Phosphate-Responsive Promoter of a Pichia pastoris Sodium Phosphate Symporter
Phosphate-Responsive Promoter of a Pichia pastoris Sodium Phosphate Symporter
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DOI：
10.1128/aem.02913-08
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发表时间：
2009-06-01
影响因子：
4.4
通讯作者：
Lee, Hongweon
中科院分区：
文献类型：
--
作者：
Ahn, Jungoh;Hong, Jiyeon;Lee, Hongweon
为了在毕赤酵母中开发功能性磷酸盐调节启动子，分离了磷酸盐响应基因 P-HO89，该基因编码推定的钠 (Na+) 偶联磷酸盐同向转运蛋白。测序分析揭示了一个 1,731 bp 的开放阅读框，编码具有 12 个假定跨膜结构域的 576 个氨基酸的多肽。使用细菌脂肪酶基因作为报告基因，在 5 升罐发酵实验中研究了 PHO89 启动子 (P-PHO89) 的特性。 P-PHO89 受到磷酸盐的严格调控，当细胞在磷酸盐限制的外部环境中生长时，P-PHO89 会被高度激活。与翻译延伸因子1α和3-磷酸甘油醛脱氢酶启动子相比，P-PHO89表现出强大的转录活性和更高的比生产率（产生的脂肪酶量/细胞/小时）。此外，还开发了一种经济高效且简单的基于 P-PHO89 的发酵工艺，用于工业应用。这些结果证明了有效利用 P-PHO89 在毕赤酵母中控制重组蛋白生产的潜力。
To develop a functional phosphate-regulated promoter in Pichia pastoris, a phosphate-responsive gene, P-HO89, which encodes a putative sodium (Na+)-coupled phosphate symporter, was isolated. Sequencing analyses revealed a 1,731-bp open reading frame encoding a 576-amino-acid polypeptide with 12 putative transmembrane domains. The properties of the PHO89 promoter (P-PHO89) were investigated using a bacterial lipase gene as a reporter in 5-liter jar fermentation experiments. P-PHO89 was tightly regulated by phosphate and was highly activated when the cells were grown in a phosphate-limited external environment. Compared to translation elongation factor 1 alpha and the glyceraldehyde-3-phosphate dehydrogenase promoter, P-PHO89 exhibited strong transcriptional activity with higher specific productivity (amount of lipase produced/cell/h). Furthermore, a cost-effective and simple P-PHO89-based fermentation process was developed for industrial application. These results demonstrate the potential for efficient use of P-PHO89 for controlled production of recombinant proteins in P. pastoris.