Abstract

Background: The fungusAcremonium chrysogenumis crucial for producing cephalosporin antibiotics. While CRISPR/Cas9 has been developed for this species, it has not been applied to first-line industrial strains, to the best of our knowledge. For example, engineering industrialA. chrysogenumfor overproducing deacetoxycephalosporin C (DAOC, an important precursor for clinically used cephalosporin antibiotics) is currently often a multi-step and inefficient process. Methods: Here, we applied CRISPR/Cas9 to create a DAOC overproducer in a single step. Our method uses a donor template to simultaneously delete and overexpress genes, offering a simple, efficient, and time-saving solution. Results: Furthermore, through methodological optimization, the final homozygous multigene-edited strain achieved an industrial-scale DAOC titer of 12.4 ± 0.2 g/L, representing a significant improvement over the initial edited strain (7.2 ± 0.22 g/L). Conclusions: This demonstrates that CRISPR/Cas9 can effectively edit industrialA. chrysogenum, providing a strategy to enhance the production of other cephalosporin precursors.