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P_GTH1-Xylanase-GCW61

VECTOR DESIGN

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 thermovulgaris6 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.

CONSTRUCTION

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.

Figure 1 | Verification of P_GTH1-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.).

FUNCTIONAL ASSAY

The transformed Pichia pastoris carrying either the P_GTH1-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 MEASUREMENTpage.

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.

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.

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.

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.

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.

Figure 3 | 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 4). This confirmed the successful surface display of xylanase in our yeast system.

Figure 4 | 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 5). The intensity of these bands indicated significant levels of protein expression, verifying the efficiency of our expression system.

Figure 5 | 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) and the higher temperatures involved in animal feed processing (70°C - 85°C).

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 6, blue line). The highest activity, observed at 65°C, was set as 100%. The optimal temperature for Xylanase activity from Streptomyces thermovulgaris was found to be 65°C, consistent with findings by Boonchuay et al.5,6. 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°C.

Figure 6 | 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 variations, 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 MEASUREMENTpage.

For the pH stability test (Figure 7, 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 7, 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.

Figure 7 | 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 Xylanase.

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 8, 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.

Figure 8 | 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.

Sequence and Features