Bian Xiaoying, Plaza Alberto, Yan Fu, Zhang Youming, Müller Rolf
Department of Microbial Natural Products, Helmholtz-Institute for Pharmaceutical Research Saarland, Helmholtz Centre for Infection Research, Campus C2 3, 66123, Saarbrücken, Germany.
Department of Pharmaceutical Biotechnology, Saarland University, Campus C2 3, 66123, Saarbrücken, Germany.
Biotechnol Bioeng. 2015 Jul;112(7):1343-53. doi: 10.1002/bit.25560. Epub 2015 Mar 2.
Cloning and engineering of natural product biosynthetic pathways followed by heterologous expression in a tractable host is a widely used approach for expression and genetic modification of microbial secondary metabolites. Herein, we employed ccdB counterselection combined with oligonucleotide-mediated recombineering to efficiently create point mutations in a complex nonribosomal peptide synthetase (NRPS) from Photorabdus luminescens directing the biosynthesis of luminmides. After in depth analysis of the luminmide production profile from the native NRPS, single and double point mutations were rationally constructed within the adenylation (A) domain from NRPS module 3 which turned out to have a broad substrate tolerance. Expression of mutated versions of the 15.6 kb NRPS gene plu3263 in E. coli led to alterations in luminmide production profiles and allowed to direct the biosynthesis towards certain derivatives. These results demonstrate the suitability of counterselection recombineering for site-directed mutagenesis of complex expression constructs, e.g., genes encoding NRPS biosynthetic pathways in multi-copy plasmids.
克隆和改造天然产物生物合成途径，随后在易处理的宿主中进行异源表达，是一种广泛用于微生物次级代谢产物表达和基因修饰的方法。在此，我们采用ccdB反选择与寡核苷酸介导的重组工程相结合的方法，在发光杆菌中负责luminmides生物合成的复杂非核糖体肽合成酶（NRPS）中高效产生点突变。在深入分析天然NRPS的luminmide生产谱后，在NRPS模块3的腺苷化（A）结构域内合理构建了单点和双点突变，结果表明该结构域具有广泛的底物耐受性。在大肠杆菌中表达15.6 kb NRPS基因plu3263的突变体导致luminmide生产谱发生改变，并能够将生物合成导向某些衍生物。这些结果证明了反选择重组工程适用于复杂表达构建体的定点诱变，例如多拷贝质粒中编码NRPS生物合成途径的基因。
