Novel methods for generating strains with increased copy number in the yeast Pichia pastoris

This thesis presents two advances for improving gene expression in Pichia pastoris: a more efficient method for selecting multicopy transformants and a new endogenous selectable marker. A posttransformational vector amplification (PTVA) strategy was developed, enabling low‑copy transformants to increase vector copy number and yielding stable tandem genomic integrations. In addition, the FLD1 gene was established as an effective selectable marker in an fld1 mutant strain, offering efficient transformation without exogenous bacterial sequences and supporting copy‑number enhancement. Together, these tools provide valuable alternatives for achieving high‑yield protein expression in P. pastoris.