Production of recombinant proteins is regarded as an important breakthrough in the field of biomedicine and industrial biotechnology. Due to the complexity of the protein secretory pathway and its tight interaction with cellular metabolism, the application of traditional metabolic engineering tools to improve recombinant protein production faces major challenges. A systematic approach is required to generate novel design principles for superior protein secretion cell factories. Here, we applied a proteome-constrained genome-scale protein secretory model of the yeast Saccharomyces cerevisiae (pcSecYeast) to simulate α-amylase production under limited secretory capacity and predict gene targets for downregulation and upregulation to improve α-amylase production. The predicted targets were evaluated using high-throughput screening of specifically designed CRISPR interference/activation (CRISPRi/a) libraries and droplet microfluidics screening. From each library, 200 and 190 sorted clones, respectively, were manually verified. Out of them, 50% of predicted downregulation targets and 34.6% predicted upregulation targets were confirmed to improve α-amylase production. By simultaneously fine-tuning the expression of three genes in central carbon metabolism, i.e. LPD1, MDH1, and ACS1, we were able to increase the carbon flux in the fermentative pathway and α-amylase production. This study exemplifies how model-based predictions can be rapidly validated via a high-throughput screening approach. Our findings highlight novel engineering targets for cell factories and furthermore shed light on the connectivity between recombinant protein production and central carbon metabolism.

中文翻译：

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模型辅助 CRISPRi/a 文库筛选揭示了增强酵母中重组蛋白生产的核心碳代谢靶标


重组蛋白的生产被认为是生物医学和工业生物技术领域的重要突破。由于蛋白质分泌途径的复杂性及其与细胞代谢的紧密相互作用，应用传统的代谢工程工具改善重组蛋白生产面临重大挑战。需要一种系统的方法为优质蛋白质分泌细胞工厂生成新的设计原则。在这里，我们应用了酵母酿酒酵母 （pcSecYeast） 的蛋白质组约束基因组规模蛋白质分泌模型来模拟有限分泌能力下的 α-淀粉酶生产，并预测下调和上调的基因靶标，以提高 α-淀粉酶的产生。使用专门设计的 CRISPR 干扰/激活 （CRISPRi/a） 文库的高通量筛选和液滴微流控筛选来评估预测靶标。从每个文库中，分别手动验证 200 和 190 个分选克隆。其中，50% 的预测下调靶点和 34.6% 的预测上调靶点被证实可以改善 α-淀粉酶的产生。通过同时微调中央碳代谢中 LPD1 、 MDH1 和 ACS1 三个基因的表达，我们能够增加发酵途径中的碳通量和 α-淀粉酶的产生。这项研究举例说明了如何通过高通量筛选方法快速验证基于模型的预测。我们的研究结果突出了细胞工厂的新工程靶点，并进一步阐明了重组蛋白生产和中心碳代谢之间的联系。