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


METHODS
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.


CONCLUSIONS
The expression system constructed may facilitate the production of large amounts of peptides with antimicrobial activity.
Department of Biotechnology, Universidad Autónoma Metropolitana-Iztapalapa, San Rafael Atlixco 186. Col. Vicentina. C.P 09340, México DF. Mexico.
Marisol
M
Roldán-Tapia
Rega Institute, Katholieke Universiteit Leuven, Laboratory of Molecular Bacteriology, Minderbroedersstraat 10,B-3000 Leuven. Belgium.
Jozef
J
Anné
Conacyt-CIBNOR, Centro de Investigaciones Biológicas del Noroeste, La Paz, BCS. Mexico.
Ana Gisela
AG
Reyes
Department of Biotechnology, Universidad Autónoma Metropolitana-Iztapalapa, San Rafael Atlixco 186. Col. Vicentina. C.P 09340, México DF. Mexico.
Ulises
U
Carrasco
Facultad de Farmacia, Universidad Autónoma del Estado de Morelos, Avenida Universidad 1001, CP 62209, Cuernavaca, Morelos. Mexico.
Cesar
C
Millán-Pacheco
Department of Biotechnology, Universidad Autónoma Metropolitana-Iztapalapa, San Rafael Atlixco 186. Col. Vicentina. C.P 09340, México DF. Mexico.
Javier
J
Barrios-González
Department of Biotechnology, Universidad Autónoma Metropolitana San Rafael Atlixco 186, Vicentina, Iztapalapa, P.O. Box: 09340. Ciudad de México. Mexico.
Armando
A
Mejía
D023181:Antimicrobial Cationic Peptides / Q000096:biosynthesis / Q000235:genetics*; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics*; D013302:Streptomyces / Q000737:chemistry / Q000235:genetics*
D023181:Antimicrobial Cationic Peptides; D001426:Bacterial Proteins; D011994:Recombinant Proteins
CAP
overexpression; Heterologous production; Streptomyces lividans; antimicrobial peptide; phytopathogens; secreted peptide