Intron <i>RPS25Ai</i>, a Novel DNA Element, Has Global Effects on Synthetic Pathway Engineering by Empowering Protein Synthesis

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 <i>RPS25Ai</i> from <i>Saccharomyces cerevisiae</i>, the expression of <i>mCherry</i> 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 <i>RPS25Ai</i> was applied to a heterologous metabolic pathway in engineered yeast, increasing β-carotene production by 4.29-fold. <i>RPS25Ai</i> functioned as a multilevel regulatory genetic element, enabling the increase in the expression of <i>crtYB</i> 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, <i>RPS25Ai</i> 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.