The Three Streptomyces lividans HtrA-Like Proteases Involved in the Secretion Stress Response Act in a Cooperative Manner.
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ABSTRACT:
SUBMITTER: Vicente RL
PROVIDER: S-EPMC5157995 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundMisfolded proteins accumulating outside the bacterial cytoplasmic membrane can interfere with the secretory machinery, hence the existence of quality factors to eliminate these misfolded proteins is of capital importance in bacteria that are efficient producers of secretory proteins. These bacteria normally use a specific two-component system to respond to the stress produced by the accumulation of the misfolded proteins, by activating the expression of HtrA-like proteases to specifically eliminate the incorrectly folded proteins.Methodology/principal findingsOverproduction of alpha-amylase in S. lividans causing secretion stress permitted the identification of a two-component system (SCO4156-SCO4155) that regulates three HtrA-like proteases which appear to be involved in secretion stress response. Mutants in each of the genes forming part of the two-genes operon that encodes the sensor and regulator protein components accumulated misfolded proteins outside the cell, strongly suggesting the involvement of this two-component system in the S. lividans secretion stress response.Conclusions/significanceTo our knowledge this is the first time that a specific secretion stress response two-component system is found to control the expression of three HtrA-like protease genes in S. lividans, a bacterium that has been repeatedly used as a host for the synthesis of homologous and heterologous secretory proteins of industrial application.
| S-EPMC3498368 | biostudies-literature
Project description:BackgroundS. lividans TK24 is a popular host for the production of small molecules and the secretion of heterologous protein. Within its large genome, twenty-nine non-essential clusters direct the biosynthesis of secondary metabolites. We had previously constructed ten chassis strains, carrying deletions in various combinations of specialized metabolites biosynthetic clusters, such as those of the blue actinorhodin (act), the calcium-dependent antibiotic (cda), the undecylprodigiosin (red), the coelimycin A (cpk) and the melanin (mel) clusters, as well as the genes hrdD, encoding a non-essential sigma factor, and matAB, a locus affecting mycelial aggregation. Genome reduction was aimed at reducing carbon flow toward specialized metabolite biosynthesis to optimize the production of secreted heterologous protein.ResultsTwo of these S. lividans TK24 derived chassis strains showed ~ 15% reduction in biomass yield, 2-fold increase of their total native secretome mass yield and enhanced abundance of several secreted proteins compared to the parental strain. RNAseq and proteomic analysis of the secretome suggested that genome reduction led to cell wall and oxidative stresses and was accompanied by the up-regulation of secretory chaperones and of secDF, a Sec-pathway component. Interestingly, the amount of the secreted heterologous proteins mRFP and mTNFα, by one of these strains, was 12 and 70% higher, respectively, than that secreted by the parental strain.ConclusionThe current study described a strategy to construct chassis strains with enhanced secretory abilities and proposed a model linking the deletion of specialized metabolite biosynthetic clusters to improved production of secreted heterologous proteins.
| S-EPMC10768272 | biostudies-literature
Project description:BACKGROUND:Streptomyces lividans has demonstrated its value as an efficient host for protein production due to its ability to secrete functional proteins directly to the media. Secretory proteins that use the major Sec route need to be properly folded outside the cell, whereas secretory proteins using the Tat route appear outside the cell correctly folded. This feature makes the Tat system very attractive for the production of natural or engineered Tat secretory proteins. S. lividans cells are known to respond differently to overproduction and secretion of Tat versus Sec proteins. Increased understanding of the impact of protein secretion through the Tat route can be obtained by a deeper analysis of the metabolic impact associated with protein production, and its dependence on protein origin, composition, secretion mechanisms, growth phases and nutrients. Flux Balance Analysis of Genome-Scale Metabolic Network models provides a theoretical framework to investigate cell metabolism under different constraints. RESULTS:We have built new models for various S. lividans strains to better understand the mechanisms associated with overproduction of proteins secreted through the Tat route. We compare models of an S. lividans Tat-dependent agarase overproducing strain with those of the S. lividans wild-type, an S. lividans strain carrying the multi-copy plasmid vector and an α-amylase Sec-dependent overproducing strain. Using updated genomic, transcriptomic and experimental data we could extend existing S. lividans models and produce a new model which produces improved results largely extending the coverage of S. lividans strains, the number of genes and reactions being considered, the predictive behaviour and the dependence on specification of exchange constraints. Comparison of the optimized solutions obtained highlights numerous changes between Tat- and Sec-dependent protein secreting strains affecting the metabolism of carbon, amino acids, nucleotides, lipids and cofactors, and variability analysis predicts a large potential for protein overproduction. CONCLUSIONS:This work provides a detailed look to metabolic changes associated to Tat-dependent protein secretion reproducing experimental observations and identifying changes that are specific to each secretory route, presenting a novel, improved, more accurate and strain-independent model of S. lividans, thus opening the way for enhanced metabolic engineering of protein overproduction in S. lividans.
