In vivo production of artificial nonribosomal peptide products in the heterologous host Escherichia coli
In vivo production of artificial nonribosomal peptide products in the heterologous host Escherichia coli
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DOI：
10.1128/aem.70.6.3282-3291.2004
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发表时间：
2004-06-01
影响因子：
4.4
通讯作者：
Stachelhaus, T
中科院分区：
文献类型：
--
作者：
Gruenewald, S;Mootz, HD;Stachelhaus, T
非核糖体多肽合成酶代表了生物合成药物相关的天然多肽，如环孢素、万古霉素和青霉素的酶流水线。由于它们的模块化结构，其中每个模块都包含一个单一氨基酸，因此可以通过生物组合的方法来构建生产新型多肽的人工流水线。一旦转移到合适的宿主，这些混合合成酶可以促进基本上任何基于多肽的分子的生物生产。在本研究中，我们描述了D-Phe-Pro-二酮基哌嗪的发酵生产，作为开发异源宿主大肠杆菌的原型，以及人工非核糖体多肽合成酶的使用。大肠杆菌为基因工程提供了巨大的潜力，在我们的研究中，通过稳定整合4‘-磷酸丙氨酸转移酶基因SFP来操纵它，以确保异源生产完全活性的全酶。D-Phe-Pro-diketopiperazine是由TycA/TycB1系统形成的，其组分代表短芽孢杆菌合成酪氨酸的前两个模块。相应基因在大肠杆菌中的共表达导致了预期的二酮哌嗪产物的产生，证明了两个模块在异源环境中的功能相互作用。此外，环二肽是稳定的，对大肠杆菌无毒，不需要任何额外的因素就可以分泌到培养基中。对影响产量的各种参数进行了综合考察，包括不同的遗传结构，以及培养基组分和温度的变化。B，这意味着，人工体系的整体生产率可以提高400%以上，以产生大约9毫克的D-苯丙酮哌嗪/升。作为一种通用工具，这种方法可以实现多肽的可持续生物生产，例如用作药物或精细化学品的多肽。
Nonribosomal peptide synthetases represent the enzymatic assembly lines for the biosynthesis of pharmacologically relevant natural peptides, e.g., cyclosporine, vancomycin, and penicillin. Due to their modular organization, in which every module accounts for the incorporation of a single amino acid, artificial assembly lines for the production of novel peptides can be constructed by biocombinatorial approaches. Once transferred into an appropriate host, these hybrid synthetases could facilitate the bioproduction of basically any peptide-based molecule. In the present study, we describe the fermentative production of the cyclic dipeptide D-Phe-Pro-diketopiperazine, as a prototype for the exploitation of the heterologous host Escherichia coli, and the use of artificial nonribosomal peptide synthetases. E. coli provides a tremendous potential for genetic engineering and was manipulated in our study by stable chromosomal integration of the 4'-phosphopantetheine transferase gene sfp to ensure heterologous production of fully active holoenzmyes. D-Phe-Pro-diketopiperazine is formed by the TycA/TycB1 system, whose components represent the first two modules for tyrocidine biosynthesis in Bacillus brevis. Coexpression of the corresponding genes in E. coli gave rise to the production of the expected diketopiperazine product, demonstrating the functional interaction of both modules in the heterologous environment. Furthermore, the cyclic dipeptide is stable and not toxic to E. coli and is secreted into the culture medium without the need for any additional factors. Parameters affecting the productivity were comprehensively investigated, including various genetic setups, as well as variation of medium composition and temperature. B, these means, the overall productivity of the artificial system could be enhanced by over 400% to yield about 9 mg of D-Phe-Pro-diketopiperazine/liter. As a general tool, this approach could allow the sustainable bioproduction of peptides, e.g., those used as pharmaceuticals or fine chemicals.