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 Acremonium chrysogenum. However, as the exclusive microbial strain used for large-scale CPC fermentation, A. chrysogenum 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 A. chrysogenum. 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 A. chrysogenum, including the driver gene. This system was subsequently applied to coexpress the key CPC biosynthetic genes, cefEF and cefG, with the driver gene of ACRE_076110, 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 A. chrysogenum.