Rational and efficient site-directed mutagenesis of adenylation domain alters relative yields of luminmide derivatives in vivo
Rational and efficient site-directed mutagenesis of adenylation domain alters relative yields of luminmide derivatives in vivo
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DOI：
10.1002/bit.25560
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发表时间：
2015-07-01
影响因子：
3.8
通讯作者：
Mueller, Rolf
中科院分区：
文献类型：
--
作者：
Bian, Xiaoying;Plaza, Alberto;Mueller, Rolf
天然产物生物合成途径的克隆和工程化以及在可调控宿主体内的异源表达是一种广泛应用于微生物次生代谢产物表达和遗传修饰的方法。在此，我们采用ccdB反选择结合寡核苷酸介导的重组，有效地在光abdus luminescens的复杂非核糖体肽合成酶（NRPS）中创建点突变，指导发光酰胺的生物合成。在对天然NRPS的发光谱进行深入分析后，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NRPS模块3具有广泛的底物耐受性。在大肠杆菌中表达15.6kb NRPS基因plu3263的突变版本导致发光胺生产谱的改变，并允许指导生物合成某些衍生物。这些结果证明了反选择重组在复杂表达结构（如编码NRPS生物合成途径的基因在多拷贝质粒中）的定点突变中的适用性。Biotechnol。Bioeng。2015;112: 1343 - 1353。(c) 2015 Wiley期刊公司
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 15.6kb 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. Biotechnol. Bioeng. 2015;112: 1343-1353. (c) 2015 Wiley Periodicals, Inc.