Rhodobacter sphaeroides is a metabolically versatile bacterium that serves as a model for analysis of photosynthesis, hydrogen production and terpene biosynthesis. The elimination of by-products formation, such as poly-β-hydroxybutyrate (PHB), has been an important metabolic engineering target for R. sphaeroides. However, the lack of efficient markerless genome editing tools for R. sphaeroides is a bottleneck for fundamental studies and biotechnological exploitation. The Cas9 RNA-guided DNA-endonuclease from the type II CRISPR-Cas system of Streptococcus pyogenes (SpCas9) has been extensively employed for the development of genome engineering tools for prokaryotes and eukaryotes, but not for R. sphaeroides. Here we describe the development of a highly efficient SpCas9-based genomic DNA targeting system for R. sphaeroides, which we combine with plasmid-borne homologous recombination (HR) templates developing a Cas9-based markerless and time-effective genome editing tool. We further employ the tool for knocking-out the uracil phosphoribosyltransferase (upp) gene from the genome of R. sphaeroides, as well as knocking it back in while altering its start codon. These proof-of-principle processes resulted in editing efficiencies of up to 100% for the knock-out yet less than 15% for the knock-in. We subsequently employed the developed genome editing tool for the consecutive deletion of the two predicted acetoacetyl-CoA reductase genes phaB and phbB in the genome of R. sphaeroides. The culturing of the constructed knock-out strains under PHB producing conditions showed that PHB biosynthesis is supported only by PhaB, while the growth of the R. sphaeroides ΔphbB strains under the same conditions is only slightly affected. In this study, we combine the SpCas9 targeting activity with the native homologous recombination (HR) mechanism of R. sphaeroides for the development of a genome editing tool. We further employ the developed tool for the elucidation of the PHB production pathway of R. sphaeroides. We anticipate that the presented work will accelerate molecular research with R. sphaeroides.

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基于Cas9的高效球形红球菌基因组编辑，用于代谢工程

球形红细菌是一种代谢广泛的细菌，可作为分析光合作用，产氢和萜烯生物合成的模型。消除副产物形成，例如聚-β-羟基丁酸酯（PHB），已经成为球形红球菌的重要代谢工程目标。然而，缺乏有效的无球藻无标记基因组编辑工具是基础研究和生物技术开发的瓶颈。来自化脓性链球菌（SpCas9）的II型CRISPR-Cas系统的Cas9 RNA引导的DNA核酸内切酶已被广泛用于原核生物和真核生物的基因组工程工具的开发，但不用于球形芽孢杆菌的基因组工程工具的开发。在这里，我们描述了一种高效的基于SpCas9的基因组DNA靶向系统，用于球形拟南芥的开发，我们将其与质粒传播的同源重组（HR）模板相结合，开发了基于Cas9的无标记且省时的基因组编辑工具。我们进一步利用该工具从球形红球菌基因组中敲除尿嘧啶磷酸核糖转移酶（upp）基因，并在改变其起始密码子的同时将其敲除。这些原理验证过程导致剔除的编辑效率高达100％，而剔除的编辑效率却不到15％。我们随后采用了发达的基因组编辑工具，用于连续删除球形红球菌基因组中的两个预测的乙酰乙酰辅酶A还原酶基因phaB和phbB。在PHB产生条件下构建的敲除菌株的培养表明，PHB的生物合成仅由PhaB支持，而R的生长则由PhaB支持。在相同条件下，红球菌ΔphbB菌株仅受到轻微影响。在这项研究中，我们将SpCas9靶向活性与球形拟南芥的天然同源重组（HR）机制相结合，用于开发基因组编辑工具。我们进一步采用开发的工具来阐明球形红球菌的PHB产生途径。我们预计，目前的工作将加速球形红球菌的分子研究。