Dataset Information

Monitoring Protein Secretion in Streptomyces Using Fluorescent Proteins.

ABSTRACT: Fluorescent proteins are a major cell biology tool to analyze protein sub-cellular topology. Here we have applied this technology to study protein secretion in the Gram-positive bacterium Streptomyces lividans TK24, a widely used host for heterologous protein secretion biotechnology. Green and monomeric red fluorescent proteins were fused behind Sec (SPSec) or Tat (SPTat) signal peptides to direct them through the respective export pathway. Significant secretion of fluorescent eGFP and mRFP was observed exclusively through the Tat and Sec pathways, respectively. Plasmid over-expression was compared to a chromosomally integrated spSec-mRFP gene to allow monitoring secretion under high and low level synthesis in various media. Fluorimetric detection of SPSec-mRFP recorded folded states, while immuno-staining detected even non-folded topological intermediates. Secretion of SPSec-mRFP is unexpectedly complex, is regulated independently of cell growth phase and is influenced by the growth regime. At low level synthesis, highly efficient secretion occurs until it is turned off and secretory preforms accumulate. At high level synthesis, the secretory pathway overflows and proteins are driven to folding and subsequent degradation. High-level synthesis of heterologous secretory proteins, whether secretion competent or not, has a drastic effect on the endogenous secretome, depending on their secretion efficiency. These findings lay the foundations of dissecting how protein targeting and secretion are regulated by the interplay between the metabolome, secretion factors and stress responses in the S. lividans model.

SUBMITTER: Hamed MB

PROVIDER: S-EPMC6292873 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Monitoring Protein Secretion in Streptomyces Using Fluorescent Proteins.

Hamed Mohamed Belal MB Vrancken Kristof K Bilyk Bohdan B Koepff Joachim J Novakova Renata R van Mellaert Lieve L Oldiges Marco M Luzhetskyy Andriy A Kormanec Jan J Anné Jozef J Karamanou Spyridoula S Economou Anastassios A

Frontiers in microbiology 20181207

Fluorescent proteins are a major cell biology tool to analyze protein sub-cellular topology. Here we have applied this technology to study protein secretion in the Gram-positive bacterium Streptomyces lividans TK24, a widely used host for heterologous protein secretion biotechnology. Green and monomeric red fluorescent proteins were fused behind Sec (SPSec) or Tat (SPTat) signal peptides to direct them through the respective export pathway. Significant secretion of f ...[more]

PMID: 30581427

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Project description:BACKGROUND:Streptomyces lividans is an appealing host for the production of proteins of biotechnological interest due to its relaxed exogenous DNA restriction system and its ability to secrete proteins directly to the medium through the major Sec or the minor Tat routes. Often, protein secretion displays non-uniform time-dependent patterns. Understanding the associated metabolic changes is a crucial step to engineer protein production. Dynamic Flux Balance Analysis (DFBA) allows the study of the interactions between a modelled organism and its environment over time. Existing methods allow the specification of initial model and environment conditions, but do not allow introducing arbitrary modifications in the course of the simulation. Living organisms, however, display unexpected adaptive metabolic behaviours in response to unpredictable changes in their environment. Engineering the secretion of products of biotechnological interest has systematically proven especially difficult to model using DFBA. Accurate time-dependent modelling of complex and/or arbitrary, adaptive metabolic processes demands an extended approach to DFBA. RESULTS:In this work, we introduce Adaptive DFBA, a novel, versatile simulation approach that permits inclusion of changes in the organism or the environment at any time in the simulation, either arbitrary or interactively responsive to environmental changes. This approach extends traditional DFBA to allow steering arbitrarily complex simulations of metabolic dynamics. When applied to Sec- or Tat-dependent secretion of overproduced proteins in S. lividans, Adaptive DFBA can overcome the limitations of traditional DFBA to reproduce experimental data on plasmid-free, plasmid bearing and secretory protein overproducing S. lividans TK24, and can yield useful insights on the behaviour of systems with limited experimental knowledge such as agarase or amylase overproduction in S. lividans TK21. CONCLUSIONS:Adaptive DFBA has allowed us to overcome DFBA limitations and to generate more accurate models of the metabolism during the overproduction of secretory proteins in S. lividans, improving our understanding of the underlying processes. Adaptive DFBA is versatile enough to permit dynamical metabolic simulations of arbitrarily complex biotechnological processes.

Dataset Information

Monitoring Protein Secretion in Streptomyces Using Fluorescent Proteins.

Publications

Monitoring Protein Secretion in <i>Streptomyces</i> Using Fluorescent Proteins.

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