1.1 Elucidation of the targeting mechanism of key methanol genes in Aureobasidium melanogenum
This study systematically revealed the targeting mechanisms of key methanol metabolism genes in the melanin-producing Aureobasidium melanogenum, establishing a research framework suitable for metabolic pathway analysis in non-model fungi. This provides clear targets for the systematic engineering of methanol metabolism pathways.
Unlike previous studies that focused only on model fungi, this study is the first to systematically apply targeting mechanisms to a non-model marine fungus, providing methodological references and inspiration for other research teams.
1.2 Engineering Aureobasidium melanogenum to enhance methanol tolerance and glucose production
The metabolic engineering strategies provided in this study serve as a reusable template and validation model for other research teams, guiding carbon flux reconstruction and pathway optimization in various non-model fungi.
Importantly, the engineered Aureobasidium melanogenum strains can stably grow and produce glucose efficiently under methanol-based conditions, providing new chassis strains for green biomanufacturing, environmental pollutant remediation, methanol waste valorization, and the development of novel microbial food sources, thus offering sustainable application avenues.
Due to the genetic intractability of melanin-producing Aureobasidium melanogenum and the lack of mature gene editing systems, this study constructed and validated a CRISPR-Cas9-Am plasmid system optimized for fungi.
The establishment of this system not only solves the low-efficiency genetic manipulation problem in Aureobasidium melanogenum but also provides a general editing framework for the study of non-model fungi. Its main scientific contributions include:
① High system universality: By incorporating the AMA1 autonomously replicating sequence, the vector can be stably maintained and exist in high copy number across various fungal hosts, providing a directly adaptable solution for fungi lacking characterized replication elements.
② Modular and replaceable design: The synthetic U6-sgRNA module allows convenient replacement of target genes. Other teams only need to substitute the sgRNA sequence to quickly achieve editing of different genes, substantially shortening construction timelines.
③ Host compatibility validation: The combination of the TEF1 promoter and CYC1 terminator exhibits excellent transcriptional activity and mRNA stability in Aureobasidium melanogenum, providing reliable references for promoter screening and vector construction in other non-model fungi.
④ Breakthrough for genetic manipulation in non-model fungi: The CRISPR-Cas9-Am system is scalable, easy to operate, and highly efficient, and can be directly applied to fungal gene function studies, metabolic pathway analysis, and synthetic biology chassis construction. For other research teams, the system offers a validated editing tool and a feasible approach for genetic modification of ecologically diverse fungi.
⑤ Feasibility validation: The knockout of the Ade2 gene was used as a proof-of-concept, providing experimental evidence for subsequent complex gene cluster editing, multi-gene regulation, and heterologous pathway introduction. Its high efficiency in Aureobasidium melanogenum suggests that the system could be extended to mangrove microbial communities and other marine-derived fungi, facilitating the transition of non-model microbes from “difficult to manipulate” to “precisely designable.”
Based on our team’s achievements in genetic engineering and metabolic optimization of Aureobasidium melanogenum P16, we systematically constructed a series of biological parts encompassing metabolic engineering, subcellular localization, gene editing, and screening systems. These modular, portable components are intended to provide a reusable toolkit for the synthetic biology field, accelerating research and application in non-model fungi and related microorganisms.
3.1 Metabolic engineering: optimizing glucose and methanol metabolism
To support pathway engineering and achieve efficient methanol utilization and glucose biosynthesis, we developed the following key functional modules:
BBa_25YHKFCT (E. coli yihX gene): Encodes the yihX gene from Escherichia coli. When heterologously expressed, it promotes the conversion of metabolic intermediates into glucose, serving as a direct tool for redirecting carbon flux and increasing glucose yield.
BBa_25Q03R3R (pNAT-LoxP-rDNA-yihX plasmid): A heterologous expression vector integrating the yihX gene, NAT resistance marker, LoxP sites, and rDNA stabilization fragments. It ensures high stability and expression efficiency of the target gene, providing a “ready-to-use” platform for metabolism-oriented experiments focused on carbohydrate biosynthesis.
