Promoter screening and identification for metabolic regulation in Acremonium chrysogenum

Acremonium chrysogenum is the major industrial producer of cephalosporin C (CPC), which is used as raw material for the production of significant cephalosporin antibiotics. Due to the lack of diverse promoter elements, the development of metabolic engineering transformation is relatively slow, resulting in a limited improvement on CPC production. In this study, based on the analysis of the transcriptome profile, 27 candidate promoters were selected to drive the expression of the reporter genes. The promoter activities of this library ranged from 0.0075 to 101 times of the control promoter PAngpdA. Simultaneously, a rapid screening method for potential bidirectional promoters was developed and 4 strong bidirectional promoters from 27 candidate options were identified and validated. Finally, the Golden Gate method was employed to combine promoter modules from the library with various target genes. Through a mixed transformation and screening process, high‐yielding strains AG‐6, AG‐18, and AG‐41 were identified, exhibiting an increase in CPC production of 30%, 35%, and 29%, respectively, compared to the control strain Ac‐∆axl2:: eGFP. Therefore, the utilization of this promoter library offers a broader range of synthetic biology toolkits for the genetic engineering transformation of A. chrysogenum, thus establishing a solid foundation for the precise regulation of gene expression. Graphical and Lay Summary Acremonium chrysogenum, a key producer of cephalosporin C (CPC), faces challenges in metabolic engineering due to limited promoter diversity. This study addressed these limitations by identifying and validating 27 candidate promoters, along with four strong bidirectional ones through transcriptome analysis. By combining these promoters with Golden Gate assembly, we achieved random combinations of genetic elements and enhance CPC production, paving the way for future genetic engineering advancements in A. chrysogenum.