iGEM 2024 — Team mingdao

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## Dec 3, 2022

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## Sep 21, 2022

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## Sep 10, 2022

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## Aug 20, 2022

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## Aug 3, 2022

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{% block title %}Engineering Success{% endblock %}
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{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
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[Problem](#Problem)

[Design concept](#Design concept)

[GCW61](#GCW61)

[PIR1](#PIR1)

[Modeling](#Modeling)

[PROOF OF CONCEPT](#PROOF OF CONCEPT)

[Conclusion](#CONCLUSION)

[Reference](#REFERENCE)

# Problem

In the agricultural sector, enhancing the digestibility and nutritional efficiency of animal feed is crucial. Our project
addresses this by using Pichia pastoris GS115 to surface-display thermostable xylanase from Streptomyces
thermovulgaris, thereby improving the breakdown of complex carbohydrates in feed. Utilizing the Pichia pastoris yeast
vector pZAHR and optimizing with the GTH1 promoter, we aim to develop a stable, efficient, and cost-effective method
for direct enzyme production in feed, enhancing both effectiveness and economic viability. Following the engineering
design cycle: Design → Build → Test → Learn, we systematically iterated through these stages to achieve our project
goals.

# Design concept

## Xylanase Choice: Streptomyces thermovulgaris

Xylanase Choice: Streptomyces thermovulgaris
Xylanases1 are enzymes that degrade xylan, a major component of plant cell walls, into simpler sugars. As feed
ylanase Choice: Streptomyces thermovulgaris
Xylanases1 are enzymes that degrade xylan, a major component of plant cell walls, into simpler sugars. As feed
additives, they play a crucial role in breaking down complex polysaccharides in animal diets, particularly for non-
ruminants like poultry and swine. This enzymatic action enhances nutrient availability and digestion, leading to improved
feed efficiency, growth performance, and overall health of the animals2. Moreover, the supplementation of xylanase in
feed efficiency, growth performance, and overall health of the animals2. Moreover, the supplementation of xylanase in
animal feed can significantly reduce feed costs and environmental impact by increasing nutrient absorption and
decreasing nutrient excretion3,4.

![](https://static.igem.wiki/teams/5049/mingdao24-engineering-1.png)

We chose the xylanase gene from Streptomyces thermovulgaris for yeast surface display due to its exceptional
properties. Research5,6 on Streptomyces thermovulgaris has shown that its crude xylanase can hydrolyze KOH-treated
corncob and produce bioactive xylooligosaccharides (XOs). The purified enzyme is highly stable across a broad pH range
(4.0-11.5) and thermostable between 50-70°C, with maximum activity at 65°C and a half-life of 90 minutes at 70°C.
Additionally, the enzyme's products act as prebiotics that promote the growth of probiotic Lactobacillus plantarum,
making it an ideal candidate for improving animal feed due to its stability, efficiency, and probiotic support.

## Host Choice: E. coli vs. P. pastoris

![](https://static.igem.wiki/teams/5049/mingdao24-engineering-2.png)

Escherichia coli is commonly used in laboratories due to its rapid growth and well-understood genetics and tools.
However, its use is limited in applications for animal or human consumption because it produces endotoxin of
lipopolysaccharides (LPS) on Gram-negative bacterial cell wall, which can trigger immune responses. This intrinsic
endotoxicity needs extensive purification to ensure product safety, thereby increasing production costs and complexity.

Pichia pastoris, a yeast recognized as generally regarded as safe (GRAS) by the U.S. Food and Drug Administration
(FDA), offers significant advantages for animal feed applications. It facilitates protein glycosylation and proper folding,
which are essential for the functional activity of enzymes7. Capable of growing in cost-effective media under high-density
conditions, it reduces overall production costs. Moreover, yeast like Pichia pastoris has potential probiotic effects,
enhancing gut health in animals8. Therefore, utilizing Pichia pastoris to express xylanase is an optimal choice for
developing effective and economical animal feed additives.

## Yeast Surface Display System: GCW61 vs. Pir1

The Yeast Surface Display System9 enables the direct use of yeast cells in products, avoiding the need for enzyme
purification. This system anchors enzymes to the yeast cell wall, simplifying production and reducing costs. In addition,
the system also ensures enzyme stability and activity for applications such as animal feed additives.

![](https://static.igem.wiki/teams/5049/mingdao24-engineering-3.png)

GCW6110, one of the glycosylphosphatidylinositol-modified cell wall proteins (GPI-CWPs) in Pichia pastoris, plays a
crucial role in maintaining normal morphology in yeast cells. This anchoring enable enzyme anchoring on the cell surface
and performing functionality throughout feed processing, ensuring consistent performance in agricultural applications.

Pir111, one of the Proteins with internal repeats (Pir) in Saccharomyces cerevisiae, another key anchor protein in
addition to GPI, increases the choices for target proteins to be displayed. It is renowned for forming strong covalent
bonds with cell wall components, enhancing enzyme durability under various conditions. This robust attachment keeps
enzymes active and intact, supporting sustained industrial processes.

In our project, we explored the application of two promising anchoring protein fusions, GCW61 and Pir1, with
xylanase in our yeast surface display system. Our goal was to evaluate their functionality and effectiveness for potential
use in animal feed additives, aiming to enhance the nutritional value and digestibility of animal feeds.