| S-EPMC6000921 | biostudies-literature
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.
| S-EPMC6815373 | biostudies-literature
Project description:The Streptomyces lividans lsp gene encodes a type II signal peptidase (Lsp) that cleaves the type II leader peptides of lipoproteins. Transcriptional profiling of the bacterium depleted of the lsp gene mainly resulted in deactivation of the sigma U regulon, as well as in downregulation of genes involved in the biogenesis and function of ribosomes and genes encoding some major secretory proteins as determined by hybridisation of commercially available S. lividans genome-wide microarrays. Almost 50% of the dowregulated genes have been described as forming part of the stringent response in streptomycetes. The gene encoding the S. lividans extracellular foldase, the lipoprotein FkpA, is equally downregulated. Therefore, the deletion of lsp from the S. livdans genome temporarily triggers a cellular stress where the stringent response is, at least, partially induced.
2014-05-22 | GSE30728 | GEO
Project description:The gene dagA encoding agarase in Streptomyces coelicolor was cloned in the multicopy plasmid pIJ486 generating plasmid pAGAs5. Plasmid pAGAs5 and pIJ486 were propagated in S. lividans TK21 to obtain S. lividans TK21(pAGAs5) and its isogenic strain S. lividans TK21(pIJ486). Transcriptional profiling of the bacterium that overproduces agarase mainly resulted in the downregulation of the genes involved in the biogenesis and function of ribosomes, together with the downregulation of the genes involved in nitrogen, aminoacids, purine/pyrimidine and central carbon metabolism as well as ABC transporters, redox processes and fatty acids biosynthesis. The number of genes upregulated in the agarase overproducer strain is lower than in the downregulated ones. Almost 50% of the dowregulated genes have been described as forming part of the stringent response in streptomycetes. Therefore, the overproduction of agarase may lead to a condition of nutrient depletion that triggers the stringent response.
2015-07-23 | GSE61447 | GEO
Project description:AdpA is a key transcriptional regulator involved in the complex growth cycle of Streptomyces. Streptomyces are Gram-positive bacteria well-known for their production of secondary metabolites and antibiotics. Most work on AdpA has been in S. griseus, and little is known about the pathways it controls in other Streptomyces spp. We recently discovered interplay between ClpP peptidases and AdpA in S. lividans. Here, we report the identification of genes directly regulated by AdpA in S. lividans.Microarray experiments revealed that the expression of hundreds of genes was affected in a S. lividans adpA mutant during early stationary phase cultures in YEME liquid medium. We studied the expression of the S. lividans AdpA-regulated genes by quantitative real-time PCR analysis after various times of growth. In silico analysis revealed the presence of potential AdpA-binding sites upstream from these genes; electrophoretic mobility shift assays indicated that AdpA binds directly to their promoter regions. This work identifies new pathways directly controlled by AdpA and that are involved in S. lividans development (ramR, SLI7885 also known as hyaS and SLI6586), and primary (SLI0755-SLI0754 encoding CYP105D5 and Fdx4) or secondary (cchA, cchB, and hyaS) metabolism.We characterised six S. lividans AdpA-dependent genes whose expression is directly activated by this pleiotropic regulator. Several of these genes are orthologous to bldA-dependent genes in S. coelicolor. Furthermore, in silico analysis suggests that over hundred genes may be directly activated or repressed by S. lividans AdpA, although few have been described as being part of any Streptomyces AdpA regulons. This study increases the number of known AdpA-regulated pathways in Streptomyces spp.
