glucose and 25 g agar, and the
squalene yield was detected. According to the experimental results, the highest squalene yield (682 mg/L)
was achieved when the RPS25Ai intron was inserted into the proximal region of the PGAL10
promoter, while
no chimeric intron was inserted into the PGAL7 promoter.

![prom-pic1](https://static.igem.wiki/teams/5843/wiki-results/change4.webp)

#### Figure 15 Comparison of squalene fermentation before and after promoter engineering modification.

The insertion of chimeric introns into promoters enhanced the expression of agarase and
neoagarobiose hydrolase genes in Saccharomyces cerevisiae to varying degrees,
leading to increased squalene yield. Through promoter engineering, the squalene yield has been
significantly improved, which is more conducive to obtaining a higher yield of the rare ginsenoside Rh1 in
subsequent experiments.

Then we replaced the promoter of the plasmid p426-AgAga-agaNash with PGAL10 and PGAL7, and used the plasmids with the replaced promoters for subsequent experiments
.

# 5 Squalene Production from Red Algal Polysaccharides

We finally used purchased red algae, which were dried and ground into powder to prepare YPDA medium for the
fermentation culture of Sq-Ag23c. As shown in the figure 23, from 0 to 24 h, Saccharomyces
cerevisiae consumed
glucose for fermentation, resulting in a decrease in reducing sugar concentration and an increase in ethanol
concentration. From 24 to 48 h, the strain began to secrete hydrolases which decomposed agar into
monosaccharides, leading to an increase in reducing sugar content and a significant increase in squalene
yield. From 48 h to 120 h, the bacterial culture transitioned to the stationary phase, with continuous
synthesis of squalene, indicating the strong squalene synthesis ability of the Sq-Ag23c strain in the
stationary phase. After five days of fermentation, the squalene
yield reached 908 mg/L. The overall fermentation process showed a reasonable metabolic and product synthesis
pattern, demonstrating the feasibility and efficiency of using red algal polysaccharides to ferment and
produce squalene.

![squa-pic19](https://static.igem.wiki/teams/5843/wiki-results/change1.webp)

#### Figure 16 Squalene yield from fermentation.

# 6 Rh1 Synthesis

The heterologous pathway enzymes for rare ginsenoside Rh1 synthesis were integrated into specific loci of
wild-type
S. cerevisiae CEN.PK2-1D and S. cerevisiae Sq-0.

## 6.1 Insert the PgDDS Gene at the X-3 Locus

### Construction of Cas9-sgRNA Plasmid (X-3 Locus)

The sgRNA 20 nt sequences were designed using CHOPCHOP, which were cloned into a Cas9 expression plasmid
for construction of individual targeting plasmids (p426-sgRNA-X-3).

### Construction of Donor DNA (X-3 Locus - PgDDS)

Homology arms for the X-3 locus were introduced via primers for PgDDS ([BBa 255LOGT6](https://registry.igem.org/parts/bba-255logt6)).
The fragment was amplified by PCR and assembled via fusion PCR.

![rh1-pic18](https://static.igem.wiki/teams/5843/wiki-results/pic23.webp)

#### Figure 17 Linear donor DNA map.

The donor was transformed into E. coli DH5α. Positive clones were
selected using ampicillin resistance. Successful ligation was confirmed by colony PCR and sequencing. The
correct construct was amplified using primers X-3-TADH1-F and X-3-PGAL1,10-R, and
the donor DNA fragment
was purified.

![rh1-pic14](https://static.igem.wiki/teams/5843/wiki-results/pic24.webp)
![rh1-pic15](https://static.igem.wiki/teams/5843/wiki-results/change-right5.webp)

#### Figure 18 (Left Panel) Successfully transformed E. coli DH5α.

#### (Right Panel) Gel electrophoresis confirming successful vector construction.

### Result Verification (X-3 Locus)

Strains were fermented for 144 h in medium containing 20 g/L glucose. Dammarenediol-II production was
measured after integrating PgDDS, with yield of 1 g/L.