## Yeast Gene Driving Promoter: AXO1 vs. GTH1

AOX1 (Alcohol oxidase 1) promoter12 is widely used in Pichia pastoris for the production of recombinant proteins due
to its strong, inducible nature with methanol as the inducer. While effective in driving high levels of protein expression,
the use of methanol introduces complications including toxicity and flammability, which pose safety risks and complicate
the production process.

![](https://static.igem.wiki/teams/5049/mingdao24-engineering-4.png)

GTH1 (Glucose transporter high affinity 1) gene promoter13, a member of the GAP (glyceraldehyde-3-phosphate
dehydrogenase) promoter family, offers a more manageable and safer alternative. Although it functions as a constitutive
promoter, its activity is finely tuned by glucose levels—it is repressed by excess glycerol and fully induced by low glucose
concentrations, specifically at 0.05 g/L (0.005%). This unique regulation allows for precise control over protein
expression, making GTH1 promoter particularly suitable for our purpose and desire with high-level expression, without
the safety concerns associated with methanol.

## Overview of Evaluation Procedures for Yeast-Based Animal Feed Additives

![](https://static.igem.wiki/teams/5049/mingdao24-engineering-5.png)

We selected Pichia pastoris as our host organism to develop animal feed additives. In our laboratory setup, we
utilized the GTH1 promoter to drive the expression of xylanase fusion proteins, comparing the functionalities of two
anchor proteins, GCW61 and Pir1, within the yeast surface display system. The comparative analysis involved DNS assays
for measuring reducing sugars and employing 3D modeling techniques such as I-TASSER, YASARA with the FoldX plugin,
and PyMOL. After identifying the more effective anchor protein, we plan to further investigate xylanase gene expression
and determine enzyme activity under optimal temperature and pH conditions. In addition, to assess the system’s
applicability for animal consumption, we will evaluate its resistance to gastric proteases and analyze the stability of feed
processing and the shelf life of our yeast-based products.

# GCW61 SURFACE DISPLAY

## Design

![](https://static.igem.wiki/teams/5049/mingdao24-engineering-6.png)

The sequences of DNA elements for the GTH1 promoter and GCW61 anchor protein were from *Pichia pastoris*. To
ensure flexibility in connecting to target fusion proteins, a GS linker ((GGSG)3) was added at the N-terminus of GCW61.
We selected a thermostable xylanase from *Streptomyces thermovulgaris*6 as our protein of interest. All DNA fragments
were synthesized by Integrated DNA Technologies (IDT) following the standard iGEM Part Registry Rule (RFC10)14, which
includes prefix cutting sites EcoRI and XbaI, and suffix cutting sites SpeI and PstI. In the issue of the assembly of the
fusion protein, we followed the rules created by the Albert-Ludwigs Universität Freiburg iGEM team in 2007 (Freiburg
assembly method, officially named by iGEM HQs as RFC25)15
. AgeI cutting site was introduced at C-terminus of the
Xylanase gene without the stop codon, and NgoMIV and AgeI sites were introduced at the either end of the GS linker-
GCW61 segment.

To create the vector for gene expression in P. pastoris, we utilized the yeast vector pZAHR, developed by Professor
Hung-Jen Liu's lab at National Chung Hsing University. This vector is a Zeocin-selectable, AOX1-based Homologous
Recombination vector designed specifically for gene knock-in applications in Pichia pastoris. It incorporates the AOX1
gene promoter and terminator to facilitate the integration of desired genes into the Pichia pastoris chromosome through
homologous recombination. This process is typically executed following electroporation-directed yeast transformation, a
method routinely employed in the Liu's laboratory.

## Build

![](https://static.igem.wiki/teams/5049/mingdao24-engineering-7.png)

The basic parts were built from DNA elements on the pUCIDT-KAN vector of IDT to the iGEM part registry standard
pSB1C3 vector, and, in basic parts, designated GTH1 as a registry number of BBa\_K5049000, GS-GCW61 as
BBa\_K5049001 and Xylanase as BBa\_K5049003.

The Xylanase-GCW61 fusion protein was connected using the Freiburg assembly method (RFC25)15 to bypass the
stop codon of TAG generated by SpeI-XbaI BioBrick scar, developed by the Albert-Ludwigs Universität Freiburg iGEM
team in 2007. The composite part was assembled firstly as a registry name of Xylanase-GCW61 and the number of
BBa\_K5049004. Then, the final composite part was constructed with GTH1 promoter within the context of the following
sequence: EcoRI-XbaI-GTH1 promoter-(SpeI/XbaI scar)-Xylanase-(AgeI/NgoMIV scar)-GS linker-GCW61-AgeI-SpeI-PstI.
This functional composite part was given an iGEM part registry name of PGTH1-Xylanase-GCW61 and the number of
BBa\_K5049006.

To express the gene in Pichia pastoris, the composite part was cloned into the pZAHR vector to create the PGTH1-
Xylanase-GCW61/pZAHR construct. This construct was verified through colony PCR, using a primer pair targeting the 5'
end of GTH1 and the 3' end of GCW61, which resulted in an approximately 2100-bp DNA fragment (Figure 1A).
Additionally, the integrity of the extracted DNA plasmids was confirmed by digestion with restriction enzymes EcoRI and
PstI, yielding DNA fragments of 2070 bp for the insert and 3210 bp for the vector (Figure 1B). Furthermore, the gene
sequence of the insert was verified through DNA sequencing performed by Genomics BioSci & Tech. Co. Ltd. in Taiwan.

