Platforms for nonribosomal peptide manipulation(RMI)

描述(由申请人提供)：这项为期三年的计划的目标是开发大肠杆菌作为DNA双嵌入物Triostin的高效异源表达系统。Triostin是一种非核糖体合成肽，是一种已知的抗生素和抗癌剂。我们将验证这样一种假设，即通过对大肠杆菌宿主进行新陈代谢工程，修改质粒载体，改变Triostin基因，可以生物合成Triostin及其类似物。这项研究有两个具体目的，(1)代谢工程大肠杆菌生产Triostin。(2)修饰Triostin NRPS以产生具有改变的序列特异性和选择性的类似物。初步结果。我们已经确定了一个四模块的非核糖体肽合成酶，它含有丝氨酸、丙氨酸、半胱氨酸和Valine氨基酸(在Triostin中发现的四种氨基酸)所特有的腺化结构域。本研究的意义在于：(A)提高了对C2对称非核糖体肽的组装机制的认识，(B)为各种非核糖体肽合成酶基因的表达提供了一个通用的宿主。

DESCRIPTION (provided by applicant): The goal of this three year proposal is to develop E. coli as an efficient heterologous expression system for the DNA bisintercalator triostin. A non-ribosomally synthesized peptide, triostin is a known antibiotic and anticancer agent. We will test the hypothesis that by metabolically engineering the E. coli host, modifying the plasmid vector, and altering the triostin gene, triostin and its analogs could be biosynthesized. There are two specific aims for this research, (1) Metabolically engineer E. coli to produce triostin. (2) Modify the triostin NRPS to produce analogs with altered sequence specificity and selectivity. Preliminary results. We have identified a four module non-ribosomal peptide synthetase that contains adenylation domains specific for Serine, Alanine, Cysteine, Valine amino acids (the four amino acids found in triostin.) The significance of this research is that it will (a) improve the mechanistic understanding of the assembly of C2-symmetric non-ribosomal peptides, (b) provide a general host for the expression of diverse non-ribosomal peptide synthetase genes.