Microfluidic screening and whole-genome sequencing identifies mutations associated with improved protein secretion by yeast
Microfluidic screening and whole-genome sequencing identifies mutations associated with improved protein secretion by yeast
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DOI：
10.1073/pnas.1506460112
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发表时间：
2015-08-25
影响因子：
11.1
通讯作者：
Nielsen, Jens
中科院分区：
文献类型：
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作者：
Huang, Mingtao;Bai, Yunpeng;Nielsen, Jens
对基于生物技术生产重组蛋白质以用作食品和饲料行业以及工业应用中的药物的需求日益增加。酵母酿酒酵母是用于重组蛋白生产的优选细胞工厂之一，并且对改善其蛋白分泌能力的兴趣日益增加。由于真核细胞分泌机制的复杂性，很难应用合理的工程来构建改良菌株。在这里，我们使用高通量微流体筛选酵母文库，产生的紫外诱变。几轮筛选和分选导致选择具有显著改善的重组α-淀粉酶分泌的八个酵母克隆。有效分泌在所选克隆中是遗传稳定的。我们对这8个克隆进行了全基因组测序，总共鉴定了330个突变。对突变基因的基因本体分析揭示了许多生物学过程，包括一些以前在蛋白质分泌背景下未被识别的过程。在这项研究中发现的突变基因可以潜在地用于反向代谢工程，目的是构建高效的蛋白质分泌细胞工厂。微流体筛选和全基因组测序的组合使用，以映射与改进的表型相关的突变，可以很容易地适用于其他产品和细胞类型，以确定新的工程目标，这种方法可以广泛地促进新的细胞工厂的设计。
There is an increasing demand for biotech-based production of recombinant proteins for use as pharmaceuticals in the food and feed industry and in industrial applications. Yeast Saccharomyces cerevisiae is among preferred cell factories for recombinant protein production, and there is increasing interest in improving its protein secretion capacity. Due to the complexity of the secretory machinery in eukaryotic cells, it is difficult to apply rational engineering for construction of improved strains. Here we used high-throughput microfluidics for the screening of yeast libraries, generated by UV mutagenesis. Several screening and sorting rounds resulted in the selection of eight yeast clones with significantly improved secretion of recombinant a-amylase. Efficient secretion was genetically stable in the selected clones. We performed whole-genome sequencing of the eight clones and identified 330 mutations in total. Gene ontology analysis of mutated genes revealed many biological processes, including some that have not been identified before in the context of protein secretion. Mutated genes identified in this study can be potentially used for reverse metabolic engineering, with the objective to construct efficient cell factories for protein secretion. The combined use of microfluidics screening and whole-genome sequencing to map the mutations associated with the improved phenotype can easily be adapted for other products and cell types to identify novel engineering targets, and this approach could broadly facilitate design of novel cell factories.