![](https://static.igem.wiki/teams/5049/mingdao24-engineering-8.png)

**Figure 1 | Verification of PGTH1-Xylanase-GCW61/pZAHR construct.**(A) Colony PCR using a GTH1-specific forward primer (5’-
CCCCAAACATTTGCTCCCCCTAG-3’) and a GCW61-specific reverse primer (5’-AATCAATAGAGCAACACCGGCTA-3’) yielded an expected 2070-bp DNA
fragment. The numbers indicate selected colonies, with lane 2 showing a control derived from a mock pick on a clear zone of the agar plate. (B)
Plasmids extracted from three successful colony PCR clones underwent a restriction enzyme analysis with EcoRI and PstI. The expected fragment
sizes are 2070 bp for the PGTH1-Xylanase-GCW61 insert and 3210 bp for the pZAHR vector. The first lane on the agarose gels features a 1kb DNA
marker (FluoroBand™ 1 KB (0.25-10 kb) Fluorescent DNA Ladder, SMOBIO Technology, Inc.).

## Test

![](https://static.igem.wiki/teams/5049/mingdao24-engineering-9.png)

The transformed Pichia pastoris carrying either the PGTH1-Xylanase-GCW61/pZAHR construct or the pZAHR vector
alone as a control were cultured in BMY media (Buffered Minimal YNB (Yeast Nitrogen Base)). To induce the GTH1
promoter, we added 0.005% (i.e., 0.05 g/L) glucose and incubated the cultures at 28°C for 48 hours to promote xylanase
expression. Simultaneously, control groups containing yeast with and without the Xylanase gene were cultured in BMY
media without glucose. After incubation, the yeasts were harvested by centrifugation and incubated in the 5% xylan
solution at 37°C for 15 minutes to produce xylose. The xylanase activities were measured by the 3,5-dinitrosalicylic acid
(DNS) assay. The amount of xylose produced was detected by the color change in the DNS reagent from yellow to
reddish-brown at 100°C for 15 minutes, which indicates the presence of reducing sugars. The xylanase activity
(Unit/mL/min), is calculated based on the quantity of xylose produced from xylan by the enzyme's catalytic action. For a
detailed protocol, including information on prepared buffers and solutions, please refer to our MEASUREMENT page.

In Figure 2, the data clearly demonstrate significant xylanase activity in Pichia pastoris carrying the Xylanase-GCW61
gene when induced with glucose. However, a low background level of enzyme activity was also detected in the absence
of glucose, which is attributed to the constitutive nature of the GTH1 promoter. In summary, the detected xylanase
activity in whole intact yeast cells implies that GCW61 is an effective anchor protein for displaying enzymes on the
surface of Pichia pastoris.

![](https://static.igem.wiki/teams/5049/mingdao24-engineering-10.png)

**Figure 2 | Xylanase-GCW61 Activity in Glucose-Induced and Non-Induced Pichia pastoris.** The yeasts were cultured in BMY media and maintained
without glucose as controls for non-induced groups (Yellow bars). For gene expression driven by the GTH1 promoter, transformed yeasts carrying
PGTH1-Xylanase-GCW61/pZAHR were induced with 0.005% glucose (right blue bar). After 48 hours at 28°C, the whole yeast cells were harvested and
subjected to reducing DNS assays using 5% xylan as the substrate. The intensity of the color change from yellow to brown, measured at OD540,
indicates the amount of xylose generated, reflecting xylanase activity. Additional details on enzyme activity calculations can be found on our
MEASUREMENT page. Controls carrying only the pZAHR vector were included for both induced (left blue bar) and non-induced conditions (left
yellow bar). Error bars represent the standard error from three independent experiments, demonstrating the reproducibility of the findings.

## Learn

We engineered Pichia pastoris to express thermostable Xylanase by integrating a genetic construct that included the
GTH1 promoter and GCW61 anchor protein. The construct, assembled into the pZAHR vector, was designed to enable
surface display of Xylanase for potential application in animal feed. Experimental results confirmed that the induced
expression under the presence of glucose significantly enhanced enzyme activity, while a baseline activity was observed
in non-induced conditions, due to the constitutive nature of the GTH1 promoter. This demonstrates the efficacy of the
GCW61 anchor protein in displaying functional enzymes on yeast cell surfaces, supporting its use in industrial
biotechnological applications.

# PIR1 SURFACE DISPLAY

## Design & Build

![](https://static.igem.wiki/teams/5049/mingdao24-engineering-11.png)

Pir1 is another anchor protein of Saccharomyces cerevisiae, widely utilized in yeast display systems. To explore its
potential compared to GCW61, we aimed to construct a Xylanase-Pir1 fusion protein. Using the same gene expression
strategy as with GCW61, the Pir1 sequence from Saccharomyces cerevisiae was synthesized by Integrated DNA
Technologies (IDT) with a GS linker at the N-terminus, flanked by EcoRI-XbaI-NgoMIV as a prefix and AgeI-SpeI-PstI as a
suffix. This basic part, GS-Pir1, was assembled into the pSB1C3 vector (BBa\_K5049002), and the composite part with
Xylanase gene, Xylanase-Pir1 (BBa\_K5049005), was generated. The final functional composite part of PGTH1-Xylanase-
Pir1/pSB1C3 was then created (BBa\_K5049007). To evaluate its performance in yeast and compare it with the GCW61
anchor, the PGTH1-Xylanase-Pir1 construct was cloned into the pZAHR vector. The construct was verified by colony PCR
(Figure 3) and confirmed by DNA sequencing.

