Stovicek Vratislav, Borodina Irina, Forster Jochen
The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Allé 6, 2970 Hørsholm, Denmark.
Metab Eng Commun. 2015 Mar 20;2:13-22. doi: 10.1016/j.meteno.2015.03.001. eCollection 2015 Dec.
There is a demand to develop 3rd generation biorefineries that integrate energy production with the production of higher value chemicals from renewable feedstocks. Here, robust and stress-tolerant industrial strains of will be suitable production organisms. However, their genetic manipulation is challenging, as they are usually diploid or polyploid. Therefore, there is a need to develop more efficient genetic engineering tools. We applied a CRISPR-Cas9 system for genome editing of different industrial strains, and show simultaneous disruption of two alleles of a gene in several unrelated strains with the efficiency ranging between 65% and 78%. We also achieved simultaneous disruption and knock-in of a reporter gene, and demonstrate the applicability of the method by designing lactic acid-producing strains in a single transformation event, where insertion of a heterologous gene and disruption of two endogenous genes occurred simultaneously. Our study provides a foundation for efficient engineering of industrial yeast cell factories.
开发第三代生物精炼厂的需求日益增长，这种生物精炼厂能将能源生产与利用可再生原料生产高价值化学品相结合。在此，强壮且耐胁迫的工业菌株将是合适的生产生物体。然而，对它们进行基因操作具有挑战性，因为它们通常是二倍体或多倍体。因此，需要开发更高效的基因工程工具。我们应用CRISPR - Cas9系统对不同工业菌株进行基因组编辑，并在几种不相关的菌株中实现了一个基因的两个等位基因的同时破坏，效率在65%至78%之间。我们还实现了报告基因的同时破坏和敲入，并通过在单个转化事件中设计产乳酸菌株证明了该方法的适用性，在此过程中，一个异源基因的插入和两个内源基因的破坏同时发生。我们的研究为工业酵母细胞工厂的高效工程改造奠定了基础。
