A fragment in the 5′ untranslated region of alcohol oxidase 1 promoter significantly influencing gene expression of Komagataella phaffii

IF 2.4 3区生物学 Q2 GENETICS & HEREDITY

Gene Pub Date : 2025-09-01 DOI:10.1016/j.gene.2025.149745

Aibo Feng, Ziwei Zhou, Wenjie Cong, Hualan Zhou, Jianguo Zhang

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Abstract

Komagataella phaffii is a famous microbial host in synthetic biology field for expressing heterologous proteins under alcohol oxidase 1 promoter (P_AOX1). This study undertook two successive rounds of gene fragment knockout, identifying a 13 bp segment, termed D3, as a negative cis-acting element in the 5′ untranslated region of P_AOX1. Notably, upon knockout of the D3 fragment, the expression of green fluorescent protein (GFP), serving as the model for heterologous protein expression, improved by 3.5-fold due to enhanced transcription levels. Subsequent transcriptomic analysis, electrophoretic mobility shift assay and DNA pull-down assays coupled with liquid chromatography-mass spectrometry corroborated efficient GFP expression, highlighting minimal alterations in transcriptional machinery subunits, 312 differentially expressed genes and non-specific interactions between nucleoproteins and P_AOX1 following the D3 fragment knockout. Consequently, D3 fragment knock out from P_AOX1 facilitates efficient expression of heterologous protein by K. phaffii.

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酒精氧化酶1启动子5'非翻译区片段显著影响法菲小松菌基因表达。

法菲Komagataella phaffii是合成生物学领域著名的在乙醇氧化酶1启动子（PAOX1）下表达异源蛋白的微生物宿主。本研究进行了连续两轮基因片段敲除，确定了一个13 bp的片段，称为D3，是PAOX1的5'非翻译区中的负顺式作用元件。值得注意的是，在敲除D3片段后，作为异源蛋白表达模型的绿色荧光蛋白（GFP）的表达由于转录水平的提高而提高了3.5倍。随后的转录组学分析、电泳迁移率转移试验和DNA下拉试验结合液相色谱-质谱法证实了高效的GFP表达，强调了转录机制亚基、312个差异表达基因以及在D3片段敲除后核蛋白与PAOX1之间的非特异性相互作用的微小变化。因此，敲除PAOX1的D3片段有助于K. phaffii高效表达异源蛋白。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Gene 生物-遗传学

CiteScore

6.10

自引率

2.90%

发文量

718

审稿时长

42 days

期刊介绍： Gene publishes papers that focus on the regulation, expression, function and evolution of genes in all biological contexts, including all prokaryotic and eukaryotic organisms, as well as viruses.