Our project focuses on the marine-derived yeast-like fungus Aureobasidium melanogenum P16, which was engineered using the CRISPR-Cas9 system to enhance methanol utilization and establish a methanol-based chassis cell factory. This strain is widely distributed in mangrove and marine environments and poses no pathogenic risk to humans, animals, or plants under standard laboratory conditions. All experiments were conducted in BSL-1 laboratories at Ocean University of China. The project does not involve pathogenic strains, toxins, animals, human samples, or any environmental release. Therefore, the overall biosafety risk of this project is minimal.
In our work, A. melanogenum P16 served as the core host strain, with Saccharomyces cerevisiae and Komagataella pastoris employed as reference controls. All of these are non-pathogenic organisms commonly used in research and industrial biotechnology. For genetic modifications, we employed a CRISPR-Cas9 editing system based on the Addgene plasmid p414-TEF1p-Cas9-CYC1t, into which the nourseothricin resistance gene (NAT) was introduced as a selectable marker, the AMA1 sequence from Aspergillus nidulans was included as an autonomous replication element, and an sgRNA expression cassette was constructed under the control of U6 promoter and terminator. Experimental chemicals were limited to standard molecular biology reagents and methanol as a carbon source. Although methanol is toxic and flammable, all operations were performed under fume hoods with proper storage in fire-proof cabinets, thereby preventing safety hazards.
To mitigate potential risks, a comprehensive set of management measures was implemented. All team members completed biosafety, chemical safety, and fire safety training organized by Ocean University of China, and passed the required examinations before entering the laboratory. In addition, specialized training was provided for handling methanol and applying CRISPR-Cas9. Experimental procedures strictly adhered to BSL-1 standards, with all researchers wearing appropriate personal protective equipment (PPE) including lab coats, gloves, and goggles. Pipetting was carried out with sterile consumables to avoid aerosol contamination, and all methanol-related experiments were conducted in fume hoods. Flammable reagents were stored in fire-safe cabinets. Importantly, all engineered strains and plasmids containing resistance markers were strictly confined to the laboratory with no environmental release. The cultivation of Aureobasidium melanogenum relies on a specific culture medium, and the strain cannot survive independently without such a medium.
Waste treatment procedures were also strictly regulated. All microbial cultures, media, and plates were sterilized by autoclaving (121 °C, 30 minutes) before disposal. Methanol-containing liquid waste was collected separately in designated chemical containers and disposed of through the university’s hazardous waste management system. These practices ensured both biosafety and environmental safety throughout the experimental process.
Our project fully complies with relevant national and institutional regulations. At the national level, we follow the Regulations on Biosafety Management of Pathogenic Microorganism Laboratories (2004), the Regulations on the Management of Human Genetic Resources (2019), and the Biosafety Law of the People’s Republic of China (2021). At the institutional level, we adhere to the Laboratory Safety Code of Conduct, the Biological Safety Management Rules, and the Hazardous Waste Disposal Regulations of Ocean University of China. These documents provide clear requirements for laboratory access, waste disposal, and emergency response. Furthermore, all experimental protocols were reviewed by our advisors and supervised by the Laboratory and Equipment Management Office and the Biosafety Office of Ocean University of China, ensuring compliance with biosafety standards.
In summary, our project has fully considered biosafety and potential risks, and ensures safety through strict laboratory protocols, systematic training, rigorous waste treatment, and compliance with national and institutional regulations. Our work does not pose significant risks to human health, other organisms, or the environment.