| S-EPMC4021200 | biostudies-literature
Project description:Proteins are the main machinery for all living processes in a cell; they provide structural elements, regulate biochemical reactions as enzymes, and are the interface to the outside as receptors and transporters. Like any other machinery proteins have to be assembled correctly and need maintenance after damage, e.g., caused by changes in environmental conditions, genetic mutations, and limitations in the availability of cofactors. Proteases and chaperones help in repair, assembly, and folding of damaged and misfolded protein complexes cost-effective, with low energy investment compared with neo-synthesis. Despite their importance for viability, the specific biological role of most proteases in vivo is largely unknown. Deg/HtrA proteases, a family of serine-type ATP-independent proteases, have been shown in higher plants to be involved in the degradation of the Photosystem II reaction center protein D1. The objective of this review is to highlight the structure and function of their cyanobacterial orthologs. Homology modeling was used to find specific features of the SynDeg/HtrA proteases of Synechocystis sp. PCC 6803. Based on the available data concerning their location and their physiological substrates we conclude that these Deg proteases not only have important housekeeping and chaperone functions within the cell, but also are needed for remodeling the cell exterior.
| S-EPMC4877387 | biostudies-literature
Project description:BackgroundProtein production as secretory-form is a powerful tool in industrial enzyme production due to the simple purification procedure. Streptomyces lividans is a versatile host for secretory production of useful proteins. In order to expand the amount of secreted protein, signal peptide sequences, which encourage protein secretion from inside cell to extracellular environment, are one of the most significant factors. In this study, we focused on Streptomyces lividans as a host strain to secrete useful proteins, and screened for signal peptides from the biomass-degradation enzymes derived from Thermobifida fusca YX and S. lividans.ResultsThree candidate signal peptides were isolated and evaluated for their protein secretion ability using β-glucosidase derived from T. fusca YX, which is a non-secreted protein, as a model protein. Using S. lividans xylanase C signal peptide, the amount of produced the β-glucosidase reached 10 times as much as that when using Streptomyces cinnamoneus phospholipase D signal peptide, which was identified as a versatile signal peptide in our previous report. In addition, the introduction of the β-glucosidase fused to xylanase C signal peptide using two kinds of plasmid, pUC702 and pTYM18, led to further protein secretion, and the maximal level of produced the β-glucosidase increased up to 17 times (1.1 g/l) compared to using only pUC702 carrying the β-glucosidase fused to S. cinnamoneus phospholipase D signal peptide.ConclusionIn the present study, we focused on signal peptide sequences derived from biomass degradation enzymes, which are usually secreted into the culture supernatant, and screened for signal peptides leading to effective protein secretion. Using the signal peptides, the hyper-protein secretion system was successfully demonstrated for the cytoplasmic β-glucosidase.
| S-EPMC3850917 | biostudies-literature
Project description:S. lividans TK24 is a popular host for the production of small molecules and for the secretion of heterologous proteins. TK24 has a large genome with at least 29 secondary metabolite gene clusters that are non-essential for viability and undergo complex regulation. To optimize heterologous protein secretion, we previously constructed ten chassis strains that are devoid of several secondary metabolite gene clusters. Genome reduction was aimed at reducing carbon flow to secondary metabolites and pigmentation in the spent growth medium and improving colony morphology. Strains RG1.0-RG1.10 contain various deletion combinations of the blue actinorhodin cluster (act), the calcium-dependent antibiotic (cda), the undecylprodigiosin (red) and coelimycin A (cpk) clusters, the melanin cluster (mel), the matAB genes that affect mycelial aggregation and the non-essential sigma factor hrdD that controls the transcription of Act and Red regulatory proteins. Two derivative strains, RG1.5 and 1.9, showed a ~15% reduction in growth rate, >2-fold increase in the total mass yield of their native secretome and altered abundance of several specific proteins compared with TK24. Metabolomics and RNAseq analysis revealed that genome reduction led to rapid cessation of growth due to aminoacid depletion and caused both redox and cell envelope stresses, upregulation of the Sec-pathway components secDF and chaperones and a cell envelope two component regulator. RG1.9 maintained elevated heterologous secretion of mRFP and mTNFα by 12-70%. An integrated model is presented linking genome reduction and enhanced secretion. Importance: S. lividans TK24 encode 29 secondary metabolite gene clusters controlled with highly complex systems. This study established an important step toward understanding how secondary metabolite clusters can be manipulated to construct a surrogate TK24 platform with optimized metabolite funnelling. Using integrative multi-omics tools with protein secretion we provide an in-depth view of this fascinating complex process and its mechanistic regulation.
2024-01-26 | PXD040146 | Pride