链霉菌作为用于异源生产抗菌肽的过表达系统。

Streptomyces as Overexpression System for Heterologous Production of an Antimicrobial Peptide.

作者信息

Roldán-Tapia Marisol, Anné Jozef, Reyes Ana Gisela, Carrasco Ulises, Millán-Pacheco Cesar, Barrios-González Javier, Mejía Armando

机构信息

Department of Biotechnology, Universidad Autónoma Metropolitana-Iztapalapa, San Rafael Atlixco 186. Col. Vicentina. C.P 09340, México DF. Mexico.

Rega Institute, Katholieke Universiteit Leuven, Laboratory of Molecular Bacteriology, Minderbroedersstraat 10,B-3000 Leuven. Belgium.

出版信息

Protein Pept Lett. 2017;24(6):483-488. doi: 10.2174/0929866524666170208154327.

DOI:10.2174/0929866524666170208154327

PMID:28183247

Abstract

BACKGROUND

Antimicrobial peptides could be used in several fields of application, and large quantities of antimicrobial peptides would be required. However, their production is very expensive; this is why a suitable production method, alternative to traditional chemical synthesis is necessary. Production of recombinant antimicrobial peptides in prokaryotic systems has demonstrated the viability of this approach. Nevertheless, expression of antimicrobial peptides in Escherichia coli an others microorganisms is potentially limited due to their toxicity to host cells and susceptibility to proteolytic degradation. As an alternative, we describe a successful antimicrobial peptide production system in Streptomyces lividans which showed to be effective for the secretion of large quantities of cationic antimicrobial peptides.

OBJECTIVE

Therefore, as a solution to the difficulties for heterologous expression of CAP we demonstrate efficient production by S. lividans.

METHOD

In this study, a strategy for CAP overexpression is presented based on the construction of an expression cassette for Streptomyces lividans TK24. For the construction of this cassette, the peptide of interest was fused to the vsi promoter and signal sequence (vsi-ss) of the subtilisin inhibitor from Streptomyces venezuelae CBS762.70, which is a signal peptide with a proven high secretion efficiency. The cloning vector used was pIJ486, which includes a transcription terminator sequence and a thiostrepton resistance marker. This system contains elements that allow the increase of the efficiency of the peptide's expression.

RESULTS

The production system allows the efficient secretion of the peptide to the growth medium, thereby simplifying its recovery and avoiding its toxic effect on the producing organism. The production obtained demonstrated the system's efficiency by achieving a peptide concentration of 11.61 mg/ml. This represents at least a 10-fold increase compared to previously established strategies.

CONCLUSION

The expression system constructed may facilitate the production of large amounts of peptides with antimicrobial activity.

摘要

背景

抗菌肽可应用于多个领域，因此需要大量的抗菌肽。然而，其生产成本非常高昂，这就是为什么需要一种替代传统化学合成的合适生产方法。在原核系统中生产重组抗菌肽已证明了这种方法的可行性。然而，由于抗菌肽对宿主细胞有毒性且易受蛋白水解降解影响，其在大肠杆菌和其他微生物中的表达可能受到限制。作为一种替代方法，我们描述了一种在淡紫链霉菌中成功的抗菌肽生产系统，该系统被证明对大量阳离子抗菌肽的分泌有效。

目的

因此，作为解决阳离子抗菌肽异源表达困难的一种方法，我们展示了淡紫链霉菌高效生产抗菌肽的能力。

方法

在本研究中，提出了一种基于构建淡紫链霉菌TK24表达盒的阳离子抗菌肽过表达策略。为构建该表达盒，将目标肽与委内瑞拉链霉菌CBS762.70枯草杆菌蛋白酶抑制剂的vsi启动子和信号序列（vsi-ss）融合，该信号肽具有经证实的高分泌效率。使用的克隆载体是pIJ486，其包含转录终止子序列和硫链丝菌素抗性标记。该系统包含可提高肽表达效率的元件。