Enhancing Cephalosporin C Biosynthesis through a 2A Peptide-Based Multigene Coexpression System

Cephalosporin C (CPC) is a natural product that serves as the key precursor for various semisynthetic cephalosporins. Its industrial production primarily relies on <i>Acremonium chrysogenum</i>. However, as the exclusive microbial strain used for large-scale CPC fermentation, <i>A. chrysogenum</i> faces challenges in metabolic engineering owing to the absence of an efficient multigene coexpression system. This study presented, for the first time, the development and application of a 2A peptide-based multigene coexpression system in <i>A. chrysogenum</i>. The self-cleavage efficiencies of ten virus-derived 2A peptides were systematically evaluated, ranging from 66.5% to 88.2%, with P2A showing the best performance. The most efficient P2A peptide was then selected, enabling both high- and low-level precalibrated expression based on the transcriptome data and supporting the coexpression of three genes in <i>A. chrysogenum</i>, including the driver gene. This system was subsequently applied to coexpress the key CPC biosynthetic genes, <i>cefEF</i> and <i>cefG</i>, with the driver gene of <i>ACRE_076110</i>, resulting in a significant 3.19-fold increase in CPC titer compared to the wild-type strain. Furthermore, the strategy was successfully extended to an industrial high-yield strain, with CPC titer increasing from 6.09 g/L to 7.45 g/L, receiving a 22.2% improvement. Overall, this study provides a valuable tool for metabolic engineering efforts aimed at enhancing CPC production in <i>A. chrysogenum</i>.