Center for Synthetic and Systems Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China.
Department of Chemistry, Tsinghua University, Beijing 100084, China.
Metab Eng. 2018 May;47:219-229. doi: 10.1016/j.ymben.2018.03.018. Epub 2018 Mar 30.
Extremophiles are suitable chassis for developing the next generation industrial biotechnology (NGIB) due to their resistance to microbial contamination. However, engineering extremophiles are not an easy task. Halomonas, an industrially interesting halophile able to grow under unsterile and continuous conditions in large-scale processes, can only be engineered using suicide plasmid-mediated two-step homologous recombination which is very laborious and time-consuming (up to half a year). A convenient approach for the engineering of halophiles that can possibly be extended to other extremophiles is therefore urgently required. To meet this requirement, a rapid, efficient and scarless method via CRISPR/Cas9 system was developed in this study for genome editing in Halomonas. The method achieved the highest efficiency of 100%. When eight different mutants were constructed via this special CRISPR/Cas9 method to study the combinatorial influences of four different genes on the glucose catabolism in H. bluephagenesis TD01, it took only three weeks to complete the deletion and insertion of up to 4.5 kb DNA. H. bluephagenesis was designed to produce a microbial copolymer P(3HB-co-3HV) consisting of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV). The CRISPR/Cas9 was employed to delete the prpC gene in H. bluephagenesis TD01. Shake flask studies showed that the 3HV fraction in the copolymers increased approximately 16-folds, demonstrating enhanced effectiveness of the ΔprpC mutant to synthesize PHBV. This genome engineering strategy significantly speeds up the studies on Halomonas engineering, opening up a wide area for developing NGIB.
极端微生物由于其对微生物污染的抗性，是开发下一代工业生物技术（NGIB）的合适底盘。然而，对极端微生物进行工程改造并非易事。盐单胞菌是一种具有工业应用价值的嗜盐菌，能够在大规模工艺中在未灭菌和连续条件下生长，但只能使用自杀质粒介导的两步同源重组进行工程改造，这非常繁琐和耗时（长达半年）。因此，迫切需要一种方便的方法来对盐单胞菌进行工程改造，该方法有可能扩展到其他极端微生物。为了满足这一要求，本研究开发了一种通过 CRISPR/Cas9 系统进行 Halomonas 基因组编辑的快速、高效、无疤痕的方法。该方法的效率最高可达 100%。当通过这种特殊的 CRISPR/Cas9 方法构建了八个不同的突变体，以研究四个不同基因对 H. bluephagenesis TD01 中葡萄糖代谢的组合影响时，仅用了三周时间就完成了长达 4.5kb 的 DNA 的缺失和插入。设计 H. bluephagenesis 生产由 3-羟基丁酸（3HB）和 3-羟基戊酸（3HV）组成的微生物共聚物 P(3HB-co-3HV)。CRISPR/Cas9 用于删除 H. bluephagenesis TD01 中的 prpC 基因。摇瓶研究表明，共聚物中的 3HV 分数增加了约 16 倍，表明 ΔprpC 突变体在合成 PHBV 方面的效果增强。这种基因组工程策略显著加快了对盐单胞菌工程的研究，为开发下一代工业生物技术开辟了广阔的领域。
