iGEM 2025 — Team links-china

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# Protocol

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* [Background](#Background)
* [Engineering Cycle 1](#Cycle1)
* [Engineering Cycle 2](#Cycle2)
* [Engineering Cycle 3](#Cycle3)
* [Engineering Cycle 4](#Cycle4)
* [Engineering Cycle 5](#Cycle5)
* [Engineering Cycle 6](#Cycle6)
* [Conclusion](#Conclusion)
* [Reference](#Reference)

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# Background

Annually, 5.3 billion tons of crop straw are generated worldwide, with production steadily increasing due to growing crop demand driven by population expansion (Bhatia et al., 2017). To fully leverage the economic potential of this agricultural residue, LINKS-China 2025 iGEM Team has developed an innovative approach to transform straw into high-performance textile fibers, promoting the efficient utilization of straw resources.

![](https://static.igem.wiki/teams/5714/wiki/engineering-success/figure1.webp)

Figure 1. Manufacturing process of regenerated cellulose fibers (A). These fibers——Viscose, Lyocell, Cupro—— are typically sourced from hardwood and produced through cellulose extraction, dissolution, and wet-spinning to form fibers and yarns. (B) Composition of straw. After removal of impurities, straw serves as an excellent source of cellulose, comparable to hardwood (Smook et al., 2016).
As shown in Figure B, the main components of straw are cellulose, hemicellulose, and lignin. Meanwhile, after impurity removal, straw can serve as a high-quality cellulose raw material for spinning.

As shown in Figure B, the main components of straw are cellulose, hemicellulose, and lignin. Meanwhile, after impurity removal, straw can serve as a high-quality cellulose raw material for spinning.

![](https://static.igem.wiki/teams/5714/wiki/engineering-success/table1.webp)

Table 1：Cellulose content of different feedstocks

Among feedstocks used for solution spinning (Lyocell, viscose, ionic liquids, etc.), a very high α-cellulose content is required (usually ≥90%; excellent feedstock for high-performance fibres can reach 95–99%) and a relatively high degree of polymerisation (Lawson et al., 2023). According to Table 1, wood has the highest α-cellulose, but bamboo and corn cob are not low. Bamboo fibre is already used in commercial spinning, and corn cob has significant potential. At the same time, hemicellulose and lignin are also important factors affecting spinning performance; maize contains less hemicellulose and lignin than wood, so using alkali and other techniques to remove hemicellulose and lignin should theoretically be more effective. Therefore, maize straw was selected in this project to explore the feasibility of straw cellulose spinning.

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# Engineering Cycle 1

## Design

Due to its high α-cellulose content and low lignin levels, we selected corn stovers to explore its potential for conversion into textile fabrics. We initially employed a rapid processing method involving cooking the corn stover powder with sodium hydroxide to remove lignin and hemicellulose.

## Build/Test

First, to conduct spinning experiments with maize straw as raw material, its powder must undergo cellulose purification, Figure 2 B: sequentially perform alkali boiling, water boiling, and sodium hypochlorite bleaching, then dry, and finally dissolve and proceed to wet spinning. During alkali boiling, NaOH can hydrolyse the ester and ether bonds between lignin and hemicellulose, leading to dissolution and removal of lignin and hemicellulose, and it can also disrupt the aromatic structure of lignin: NaOH treatment can break aromatic structures in lignin, causing its cleavage and dissolution (Jin et al., 2019).

![](https://static.igem.wiki/teams/5714/wiki/engineering-success/figure2.webp)

Figure 2. Cellulose Extraction from Straw.(A) Schematic of cellulose purification from straw powder through chemical and enzymatic methods. (B) Procedure for obtaining cellulose powder: straw powder is treated with NaOH boiling, water boiling, NaClO bleaching, and drying. (C) The cellulose obtained is dissolved in NMMO solvent and processed via wet-spinning; however, the resulting filaments are dry, brittle, and prone to breakage. Uniform fine fibers can only be spun after blending this cellulose with 50% wood cellulose.

