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

Classical genetic components in synthetic biology encompass\nessential\nelements of promoters, transcription factors, protein-coding genes,\nand terminators while both academic and industrial needs require novel\nengineering tools. Our study explores the potential of introns as\nversatile, novel biological DNA elements. Using intron <i>RPS25Ai</i> from Saccharomyces cerevisiae, the\nexpression of <i>mCherry</i> was enhanced by 18.4-fold,\ndemonstrating spatiotemporal regulatory patterns at both transcriptional\nand translational levels. A molecular mechanism study shows that this\ndistinctive fine-tuning control relies on correct splicing events\nand extends to post-transcriptional processes. Intron <i>RPS25Ai</i> was applied to a heterologous metabolic pathway in engineered yeast,\nincreasing β-carotene production by 4.29-fold. <i>RPS25Ai</i> functioned as a multilevel regulatory genetic element, enabling\nthe increase in the expression of <i>crtYB</i> both at the\npre-mRNA (99%) and mature RNA level (64%), with a splicing efficiency\nof 82%. Furthermore, the intron-engineered strain achieved a genome-scale\nregulation, upregulating 67% of “intron-containing”\ngenes, with an average expression increase of 27%, compared with the\nupregulation of only 37% of “no-intron” genes. In addition, <i>RPS25Ai</i> induced a comprehensive rearrangement of ribosomal\ncomponents, with the expression of 89% of ribosomal genes being upregulated,\nfurther empowering protein synthesis in the β-carotene-producing\nyeast cell factory.