RNA polymerase II-driven CRISPR-Cas9 system for efficient non-growth-biased metabolic engineering of Kluyveromyces marxianus

IF 3.7 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Danielle Bever , Ian Wheeldon , Nancy Da Silva
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引用次数: 2

Abstract

The thermotolerant yeast Kluyveromyces marxianus 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 K. marxianus, 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 K. marxianus 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 K. marxianus 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 K. marxianus 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.

RNA聚合酶ii驱动的CRISPR-Cas9系统高效非生长偏倚克卢维酵母代谢工程
近年来,耐热酵母菌马氏克鲁维菌(Kluyveromyces marxianus)作为一种有前景的工业生物制造微生物候选者受到了极大的关注。尽管对K. marxianus的基因表达和代谢工程的分子工具箱有了一些贡献,但仍然需要一个更高效、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,该系统可以在NHEJ功能性和非功能性马氏k.m arxianus菌株中使用仅40 bp的同源臂进行高效的无标记基因破坏和整合。使用强大的RNA聚合酶II启动子,可以从一个共表达密码子优化的Cas9的质粒上有效地表达带有自切割RNA结构、tRNA和HDV核酶的gRNAs。该系统对nhej功能菌株和nhej缺陷菌株的基因破坏效率均接近100%,两株菌株的供体整合效率分别达到50%和100%。高gRNA靶向性能也被证明有助于选择生长速度较低但通过避免延长生长周期而改善聚酮生物合成的工程菌株,这是一种用于富集编辑细胞的常用方法,但无法恢复适应性略有受损的有利突变体。最后,我们首次展示了利用2.6 kb和7.6 kb的供体在马氏K. marxianus的多个位点上同时进行无标记整合,在缺乏nhej的菌株中实现了25.5%的双整合效率。这些结果突出了该系统的易用性和总体稳健性,可用于这种非常规酵母的快速和灵活的代谢工程。
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来源期刊
Metabolic Engineering Communications
Metabolic Engineering Communications Medicine-Endocrinology, Diabetes and Metabolism
CiteScore
13.30
自引率
1.90%
发文量
22
审稿时长
18 weeks
期刊介绍: Metabolic Engineering Communications, a companion title to Metabolic Engineering (MBE), is devoted to publishing original research in the areas of metabolic engineering, synthetic biology, computational biology and systems biology for problems related to metabolism and the engineering of metabolism for the production of fuels, chemicals, and pharmaceuticals. The journal will carry articles on the design, construction, and analysis of biological systems ranging from pathway components to biological complexes and genomes (including genomic, analytical and bioinformatics methods) in suitable host cells to allow them to produce novel compounds of industrial and medical interest. Demonstrations of regulatory designs and synthetic circuits that alter the performance of biochemical pathways and cellular processes will also be presented. Metabolic Engineering Communications complements MBE by publishing articles that are either shorter than those published in the full journal, or which describe key elements of larger metabolic engineering efforts.
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