Phylogenetic debugging of a complete human biosynthetic pathway transplanted into yeast

Area of Science:

• Synthetic biology
• Metabolic engineering
• Biochemistry

Background:

• Cross-species pathway transplantation is a powerful tool for understanding biological processes.
• The adenine de novo biosynthesis pathway is essential for cell growth and viability.

Purpose of the Study:

• To replace the yeast adenine de novo biosynthesis pathway with its human counterpart.
• To investigate the functional complementation and identify limitations in a humanized yeast system.
• To explore methods for improving the stability and function of transplanted pathways.

Main Methods:

• Enzyme replacement of the Saccharomyces cerevisiae adenine de novo biosynthesis pathway with human enzymes.
• Phenotypic analysis of the 'humanized' yeast, including growth assays.
• Suppressor analysis to identify regulatory pathways.
• Phylogenetic analysis of enzyme regulation.
• DNA shuffling to engineer protein stability.

Main Results:

• The humanized yeast exhibited poor growth in the absence of adenine, indicating incomplete functional complementation.
• The human enzyme PPAT (ortholog of yeast ADE4) showed only partial function.
• Suppressor analysis revealed regulatory roles of other pathways in adenine biosynthesis.
• Phylogenetic analysis suggested enzyme regulation is adapted to organism-specific metabolite levels.
• DNA shuffling identified amino acid combinations that stabilize the human enzyme in yeast.

Conclusions:

• Cross-species metabolic pathway transplantation is feasible but presents functional challenges.
• Enzyme-specific limitations and organismal regulatory networks impact pathway function.
• Engineering strategies like DNA shuffling can enhance the performance of transplanted pathways.
• The developed methods provide a framework for transplanting diverse metabolic pathways into yeast for research and engineering.