Bever Danielle, Wheeldon Ian, Da Silva Nancy
Department of Chemical and Biomolecular Engineering, University of California, Irvine, CA, 92697, USA.
Department of Chemical and Environmental Engineering, University of California, Riverside, CA, 92521, USA.
Metab Eng Commun. 2022 Sep 24;15:e00208. doi: 10.1016/j.mec.2022.e00208. eCollection 2022 Dec.
The thermotolerant yeast has gained significant attention in recent years as a promising microbial candidate for industrial biomanufacturing. Despite several contributions to the expanding molecular toolbox for gene expression and metabolic engineering of , there remains a need for a more efficient and versatile genome editing platform. To address this, we developed a CRISPR-based editing system that enables high efficiency marker-less gene disruptions and integrations using only 40 bp homology arms in NHEJ functional and non-functional strains. The use of a strong RNA polymerase II promoter allows efficient expression of gRNAs flanked by the self-cleaving RNA structures, tRNA and HDV ribozyme, from a single plasmid co-expressing a codon optimized Cas9. Implementing this system resulted in nearly 100% efficiency of gene disruptions in both NHEJ-functional and NHEJ-deficient strains, with donor integration efficiencies reaching 50% and 100% in the two strains, respectively. The high gRNA targeting performance also proved instrumental for selection of engineered strains with lower growth rate but improved polyketide biosynthesis by avoiding an extended outgrowth period, a common method used to enrich for edited cells but that fails to recover advantageous mutants with even slightly impaired fitness. Finally, we provide the first demonstration of simultaneous, markerless integrations at multiple loci in using a 2.6 kb and a 7.6 kb donor, achieving a dual integration efficiency of 25.5% in a NHEJ-deficient strain. These results highlight both the ease of use and general robustness of this system for rapid and flexible metabolic engineering in this non-conventional yeast.
近年来，耐热酵母作为工业生物制造中一个有前景的微生物候选者受到了广泛关注。尽管在扩展用于基因表达和代谢工程的分子工具包方面做出了一些贡献，但仍需要一个更高效、更通用的基因组编辑平台。为了解决这个问题，我们开发了一种基于CRISPR的编辑系统，该系统能够在NHEJ功能型和非功能型菌株中仅使用40 bp的同源臂实现高效的无标记基因破坏和整合。使用强RNA聚合酶II启动子可以从共表达密码子优化的Cas9的单个质粒中高效表达两侧带有自我切割RNA结构、tRNA和HDV核酶的gRNA。实施该系统后，在NHEJ功能型和NHEJ缺陷型菌株中基因破坏效率均接近100%，在这两种菌株中供体整合效率分别达到50%和100%。高gRNA靶向性能还被证明有助于选择生长速率较低但聚酮化合物生物合成得到改善的工程菌株，通过避免延长的生长后期，这是一种用于富集编辑细胞的常用方法，但无法回收适应性略有受损的有利突变体。最后，我们首次证明了使用2.6 kb和7.6 kb的供体在多个位点同时进行无标记整合，在NHEJ缺陷型菌株中实现了25.5%的双整合效率。这些结果突出了该系统在这种非常规酵母中进行快速灵活的代谢工程时的易用性和总体稳健性。