In the purification process, straw powder was placed in 5% NaOH solution at a solid-to-liquid ratio of 1:8 (e.g., 400 g cellulose added to 3200 mL 5% NaOH), boiled for 1 h with an induction cooker; then filtered with gauze and repeatedly rinsed with water. The solid residue was subsequently treated with 6% NaClO solution at room temperature for 3 h, filtered again, and repeatedly rinsed with water until neutral pH was achieved. The purified material was finally dried to obtain cellulose powder.

Subsequently, the cellulose dissolution system was prepared: for every 7.5 g cellulose, 120 g NMMO was added to make a 5% (w/w) solution, then 0.128 g propyl gallate was added as an antioxidant; the mixture was heated by a water bath using a round bottom flask until cellulose was completely dissolved and the solution appeared dark brown.

Next, fibre spinning was carried out: as shown in Figure 1A, the spinning dope was extruded through a heated tube to reduce viscosity and improve rheological properties; the nascent fibre entered a coagulation bath to solidify and was drawn by a roller at the end of the device at 100 rpm. The obtained filament was then washed in 65 °C warm water to remove residual NMMO on the fibre surface.

Pure straw-cellulose fibre (designated as V0) appeared dark brown, was hard and brittle, and broke easily, completely failing to meet apparel-fibre requirements. We then implemented our V1 formulation using 50% wood cellulose blended with 50% straw cellulose for spinning. As shown in Figure 2D, the resulting fibre exhibited a significantly lighter colour and noticeably improved mechanical properties, achieving a strength of 0.60 ± 0.07 cN/dtex and a breaking elongation of 4.51 ± 0.93%.

## Learn

Collectively, the above data demonstrate that although we successfully spun corn straw into fibers, the resulting material lacks the mechanical strength required for apparel applications. To transform straw into textile-grade fibers, its tensile strength must be enhanced to meet or exceed industry standards (≥2 cN/dtex)(Jiang et al., 2020). Blending straw cellulose with wood cellulose presents a viable strategy for improving the mechanical properties of the regenerated fibers, offering a promising direction for further technical optimization.

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# Engineering Cycle 2

## Design

To develop straw-based fibers meeting the textile strength standard of 2 cN/dtex, we drew inspiration from the LINKS-China 2021 iGEM project (https://2021.igem.org/Team:LINKS\_China/Engineering) and previous research on spider silk-enhanced cellulose fibers (Pezhman et al., 2019). Inspired by LINKS-China 2021's approach of enhancing bacterial cellulose using spider silk fibroin, we evaluated various high-performance proteins—including squid beak protein, octopus sucker ring teeth protein, and springtail elastin. We were particularly drawn to squid beak-derived histidine-rich proteins (HBP) due to their natural chitin-binding domains that enable rigid structure formation through cross-linking with chitin (Tan et al., 2015), suggesting potential for cellulose reinforcement when modified with cellulose-binding modules. Additionally, we selected bagworm silk fibroin (EV1) inspired by the larva's ability to construct sturdy shelters from plant debris, with reported performance surpassing spider silk (Yoshioka et al., 2019).

![](https://static.igem.wiki/teams/5714/wiki/engineering-success/engineering-fig31.avif)

Figure 3. A Structural Protein-Based Approach for Cellulose Reinforcement. (A) Schematic representation showing structural proteins fused with carbohydrate-binding modules at both termini, enabling cellulose binding and enhancing its mechanical toughness. (B) The two structural proteins selected for this study: HBP derived from squid beak and EV1 sourced from bagworm silk. (C, D) AlphaFold-predicted structures of HBP (C) and EV1 (D). (E) Protein expression using E. coli BL21 (DE3) strain and pET28a plasmid. (F-H) Protein expression and purification profiles: M, protein molecular weight marker; S, supernatant after cell lysis; P, pellet; FT, flow-through; W, wash; E1-E6, elution fractions collected with increasing imidazole concentrations.