![](https://static.igem.wiki/teams/5049/mingdao24-engineering-12.png)

**Figure 3 | Verification of PGTH1-Xylanase-Pir1** Colony PCR using a GTH1-specific forward primer (5’-CCCCAAACATTTGCTCCCCCTAG-3’) and a Pir1-specific reverse primer (5’-AGAAGTTAAAGTTGTGGCTTG-3’) yielded an expected 2346-bp DNA
fragment. The numbers indicate selected colonies, with lane 1 showing a 1kb DNA marker on 1% agarose gel in 0.5x TAE buffer (FluoroBand™ 1 KB
(0.25-10 kb) Fluorescent DNA Ladder, SMOBIO Technology, Inc.).

## Modeling

In order to display xylanase on the yeast surface, the choice of anchor protein is crucial for the beneficial
presentation of the target protein, which can determine the application and characteristics of the target protein. Yeast
anchor proteins have signal sequences that can guide the transportation of target proteins to the cell surface, thereby
anchoring them to the cell wall surface. Before comparing the effect of GCW61 and Pir1 anchor proteins on xylanase
activities, we want to understand the 3D structures of wild-type xylanase, xylanase-GCW61, and xylanase-Pir1, along
with their ligand binding, active sites, and protein stability features. For this purpose, I-TASSER, YASARA with the FoldX
plugin, and PyMOL were utilized as modeling tools. I-TASSER predicts the 3D coordinates of protein models based on
sequence-to-structure-to-function predictions. YASARA, enhanced with the FoldX plugin, provides detailed stability
analysis and predicts the effects of mutations on protein stability and interactions. PyMOL enables the visualization and
analysis of the predicted structures to assess structural changes and interaction sites. These analyses provide critical
insights into the functional impacts of the fusion proteins on enzymatic activity and stability. For details, please check our
MODELING page.

### **3D protein structure modeling tools and references:**

1. TASSER16,17,18 was utilized for predicting and scoring ligand binding site residues and active site residues. Each
   residue set includes a C-SCORE, which indicates the confidence of the predicted interactions and site specifications.
2. YASARA19/FoldX20 was used for calculating the stability of each variant, expressed in kcal/mol, which helps in
   understanding the structural integrity and potential functional efficiency of each variant under different conditions.
3. I-TASSER & PyMOL21 were employed for generating and visualizing the 3D structure images of each variant, allowing
   detailed observation of molecular architecture and potential functional sites.

### **Amino acid sequences of xylanase variants with anchor proteins**

![](https://static.igem.wiki/teams/5049/mingdao24-engineering-13.png)

## Xylanase-WT

![](https://static.igem.wiki/teams/5049/mingdao24-engineering-14.png)

## Xylanase-GCW61

![](https://static.igem.wiki/teams/5049/mingdao24-engineering-15.png)

## Xylanase-Pir1

![](https://static.igem.wiki/teams/5049/mingdao24-engineering-16.png)

### **Result of 3d protein structure modeling**

**Table 1 | Comparison of Xylanase Variants in Terms of Ligand Binding Sites, Active Sites, Stability, and 3D Structures**

![](https://static.igem.wiki/teams/5049/mingdao24-engineering-17.png)

\* Ligand binding site residues and active site residues predicted by I-TASSER with C-SCORE representing a confidence score for estimating the quality
of predicted models.

\*\* Stability in terms of free energy (kcal/mol) predicted by YASARA with FoldX plugin using models from I-TASSER

\*\*\* Protein 3D structure output generated by PyMOL using models from I-TASSER

![](https://static.igem.wiki/teams/5049/mingdao24-engineering-18.png)

**Figure 4 | (A) Xylanase (B) Xylanase-GCW61 (C) Xylanase-Pir1.**The 3D protein models were generated by I-TASSER and imported into PyMOL for
visualization. The models are colored by secondary structures: turquoise for alpha-helices, purple for beta-sheets, and pink for unstructured or
flexible loops. Sphere colors: blue for GS linkers, and red for either GCW61 or Pir1 anchor proteins. Glowing residues highlight: yellow for the
predicted active sites, and green for the original catalytic triad.

Xylanase from Streptomyces thermovulgaris has a conserved catalytic dyad consisting of two glutamate (Glu)
residues (E82-E171), similar to other xylanases22,23. We modeled wild-type xylanase (Xylanase-WT), Xylanase-GCW61,
and Xylanase-Pir1 using I-TASSER to predict their 3D structures. The modeling results and 3D simulated protein structure
were shown in Table 1 and Figure 4. The active site residues in Xylanase-WT matched published data22,23, with residues
82 and 171 showing high confidence scores (C-SCORE: 0.637). The stability of Xylanase-WT was calculated to be 182.1
kcal/mol.

For Xylanase-GCW61, the ligand binding sites were consistent with the wild-type, but the active site residues showed
a lower confidence score (C-SCORE: 0.459). The stability decreased to 351.7 kcal/mol. Similarly, Xylanase-Pir1 exhibited
different ligand binding sites and a lower confidence score for the active site residues (C-SCORE: 0.218), with the lowest
stability of 951.93 kcal/mol.

These findings indicate that while Xylanase-GCW61 and Xylanase-Pir1 maintain the essential active sites, their overall
stability is compromised compared to the wild-type. This suggests potential challenges in protein expression levels and
stability, which require further experimental validation.

