Our project makes multiple reusable and experimentally validated contributions across genetic tool development, microbial engineering, and sustainable biomaterial production, all aligned with iGEM's spirit of open science and collaborative innovation.

We have validated and enhanced existing iGEM parts through practical implementation in our yeast engineering systems. While the original α-signal peptide (BBa_K3711016) showed limited secretion efficiency in our tests, we developed and characterized improved signal peptide variants that significantly enhance protein secretion. Additionally, we successfully implemented the AGA2 surface display scaffold (BBa_M45076) to assemble a functional three-enzyme complex on yeast surface. These implementations demonstrate the practical utility of existing registry parts while providing performance data and optimization strategies that expand their application potential. Our work contributes to strengthening the iGEM Registry by validating part functionality in complex systems and offering enhanced alternatives where needed, providing future teams with reliable implementation blueprints for yeast surface display and protein secretion applications.

Table 1：New Basic Part name,number and link

Our contributions to the iGEM community include two extensively characterized genetic toolkits designed to advance synthetic biology capabilities. The Pichia pastoris signal peptide collection offers eight experimentally validated secretion signals capable of overcoming the traditional bottleneck in recombinant protein expression. This resource enables teams to bypass tedious screening processes and directly enhance protein secretion efficiency. The modular multi-enzyme display system provides a programmable platform for constructing scaffold-based enzyme complexes through orthogonal cohesin-dockerin interactions. This versatile system supports diverse applications including enhanced enzymatic cascades for biosynthetic pathways and improved enzyme stability through scaffold-mediated immobilization. Both collections deliver standardized, reusable components that can significantly accelerate project development across various domains including biocatalysis, metabolic engineering, and environmental applications.

Table 2：Part collection name and UUID

We have established a complete and reproducible pathway for converting corn straw into textile-grade fibers, demonstrating a viable approach for enhancing the value of agricultural waste. Through systematic process optimization and the application of engineered protein reinforcement, we achieved fiber strength of 2.56 cN/dtex using 100% straw cellulose – meeting industry standards for textile applications. Our methodology encompasses the entire production chain from straw pretreatment to fiber spinning, providing comprehensive documentation of material processing parameters, protein incorporation protocols, and quality assessment criteria. This work provides future iGEM teams with a validated foundation for developing sustainable textile production systems, offering detailed methodologies that can be directly implemented and further optimized for various biomass valorization projects.

Figure 1. Purification of Straw Fibers through Multi-stage Chemical Treatment. (A) Relatively pure cellulose powder was obtained after four cycles of NaOH alkaline boiling, water boiling, and filtration. (B) Physical states 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 treated straw cellulose. (D) Fiber spun from wood pulp cellulose. (F) Fiber spun from V3 straw cellulose supplemented with 5% HBP. (G) Fiber spun from V3 straw cellulose supplemented with 5% EV1.

We established a novel selection method that eliminates dependency on high-concentration antibiotics for identifying high-expression P. pastoris strains. This phenotype-based approach uses chromogenic substrates to simultaneously detect Lac and LPMO activity, enabling rapid screening of transformants without antibiotic resistance markers. The method is particularly valuable given the broad industrial applications of these enzymes: laccase is widely used in food processing, textile manufacturing, and paper industries, while LPMO plays crucial roles in hemicellulose degradation and enhancing cellulase activity in biomass conversion. This sustainable screening strategy provides a biosafety-enhanced alternative to conventional methods and is readily adaptable to automated systems, offering future teams an efficient tool for developing enzyme production strains with significant industrial potential.

Figure 2. Rapid Screening Method for Laccase- and LPMO-Producing Pichia pastoris. (A) Traditional high-copy selection of P. pastoris using antibiotic resistance. (B) Antibiotic-independent plate phenotyping for identifying high-expression strains of Lac and LPMO.（C) Schematic of the phenotype-based screening strategy without antibiotic dependence.

We present a novel strategy for reducing antibiotic dependency in synthetic biology systems through a Cre/lox-mediated marker excision system for P. pastoris, following the principle of marker excision established by Zheng et al. (2024). This approach demonstrates the feasibility of removing antibiotic resistance genes after strain selection, significantly lowering the risk of environmental gene transfer. While further optimization may be needed for different applications, this system provides future teams with a validated starting point for developing safer microbial engineering platforms. All genetic designs and implementation protocols are available in the Registry, inviting the iGEM community to build upon this foundation for more responsible biotechnology development.

Figure 3. Yeast Expression Vector with Self-Excision of Resistance Markers. (A) DNA design: After plasmid integration into the yeast genome, methanol induction triggers Cre recombinase expression, which recognizes and excises the region between lox71 and lox66 containing zeocin and kanamycin resistance genes, while retaining the AOX1-signal peptide-gene of interest-terminator expression cassette. (B) Validation of the excision system: Colony PCR of αSP-Lac/VP/LPMO expression strains before and after methanol induction shows fragment size reduction to 1194 bp after induction, confirming precise excision of the antibiotic resistance cassette by Cre recombinase.

Our project delivers six key contributions to advance synthetic biology capabilities: novel structural proteins for textile-grade straw fibers; two modular yeast engineering toolkits for enzyme display and protein secretion; a complete straw-to-textile manufacturing methodology; antibiotic-free screening systems; enhanced biosecurity through marker excision technology; and practical enhancement of existing Registry parts through validation and optimization. Together, these resources provide comprehensive, ready-to-use solutions for sustainable biomaterial production while strengthening the iGEM part ecosystem for future teams.

1. Li, D., Zhang, B., Li, S., Zhou, J., Cao, H., Huang, Y., & Cui, Z. (2017). A novel vector for construction of markerless multicopy overexpression transformants in Pichia pastoris. Frontiers in microbiology, 8, 1698.