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中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2023, Vol. 43 Issue (11): 78-91    DOI: 10.13523/j.cb.2307007
    
Research Progress of the Promoter Engineering in Saccharomyces cerevisiae
JIANG Hui-hui1,2,WANG Qiang2,RAO Zhi-ming2,ZHANG Xian2,**()
1 School of Biological and Environmental Engineering, Chaohu University, Hefei 238024, China
2 The Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China
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Abstract  

Promoter is the most important element of initiation gene transcription and its function is closely related to the gene expression level. The promoter engineering aims to study the functional modification and directed evolution of promoters, which can expand and deepen the application of Saccharomyces cerevisiae promoters in synthetic biology. Based on the structural characteristics of S. cerevisiae promoters, this review focuses on the strategies and applications of S. cerevisiae promoter engineering, including regulatory sequence knockout, random mutation of traditional promoters, saturated mutation, promoter hybridization, synthesis of minimum promoter skeleton, and modification of transcription factor binding sites. In addition, the latest progress of CRISPR/dCas9 and artificial intelligence tools in the field of S. cerevisiae promoter engineering is introduced. The future development prospects of promoter engineering in the field of synthetic biology are also discussed.

Key words： Saccharomyces cerevisiae      Transcriptional regulation      Promoter modification      CRISPR/dCas9      Artificial intelligence     
Received: 06 July 2023      Published: 01 December 2023
ZTFLH:  Q815  
Corresponding Authors: **Xian ZHANG     E-mail: zx@jiangnan.edu.cn
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Cite this article:

JIANG Hui-hui, WANG Qiang, RAO Zhi-ming, ZHANG Xian. Research Progress of the Promoter Engineering in Saccharomyces cerevisiae. China Biotechnology, 2023, 43(11): 78-91.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2307007     OR     https://manu60.magtech.com.cn/biotech/Y2023/V43/I11/78

