title: Rational and efficient site-directed mutagenesis of adenylation domain alters relative yields of luminmide derivatives in vivo.
abstract: 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.
authors: 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.|Shandong University-Helmholtz Institute of Biotechnology, State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Zhuzhou Road 168, 266101, Qingdao, P. R. China.
authors: Xiaoying
authors: X
authors: Bian
authors: 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.
authors: Alberto
authors: A
authors: Plaza
authors: 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.
authors: Fu
authors: F
authors: Yan
authors: Shandong University-Helmholtz Institute of Biotechnology, State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Zhuzhou Road 168, 266101, Qingdao, P. R. China. zhangyouming@sdu.edu.cn.
authors: Youming
authors: Y
authors: Zhang
authors: Department of Microbial Natural Products, Helmholtz-Institute for Pharmaceutical Research Saarland, Helmholtz Centre for Infection Research, Campus C2 3, 66123, Saarbrücken, Germany. rolf.mueller@helmholtz-hzi.de.|Department of Pharmaceutical Biotechnology, Saarland University, Campus C2 3, 66123, Saarbrücken, Germany. rolf.mueller@helmholtz-hzi.de.
authors: Rolf
authors: R
authors: Müller
mesh_terms: D001688:Biological Products / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D010453:Peptide Synthases / Q000235:genetics* / Q000378:metabolism*; D020637:Photorhabdus / Q000201:enzymology*; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D001688:Biological Products; D050505:Mutant Proteins; D011994:Recombinant Proteins; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase
keywords: biosynthesis; ccdB counterselection recombineering; heterologous expression; luminmide; natural products; point mutation