Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, USA.
Waunakee High School, Waunakee, USA.
J Ind Microbiol Biotechnol. 2018 Jul;45(7):517-527. doi: 10.1007/s10295-017-2001-5. Epub 2018 Jan 3.
Pseudomonas putida is a promising bacterial host for producing natural products, such as polyketides and nonribosomal peptides. In these types of projects, researchers need a genetic toolbox consisting of plasmids, characterized promoters, and techniques for rapidly editing the genome. Past reports described constitutive promoter libraries, a suite of broad host range plasmids that replicate in P. putida, and genome-editing methods. To augment those tools, we have characterized a set of inducible promoters and discovered that IPTG-inducible promoter systems have poor dynamic range due to overexpression of the LacI repressor. By replacing the promoter driving lacI expression with weaker promoters, we increased the fold induction of an IPTG-inducible promoter in P. putida KT2440 to 80-fold. Upon discovering that gene expression from a plasmid was unpredictable when using a high-copy mutant of the BBR1 origin, we determined the copy numbers of several broad host range origins and found that plasmid copy numbers are significantly higher in P. putida KT2440 than in the synthetic biology workhorse, Escherichia coli. Lastly, we developed a λRed/Cas9 recombineering method in P. putida KT2440 using the genetic tools that we characterized. This method enabled the creation of scarless mutations without the need for performing classic two-step integration and marker removal protocols that depend on selection and counterselection genes. With the method, we generated four scarless deletions, three of which we were unable to create using a previously established genome-editing technique.
铜绿假单胞菌是一种很有前途的生产天然产物的细菌宿主，如聚酮和非核糖体肽。在这些类型的项目中，研究人员需要一个由质粒、特征启动子和快速编辑基因组的技术组成的遗传工具包。过去的报告描述了组成型启动子文库，这是一套在铜绿假单胞菌中复制的广谱宿主范围质粒，以及基因组编辑方法。为了扩充这些工具，我们对一组诱导型启动子进行了表征，并发现 IPTG 诱导型启动子系统由于 LacI 阻遏物的过表达而动态范围较差。通过用较弱的启动子替换驱动 lacI 表达的启动子，我们将 IPTG 诱导型启动子在铜绿假单胞菌 KT2440 中的诱导倍数提高到 80 倍。在发现使用 BBR1 原点的高拷贝突变体时，质粒基因表达不可预测后，我们确定了几种广谱宿主范围原点的拷贝数，并发现质粒拷贝数在铜绿假单胞菌 KT2440 中明显高于合成生物学的主要工具大肠杆菌。最后，我们使用我们所描述的遗传工具，在铜绿假单胞菌 KT2440 中开发了一种 λRed/Cas9 重组酶法。该方法能够在不需要进行依赖于选择和反选择基因的经典两步整合和标记去除方案的情况下，实现无痕突变。使用该方法，我们生成了四个无痕缺失，其中三个我们无法使用以前建立的基因组编辑技术来创建。
