Abstract
As a fundamental tool in synthetic biology, promoters are pivotal in regulating gene expression, enabling precise genetic control and spurring innovation across diverse biotechnological applications. However, most advances in engineered genetic systems rely on host-specific regulation of the genetic portion. With the burgeoning diversity of synthetic biology chassis cells, there emerges a pressing necessity to broaden the universal promoter toolkit spectrum, ensuring adaptability across various microbial chassis cells for enhanced applicability and customization in the evolving landscape of synthetic biology. In this study, we analyzed and validated the primary structures of natural endogenous promoters from Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Saccharomyces cerevisiae, and Pichia pastoris, and through strategic integration and rational modification of promoter motifs, we developed a series of cross-species promoters (P(sh)) with transcriptional activity in five strains (prokaryotic and eukaryotic). This series of cross species promoters can significantly expand the synthetic biology promoter toolkit while providing a foundation and inspiration for standardized development of universal components The combinatorial use of key elements from prokaryotic and eukaryotic promoters presented in this study represents a novel strategy that may offer new insights and methods for future advancements in promoter engineering.