Tuning interdomain conjugation to enable in situ population modification in yeasts

Area of Science:

• Microbiology
• Synthetic Biology
• Biotechnology

Background:

• Modifying fungal members of microbiomes is crucial for biotechnology and biomedicine but technologically challenging.
• Interdomain conjugation (IDC), DNA transfer from bacteria to yeast, offers a method for in situ microbiome modification.
• Current IDC techniques face efficiency limitations, hindering their widespread application.

Purpose of the Study:

• To enhance the efficiency and control of interdomain conjugation (IDC) for modifying fungal populations within microbiomes.
• To identify and manipulate key interactions between bacterial donors and yeast recipients to optimize DNA transfer.
• To demonstrate the application of controlled IDC for irreversible alteration of yeast populations, including rescue and collapse.

Main Methods:

• Manipulating metabolic and physical interactions between Escherichia coli (bacterial donor) and Saccharomyces cerevisiae (yeast recipient).
• Testing various population interaction landscapes to identify conditions maximizing IDC.
• Utilizing cell-to-cell binding via mannoproteins to enhance both IDC and bacterial commensalism.
• Modeling tunable controls to predictably yield a range of IDC outcomes.
• Applying a novel IDC-mediated CRISPR/Cas9 system for population alteration.

Main Results:

• Bacterial commensalism was found to maximize IDC efficiency in both culture and mixed colonies.
• Cell-to-cell binding via mannoproteins significantly assisted both IDC incidence and bacterial commensalism.
• Tunable controls were modeled to predictably yield a range of IDC outcomes.
• Controlled IDC was successfully used to irreversibly alter yeast populations, including rescuing a poor-growing population and collapsing a stable population via CRISPR/Cas9.

Conclusions:

• Optimized metabolic and physical interactions between bacteria and yeast provide effective controls for interdomain conjugation (IDC).
• These findings offer building blocks for in situ mycobiome editing, with implications for treating fungal pathogens and engineering fungal systems.
• The developed methods enable precise and irreversible modification of yeast populations, advancing microbiome engineering capabilities.