A fragment in the 5′ untranslated region of alcohol oxidase 1 promoter significantly influencing gene expression of Komagataella phaffii

Komagataella phaffii is a famous microbial host in synthetic biology field for expressing heterologous proteins under alcohol oxidase 1 promoter (P_AOX1). This study undertook two successive rounds of gene fragment knockout, identifying a 13 bp segment, termed D3, as a negative cis-acting element in the 5′ untranslated region of P_AOX1. Notably, upon knockout of the D3 fragment, the expression of green fluorescent protein (GFP), serving as the model for heterologous protein expression, improved by 3.5-fold due to enhanced transcription levels. Subsequent transcriptomic analysis, electrophoretic mobility shift assay and DNA pull-down assays coupled with liquid chromatography-mass spectrometry corroborated efficient GFP expression, highlighting minimal alterations in transcriptional machinery subunits, 312 differentially expressed genes and non-specific interactions between nucleoproteins and P_AOX1 following the D3 fragment knockout. Consequently, D3 fragment knock out from P_AOX1 facilitates efficient expression of heterologous protein by K. phaffii.