Modifying the upstream open reading frames of cellulase gene enhances cellulase production in Penicillium oxalicum

Cellulase plays an irreplaceable role in biomanufacturing using plant biomass as feedstock. However, improving cellulase production by fungi through manipulation of upstream open reading frames (uORFs) in the 5'-untranslated regions (5'-UTR) of cellulase genes has been less frequently explored. This study aimed to screen uORFs in the 5'-UTR of cellulase genes in <i>Penicillium oxalicum</i>, identify functional uORFs in the 5'-UTR of the <i>eg1</i> gene which encodes a key endo-β-1,4-glucanase (EG) in <i>P</i>. <i>oxalicum</i>, and enhance fungal cellulase production through uORF modifications. Among the 25 cellulase genes examined in <i>P</i>. <i>oxalicum</i> strain HP7-1, 23 contained uORFs in their 5'-UTR. Seven uORFs were annotated in the 5'-UTR of the <i>eg1</i> gene. A uORF-green fluorescent protein (GFP) reporter system demonstrated that uORF1 and uORF3 inhibited, while uORF7 enhances, GFP abundance. Overexpression of <i>eg1</i> containing uORF1 or uORF3 variants where the start codon of the uORF was mutated to AAG in <i>P</i>. <i>oxalicum</i> led to a significant 91.7 % and 62.1 % average increase in carboxymethyl cellulase production after 4 days of induction compared to the start strain Δ<i>PoxKu70</i>. Real-time quantitative reverse transcription-polymerase chain reaction, mRNA stability determination, and <i>in vitro</i> translation experiments collectively revealed that these three uORFs influence the mRNA stability of the downstream mORF, but not translation efficiency. These findings highlight the critical role of uORFs in regulating gene expression during fungal enzyme biosynthesis and offer a valuable alternative strategy for improving enzyme production.