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 KZ