Rütering Marius, Cress Brady F, Schilling Martin, Rühmann Broder, Koffas Mattheos A G, Sieber Volker, Schmid Jochen
Chair of Chemistry of Biogenic Resources, Technical University of Munich, Straubing, Germany.
Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, USA.
Synth Biol (Oxf). 2017 Dec 21;2(1):ysx007. doi: 10.1093/synbio/ysx007. eCollection 2017 Jan.
Application of state-of-the-art genome editing tools like CRISPR-Cas9 drastically increase the number of undomesticated micro-organisms amenable to highly efficient and rapid genetic engineering. Adaptation of these tools to new bacterial families can open up entirely new possibilities for these organisms to accelerate as biotechnologically relevant microbial factories, also making new products economically competitive. Here, we report the implementation of a CRISPR-Cas9 based vector system in , enabling fast and reliable genome editing in this host. Homology directed repair allows for highly efficient deletions of single genes and large regions as well as insertions. We used the system to investigate the yet undescribed biosynthesis machinery for exopolysaccharide (EPS) production in DSM 365, enabling assignment of putative roles to several genes involved in EPS biosynthesis. Using this simple gene deletion strategy, we generated EPS variants that differ from the wild-type polymer not only in terms of monomer composition, but also in terms of their rheological behavior. The developed CRISPR-Cas9 mediated engineering approach will significantly contribute to the understanding and utilization of socially and economically relevant species and extend the polymer portfolio.
应用诸如CRISPR-Cas9等最先进的基因组编辑工具，极大地增加了适合进行高效快速基因工程的未驯化微生物的数量。使这些工具适用于新的细菌家族，可为这些生物开辟全新的可能性，使其作为具有生物技术相关性的微生物工厂加速发展，也使新产品在经济上具有竞争力。在此，我们报告了一种基于CRISPR-Cas9的载体系统在[具体宿主]中的实施情况，该系统能够在该宿主中实现快速可靠的基因组编辑。同源定向修复允许高效删除单个基因和大片段区域以及进行插入操作。我们利用该系统研究了DSM 365中尚未描述的胞外多糖（EPS）生物合成机制，从而能够确定几个参与EPS生物合成的基因的推定作用。使用这种简单的基因删除策略，我们生成了EPS变体，这些变体不仅在单体组成方面与野生型聚合物不同，而且在流变行为方面也有所不同。所开发的CRISPR-Cas9介导的工程方法将对理解和利用具有社会和经济相关性的[具体物种]做出重大贡献，并扩展聚合物产品组合。