BBa_254B2L3Q (Pichia pastoris Das gene): The Das gene from Pichia pastoris. Heterologous expression enhances methanol oxidation and assimilation in P16, serving as a core component for improving methanol utilization efficiency.
BBa_25D4LV5G (pNAT-LoxP-rDNA-P.p Das plasmid): An overexpression plasmid carrying the P. pastoris Das gene. It effectively enhances methanol metabolism in P16 and provides a plug-and-play solution for research teams working on methanol-driven biomanufacturing.
3.2 Subcellular localization: a fundamental tool for studying protein spatial function
Subcellular localization vectors are essential tools for investigating protein function and intracellular distribution. This study provides a range of localization vectors suitable for non-model fungi:
BBa_25TNPOI3 (pHPT-LoxP-rDNA plasmid): A backbone plasmid containing the HPT resistance gene, LoxP sites, and rDNA sequences. It is designed for fluorescence labeling and localization experiments, significantly simplifying vector construction.
BBa_25DC5QOI (pHPT-LoxP-rDNA-mCherry-SKL plasmid): A standard localization plasmid carrying the mCherry-SKL peroxisome-targeting sequence. It can serve as a positive control for verifying whether the target protein localizes to the peroxisome.
BBa_25QPWWAX (pNAT-LoxP-rDNA plasmid): Functionally similar to the above, but uses NAT as the selection marker. It is compatible with different host backgrounds and supports flexible modifications and insertion of fluorescent tags.
3.3 Gene editing and screening: auxiliary tools for non-model fungal genetic manipulation
To advance genetic manipulation in Aureobasidium melanogenum P16 and related fungi, this study established an efficient toolkit for selection and editing:
BBa_25MXN531 (fl4a-nat-loxp plasmid): A template plasmid for amplification and acquisition of the nourseothricin (NAT) resistance gene, supporting the construction of diverse fungal vectors using NAT as a selection marker.
BBa_256AEFB2 (Aureobasidium melanogenum Ade2 sgRNA): An sgRNA targeting the Ade2 gene in P16, which can directly validate the CRISPR-Cas9 system, serving as an ideal test locus for establishing gene editing systems.
These modular components fill critical technical gaps in metabolic pathway optimization (glucose/methanol utilization), protein localization studies, and gene editing in non-model fungi.
By providing a series of standardized, reusable “biological modules,” this work significantly reduces the time and cost other research teams would need for basic part design and validation. Researchers can thus focus directly on higher-level system construction, such as industrial methanol biomanufacturing, environmental pollutant removal, and production of alternative biomass-based compounds.
These modules can be regarded as “functional building blocks” in synthetic biology. They can be flexibly integrated into multiple experimental systems, laying a solid foundation for future cross-species metabolic engineering and genome editing studies.
By replacing weeks of trial-and-error cloning with a <45-second, structure-aware localization prediction, the pipeline shortens the design–build–test cycle for protein drugs that must reach specific organelles (e.g., lysosomal enzymes for LSD therapies or mitochondrial antioxidants). Faster iteration translates directly into lower R&D costs and quicker delivery of life-saving biologics to patients.
The entire workflow runs on a single laptop GPU, requires no experimental structures or MSAs, and is released open-source. This removes the computational and financial barriers that typically exclude small academic labs, start-ups, and under-funded institutes from engineering organelle-targeted proteins, thereby broadening participation in advanced biotechnology.
LocAgent ranks cloning strategies by predicted success probability and supplies ready-to-order primers, enabling researchers to test only the most promising constructs. In the validated hydratase case a single amino-acid change achieved 87% peroxisomal relocation, illustrating how in-silico guidance can replace multiple rounds of cellular assays and transgenic animal work.
