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首页> 外文期刊>Applied Microbiology and Biotechnology >Hybrid-architectured double-promoter expression systems enhance and upregulate-deregulated gene expressions inPichia pastorisin methanol-free media
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Hybrid-architectured double-promoter expression systems enhance and upregulate-deregulated gene expressions inPichia pastorisin methanol-free media

机译：杂交架构的双启动子表达系统增强和上调甲醇无甲醇介质中的令人讨厌的基因表达

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Double-promoter expression system (DPES) design as de novo metabolic engineering strategy enables fine-tuned and enhanced gene expression. We constructed a collection of monodirectional hybrid-architectured DPESs with engineered promoter variants P(ADH2-Cat8-L2)and P(mAOX1)and with the naturally occurring promoter P(GAP)to enhance and upregulate-deregulated gene expressions inPichia pastorisin methanol-free media. Reporter red fluorescent protein (mApple) and enhanced green fluorescent protein (eGFP) were expressed under P(ADH2-Cat8-L2)and P(mAOX1)or P-GAP, respectively, enabling the determination of the transcription period and strength of each constituent in the DPESs. We determined fluorescent protein expressions in batch cultivations on 2% (v/v) ethanol, excess glucose, and excess glycerol, and compared them with single-promoter expression systems constructed with PADH2-Cat8-L2, P-mAOX1, and P-GAP. The transcription- and expression-upregulation power of bifunctional DPESs was higher than that of twin DPESs (two-copy expression systems). Our findings answer long-standing questions regarding the high- (or multi-) copy clone results in the literature. Our first conclusion is that increasing identical components in the DPES architectures linearly increases the concentrations ofcis-acting DNA sites and increases the demand for key transcription factors (TFs) that perturb their good coupling of supply and demand. The next is that the synthesis of some amino acids may create bottleneck(s) as rate-limiting amino acid(s) in recombinant protein synthesis. With bifunctional DPESs, each constituent upregulated the transcription and increased the expression and reduced the demand for the same TF(s) in the generation of novel regulatory circuits, due to the increased number of nonidenticalcis-acting DNA sites. We tested superior DPES performances in extracellular human growth hormone (rhGH) production. Thereby, the indications related to the rate-limiting amino acids were verified. Compared with its constituents P(ADH2-Cat8-L2)and P-mAOX1, the bifunctional DPES(4)enhanced rhGH production by 1.44- and 2.02-fold, respectively. The DPES design method, with its constraint and parameters, enables the generation of promising r-protein production platforms with high impact on industrial-scale production processes and opens up new avenues for research in yeasts.
机译：De Novo代谢工程策略的双启动子表达系统（DPES）设计能够进行微调和增强的基因表达。我们构建了一系列单向杂种架构的DPES，具有工程化启动子变体P（ADH2-CAT8-L2）和P（MAOX1），并且具有天然存在的启动子P（间隙），以增强和上调令人讨厌的基因表达Inpichia Pastorisin甲醇的甲醇媒体。记者红荧光蛋白（Mapple）和增强的绿色荧光蛋白（EGFP）分别在P（ADH2-CAT8-L2）和P（MAOX1）或P差间隙下表达，从而能够测定每个组分的转录周期和强度在dpess。我们在2％（v / v）乙醇，过量葡萄糖和过量甘油上的分批培养中确定荧光蛋白表达，并将其与用PADH2-CAT8-L2，P-MAOX1和P-GAP构建的单启动子表达系统进行比较。双功能DPES的转录和表达 - 上调功率高于双DPESS（两拷贝表达系统）。我们的研究结果应对文献中的高（或多元）复制克隆产生的长期问题回答了重要的问题。我们的第一个结论是，增加DPES架构中的相同组分线性地增加了CIS作用DNA位点的浓度，并增加了对扰动其良好耦合供需耦合的关键转录因子（TFS）的需求。接下来的是，一些氨基酸的合成可以在重组蛋白合成中产生瓶颈作为速率限制氨基酸。对于双功能DPES，每个组成部分上调转录并增加了表达，并且由于非识别的基于次数的数量增加，对新型监管电路的产生中相同的TF的需求降低。我们在细胞外人生长激素（RHGH）生产中测试了优异的DPES性能。由此，验证了与速率限制氨基酸有关的适应症。与其成分P（ADH2-CAT8-L2）和P-MAOX1相比，双功能DPES（4）分别增强了rhGH产量1.44-和2.02倍。具有约束和参数的DPES设计方法使得能够产生具有高影响工业规模生产过程的r-蛋白生产平台，并开辟了酵母研究的新途径。

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