### **Using Experimental Data to Validate and Develop the Xylanase-GCW61 Model**

To develop and validate our model, we used precise measurements of the xylanase-GCW61 enzyme and its mutants.
We created mutants of Xylanase-GCW61 (E82A and E171A) to address the predicted catalytic glutamate residues' role in
xylanase activity, with the assistance of Prof. Hung-Jen Liu at National Chung Hsing University. DNS assays were
conducted to quantify the enzyme activity, as detailed in our ENGINEERING and MEASUREMENT pages. The results
showed in Table 2 that the activity levels of Xylanase-GCW61 (E82A) and Xylanase-GCW61 (E171A) were below the
detection threshold, in contrast to the high activity of wild-type Xylanase-GCW61 at 106±14 Unit/mL/min. These
measurements were crucial for validating our model, as they confirmed the essential role of the catalytic dyad in
xylanase activity. This empirical data directly influenced the accuracy and reliability of our 3D structural models and
stability predictions, ensuring that our computational predictions aligned with observed enzymatic functions. These
findings are consistent with published research on xylanase engineering24,25, thereby reinforcing the robustness of our
model.

**Table 2 | Comparison of Xylanase Variants in Terms of Ligand Binding Sites,
Active Sites, Stability, and 3D Structures**

![](https://static.igem.wiki/teams/5049/mingdao24-engineering-19.png)

\*Note: N.D. indicates no detectable activity.

In our modeling, we utilized computational tools such as I-TASSER, YASARA with FoldX, and PyMOL to model the 3D
structures and assess the stability of xylanase variants from Streptomyces thermovulgaris. Xylanase-GCW61 and
Xylanase-Pir1 were modeled and compared against wild-type xylanase. Despite maintaining essential active sites, the
stability of the engineered variants was compromised. Experimental validation using DNS assays confirmed the critical
role of the catalytic dyad in xylanase activity. Our findings highlight the importance of selecting suitable anchor proteins
for functional enzyme display in yeast systems, while also addressing challenges related to protein stability.

## Test

In order to verify the activity of xylanase displayed on the yeast surface, we conducted a DNS assay to compare the
activities of Xylanase-GCW61 and Xylanase-Pir1. Following the same procedure as in previous experiments, glucose-
induced whole cells of Pichia pastoris harboring the respective plasmid DNAs were collected at 24-hour intervals over a
96-hour period. The cells were mixed with 5% xylan and incubated at 37°C for 15 minutes. Subsequently, DNS solution
was added, and the mixture was heated at 100°C for 15 minutes to halt the enzyme activities and facilitate color
development. After cooling the samples on ice for 5 minutes, the extent of color change was measured at OD540. The
results were converted to enzyme activity, expressed as Units/mL/min, consistent with our prior measurements.

As shown in Figure 5, the enzyme activities of Xylanase-GCW61 and Xylanase-Pir1 displayed on the surface of Pichia
pastoris exhibited distinct trends. Xylanase-GCW61 activity peaked at approximately 109 Units/mL/min after 48 hours
but rapidly declined over the subsequent two days. In contrast, Xylanase-Pir1 activity showed a gradual increase,
reaching around 91 Units/mL/min at 72 hours before slightly decreasing. And the activities of wild-type xylanase
(Xylanase-WT) without an anchor protein showed a background level of around 22-24 Units/mL/min, demonstrating the
effectiveness of using either GCW61 or Pir1 as anchor proteins in the yeast surface display system. Given our objective of
using yeast carriers as animal feed additives, where rapid protein induction and enzymatic activity are critical for cost
efficiency, we selected Xylanase-GCW61 for further experimentation. This upcoming study is designed to serve as a
proof-of-concept, demonstrating the practical application and effectiveness of this approach in real-world feed additive
scenarios.

![](https://static.igem.wiki/teams/5049/mingdao24-engineering-20.png)

Figure 5 | Comparison of Xylanase Activities in Yeast Surface Display System. This figure illustrates the xylanase activities of wild-type xylanase
(Xylanase-WT) without an anchor protein (yellow line), Xylanase-GCW61 (blue line), and Xylanase-Pir1 (red line) expressed in Pichia pastoris. The
yeast strains carrying these genes were cultured in BMY media and induced with 0.005% glucose (0.05g/L) for 96 hours, with samples collected at
24-hour intervals. Xylanase activity was determined using DNS assays, with methods and calculations as previously described. Error bars represent
the standard error from three independent experiments, demonstrating the reproducibility of the findings.

## Learn

Xylanase-Pir1 exhibited measurable enzymatic activity in our assays, despite 3D modeling data suggesting a less than
optimal ligand binding sites geometry and unfavorable free energy dynamics. This discrepancy underscores the critical
importance of using biochemical assays to validate computational models, as theoretical predictions can occasionally
diverge from observed enzyme functionality.

For Xylanase-GCW61, both structural modeling and kinetic assays yielded positive results, confirming an ideal active
site conformation and significant catalytic efficiency. In addition, the biochemical enzyme activity assays further
supporting these findings, reinforcing the observed high levels of enzyme functionality. These robust outcomes
enhanced our confidence as we proceed to the proof-of-concept study, strongly providing the potential of Xylanase-
GCW61 for effective real-world application in enzyme-mediated processes.

