To systematically evaluate the performance of our engineered systems, we established a set of rapid measurement methods. This included the development of a low-dosage antibiotic screening method for rapid selection of Pichia pastoris transformants expressing laccase (Lac) and lytic polysaccharide monooxygenase (LPMO), a series of enzymatic activity assays to quantify Lac, versatile peroxidase (VP), and LPMO functionality, a spectrophotometric assay to measure lignin degradation efficiency by our engineered yeast consortium, and textile strength tests to validate the enhanced mechanical properties of protein-enhanced straw fibers. Our work provides a standardized and reproducible framework for performance verification and material characterization, offering a useful toolkit for the iGEM community.

Antibiotic-Independent High-Copy Screening Method for Pichia pastoris

Pichia pastoris as a second-generation yeast expression system offers distinct advantages including simpler genetic manipulation, higher protein expression levels, and enhanced protein modification capabilities—such as O-glycosylation, N-glycosylation, and disulfide bond formation. Various therapeutic proteins, including human serum albumin, hepatitis B vaccine, interferon, and trypsin, have been successfully produced using P. pastoris as the host(Shemesh et al,. 2024, Pochini et al,. 2022, Mohajeri et al,. 2017).

As an obligate aerobic yeast, P. pastoris can utilize methanol as a carbon source, employs the inducible AOX1 promoter system, achieves high recombinant titers, and does not lose carbon source to ethanol formation under respiratory conditions—leading to higher biomass yield and increased recombinant protein production.

However, screening for high-yield P. pastoris strains is challenging due to the low probability (1–5%) of multi-copy gene integration (Mariz et al., 2015). Obtaining a multi-copy strain thus requires either screening tens of thousands of single clones, or pre-assembling multiple copies into an expression vector prior to transformation (Li et al., 2017)—both of which are highly tedious. To isolate these rare high-copy transformants, it is necessary to use progressively higher antibiotic concentrations to selectively inhibit the growth of low-copy or false-positive colonies. As demonstrated in our pre-project testing by our secondary PI, He Zhengyu, substantial colony growth was still observed even at 800 μg/mL Zeocin (Figure 1A), indicating a requirement for concentrations potentially as high as 5 mg/mL for effective selection(Eissazadeh et al., 2017).

This reliance on high-dose antibiotics creates two significant issues. First, it incurs a substantial financial cost, as a single 50 mL vial of Thermo Fisher's 100 mg/mL Zeocin is priced at approximately ¥21,022. More critically, this practice raises the serious risk of antibiotic misuse and environmental release, potentially contributing to the spread of antibiotic resistance genes. To address these concerns, we implemented a resistance gene self-excision strategy using the Cre/lox system in our expression vectors, which effectively removes antibiotic resistance genes after genomic integration. For detailed description of this safety strategy, please refer to our Safety page.

This year, our project aims to use P. pastoris to express three lignin-degrading enzymes, with the potential future use of additional enzyme combinations in industrial applications. To simplify this process, we propose introducing a phenotype-based screening method—analogous to the blue-white screening in E. coli—where expression levels can be visually estimated based on color. Coupled with high-throughput screening equipment, this approach could significantly reduce screening costs and improve efficiency.

Lac and VP are common lignin-degrading enzymes capable of oxidizing a broad spectrum of phenolic substrates, leading to visible color reactions. For laccase, we selected the widely used substrate ABTS, which turns green upon oxidation. For VP, we chose 2,6-DMP due to its strong chromogenic property, yielding a yellow product when oxidized (Teo et al,. 2024).

LPMO is typically assayed using the DNS method in combination with cellulases. However, this method requires boiling for color development and is unsuitable for plate-based phenotypic screening. Although the underlying mechanism remains unclear, it has been reported that LPMO can also generate a color reaction with 2,6-DMP. (Breslmayr et al,. 2018) This principle has been developed into a rapid activity assay for LPMO, making 2,6-DMP a suitable substrate for our plate screening approach.

Our strategy involves using MM medium instead of MD medium for auxotrophic selection after yeast electrotransformation, where the glucose in MD medium is replaced with methanol in MM medium to enable direct induction of expression. We supplemented the MM medium with necessary cofactors and substrates for the three enzymes: for Lac, we added 0.5 mM ABTS and 1 mg/mL CuSO₄; for LPMO, we added 2.4 µM 2,6-DMP ,1 mg/mL CuSO₄, and 3 mM H2O2; and for VP, we added 2.4 µM 2,6-DMP, 1 mg/mL MnSO₄, 1 mg/mL CaCl₂ ,3 mM hydrogen peroxide, and 100 µg/mL hemin(Breslmayr et al,. 2018). Due to the volatile nature of methanol, we supplemented each plate daily with 1 mL of methanol to prevent carbon source depletion in the MM medium. A replica plate was prepared using YPD medium supplemented with 100 µg/mL Zeocin (YPDZ), as shown in Fig. 1C.

After two days of incubation, we successfully identified yeast strains expressing Lac and LPMO as shown in Figure 1B By observing the intensity of the color development, we were able to directly select strains with high expression levels. Unfortunately, VP is a hemin-dependent enzyme, and the addition of hemin turns the medium brown, which severely interferes with the yellow color reaction. Therefore, VP is currently not suitable for this screening method, though substituting the substrate—for example, with ABTS—or reducing the amount of hemin might provide viable alternatives, pending further optimization in future studies.

Activity Assays for Lac, VP, and LPMO

We produced Lac, VP, and LPMO in Pichia pastoris with the objective of displaying these enzymes on the surface of Saccharomyces cerevisiae EBY100 cells. To verify enzymatic functionality, we measured the enzyme activity in the fermentation supernatant after P. pastoris fermentation using the assay methods illustrated in Figure 2. One unit of Lac activity was defined as the amount of enzyme required to convert 1 mM ABTS per minute per microliter of supernatant, while one unit of VP/LPMO activity was defined as the amount required to convert 1 μM 2,6-DMP per minute per microliter of supernatant.

In summary, we have designed and executed a comprehensive measurement strategy that spans from the molecular level to the macro-scale. Our high-throughput screening method provides a valuable protocol for future iGEM teams working with yeast and enzyme engineering. The quantitative data on lignin degradation robustly supports the efficacy of our engineered multi-enzyme consortium. Finally, the standardized textile tests concretely demonstrate the success of our approach in creating a stronger, more flexible bio-based fiber.

This suite of measurements not only validates our project's core hypotheses but also provides a reproducible and shareable framework for the community. We hope our methods, particularly the screening and lignin assay protocols, will serve as a resource for future teams tackling similar challenges in synthetic biology and sustainable material design.

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