Contribution

We successfully applied the PGASO (Promoter-based Gene Assembly and Simultaneous Overexpression) system to assemble and integrate multiple genes into Kluyveromyces marxianus in a single step.

• The A2 β-casein gene (CSN2) from Bos taurus and Bos grunniens.
• RuBisCO Form I and II gene sets for carbon fixation.

This is among the first attempts to apply PGASO to food-related protein synthesis in yeast, linking nutritional enhancement and carbon mitigation. Successful gene assembly was confirmed by restriction digestion, PCR, and sequencing. All primers, plasmid maps, and cassette designs were optimized for K. marxianus and documented for future teams.

We introduced the RuBisCO-based CO₂ fixation module into K. marxianus to improve carbon utilization efficiency. Comparative cultivation under aerobic and anaerobic conditions showed:

• Strains with RuBisCO genes maintained more stable OD₆₀₀ values in stationary phase.
• CO₂ emission rates measured by gas chromatography indicated altered carbon metabolism and partial carbon recapture.

Though not all data met initial hypotheses, these results suggest co-integration of carbon-fixation and protein production pathways can reduce net carbon emissions, important for sustainable biotechnology.

We developed a complete heterologous protein expression workflow in yeast including:

• Optimization of cell disruption via enzymatic digestion and freeze–thaw cycles.
• SDS-PAGE protocols for target protein confirmation.
• Elemental analysis (C, H, N) to assess biomass composition changes under genetic modifications.

Though the A2 β-casein band was not clearly visible, our standardized methods and troubleshooting plan (including GFP marker integration) provide a detailed framework for future teams facing similar challenges.

We created a combined analysis model integrating optical density (OD₆₀₀), gas chromatography CO₂ trends, and elemental analysis to evaluate metabolic efficiency:

Efficiencyn = ΔCO₂n / ((ODn + ODn-1/2) Δt)

This model assesses the relationship between carbon utilization and growth rate in engineered microorganisms, adaptable to other fermentation and carbon-fixation projects.

All experimental data, gene sequences, and PGASO assembly protocols are documented and available on our wiki for open access.

Our work lays the foundation for:

• Carbon-neutral dairy alternatives produced microbially.
• Integrating metabolic engineering with sustainability goals.
• Reusable PGASO-based gene assembly workflows for future iGEM teams.

This project demonstrates how synthetic biology can marry environmental responsibility with industrial biotechnology, advancing microbially produced, low-emission dairy proteins.