# PROOF OF CONCEPT

In order to create an effective animal feed additive, especially for pigs, we have demonstrated the activities of the
Xylanase-GCW61 fusion protein within the Pichia pastoris surface display system. Based on these promising results, the
following sections will further explore the feasibility of this application across a range of practical parameters. We aim to
evaluate the optimal temperature and pH levels for enzyme activity, ensuring functionality in the specific environments
of a pig's stomach and small intestine26. Additionally, we tested the enzyme's resistance to gastric proteases, including
pepsin and trypsin, to assess its durability and functionality within the animal's digestive system. For processing as
animal feeds27, we explored the temperature tolerances necessary for maintaining enzymatic activity. Following this, we
evaluated the shelf-life in a freeze-dried form28, which is crucial for ensuring the long-term viability and efficacy of the
enzyme when used in commercial feed additives. These comprehensive analyses are designed to provide a convincing
proof-of-concept for the application of Xylanase-GCW61, highlighting its practical viability and operational benefits.

![](https://static.igem.wiki/teams/5049/mingdao24-engineering-21.png)

## Xylanase Protein Expression

### Electroporation Transformation

We transformed Pichia pastoris GS115 with the linearized plasmid PGTH1-Xylanase-GCW61/pZAHR using
electroporation. The plasmid DNA was prepared by digesting 10 μg of the vector with SacI and dissolving it in sterile
water. This linear plasmid DNA was then added to competent cells and kept on ice before electroporation at 25 μF, 1.5
kV, and 0.5 msec using the ECM 830 Square Wave Mode in the BTX Gemini System. Following electroporation, cells were
recovered in 1 M sorbitol and incubated at 28°C for 2-4 hours. The cells were then plated on YPDS (Yeast Peptone
Dextrose Sorbitol) agar plates containing 2000 μg/ml Zeocin and incubated at 28°C for 3-5 days until colonies were
visible (Figure 6). This high concentration of Zeocin ensured the selection of successfully transformed colonies.

![](https://static.igem.wiki/teams/5049/mingdao24-engineering-22.png)

**Figure 6 | Electroporation Transformation of Pichia pastoris
GS115.**Selection of Pichia pastoris GS115 transformants carrying
the PGTH1-Xylanase-GCW61/pZAHR plasmid on YPDS agar plates
containing 2000 μg/ml Zeocin. Colonies were visible after 3-5 days
of incubation at 28°C (marked with red circles).

### Immunofluorescence Assay (IFA)

To confirm the expression of the Xylanase-GCW61 fusion protein, we performed an immunofluorescence assay.
Induced cultures of transformed Pichia pastoris were collected, and cells were fixed with formaldehyde on microscope
slides. Following fixation, the cells were blocked and incubated with a primary antibody (Anti-6×His) and a secondary
antibody (Goat anti-mouse IgG-FITC). The microscope slides were then washed and mounted for fluorescence
microscopy. The immunofluorescence images showed significant expression of the Xylanase-GCW61 fusion protein on
the cell surface after induction with 0.005% glucose for 24 hours (Figure 7). This confirmed the successful surface display
of xylanase in our yeast system.

![](https://static.igem.wiki/teams/5049/mingdao24-engineering-23.png)

**Figure 7 | Immunofluorescence Assay for Transformed P. pastoris Displaying
Xylanase-GCW61.**Immunofluorescence detection of Xylanase-GCW61 expression
in Pichia pastoris GS115 transformants. The images include bright field, fluorescent
field, and merged pictures for both control and xylanase-GCW61 expressing cells.
Cells were induced with 0.005% glucose for 24 hours and stained with Anti-6×His
primary antibody and Goat anti-mouse IgG-FITC secondary antibody.

### SDS-PAGE & Coomassie Blue Analysis

To further analyze the expression and molecular weight of the Xylanase-GCW61 fusion protein, we performed SDS-
PAGE. Transformed Pichia pastoris cells were lysed using glass beads and the protein extracts were prepared. These
samples were run on a 10% SDS-PAGE gel and stained with Coomassie Brilliant Blue. The expected size of the Xylanase-
GCW61 fusion protein is 37.75 kDa. The SDS-PAGE analysis revealed distinct bands corresponding to this molecular
weight, confirming the expression of the fusion protein in the transformed yeast strains (Figure 8). The intensity of these
bands indicated significant levels of protein expression, verifying the efficiency of our expression system.

![](https://static.igem.wiki/teams/5049/mingdao24-engineering-24.png)

**Figure 8 | SDS-PAGE analysis of xylanase-GCW61 expression in Pichia pastoris
GS115 transformants.**Lane M: protein marker; Lane 1: untransformed control;
Lanes 2-4: three different clones expressing Xylanase-GCW61. The expected size of
the Xylanase-GCW61 fusion protein is 37.75 kDa.

## Xylanase Stability and Optimal Temperature

As potent animal feeds using yeast cells carrying Xylanase on the surface, the products must tolerate the food
manufacturing process and endure variations in temperature and pH levels. After verifying the surface display and
protein expression of Xylanase, we want to investigate the enzyme's stability and determine the optimal temperature
and pH values for its activity.

For the temperature stability test (Figure 9, red line), Xylanase-displayed whole yeast cells were dissolved in 50 mM
Tris-HCl buffer (pH 8) and subjected to various temperatures ranging from 15°C to 85°C for 10 min. The untreated group
was set as the 100% enzyme activity baseline, measured by the DNS assay as previously described. The data indicated
that high activities were maintained up to 45°C, with over 90% activity. Between 55°C and 85°C, the enzyme activity
remained around 70% to 90%, suggesting that the yeast-displayed enzyme could endure both the body temperature of
livestock (38°C - 40°C)26 and the higher temperatures involved in animal feed processing (70°C - 85°C)27
.