We innovatively engineered these proteins by fusing CBM3 domains at both termini, creating CBM3-HBP-CBM3 and CBM3-EV1-CBM3 (hereafter referred to as HBP and EV1), as illustrated in Figure 3A. These constructs are designed to cross-link with cellulose fibers and enhance mechanical strength. To evaluate this strategy, we established a 50:50 straw/wood cellulose system (V1) and incorporated the expressed HBP and EV1 proteins to assess the feasibility of producing industry-standard regenerated fibers.

## Build

We first employed AlphaFold3 to predict the structures of the CBM3-fused HBP and EV1 proteins. The results showed that both the dual-CBM3-HBP and dual-CBM3-EV1 fusion proteins maintained structural conformations consistent with their native forms (Figure 3C). Using the pET28a-CBM3-2REP-CBM3 vector backbone provided by the LINKS 2021 iGEM Team, we synthesized the E. coli expression vectors pET28a-CBM3-HBP1-CBM3 and pET28a-CBM3-EV1-CBM3 through Atantares in Suzhou, China (Figure 3E). These vectors were subsequently transformed into E. coli BL21(DE3) to generate expression strains.

## Test

After constructing the high-performance protein expression strains, we performed fermentation in 400 mL of LB medium. Upon induction with 0.3 mM IPTG, expression of both CBM3 fusion proteins was observed after 16 hours. As shown in Figure 3F, the two CBM3-fused proteins were abundantly present in the cell lysate supernatant, indicating their high solubility. We then performed purification using Ni-NTA affinity chromatography, with the results presented in Figures 3G and 3H. Following protein dialysis, we quantified the protein concentration using a BCA assay kit, which indicated a yield of 373.3 mg/L for the EV1-CBM3 protein and 607.5 mg/L for the HBP-CBM3 protein. The proteins were subsequently lyophilized, ultimately obtaining 154 mg and 260 mg of the respective proteins in lyophilized form.

We subsequently evaluated whether the incorporation of CBM3-fused protein could enhance the performance of straw-based fibers. Using our V1 system (50:50 straw-to-wood pulp cellulose), we added HBP-CBM3 and EV1-CBM3-fused proteins at a loading of 5% by mass relative to cellulose (i.e., 5 g protein per 100 g cellulose), respectively, followed by dissolution in NMMO and wet spinning using the V1 formulation (Figure 4 D, E, F). As summarized in Table 2, the addition of HBP improved the fiber strength by approximately 1.2-fold, though the resulting strength still fell short of textile requirements (≥2 cN/dtex). In contrast, EV1 incorporation did not yield significant enhancement, which is likely attributed to the notably larger fiber diameter compared to the control. This outcome is speculated to be due to insufficient optimization of the spinning process, particularly the lack of adequate drawing treatment

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Table 2：Physical performances of 1st version straw fibers

## Learn

The addition of 5% HBP to the V1 system significantly enhanced the mechanical properties of the straw-based fibers. However, the resulting strength still failed to meet textile requirements (≥2 cN/dtex). Due to limited protein yield and considering the costs associated with wood cellulose procurement and protein production, we did not further increase the protein proportion. Given our preliminary findings in Cycle 1 that increasing the wood cellulose content improved the spinnability of straw fibers, we plan to explore whether further elevating the proportion of wood cellulose can help achieve the required 2 cN/dtex strength standard.

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# Engineering Cycle 3

## Design

Since adding high-performance proteins alone was insufficient to meet the 2 cN/dtex requirement, we sought to achieve this target by further reducing the straw proportion. This decision was supported by microscopic examination (Figure 4 B, C), which revealed that straw fibers exhibited larger diameters, rough surfaces, and fractured morphology, whereas wood fibers showed smaller diameters with smooth, continuous surfaces. Consequently, we increased the wood cellulose content from 50% to 70% and defined this new formulation as V2.