Fig.1 Structure of initiating transcription elements in S. cerevisiae
启动子 启动子来源 英文全称 参考文献
组成型启动子
pPGK1 磷酸甘油酸激酶 Phosphoglycerate kinase [9,12-13]
pTEF1 转录延伸因子 Transcriptional elongation factor [10, 14, 15,16,17]
pCCW14 细胞壁甘露糖蛋白 Cell wall glycoprotein [18]
pHXT7 己糖转运蛋白 Hexose transporter [19]
pTPI 磷酸丙糖异构酶 Triose phosphate isomerase [19]
pFBA1 果糖-二磷酸醛缩酶 Fructose-bisphosphate aldolase [20]
pADH1 乙醇脱氢酶 Alcohol dehydrogenase [14]
pENO2 烯醇化酶II Gamma-enolase [21]
pPDC2 丙酮酸脱羧酶 Pyruvate decarboxylase [21]
pPFY1 细胞骨架组织蛋白脯氨酸 Cytoskeletal tissue protein proline [22]
pCYC1 细胞色素c Cytochrome c [23]
pTDH3 甘油醛-3-磷酸脱氢酶 Glyceraldehyde-3-phosphate dehydrogenase [24-25]
pExp1 跨膜输出蛋白 Expansin [26]
诱导型启动子
pGAL1 半乳糖激酶 Galactokinase [23, 27-28]
pGAL7 半乳糖转移酶 Hexose-1-phosphate uridylyltransferase [28]
pGAL10 半乳糖差向异构酶 Bifunctional UDP-glucose 4-epimerase/aldose 1-epimerase [29]
pGPD 3-磷酸甘油脱氢酶 Glycerol 3-phospahte dehydrogenase [23, 26, 30]
pCUP1 金属硫蛋白 Metallothionein [31]
pDAN1 DNA/RNA结合蛋白 Alba DNA/RNA-binding protein [24]
pPHO5 酸性磷酸酶 Acid phosphatase [32]
Table 1 Promoters of Saccharomyces cerevisiae commonly used in synthetic biology
Fig.2 The structure of the pGAL promoter and schematic diagram of transcriptional regulation ①Transcriptional activation: The transcriptional activator Gal4p binds to the upstream activator sequence of the pGAL promoter; ②Transcriptional repression: The negative regulator Gal80p directly binds to Gal4p and inhibits Gal4p function under non-galactose conditions; ③In the presence of galactose, the inhibitory effect of Gal80p on Gal4p is relieved by Gal3p[33]
Fig.3 Research of the promoter engineering in S. cerevisiae
应用分类 启动子 应用实例 参考文献
起始蛋白质表达 pHXT7 在重组酿酒酵母中起始戊二烯合成酶基因CnVS过表达,提高瓦伦烯产量 [19]
pPGK 组成性启动子pPGK驱动艾利希途径(Ehrlich pathway)中关键酶基因ARO8、ARO10和ADH2表达,提高2-苯乙醇产量 [12]
pGAL1 起始SARS-CoV-2刺突蛋白的全长受体结合域(RBD)表达,制备出基于S.cerevisiae的SARS-CoV-2疫苗 [27]
调控细胞代谢水平 pGAL 构建pGAL启动子的负/正转录调控回路,使用四环素触发对pGAL的抑制,升高温度触发pGAL的去抑制,并应用于酵母中异源倍半萜烯生产 [29]
开发与优化生物传感器 pPGK1 筛选出对葡萄糖二酸响应最显著的启动子pYCR012W(pPGK1),并通过截短改造确定相应的转录因子,用于构建酿酒酵母葡萄糖二酸生物传感器 [13]
pCCW14 通过修改启动子上游激活序列中的转录因子结合位点,实现启动子pCCW14对低pH的响应 [18]
增强宿主细胞对环境的耐受性 pFBA1 从嗜热栖热菌和酿酒酵母中挖掘出13种潜在的抗氧化耐热功能基因,分别与酿酒酵母强组成型启动子pFBA1重组,构建出基于抗氧化防御系统的耐热性酿酒酵母 [20]
pCYR1 通过改变腺苷酸环化酶编码基因CYR1启动子的长度,实现微调控CYR1基因的表达水平,使酿酒酵母细胞具有良好耐热和耐乙醇性能 [34]
Table 2 Applications of Saccharomyces cerevisiae promoter in the field of synthetic biology
方法 改造策略 优点 缺点 参考文献
调控序列敲除 利用同源重组或其他手段使启动子特定的调控序列失活或缺失 技术成熟,改造针对性强 需要对目标功能序列了解清楚,仅适用于诱导型启动子 [38-39]
传统启动子随机突变 通过易错PCR等随机改变传统启动子区的个别碱基 技术成熟,可批量处理,改造后启动子活性范围广 实验结果不可预测,突变后筛选工作量很大 [15,21,40-41]
饱和突变 利用简并引物针对启动子间隔区序列进行改造 可以半理性的对启动子非保守区进行定向设计改造 需要对目标启动子序列的保守区和间隔区进行区分,筛选工作量较大 [22, 31]
启动子杂交 将核心启动子序列与新增强子元件组合,调节并增强启动子活性 可根据需要选择增强子,以单个序列或多个序列串联的方式重组,改造后转录活性可控 需要建立杂交启动子库,实验工作量大,难以实现高通量筛选 [16,23-24,42-47]
合成最小化启动子骨架 敲除或替换内源启动子中非必需碱基 具有最小功能单位的启动子骨架,可以满足大规模合成生物学需要 对研究人员专业要求较高,实验前的序列分析工作量大 [17,26,32,48]
转录因子结合位点的修饰和改造 新增外源转录因子结合位点,或对原有结合位点进行突变或替换 可以结合转录因子的定制和修饰,来定向改变天然启动子功能和表达水平。 真核转录因子参与调控的过程复杂,难以实现动态精确调控 [18,31,46]
Table 3 Comparison of methods for promoter modification and directed evolution
Fig.4 Schematic diagram of regulation by CRISPR/dCas9 A. Artificially synthesized of gRNA B. Fusion of dCas9 with specific transcription factors C. Complex formation with dCas9 and gRNA D. The dCas9 protein guides the complex to the vicinity of the PAM site, gRNA is precisely located by base complementarity, and transcription factors participate in transcriptional regulation
Fig.5 Schematic diagram of machine learning for the engineering of promoters in S.cerevisiae A. Model selection B. Feature extraction C. Database building and trainin D. Prediction and validation E. Rational design of artificial promoters
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