![rh1-pic23](https://static.igem.wiki/teams/5843/wiki-results/change3.webp)

#### Figure 19 (Left Panel) Dammarenediol-II HPLC standard curve.

#### (Right Panel) Dammarenediol-II production at 144 h after PgDDS integration.

## 6.2 Insert the CYP716A47 and PgCPR1 Genes at the XI-3 Locus

### Construction of Cas9-sgRNA Plasmid (XI-3 Locus)

Design of sgRNA 20 nt sequences was performed using CHOPCHOP, which were cloned into a Cas9 expression
plasmid for the construction of individual targeting plasmids (p426-sgRNA-XI-3).

### Construction of Donor DNA (XI-3 Locus - CYP716A47, PgCPR1)

Homology arms for the XI-3 locus were introduced via primers for CYP716A47
([BBa\_25RY2RMX](https://registry.igem.org/parts/bba-25ry2rmx))
and PgCPR1. Fragments were amplified by PCR and assembled via fusion PCR.

![rh1-pic25](https://static.igem.wiki/teams/5843/wiki-results/pic028.webp)

#### Figure 20 Linear donor DNA map.

The donor was transformed into E. coli DH5α. Positive clones were
selected using ampicillin
resistance. Successful ligation was confirmed by colony PCR and sequencing. The correct construct was
amplified using primers XI-3-PGAL1,10-F and XI-3-TCYC1-R, and the donor DNA fragment
was purified.

![utili-pic14](https://static.igem.wiki/teams/5843/wiki-results/pic46.webp)

![utili-pic15](https://static.igem.wiki/teams/5843/wiki-results/change-right6.webp)

#### Figure 21 (Left Panel) Successfully transformed E. coli DH5α.

#### (Right Panel) Gel electrophoresis confirming successful vector construction.

### Result Verification (XI-3 Locus)

Strains were fermented for 144 h in medium containing 20 g/L glucose. Protopanaxadiol (PPD) production
was measured after integrating CYP716A47 and PgCPR1.
PPD yield reached 273.8 mg/L.

![rh1-pic23](https://static.igem.wiki/teams/5843/wiki-results/change5-2.webp)

#### Figure 22 (Left Panel) PPD HPLC standard curve.

#### (Right Panel) PPD production at 144 h after CYP716A47 and PgCPR1 integration.

### Construction of Cas9-sgRNA Plasmid (LPP1 Locus)

Design of sgRNA 20 nt sequences are conducted by using CHOPCHOP, which were cloned into a Cas9 expression
plasmid to construct the individual targeting plasmids (p426-sgRNA-LPP1).

## 6.3 Insert the CYP716A53v2, UGTPg100 Genes at the LPP1 Locus

### Construction of Donor DNA (LPP1 Locus - CYP716A53v2, UGTPg100)

Homology arms for the LPP1 locus were introduced via primers for CYP716A53v2
and UGTPg100. Fragments were amplified by PCR and assembled via fusion PCR.

![rh1-pic30](https://static.igem.wiki/teams/5843/wiki-results/pic007.webp)

#### Figure 23 Linear donor DNA map.

The donor was transformed into E. coli DH5α. Positive clones were
selected using ampicillin
resistance. Successful ligation was confirmed by colony PCR and sequencing. The correct construct was
amplified using primers LPP1-TADH1-F and LPP1-TALT1-R, and the donor DNA fragment
was purified.

![utili-pic14](https://static.igem.wiki/teams/5843/wiki-results/pic34.webp)

![utili-pic15](https://static.igem.wiki/teams/5843/wiki-results/change-right7.webp)

#### Figure 24 (Left Panel) Successfully transformed E. coli DH5α.

#### (Right Panel) Gel electrophoresis confirming successful vector construction.

### Result Verification (LPP1 Locus)

Strains were fermented for 144 h in medium containing 20 g/L glucose. Rare ginsenoside Rh1 production was
measured after integrating CYP716A53v2 ([BBa\_25NSM6TW](https://registry.igem.org/parts/bba-25nsm6tw)) and
UGTPg100 ([BBa\_255ROLW2](https://registry.igem.org/parts/bba-255rolw2)). Rh1 yield reached 170.8 mg/L.