![](https://static.igem.wiki/teams/5714/wiki/engineering-success/engineering-fig41.avif)

Figure 4. Hybrid Fibers of Straw Cellulose and Wood Cellulose.(A) Failed spinning attempt of pure straw fibers, which were brittle, rigid, and fragile.(B, C) Microscopic structures of pure straw cellulose fibers (B) and wood cellulose fibers (C) under microscope. (D-F) Photographs of fibers composed of 50% straw cellulose and 50% wood cellulose: (D) baseline blend, (E) supplemented with 5% HBP, (F) supplemented with 5% EV1. (G-I) Photographs of fibers composed of 30% straw cellulose and 70% wood cellulose: (G) baseline blend, (H) supplemented with 5% HBP, (I) supplemented with 5% EV1.

## Build/Test

We conducted spinning using the V2 system with the incorporation of two types of CBM3-fused proteins (Figure 4 G, H, I). As shown in Table 3, after increasing the wood cellulose proportion, the V3 formulation demonstrated improved performance compared to V2 across all parameters. The fiber strength reached 1.59 cN/dtex with HBP addition and 1.79 cN/dtex with EV1 incorporation. Notably, the EV1-enhanced fiber strength has approached the target value of 2 cN/dtex.

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Table 3：Physical performances of 2nd version straw fibers

## Learn

After increasing the wood cellulose ratio to 70%, the EV1-enhanced fiber strength approached the target value remarkably close. However, further increasing the wood cellulose content would contradict our original intent of primarily utilizing straw rather than creating a partial substitution, which would still yield limited improvement in straw's added value. This limitation may stem from defects in our current straw cellulose extraction method, where excessive residual lignin potentially compromises fiber performance. Thus, to further optimize the process, implementing repeated alkaline and aqueous boiling treatments appears necessary.

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# Engineering Cycle 4

## Design

To achieve the goal of spinning pure straw-based cellulose fibers and fully enhancing the value-added potential of straw, we further improved the purity of cellulose extracted from straw by repeating the NaOH boiling-water boiling-NaClO bleaching cycle four times. Although this process leads to a higher loss of straw mass, it remains more economically advantageous than using straw merely as a partial substitute for wood cellulose.

## Build/Test

We designated the protocol involving four cycles of treatment followed by spinning with 100% straw cellulose as the V3 process. As shown in Figure 5B, the straw subjected to the V3 treatment became noticeably whiter and closely resembled wood cellulose in appearance (Figure 5A), indicating significantly higher purity compared to the single-treatment V0 process. We then incorporated two types of CBM3-fused proteins into the V3 system, dissolved the mixture in NMMO, and proceeded with fiber spinning. The resulting fibers (shown in Figures 5C, F, and G) still exhibited a yellowish tint compared to wood cellulose fibers (Figures 5D).

![](https://static.igem.wiki/teams/5714/wiki/engineering-success/engineering-fig51.avif)

Figure 5. Purification of Straw Fibers through Multi-stage Chemical Treatment. (A) Relatively pure cellulose powder was obtained after four cycles of NaOH boiling and water boiling. (B) Photos of cellulose at different purification stages, showing the final product as whitish and clean, similar in appearance to wood pulp cellulose (E). (C) V3 fiber spun from the 100% purified straw cellulose. (D) Fiber spun from 100% wood cellulose. (F) Fiber spun from V3 straw cellulose supplemented with 5% HBP. (G) Fiber spun from V3 straw cellulose supplemented with 5% EV1.

We subsequently measured the mechanical strength of the V3 fibers, with the results summarized in Table 4. All fiber formulations demonstrated further improvement in performance. Notably, the EV1-enhanced fibers achieved a breakthrough strength of 2.56 cN/dtex, exceeding the 2 cN/dtex threshold and matching the performance of conventional viscose fibers(Jiang et al., 2020), thus confirming their suitability for textile applications.

