Enhancing mycophenolic acid production in Penicillium brevicompactum through Kozak-optimized 2A peptide multi-gene expression system

Although Penicillium brevicompactum is widely used for industrial mycophenolic acid (MPA) production, research on its metabolic engineering and gene regulation remains limited. Efficient, coordinated expression of multiple genes is crucial for optimizing biosynthetic circuits and metabolic pathways, yet current strategies often suffer from inefficiencies and imbalances. These challenges not only limit the production of the desired metabolic products but can also result in wasted resources and inhibited cell growth. In this study, we optimized the 2A peptide multi-gene expression system in P. brevicompactum by introducing the Kozak sequence. This modification significantly enhanced the transcription of two key genes in the mevalonate (MVA) pathway precursor farnesyl pyrophosphate (FPP): the Acetyl-CoA acetyltransferase gene (ERG10) and the HMG-CoA synthetase gene (ERG13). In the PP-K10K13 strain, the transcription levels of the ERG10 and ERG13 genes increased by 77.02 % and 67.63 %, respectively, compared to the PP-1013 strain, which lacked the Kozak sequence. Consequently, the mycophenolic acid (MPA) production reached 4.30 g/L, representing a 49.31 % increase relative to the wild-type strain (WT). Additionally, observations of the engineered strains incorporating the introduced Kozak sequence showed improved growth, evident in an increase in mycelial dry weight, indicating reduced growth inhibition from metabolic engineering modifications. These results demonstrated that the optimized 2A peptide expression system not only effectively enhanced product synthesis efficiency but also helped restore the normal growth state of the engineered strains. This system is poised to serve as an effective tool for multi-gene expression and further genetic engineering modifications in P. brevicompactum. The study provides a new strategy for constructing more efficient 2A peptide multi-gene expression systems in Penicillium or filamentous fungi in future research endeavors.