DNA Sequence Changes Resulting from Codon Optimization Affect Gene Expression in Pichia pastoris by Altering Chromatin Accessibility

Codon optimization is a widely employed strategy to enhance protein expression. However, it occasionally leads to unexpected transcriptional repression despite preserving amino acid sequences. This study investigates the mechanistic basis of such transcriptional attenuation by analyzing two gene candidates (<i>0432</i> and <i>Fluc</i>) in the common expression chassis <i>P. pastoris</i>. Both genes experienced severe mRNA reduction following codon optimization. Evidenced by histone H3 chromatin immunoprecipitation (ChIP) and a DNase I hypersensitivity assay, gene sequences with transcriptional repression displayed elevated nucleosome occupancy and reduced chromatin accessibility. The above change was caused by an ORF sequence change independent of the promoter, since transcriptional attenuation and compromised chromatin accessibility were still observed after replacing the strong promoter P<i>_GAP</i> with P<i>_por1</i> or P<i>_rps8b</i>. Our findings challenge the conventional view of codon optimization as solely translation-centric, revealing its capacity to preemptively modulate transcription through chromatin accessibility. This work underscores the necessity of integrating chromatin-level considerations into synthetic gene design to avoid unintended transcriptional silencing and optimize expression outcomes.