![](https://static.igem.wiki/teams/5714/wiki/engineering-success/table4.webp)

Table 4：Physical performances of 3rd version straw fibers

## Learn

After purifying corn straw cellulose through four rounds of treatment, we successfully achieved fiber spinning using 100% straw cellulose. With the addition of 5% EV1 protein, the resulting fiber strength exceeded 2 cN/dtex, reaching 2.56 cN/dtex—comparable to conventional viscose fibers.

However, we identified two notable observations. First, the incorporation of EV1 deepened the fiber color (Figure 5G), likely due to the intrinsic hue of the protein itself, which may pose challenges for subsequent dyeing processes. To explore this characteristic, we consulted sustainable material designer Ms. Julie [（Human Practices）](https://2025.igem.wiki/links-china/human-practices) , who suggested that this natural, straw-like color could be perceived as an aesthetic advantage by certain consumers who appreciate bio-based material qualities. Additionally, we experimented with indigo dyeing using a fermentation-based indigo production system developed by LINKS-China 2021 [（Implementation）](https://2025.igem.wiki/links-china/implementation), successfully achieving shibori-style dyeing effects on our straw-based fibers.

However, our repeated processing method consumes substantial amounts of water. Taking the treatment of 400g of straw as an example, each cycle requires 38 L of water, 640 g of NaOH, and 768 g of sodium hypochlorite, along with significant electricity consumption for boiling. Although industrial water, electricity, alkali, and sodium hypochlorite are relatively inexpensive in China, this approach is neither energy-efficient nor environmentally friendly. Moreover, it runs counter to our original intention of sustainable straw utilization. Therefore, we are actively exploring greener and more environmentally sustainable methods for purifying straw cellulose.

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# Engineering Cycle 5

## Design

Building upon the success of the chemical treatment method that significantly improved cellulose purity and fiber performance, we sought to address its environmental drawbacks, including high consumption of water and chemicals. To establish a more sustainable and environmentally friendly pretreatment process, we focused on developing a biological alternative leveraging the synergism of lignin-degrading enzymes. We selected two lignin-degrading enzymes, Laccase (Lac) lcc1 from Coriolopsis trogii and Versatile Peroxidase (VP) vpl2 from Pleurotus eryngii, along with an auxiliary enzyme, Lytic Polysaccharide Monooxygenase (LPMO) gh61-3 from Neurospora crassa. Their synergistic cooperation is designed to enhance residual lignin degradation efficiency: Laccase oxidizes phenolic subunits in lignin, generating radical intermediates that react with oxygen to produce hydrogen peroxide, which serves as the essential co-substrate for VP to cleave more recalcitrant lignin bonds. Simultaneously, LPMO acts as an electron sink for Lac and VP reactions and is activated by hydrogen peroxide to cleave hemicellulose and disrupt lignin-carbohydrate complexes(Ye et al., 2024). This coordinated action promotes thorough lignin removal and aids hemicellulose degradation, thereby purifying straw cellulose for spinning. Since these enzymes are prokaryotic-derived, we employed Pichia pastoris for secretory expression. To further enhance synergy, we engineered a synthetic multi-enzyme complex inspired by natural cellulosomes, utilizing a mini-scaffold protein containing three distinct cohesin domains from Acetivibrio thermocellus CipA, Clostridium cellulolyticum ScaB, and Clostridium cellulolyticum CipC, displayed on Saccharomyces cerevisiae surface. The enzymes, each fused to a specific dockerin domain, were secreted from P. pastoris and assembled onto the yeast-surface scaffold via mutually exclusive cohesin-dockerin interactions. Specifically, Lac was fused to a dockerin from Ruminiclostridium cellulolyticum for specific binding to the CipCcoh2 cohesin domain; VP was fused to a dockerin from Ruminococcus flavefaciens for binding to the ScaBcoh4 cohesin domain; and LPMO was fused to a dockerin from Acetivibrio thermocellus for binding to the CipAcoh2 cohesin domain(Ding et al., 2001, You et al., 2012, Pinheiro et al., 2008). This spatial co-localization creates a concentrated catalytic platform to maximize synergistic effects and minimize enzyme loss (Tian et al., 2019)