The modular LangChain architecture couples a 150M-parameter structure-biased encoder with extensible signal-prediction tools, establishing a reusable framework that can be upgraded as new structure or language models emerge. This provides the research community with an evolving platform for tackling other trafficking-related diseases—such as neurodegeneration or metabolic disorders—without retraining large models from scratch.
To address the industry pain points in methanol concentration monitoring and regulation during the cultivation of Pichia pastoris in laboratories—specifically, the issues of traditional liquid sensors being prone to contamination, having a short lifespan, being high in cost, and requiring intrusive installation—the Hardware Team has developed a real-time methanol monitoring and automatic feeding device based on the gas-phase detection and gas-liquid conversion model. It has established a technical chain of “non-intrusive sensing - precision calculation - adaptive control,” providing a low-cost and high-reliability solution for microbial fermentation monitoring with significant social value.
This device takes the gas-phase methanol concentration as the detection target and infers the liquid-phase concentration through an independently developed gas-liquid conversion model, thus avoiding the problem of direct contact between the sensor and the fermentation broth. Equipped with the GT-CX gas sensor and STM32G070 microcontroller, it achieves a response time of <30 seconds and a repeatability error of <±3%. The cost per unit is controlled within 1,600 RMB, which is only 1/3 to 1/5 of the price of commercial liquid sensors. In addition, it complies with multiple national standards, has an explosion-proof rating of Exd IIC T6 Gb, and is suitable for the working conditions of 10L fermenters. This greatly lowers the monitoring threshold for small and medium-sized research teams and promotes the popularization of research on methanol-induced expression systems such as Pichia pastoris. Meanwhile, the device is built with a three-stage gas-liquid conversion model integrating “basic balance - environmental correction - metabolic compensation” (with an error controllable within ±5%) and embeds a fuzzy control algorithm (reducing the response time to 10 seconds). It accurately matches the methanol demand of Pichia pastoris, avoids toxic inhibition caused by excessive methanol or reduced expression efficiency due to insufficient methanol, and ensures the high-efficiency expression of heterologous proteins.
Furthermore, the device adopts a “sensing - control - auxiliary” modular hardware architecture and a standardized software system. It supports independent replacement and upgrading, as well as expansion of temperature/pH detection functions, and is applicable to fermentation scenarios with volatile substrates such as ethanol and acetone, providing a replicable non-intrusive monitoring framework. It not only meets the current application needs of 10L fermenters but also lays a foundation for pilot-scale expansion. In the long run, it can promote the resource utilization of waste methanol and green biomanufacturing, providing technical support for the sustainable bio-industry under the “dual carbon” goal.
1.1 Primary School Outreach — Huancheng Town Experimental Primary School
Content & Format: Focused on storytelling and interactive experiments, this activity introduced concepts such as genetic modification and carbon neutrality to primary school students through vivid examples and hands-on sessions, accompanied by posters and video displays.
Our contribution to the future is reflected in the fact that we have cultivated the next generation of our motherland, brought the knowledge we have learned to children in poverty-stricken areas, and imparted the mysteries of biology in the form of stories. We believe that whether they remember it or not, a seed of biology will surely be planted in the hearts of children.
1.2 High-School Summer Camp — “Exploring the World of Biological Experiments”
Content & Format: Included guest lectures by professors, thematic experiments (leaf observation, microbiology experiments, and sea urchin dissection), as well as knowledge competitions and forums. The aim was to spark students’ scientific curiosity and teach basic experimental skills.
We have created a platform for high school students to explore marine life and research methods, and it has received unanimous praise from the teachers and students of the corresponding high schools. This project is inheritable. We hope that in the years to come, we can continuously communicate with high school students, provide them with more cutting-edge biological knowledge, and call on more students with aspirations to devote themselves to scientific research.
1.3 University-Level Recruitment & Training
Content & Format: Recruitment was conducted through campus fairs and social media (WeChat official account and online groups). A multi-week pre-training program was organized, including literature reading, team division (wet lab, dry lab, modeling, hardware, design), and one-on-one interviews. Participants received pre-reading materials and mentorship-style guidance.
