Work

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

Year: 2025

Type: article

Abstract: 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 (0432 and Fluc) in the common expression chassis P. pastoris. 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 PGAP with Ppor1 or Prps8b. 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.

Source: Journal of Fungi

DOI: 10.3390/jof11040282

Authors: Chaoyu Lu, Linna Guo, Bohao Fang, Jiacheng Shi, Mian Zhou

Institutions: East China University of Science and Technology, State Key Laboratory of Bioreactor Engineering

Language: English

Topic: Fungal and yeast genetics research

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: gold

Estimated APC paid: $3,137

Funder: Natural Science Foundation of Shanghai