![](https://static.igem.wiki/teams/5714/wiki/engineering-success/figure6.webp)

Figure 6. Enzymatic Purification of Cellulose from Straw.(A) Schematic of cellulose purification through enzymatic decomposition of straw. (B) An effective enzyme combination includes Laccase (Lac), Versatile Peroxidase (VP), and Lytic Polysaccharide Monooxygenase (LPMO). Specifically, laccase degrades lignin while generating hydrogen peroxide; VP simultaneously cleaves lignin by consuming hydrogen peroxide to produce radical electrons; and LPMO facilitates electron transfer between the two enzymes while additionally degrading hemicellulose.

## Build

The plasmids for the three dockerin-fused enzymes (Lac, VP, LPMO) and the mini-scaffold were constructed using E. coli DH5α. For each enzyme, the gene sequences were cloned into P. pastoris expression vectors under the control of the AOX1 promoter with an α-factor signal peptide. The plasmids were linearized and transformed through electrotransformation into P. pastoris GS115. We then implemented an antibiotic-independent, phenotype-based screening strategy to identify high-expression clones (See Measurement for development and validation of this method). Transformants were selected on MM minimal medium plates where methanol replaced glucose as the carbon source, enabling direct induction of expression. The screening medium was supplemented with enzyme-specific cofactors and chromogenic substrates: for Lac screening, plates contained 0.5 mM ABTS and 1 mg/mL CuSO₄; for LPMO, 2.4 µM 2,6-DMP, 1 mg/mL CuSO₄, and 3 mM H₂O₂; and for VP, 2.4 µM 2,6-DMP, 1 mg/mL MnSO₄, 1 mg/mL CaCl₂, 3 mM H₂O₂, and 100 µg/mL hemin. To maintain induction, 1 mL of methanol was added daily to each plate. After two days of incubation, we successfully identified high-expression clones of Lac and LPMO by assessing color development intensity, though VP screening was compromised due to hemin-induced background coloration. Positive clones were further verified by colony PCR. For the mini-scaffold, the construct was transformed into S. cerevisiae EBY100, with transformants selected on SD/-Trp medium and expression induced in SG-CAA medium. For the mini-scaffold, the gene was cloned into a S. cerevisiae surface display vector and transformed into EBY100 competent cells. Transformants were selected on SD/-Trp medium, and scaffold expression was induced in SG-CAA medium. Protein expression was verified through SDS-PAGE and Western blot.

## Test

To test protein expression, the transformed P. pastoris strains were cultured in a stepwise fermentation process. Selected clones were first grown in YPD medium overnight, then transferred to BMGY medium for 14-16 hours until the OD₆₀₀ reached 2-6. The cells were harvested by centrifugation and resuspended in BMMY induction medium containing 0.5% methanol. The cultures were maintained at 30°C with shaking for 120 hours, with additional methanol added every 24 hours to maintain a final concentration of 0.5%. Culture samples were collected at 24-hour intervals, and the supernatants were analyzed by SDS-PAGE. The results showed only very faint bands at the expected molecular weights for all three enzymes, indicating extremely low secretion levels into the culture medium (Figure 7C).

Meanwhile, analysis of S. cerevisiae lysates expressing the mini-scaffold confirmed successful protein expression. We used NetNGlyc 1.0 to predict N-glycosylation sites on the mini-scaffold and identified seven potential modification sites. Given that S. cerevisiae typically mediates substantial glycosylation, SDS-PAGE alone could not accurately determine the protein's molecular size. Therefore, after lysing the mini-scaffold expression strains with 5× SDS loading buffer, we utilized the V5 tag fused to the mini-scaffold and performed Western blot analysis, which clearly detected the target protein expression (Figure 7D).

