Genome-Wide Landscape of Position Effect Variegation in Pichia pastoris

Exogenous genes are generally expressed by integration into the chromosomes of <i>Pichia pastoris</i>. 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 <i>P. pastoris</i> was successfully constructed using the <i>piggyBac</i> (<i>PB</i>) transposon system. Through sequencing, the sequence TTAA was identified as the major recognition site of the <i>PB</i> transposon, which exhibited relatively high coverage on <i>P. pastoris</i> chromosomes, making it a valuable tool for studying position effect variegation in <i>P. pastoris</i>. Using the enhanced green fluorescent protein gene (<i>eGFP</i>) 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 <i>P. pastoris</i>. Additionally, it has provided novel insights into high-throughput screening strategies for strains with high exogenous gene expression.