For society, we have established an interdisciplinary team and learning path, strengthened the team inheritance and talent cultivation mechanism, and also provided a valuable opportunity for students of Ocean University of China who want to participate in iGEM in the future. We believe that the iGEM competition will be known and participated in by more students.
1.4 Social Media & Continuous Outreach
Content: The team managed official WeChat and Xiaohongshu (RED) accounts, documenting every outreach activity, camp, and lecture to broaden the audience and maintain community engagement.
Impact: Extended offline education into sustained online influence, facilitating long-term science communication and recruitment.
2.1 Academic and Technical Communication
Through multidisciplinary collaboration on key issues such as enzyme subcellular localization, metabolic flux distribution, and knockout efficiency, we successfully translated academic insights into verifiable engineering practices. With support from expert input and technical partners, we proposed the strategy of “weakening consumption pathways while enhancing synthesis pathways,” leading to the construction of the Δpfk engineered strain. Additionally, by optimizing replication sequences, promoters, and sgRNA design in the gene editing system, we significantly improved knockout efficiency and reduced experimental time and cost.
These methodological and practical experiences can provide reusable strategies and technical references for other iGEM teams working on metabolic engineering and gene editing system optimization, promoting the transition of synthetic biology research from theoretical design to efficient engineering implementation.
2.2 Industrial, Design, and Public Communication
Based on our practices in industry integration, hardware optimization, and science communication, we have developed a research model that combines technical feasibility, engineering thinking, and public engagement. Industry advisors and sector exchanges guided us to adopt a “technology as the core, storytelling as the bridge” approach, incorporating narrative expression into our project Wiki and outreach materials to enhance the comprehensibility and communicability of scientific content. Hardware and process experts helped us make practical trade-offs in areas such as gas–liquid modeling and fermentation exhaust treatment, strengthening the implementability of our solutions. Meanwhile, visual elements, such as mascots, effectively improved cognitive familiarity and brand recognition of synthetic biology projects among non-specialist audiences.
These experiences provide a reusable reference for integrated “research–communication–application” in science communication and public engagement, facilitating the transformation of laboratory achievements into solutions that are socially perceivable and industrially applicable.
3.1 Educational Support in Remote and Underdeveloped Areas
During a one-week volunteer teaching program, we engaged local primary and middle school students through hands-on experiments and drawing activities centered on biology and environmental science.
This initiative helped supplement regional educational resources and expanded the reach of science education. By adopting an interdisciplinary and engaging approach, it sparked curiosity and interest in scientific topics among young learners, contributing to their scientific literacy and inspiration. This educational model also offers a replicable and scalable example for future grassroots science outreach activities.
3.2 “Voices of the Elderly” — Survey and Interviews at Qingdao Xingfu Zhi Jia Elderly Apartment
Through interviews and questionnaires with nearly 30 elderly residents aged 60–100, we gained in-depth insights into their perceptions of climate warming. The survey revealed that approximately 60% of elderly participants felt summers have become hotter, 75% perceived increased humidity and longer summer durations, and 70% preferred receiving information through face-to-face communication with staff.
This study incorporated the often-overlooked elderly population into climate awareness research and developed an age-friendly science communication model centered on “face-to-face engagement, supplemented by short videos and posters,” based on their cognitive characteristics and communication preferences. This approach not only enhanced the sense of involvement and expressive capacity of the elderly in climate change discussions but also provided a replicable methodology and strategic reference for implementing age-inclusive environmental communication initiatives.
Our Human Practices work built a vital bridge between science and society. Through education, we inspired learners from primary school to university and fostered interdisciplinary understanding. Through communication, we transformed expert and industrial insights into concrete experimental improvements and clearer public narratives. Through inclusivity, we engaged diverse groups—from students in rural areas to elderly residents—ensuring multiple perspectives were reflected in our project.
Together, these efforts formed a continuous feedback loop that guided our design, experimentation, and outreach, making our project both scientifically rigorous and socially meaningful.