![](https://static.igem.wiki/teams/5714/wiki/engineering-success/engineering-fig71.avif)

Figure 7. Expression of Three Enzymes in Pichia pastoris. (A) The three enzymes were secreted extracellularly via the α-mating factor secretion signal and fused with distinct dockerin domains. (B) These dockerin domains specifically interact with cohesin modules displayed on mini-scaffold proteins expressed by engineered Saccharomyces cerevisiae, forming stable enzyme complexes.(C) SDS-PAGE analysis of yeast fermentation supernatant confirmed protein expression, though the bands were barely detectable.(D) Expression of the mini-scaffold proteins was verified by both SDS-PAGE and Western blot analysis.

## Learn

We successfully confirmed the expression of the mini-scaffold in S. cerevisiae. However, the extremely weak bands on the SDS-PAGE gel indicated that the secretion expression of all three enzymes in P. pastoris was insufficient using the original α-factor signal peptide. This low yield of enzymes would severely limit the assembly efficiency and catalytic performance of our designed multi-enzyme complex, thus enhancing the secretion efficiency of the enzymes was the critical next step. We decided to systematically optimize the signal peptides to address this bottleneck.

![](https://static.igem.wiki/teams/5714/wiki/section-heading.webp)

# Engineering Cycle 6

## Design

To improve the secretion efficiency of the enzymes from P. pastoris, we constructed a library of eight different signal peptides to replace the original α-factor signal peptide (Figure 8A), including MF4I, α-factor Δ57–70, α-factor Δ57–70 Plus HL28, nSB, Dse4, Msb2, Gas1, and gh61-3 sp. These signal peptides were selected based on their documented abilities to enhance secretory expression of heterologous proteins in yeast systems (Zheng et al., 2024, Rieder et al., 2021). To enable definitive verification of the multi-enzyme complex assembly on the yeast surface—a critical step that could not be confirmed in the previous cycle—we incorporated specific epitope tags into our design. The mini-scaffold protein was engineered with a C-terminal V5-tag, while each of the three dockerin-fused enzymes (Lac, VP, LPMO) was designed with a C-terminal His-tag. This strategic tagging allowed for the subsequent use of immunofluorescence staining with specific antibodies to simultaneously detect the surface-displayed scaffold (via anti-V5) and the bound enzymes (via anti-His), thereby providing direct visual confirmation of successful complex formation.

![](https://static.igem.wiki/teams/5714/wiki/engineering-success/engineering-fig81.avif)

Figure 8. Enhanced Enzyme Secretion through Signal Peptide Engineering. (A) A yeast signal peptide library was constructed by designing eight novel signal peptides to replace the original α-factor signal. (B-D) SDS-PAGE analysis of Lac (B), VP (C), and LPMO (D) expression. Distinct protein bands were observed for Lac, indicating successful expression. (E-G) Enzymatic activity assessment: absorbance changes of catalytic products reflect the enzymatic activity in supernatants containing Lac (E), VP (F), and LPMO (G), indirectly indicating the impact of different signal peptides on the expression levels of the three enzymes.

## Build

The signal peptide library was incorporated into the P. pastoris expression vectors for each enzyme. Plasmids were constructed using E. coli DH5α through standard molecular cloning techniques involving PCR, ligation, and transformation. The engineered plasmids were then transformed into P. pastoris GS115. Successful transformations were confirmed for Lac with signal peptides S1, S2, S3, S6, S7; for VP with S1, S2, S4, S6, S7; and for LPMO with S1, S2, S3, S5, S8. The sequences of all signal peptides are listed in Table 5.