To determine the optimal temperature for Xylanase activity, we performed DNS assays at various temperatures
ranging from 15°C to 85°C, instead of the standard DNS assays conducted at 37°C (Figure 9, blue line). The highest
thermovulgaris was found to be 65°C, consistent with findings by Boonchuay et al.5,6
activity, observed at 65°C, was set as 100%. The optimal temperature for Xylanase activity from Streptomyces. In addition, our analysis
demonstrated that Xylanase activity remained high, between 75% and 100%, across a temperature range of 35°C to
65°C. This suggests that the enzyme functions effectively at the pig's intestinal temperature of around 38°C to 40°C26.

![](https://static.igem.wiki/teams/5049/mingdao24-engineering-25.png)

**Figure 9 | Temperature Stability and Optimal Temperature of Xylanase Activity. Red line:**For stability testing, Xylanase-displayed yeast cells were
dissolved in 50 mM Tris-HCl buffer (pH 8) and subjected to various temperatures from 15°C to 85°C for 10 minutes before being measured by the
DNS assay at 37°C. The untreated group was set as the 100% enzyme activity baseline. **Blue line:** The optimal temperature was determined by
performing DNS assays at different temperatures as indicated, with the highest activity observed at 65°C set as 100% enzyme activity baseline. Error
bars represent the standard error from three independent experiments, demonstrating the reproducibility of the findings.

## Xylanase Stability and Optimal pH Level

Food animals experience pH variations26, including pH 3-4 in the stomach and pH 6-7.5 in the intestine. In addition to
temperature stability, we evaluated the stability and optimal pH for Xylanase activity to ensure its functionality under
varying pH levels encountered in the animal digestive system and during feed processing. To prepare the different pH
buffers for the following studies, please refer to our [MEASUREMENT](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27measurement%27%29%20%7D%7D) page.

For the pH stability test (Figure 10, red line), Xylanase-displayed whole yeast cells were dissolved in 50 mM sodium
phosphate buffer for pH 6-8 and 50 mM citric acid-sodium buffer for pH 3-5, then treated at 37°C for 10 minutes. The
treated lysates were centrifuged, and the supernatants were discarded. The resulting pellets were resuspended in 50
mM Tris-HCl buffer (pH 8), followed by DNS assays. The untreated group was set as the 100% enzyme activity baseline.
The data showed that the yeast cell-displayed Xylanase maintained over 50% activity across a pH range of 3 to 8, with the
highest activity at pH 5 (81.25%). Although some Xylanase activity was lost, the enzyme remained sufficiently active to be
effective in the stomach and intestine.

On the other hand, we tested the optimal pH level for yeast cell-displayed Xylanase by DNS assays at 37°C for 15
minutes at different pH levels in 50 mM sodium phosphate buffer for pH 6-8, and 50 mM citric acid-sodium buffer for pH
3-5 (Figure 10, blue line). The highest activity, observed at pH 5, was set as 100%. Although activities may reduce to less
than 50% under pH 4, over 80% of Xylanase activities were maintained between pH 5-7, suggesting its feasibility in the
pig intestine environment, which typically has a pH around 6-7.

![](https://static.igem.wiki/teams/5049/mingdao24-engineering-26.png)

**Figure 10 | pH Stability and Optimal pH level of Xylanase Activity.** Red line: Xylanase-displayed whole yeast cells were dissolved in 50 mM sodium
phosphate buffer for pH 6-8 and 50 mM citric acid-sodium buffer for pH 3-5, then treated at 37°C for 10 minutes. The treated lysates were
centrifuged, and the supernatants were discarded. The resulting pellets were resuspended in 50 mM Tris-HCl buffer (pH 8), followed by DNS assays.
The untreated group was set as the 100% enzyme activity baseline. **Blue line:** The optimal pH was determined by performing DNS assays at different
pH buffers ranging from pH 3-8 as indicated, with the highest activity observed at pH 5 set as 100% enzyme activity baseline. Error bars represent
the standard error from three independent experiments, demonstrating the reproducibility of the findings.

## Xylanase Resistance to Gastric Proteases

Pepsinogen is secreted by the stomach lining and is activated to pepsin in the acidic environment of the stomach.
Trypsinogen, secreted by the pancreas, is converted to trypsin in the small intestine, where it operates in a more alkaline
environment. Pepsin and trypsin are protease, which may break down the feeds supplemented with yeast-displayed
Xylanase29
.

Therefore, we treated whole yeast lysates containing Xylanase at 37°C for 2 hours in 50 mM citric acid-sodium buffer
(pH 2.0) with 0.5% pepsin and in 50 mM sodium phosphate buffer (pH 8.0) with 0.5% trypsin, respectively. The reactions
were then centrifuged, and the supernatants were removed. The resultant pellets were dissolved in Tris-HCl (pH 8.0) for
DNS assays. Mock controls were treated under the same conditions but without pepsin or trypsin, and the Xylanase
activities from these controls were set as the 100% baseline. As demonstrated in Figure 11, both pepsin and trypsin-
treated groups retained nearly 50% of enzyme activities, indicating the effectiveness of yeast-displayed Xylanase in the
physiological conditions of pig bodies.

