Intron RPS25Ai, a Novel DNA Element, Has Global Effects on Synthetic Pathway Engineering by Empowering Protein...

Classical genetic components in synthetic biology encompass essential elements of promoters, transcription factors, protein-coding genes, and terminators while both academic and industrial needs require novel engineering tools. Our study explores the potential of introns as versatile, novel biological DNA elements. Using intron RPS25Ai from Saccharomyces cerevisiae, the expression of mCherry was enhanced by 18.4-fold, demonstrating spatiotemporal regulatory patterns at both transcriptional and translational levels. A molecular mechanism study shows that this distinctive fine-tuning control relies on correct splicing events and extends to post-transcriptional processes. Intron RPS25Ai was applied to a heterologous metabolic pathway in engineered yeast, increasing β-carotene production by 4.29-fold. RPS25Ai functioned as a multilevel regulatory genetic element, enabling the increase in the expression of crtYB both at the pre-mRNA (99%) and mature RNA level (64%), with a splicing efficiency of 82%. Furthermore, the intron-engineered strain achieved a genome-scale regulation, upregulating 67% of "intron-containing" genes, with an average expression increase of 27%, compared with the upregulation of only 37% of "no-intron" genes. In addition, RPS25Ai induced a comprehensive rearrangement of ribosomal components, with the expression of 89% of ribosomal genes being upregulated, further empowering protein synthesis in the β-carotene-producing yeast cell factory.