![rh1-pic23](https://static.igem.wiki/teams/5843/wiki-results/change6-2.webp)

#### Figure 25 (Left Panel) Rh1 HPLC standard curve.

#### (Right Panel) Rh1 production at 144 h after CYP716A53v2 and UGTPg100 integration.

### Screening Engineered Strains (Rh1 Pathway)

Plasmids were transformed into prepared competent S. cerevisiae CEN.PK2-1D.
Primary and counter-selection were performed to obtain strains with successful genomic integration at the
X-3, XI-3, and LPP1
loci.

![utili-pic14](https://static.igem.wiki/teams/5843/wiki-results/pic39.webp)

![utili-pic15](https://static.igem.wiki/teams/5843/wiki-results/change-right02.webp)

#### Figure 26 (Left Panel) Primary selection plate results.

#### (Right Panel) Gel electrophoresis results showing bands of expected length, indicating successful genomic integration.

Counter-selection was used to remove the URA3 selectable marker from positive clones.

![utili-pic14](https://static.igem.wiki/teams/5843/wiki-results/pic42.webp)

![utili-pic15](https://static.igem.wiki/teams/5843/wiki-results/change-right03.webp)

#### Figure 27 (Left Panel) Counter-selection plate results.

#### (Right Panel) Gel electrophoresis results confirming successful excision of the URA3 marker.

The final engineered strains with successful genomic integration and marker excision were obtained: S. cerevisiae SC0Rh1 (control, based on CEN.PK2-1D) and S.
cerevisiae Rh1-con (based on Sq-0).

## 6.4 Rh1 Synthesis Using Red Algal Polysaccharides

Plasmid p426-AqAga-agaNash was transformed into engineered strains S.
cerevisiae SC0Rh1 and S. cerevisiae Rh1-con using the lithium acetate
method, yielding engineered strains S. cerevisiae SC0Rh1-Ag and S. cerevisiae Rh1-Ag.

Strains were fermented in YPDA medium with 10 g/L glucose + 25 g/L agar at 30°C, 220 rpm for 144 h.
Samples were taken at 0, 12, 24, 48, 72, 96, 120, and 144 h to measure sugar consumption, cell growth, and
Rh1 production. Results showed that compared to control strain Rh1-con, Rh1 production in Rh1-Ag
significantly increased during the later fermentation stage (72-144 h), reaching a maximum yield of 141.78
mg/L at 144 h. No Rh1 was detected in S. cerevisiae SC0Rh1-Ag.

![rh1-pic38](https://static.igem.wiki/teams/5843/wiki-results/pic45.webp)

#### Figure 28 Results showing sustained utilization of red algal polysaccharides for Rh1 synthesis by the engineered strain.

---

# 7 References

###### [1] Mumberg D, Müller R, Funk M. Regulatable promoters of Saccharomyces cerevisiae: comparison of transcriptional activity and their use for heterologous expression. Nucleic Acids Res. 1994 Dec 25;22(25):5767-8. doi: 10.1093/nar/22.25.5767. PMID: 7838736; PMCID: PMC310147.

###### [2] FU W D. Screening of high-yield agarase-producing strains, optimization of fermentation conditions and study on enzymatic properties of agarase[Dissertation]. Qingdao: Ocean University of China, 2006.

###### [3] PAN Y Q, ZHOU H, WANG C, et al. Metabolic engineering of Yarrowia lipolytica for high-efficiency synthesis of squalene[J/OL]. Journal of Biology, [2025-09-22]. https://link.cnki.net/urlid/34.1081.Q.20250825.1043.004.

###### [4] JIANG H H, WANG Q, RAO Z M, et al. Research progress on promoter engineering of Saccharomyces cerevisiae[J]. China Biotechnology, 2023, 43(11): 78-91. DOI: 10.13523/j.cb.2307007.

{% endblock %}