![](https://static.igem.wiki/teams/5049/mingdao24-engineering-27.png)

**Figure 11 | Enzyme Activity of Yeast-Displayed Xylanase After Protease Treatment.** Whole yeast lysates containing Xylanase were treated at 37°C
for 2 hours in 50 mM citric acid-sodium buffer (pH 2.0) with 0.5% pepsin and in 50 mM sodium phosphate buffer (pH 8.0) with 0.5% trypsin,
respectively. The reactions were then centrifuged, and the supernatants were removed. The resultant pellets were dissolved in Tris-HCl (pH 8.0) for
DNS assays. Mock controls were treated under the same conditions but without pepsin or trypsin, and the Xylanase activities from these controls
were set as the 100% baseline. Error bars represent the standard error from three independent experiments, demonstrating the reproducibility of
the findings.

## Freeze-Dried Xylanase Shelf Life

To enhance the preservation of the enzyme for commercial applications, we aimed to freeze-dry *β*-xylanase and
store it at room temperature for extended periods. We consulted Prof. Hung-Jen Liu at National Chung Hsing University
for guidance on this issue. With his lab’s help, we concentrated the culture broth containing yeast-displayed Xylanase
tenfold, freeze-dried it, and stored it at 37°C for various durations. The enzyme was rehydrated with sterile distilled water
and subjected to DNS assays. The activity of the untreated culture broth was set as the 100% baseline. The relative
activity was plotted over time. Figure 12 showed that the Xylanase expressed by *Pichia pastoris* GS115 carrying Xylanase-
GCW61 retained over 80% activity after 16 weeks at 37°C, demonstrating consistent trends. This result suggests that our
production method yields a stable product with significant potential for marketing.

![](https://static.igem.wiki/teams/5049/mingdao24-engineering-28.png)

**Figure 12 | Long-Term Stability of Freeze-Dried Xylanase.** The 10-fold concentrated yeast broth containing Xylanase was freeze-dried into powders.
The powders were incubated at 37°C for 2, 4, 6, 8, and 16 weeks, then resuspended in sterile ddH2O prior to DNS assays. The control was the yeast-
displayed Xylanase lysates without freeze-dry treatment, with enzyme activity from the control set as the 100% baseline. Error bars represent the
standard error from three independent experiments, demonstrating the reproducibility of the findings.

# CONCLUSION

Our project, aimed at improving animal feed digestibility and nutritional efficiency, utilizes Pichia pastoris GS115 to
surface-display thermostable xylanase from Streptomyces thermovulgaris. This approach targets the Sustainable
Development Goals (SDGs) 2 (Zero Hunger) and 12 (Responsible Consumption and Production) by enhancing the
breakdown of complex carbohydrates in feed. We systematically followed the engineering design cycle (Design → Build
→ Test → Learn) to develop a stable and efficient method for enzyme production.

The yeast surface display system compared two anchor proteins, GCW61 and Pir1, with the GTH1 promoter driving
xylanase expression. 3D modeling tools (I-TASSER, YASARA with FoldX, and PyMOL) and DNS assays validated the
functionality and stability of the xylanase variants. Experimental results showed significant xylanase activity and stability,
particularly with the GCW61 anchor protein.

Further testing confirmed the enzyme's stability under various temperature and pH conditions, essential for its
functionality in the animal digestive system and during feed processing. Resistance to gastric proteases (pepsin and
trypsin) and long-term stability in a freeze-dried form demonstrated the enzyme's potential for commercial applications.

Our project successfully demonstrated the engineering success and practical viability of using Pichia pastoris GS115
to surface-display thermostable xylanase for enhancing animal feed digestibility. The findings highlight the potential of
this method to improve livestock feed efficiency safely and cost-effectively, aligning with the goals of sustainable
agriculture and responsible consumption. The robustness of our approach, validated through comprehensive
experimental and modeling analyses, supports its application in real-world feed additive scenarios.

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15. iGEM Registry of Standard Biological Parts: Assembly standard 25
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16. Wei Zheng, Chengxin Zhang, Yang Li, Robin Pearce, Eric W. Bell, Yang Zhang. Folding non-homology
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    Applications. Catalysts. 2023; 13(11):1405. doi:10.3390/catal13111405
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    10.3389/fmicb.2020.00545. PMID: 32390953; PMCID: PMC7193231.
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25. Ko EP, Akatsuka H, Moriyama H, Shinmyo A, Hata Y, Katsube Y, Urabe I, Okada H. Site-directed mutagenesis at
    aspartate and glutamate residues of xylanase from Bacillus pumilus. Biochem J. 1992 Nov 15;288 ( Pt 1)(Pt
    1):117-21. doi: 10.1042/bj2880117. PMID: 1359880; PMCID: PMC1132087.
26. Jan Bureš, Věra Radochová, Jaroslav Květina, Darina Kohoutová, Martin Vališ, Stanislav Rejchrt, Jana Žďárová
    Karasová, Ondřej Soukup, Štěpán Suchánek, Miroslav Zavoral. Wireless Monitoring of Gastrointestinal Transit
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27. Kiarie EG, Mills A. Role of Feed Processing on Gut Health and Function in Pigs and Poultry: Conundrum of
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    doi: 10.1017/S0022029910000610. Epub 2010 Sep 8. PMID: 20822567.
29. Low AG. The activity of pepsin, chymotrypsin and trypsin during 24 h periods in the small intestine of growing
    pigs. Br J Nutr. 1982 Jul;48(1):147-59. doi: 10.1079/bjn19820097. PMID: 6809038.

{% endblock %}

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

## What should this page contain?

---

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

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

## Inspirations

---

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

{% endblock %}

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

## What should this page contain?

---

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

## Describe what your results mean

---

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

## Project Achievements

---

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

## Inspirations

---

* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)

{% endblock %}