Whole-genome combinatorial gene fusions generate novel genes for advanced microbial trait development

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Gene fusion is an evolutionary mechanism driving new protein functions and architectures.
• Gene fusion is a valuable tool in biotechnology for protein tagging and activity modification.

Purpose of the Study:

• To broadly demonstrate and harness the gain-of-function capabilities of fusion genes using a whole-genome approach.
• To construct a comprehensive fusion gene library in Saccharomyces cerevisiae.

Main Methods:

• Created a "Function Generator™" library with pairwise combinations of 5,019 yeast protein-coding sequences (ORFs).
• PCR amplified ORFs from the S288C yeast genome and cloned them into a centromeric expression vector.
• Screened the yeast library for resistance to toxic heavy metal ions (Cd⁺², Co⁺², Cu⁺², Ni⁺²).

Main Results:

• Identified active fusion genes conferring complex phenotypes, including heavy metal resistance.
• Sequencing revealed diverse biological functions in active fusions: transcription, translation, metal ion binding/transport, cell cycle control, and unknown functions.
• Confirmed the gain-of-function principle by comparing fusion gene activity to constituent single ORFs.

Conclusions:

• The Function Generator™ library effectively demonstrates the gain-of-function potential of gene fusions.
• This approach accelerates the discovery of novel biological functions and aids in strain development.
• Gene fusions represent a powerful strategy for exploring phenotypic diversity and overcoming evolutionary bottlenecks.