RNAi-assisted genome evolution in Saccharomyces cerevisiae for complex phenotype engineering.

Area of Science:

• Synthetic biology
• Genomics
• Molecular biology

Background:

• Cellular reprogramming for improved traits is a key challenge in biology.
• Current genome-scale reprogramming is limited, primarily to bacterial cells.
• Developing scalable methods for eukaryotic cell engineering is crucial.

Purpose of the Study:

• To introduce RNA interference (RNAi)-assisted genome evolution (RAGE) as a broadly applicable method for genome-scale engineering in yeast.
• To demonstrate the utility of RAGE for improving specific cellular traits, such as acetic acid tolerance.
• To validate the RAGE methodology through functional screening in Saccharomyces cerevisiae.

Main Methods:

• Construction of an RNAi library using yeast genomic DNA and convergent-promoter expression cassettes.
• Iterative cycles of RNAi-induced reduction-of-function mutant creation.
• High-throughput screening and selection for desired trait improvements.
• Application of RAGE for enhancing acetic acid tolerance in yeast.

Main Results:

• Successful RNAi screening in Saccharomyces cerevisiae, identifying suppressors of a telomerase-deficient mutation.
• Demonstration of RAGE for improving acetic acid tolerance through iterative screening.
• Identification of three synergistic gene knockdown targets conferring enhanced acetic acid tolerance.
• Engineering of a yeast strain with substantially improved acetic acid tolerance.

Conclusions:

• RAGE is a versatile and effective platform for genome-scale engineering in yeast.
• The RAGE method accelerates the design and evolution of organisms with novel or enhanced traits.
• This approach offers new insights into genome structure, function, and evolution.