![](https://static.igem.wiki/teams/5714/wiki/engineering-success/table5.webp)

Table 5：Signal peptides for protein secretion in Pichia pastoris

## Test

SDS-PAGE analysis confirmed the secretion of each enzyme using the respective subset of signal peptides, with Lac showing particularly obvious expression bands (Figure 8B-D). Enzyme activity assays of the supernatants revealed that specific signal peptides significantly enhanced functional expression: SP3 was most effective for Lac, SP4 for VP, and SP3 for LPMO (Figure 8E-G).

Immunofluorescence staining was employed to verify the functional assembly of our system, leveraging the His tag engineered at the C-terminus of each enzyme and the V5 tag fused to the C-terminus of the mini-scaffold. To confirm surface display of the scaffold, yeast cells after 3-day fermentation were incubated with mouse anti-V5 primary antibody, followed by Goat anti-Mouse IgG(H+L) AF488 Conjugate. AF488 emits green fluorescence when excited by blue light, allowing visualization of cells successfully displaying the mini-scaffold (Figure 9B). For enzyme binding assessment, the dockerin-fused enzymes were incubated with scaffold-displaying yeast to enable specific cohesin-dockerin interaction. Complexes were then sequentially treated with rabbit anti-His primary antibody and Goat anti-Rabbit IgG(H+L) AF647 Conjugate. Red fluorescence under green light excitation confirmed successful enzyme binding to the scaffold-displaying yeast (Figure 9C). Subsequent treatment of straw cellulose with yeast displaying the full three-enzyme complex (LVP) for five days resulted in significant lignin reduction, confirming successful enzymatic synergism (Figure 9D).

![](https://static.igem.wiki/teams/5714/wiki/engineering-success/figure9.webp)

Figure 9. Analysis of Enzyme-Scaffold Assembly and Lignin Degradation. (A) Schematic of the experimental design: the three enzymes were incubated with mini-scaffold-displaying yeast cells, and binding efficiency was analyzed using co-immunofluorescence. (B) Detection of mini-scaffold expression: cells were labeled with an anti-V5 primary antibody and a green-fluorescent secondary antibody. Green fluorescence on the cell surface confirms successful mini-scaffold display. (C) Verification of enzyme binding: samples were labeled with an anti-His primary antibody and a red-fluorescent secondary antibody. Red fluorescence on the cell surface indicates specific enzyme binding to the scaffold. (D) Lignin degradation efficiency: residual lignin content after treatment with either single enzymes or the multi-enzyme complex (LVP) surface-displayed on S. cerevisiae. CT represents the untreated control; LVP denotes the ternary enzyme complex.

## Learn

The engineered yeast system was successfully constructed and effectively degraded lignin in straw, with the LVP complex reducing lignin content to approximately 2.6%, a substantial improvement from the 6.6% in the control. This system demonstrates a proof-of-concept for a consolidated, sustainable, cell-based pretreatment platform that minimizes enzyme loss and avoids the environmental burden of chemical methods. While time constraints prevented full optimization for greater synergy, it establishes a robust and eco-friendly foundation for future development of straw-based textile production.

![](https://static.igem.wiki/teams/5714/wiki/section-heading.webp)

# Conclusion

Our project exemplifies the power of synthetic biology to bridge natural inspiration and engineering execution. By designing and characterizing novel CBM3-fused structural proteins, we successfully enhanced straw-based fibers to meet textile strength requirements (2.56 cN/dtex) through rational protein engineering. Furthermore, we established a modular enzyme display platform using standardized biological parts, creating a programmable system for efficient straw bioprocessing. This integrated approach—combining engineered biological systems with industrial manufacturing processes—demonstrates how synthetic biology can transform agricultural waste into valuable materials while addressing sustainability challenges. Our work validates the engineering principles central to iGEM, showing how predictable biological design can create practical solutions for real-world problems in the textile industry.

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# Reference

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2. Jin, D., Dong, W., Zhang, H., Ci, Y., Hou, L., Zang, L., ... & Lucia, L. (2019). Comparison of structural characteristics of straw lignins by alkaline and enzymatic hydrolysis. BioResources, 14(3), 5615-5629.
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