Nucleotide distribution analysis of 5'UTRs in genome-scale directs their redesign and expression regulation in yeast

Non-conventional yeasts have emerged as important sources of valuable products in bioindustries. However, tools for the control of expression are limited in these hosts. In this study, we aimed to excavate the tools for the regulation of translation that are often overlooked. 5'UTR analysis of genome-scale annotated genes of four yeast species revealed a distinct decreasing 'G' frequency in -100 ∼ -1 region from 5040 5'UTRs in Komagataella phaffii. New 5'UTRs were regenerated by base substitutions in defined regions, and replacement of 'G' by 'A' or 'T' in the -50 ∼ -1 region highly facilitated gene expression. Preference analysis of all nucleotide triplets in 5'UTRs revealed a KZ₃ (-3 ∼ -1) that dominantly affected gene expression. A total of 128 KZ₃ variants were constructed to work with promoters of methanol-inducible P_AOX1 and constitutive P_GAP, of which 58 KZ₃ variants increased gene expression and maximum difference in strength was 15-fold among all variants. Polysome profiling analysis clarified that 5'UTR-KZ₃ enhanced gene expression at translational but not transcriptional levels. Finally, improved production of three industrial proteins and one platform compound were achieved by ready-made 5'UTR-KZ₃ or in situ modification of the 5'UTR. This study provides new references and tools for the fine-tuning of translational regulation in yeast and other fungi.