Platforms for nonribosomal peptide manipulation

描述（由申请人提供）：这个为期三年的提案的目标是开发大肠杆菌作为DNA双插层子triostin的高效异源表达系统。triostin是一种非核糖体合成的肽，是一种已知的抗生素和抗癌剂。我们将验证这个假设，即通过代谢工程的大肠杆菌宿主，修改质粒载体，改变triostin基因，可以生物合成triostin及其类似物。本研究有两个具体目的：(1)对大肠杆菌进行代谢工程，使其产生triostin。(2)对triostin NRPS进行修饰，产生序列特异性和选择性改变的类似物。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，包含特定的丝氨酸，丙氨酸，半胱氨酸，缬氨酸氨基酸的腺苷化结构域（在triostin中发现的四种氨基酸）。本研究的意义在于：(a)提高了对c2对称非核糖体多肽组装机制的理解，(b)为多种非核糖体多肽合成酶基因的表达提供了一个通用宿主。

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.