Random mutagenesis platform for accelerated genome evolution (RAMPAGE) of Pichia pastoris.

The biological conversion of methanol into valuable chemicals represents a promising route for achieving global carbon neutrality. However, the rational engineering of complex industrial traits in microbial cell factories is hindered by an incomplete understanding of cellular metabolism and regulation. In this study, we developed a Random Mutagenesis Platform for Accelerated Genome Evolution (RAMPAGE) for the yeast Pichia pastoris (Komagataella phaffii). This platform enables continuous genome-wide diversification and facilitates the rapid evolution of superior phenotypes under defined selective pressures. Using RAMPAGE, we evolved yeast strains with significantly enhanced tolerance to extreme industrial conditions, including growth in 18% methanol, survival at 40 °C, and resistance to methanol and thermal stresses. Genome resequencing of these strains revealed a collection of genes associated with methanol tolerance and thermotolerance. In addition, we used RAMPAGE to evolve a glycoengineered yeast strain with improved growth and stress tolerance. Subsequent genome resequencing and functional validation identified novel genetic engineering targets to improve the cellular fitness of glycoengineered yeast strains. By providing a simple, efficient, and programmable tool for continuous genome evolution, RAMPAGE expands the synthetic biology toolkit for P. pastoris, enabling its broad application in industrial biotechnology.