# Genome-Wide Landscape of Position Effect Variegation in Pichia pastoris

## TL;DR

This study maps where genes inserted into the yeast Pichia pastoris are likely to be highly expressed or silenced, offering insights for genetic engineering.

## Contribution

The study provides the first genome-wide analysis of position effect variegation in Pichia pastoris using transposon-based mutagenesis.

## Key findings

- High-expression gene insertion sites are enriched in gene interiors and chromatin remodeling pathways.
- Low-expression sites cluster near promoter and terminator regions and are linked to autophagy and signaling pathways.
- TTAA is the main transposon insertion site, enabling broad genomic exploration of expression variation.

## Abstract

Exogenous genes are generally expressed by integration into the chromosomes of Pichia pastoris. However, systematic studies on the chromosomal position effect are lacking, and locations that are conducive to the high expression of foreign genes are rarely reported. In this study, a genomic random insertion mutagenesis library for P. pastoris was successfully constructed using the piggyBac (PB) transposon system. Through sequencing, the sequence TTAA was identified as the major recognition site of the PB transposon, which exhibited relatively high coverage on P. pastoris chromosomes, making it a valuable tool for studying position effect variegation in P. pastoris. Using the enhanced green fluorescent protein gene (eGFP) as a reporter, two libraries including low-expression positions and high-expression positions were obtained by flow cytometry. The low-expression sites were mainly located upstream of ORFs around the promoter region and downstream near the terminator region, while the high-expression sites were predominantly located at the gene interior. KEGG and GO analyses showed that genes in high-expression positions were significantly enriched in the ATP-dependent chromatin remodeling and histone binding pathways, and genes in low-expression positions were significantly enriched in the MAPK signaling pathway, autophagy, mitochondrial autophagy, ABC transporters, and the arginine synthesis pathway. This study has clarified the genome-wide landscape of position effect variegation in P. pastoris. Additionally, it has provided novel insights into high-throughput screening strategies for strains with high exogenous gene expression.