Zhou Hangcheng, Wang Zhipeng, Qian Jiangchao
State Key Laboratory of Bioreactor Engineering, East China University of Science & Technology, 130 Meilong Road, Shanghai 200237, PR China.
State Key Laboratory of Bioreactor Engineering, East China University of Science & Technology, 130 Meilong Road, Shanghai 200237, PR China.
J Biotechnol. 2023 Nov 10;376:24-32. doi: 10.1016/j.jbiotec.2023.09.002. Epub 2023 Sep 9.
Hypoxia-inducible promoters of a wide range of activities are desirable for fine-tuning gene expression in response to oxygen limitation, especially for the Crabtree negative yeast Pichia pastoris (Komagataella phaffii) with a high oxygen consumption rate in large-scale fermentations. Here we constructed a hypoxia-inducible promoter library for P. pastoris through error-prone PCR of Pichia stipitis ADH2 promoter (Ps). The library of 30 selected promoters showing 0.4- to 5.5-fold of the Ps activity was obtained through high-throughput screening in microplates using the reporter yeast-enhanced green fluorescent protein. Two strong promoters, AM23 and AM30, were further characterized in shake flask cultures at high and low dissolved oxygen levels. They responded more sensitively to the low dissolved oxygen level, achieving a 4.6-, 7.9-fold and 3.6-, 7.7-fold higher fluorescence intensity and transcript level, respectively, than the wild-type Ps. Their hypoxia-inducible properties were confirmed with two additional reporters: β-galactosidase and Vitreoscilla hemoglobin, to demonstrate the broad applicability of the promoter library. During the typical fermentation process in shake flasks, the promoter AM30 showed strong expression with cell growth and decreased oxygen levels, without any additional chemical inducers or operations. Since the potent industrial host P. pastoris is recognized as an easy to scale-up system, it is reasonable to expect that the obtained hypoxia-inducible promoter library may have great potential to enable convenient regulation of gene expression under industrial fermentations which are usually run under oxygen limitation due to high cell density cultivations.
广泛活性的缺氧诱导型启动子对于响应氧限制来微调基因表达是很有必要的，特别是对于在大规模发酵中具有高耗氧率的克勒克酵母阴性酵母毕赤酵母（Komagataella phaffii）。在这里，我们通过对树干毕赤酵母ADH2启动子（Ps）进行易错PCR，构建了毕赤酵母的缺氧诱导型启动子文库。通过使用报告基因酵母增强绿色荧光蛋白在微孔板中进行高通量筛选，获得了30个选定启动子的文库，其活性为Ps活性的0.4至5.5倍。在高低溶解氧水平的摇瓶培养中进一步表征了两个强启动子AM23和AM30。它们对低溶解氧水平的反应更敏感，荧光强度和转录水平分别比野生型Ps高4.6倍、7.9倍和3.6倍、7.7倍。用另外两个报告基因β-半乳糖苷酶和透明颤菌血红蛋白证实了它们的缺氧诱导特性，以证明启动子文库的广泛适用性。在摇瓶中的典型发酵过程中，启动子AM30在细胞生长和氧水平降低时表现出强表达，无需任何额外的化学诱导剂或操作。由于强大的工业宿主毕赤酵母被认为是一个易于放大的系统，因此可以合理预期，所获得的缺氧诱导型启动子文库可能具有巨大潜力，能够在通常由于高细胞密度培养而在氧限制下进行的工业发酵中方便地调节基因表达。
