使用异源 GAL 启动子和转录激活子在酿酒酵母中进行高效表达,ACS Synthetic Biology

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使用异源 GAL 启动子和转录激活子在酿酒酵母中进行高效表达
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2023-05-24 , DOI: 10.1021/acssynbio.3c00243
Yanling Wu _{1,

2,

3,

4} , Jiliang Deng ₄ , Zhaohui Zheng ₄ , Nanzhu Chen ₄ , Xiaozhou Luo _{1,

2,

3,

4} , Hongting Tang _{1,

3,

4}

Affiliation

半乳糖诱导型 ( GAL ) 启动子已广泛应用于酿酒酵母的代谢工程中，以生产有价值的产品。内源GAL启动子和GAL转录因子通常经过改造以提高GAL启动子活性。异源GAL启动子和GAL激活子（Gal4p 样转录激活子）虽然存在于其他酵母或真菌中，但尚未得到充分研究。在这项研究中，我们全面表征了来自不同酵母或真菌的 Gal4p 激活剂对GAL启动子变体的激活作用。_{P HHF1}驱动的内源 Gal4p 过表达使天然 P _GAL1和异源 P _{Sk GAL2}的活性分别增加了 131.20% 和 72.45%。此外，还对来自不同生物体的八种转录激活因子进行了表征，其中大多数表现出与Sc Gal4p 一致的功能。与Sc Gal4p表达相比，来自乳酸克鲁维酵母的Kl Lac9p的表达进一步使P _Sc_GAL1和P _Sk_GAL2的活性分别增加41.56%和100.63%，并且能够逃避Gal80p抑制。这种优化的GAL表达系统可用于将酿酒酵母中 β-胡萝卜素的产量提高 9.02 倍。我们的研究表明，异源转录激活子和GAL启动子的组合为GAL表达系统的优化提供了新的见解。

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Engineering an Efficient Expression Using Heterologous GAL Promoters and Transcriptional Activators in Saccharomyces cerevisiae

Galactose-inducible (GAL) promoters have been widely used in metabolic engineering in Saccharomyces cerevisiae for production of valuable products. Endogenous GAL promoters and GAL transcription factors have often been engineered to improve GAL promoter activities. Heterologous GAL promoters and GAL activator (Gal4p-like transcriptional activators), although existing in other yeasts or fungi, have not been well explored. In this study, we comprehensively characterized the activation effects of Gal4p activators from different yeasts or fungi on a variant of GAL promoters. Overexpressing endogenous Gal4p driven by P_HHF1 increased the activities of native P_GAL1 and heterologous P_SkGAL2 by 131.20% and 72.45%, respectively. Furthermore, eight transcriptional activators from different organisms were characterized and most of them exhibited functions that were consistent with ScGal4p. Expression of KlLac9p from Kluyveromyces lactis further increased the activity of P_ScGAL1 and P_SkGAL2 by 41.56% and 100.63%, respectively, compared to ScGal4p expression, and was able to evade Gal80p inhibition. This optimized GAL expression system can be used to increase the production of β-carotene by 9.02-fold in S. cerevisiae. Our study demonstrated that a combination of heterologous transcriptional activators and GAL promoters provided novel insights into the optimization of the GAL expression system.

更新日期：2023-05-24

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