29 research outputs found
Germination and Growth Analysis of Streptomyces lividans at the Single-Cell Level Under Varying Medium Compositions
Koepff J, Sachs CC, Wiechert W, et al. Germination and Growth Analysis of Streptomyces lividans at the Single-Cell Level Under Varying Medium Compositions. FRONTIERS IN MICROBIOLOGY. 2018;9: 2680.Quantitative single-cell cultivation has provided fundamental contributions to our understanding of heterogeneity among industrially used microorganisms. Filamentous growing Streptomyces species are emerging platform organisms for industrial production processes, but their exploitation is still limited due to often reported high batch-to-batch variations and unexpected growth and production differences. Population heterogeneity is suspected to be one responsible factor, which is so far not systematically investigated at the single-cell level. Novel microfluidic single-cell cultivation devices offer promising solutions to investigate these phenomena. In this study, we investigated the germination and growth behavior of Streptomyces lividans TK24 under varying medium compositions on different complexity levels (i.e., mycelial growth, hyphal growth and tip elongation) on single-cell level. Our analysis reveals a remarkable stability within growth and germination of spores and early mycelium development when exposed to constant and defined environments. We show that spores undergo long metabolic adaptation processes of up to > 30 h to adjust to new medium conditions, rather than using a "persister" strategy as a possibility to cope with rapidly changing environments. Due to this uniform behavior, we conclude that S. lividans can be cultivated quite robustly under constant environmental conditions as provided by microfluidic cultivation approaches. Failure and non-reproducible cultivations are thus most likely to be found in less controllable larger-scale cultivation workflows and as a result of environmental gradients within large-scale cultivations
Germination and Growth Analysis of Streptomyces lividans at the Single-Cell Level Under Varying Medium Compositions
Quantitative single-cell cultivation has provided fundamental contributions to our understanding of heterogeneity among industrially used microorganisms. Filamentous growing Streptomyces species are emerging platform organisms for industrial production processes, but their exploitation is still limited due to often reported high batch-to-batch variations and unexpected growth and production differences. Population heterogeneity is suspected to be one responsible factor, which is so far not systematically investigated at the single-cell level. Novel microfluidic single-cell cultivation devices offer promising solutions to investigate these phenomena. In this study, we investigated the germination and growth behavior of Streptomyces lividans TK24 under varying medium compositions on different complexity levels (i.e., mycelial growth, hyphal growth and tip elongation) on single-cell level. Our analysis reveals a remarkable stability within growth and germination of spores and early mycelium development when exposed to constant and defined environments. We show that spores undergo long metabolic adaptation processes of up to > 30 h to adjust to new medium conditions, rather than using a “persister” strategy as a possibility to cope with rapidly changing environments. Due to this uniform behavior, we conclude that S. lividans can be cultivated quite robustly under constant environmental conditions as provided by microfluidic cultivation approaches. Failure and non-reproducible cultivations are thus most likely to be found in less controllable larger-scale cultivation workflows and as a result of environmental gradients within large-scale cultivations
Characterization of Sigma Factor Genes in Streptomyces lividans TK24 Using a Genomic Library-Based Approach for Multiple Gene Deletions
Alternative sigma factors control numerous aspects of bacterial life, including adaptation to physiological stresses, morphological development, persistence states and virulence. This is especially true for the physiologically complex actinobacteria. Here we report the development of a robust gene deletions system for Streptomyces lividans TK24 based on a BAC library combined with the λ-Red recombination technique. The developed system was validated by systematically deleting the most highly expressed genes encoding alternative sigma factors and several other regulatory genes within the chromosome of S. lividans TK24. To demonstrate the possibility of large scale genomic manipulations, the major part of the undecylprodigiosin gene cluster was deleted as well. The resulting mutant strains were characterized in terms of morphology, growth parameters, secondary metabolites production and response to thiol-oxidation and cell-wall stresses. Deletion of SLIV_12645 gene encoding S. coelicolor SigR1 ortholog has the most prominent phenotypic effect, resulted in overproduction of actinorhodin and coelichelin P1 and increased sensitivity to diamide. The secreted proteome analysis of SLIV_12645 mutant revealed SigR1 influence on trafficking of proteins involved in cell wall biogenesis and refactoring. The reported here gene deletion system will further facilitate work on S. lividans strain improvement as a host for either secondary metabolites or protein production and will contribute to basic research in streptomycetes physiology, morphological development, secondary metabolism. On the other hand, the systematic deletion of sigma factors encoding genes demonstrates the complexity and conservation of regulatory processes conducted by sigma factors in streptomycetes
Monitoring Protein Secretion in Streptomyces Using Fluorescent Proteins
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
Filamentous organisms catching up: Parallelized, miniaturized and automated characterization methods for Streptomyces lividans
Establishment and application of accelerated bioprocess development for filamentous organisms
Filamentous organisms of the genus Streptomyces are key players in industrial production processes. Particularly their ability to synthezise an extensive bandwidth of bio-active compounds such as antibiotics and their efficiency in (heterologous) protein production have put these species into focus of researchers and biopharmaceutical companies, already for decades. While genetic engineering tools are available to rapidly build up large strain libraries, the subsequent strain screening and bioprocess development still constitutes a bottleneck. To tackle this issue, an integrated phenotyping pipeline for Streptomyces lividans was developed and validated on the basis of shaken, parallelized 1000 µL microtiter-plate (MTP) cultivation. Starting from a standardized pre-culture procedure, highly reproducible results ((CV) ≤ 3.2 %) were obtainedfor biological independent batches. Even in comparison to a lab-scale bioreactor system with 1000-fold larger working volume, high comparable cultivation behavior was observed. This conformity could be further confirmed using state-of-the-art transcriptomics and proteomics. Remarkably, the optical biomass-related scattered light intensity signal revealed to be of great value, as it enabled to reliable deduce relevant parameters such as maximum specific growth rate and batch duration. Furthermore, tailored solutions for additional atline signals, such as robot-assisted cell-dry-weight and automated morphology analysis were successfully developed and validated. Hereby, the frame conditions of µL-scale cultivation and the special needs of the filamentous biological system were always considered.The developed accelerated cultivation workflow was subsequently employed and tested towards its applicability for further filamentous organisms, where it revealed excellent results for S. fradiae. Even for the filamentous fungi T. reesei useful information could be obtained, even though to a limited extend. Additionally, two S. lividans deletion strain libraries were phenotyped towards growth and production related parameters to investigate the function of relevant σ-factors and proteases in this filamentous Streptomycete.Embedded within the presented cultivation workflow, the integrated MTP-based phenotyping approach seems to be a suitable screening tool for filamentous and industrial relevant organisms like Streptomyces. The results of this thesis can thereby contribute to widen the field of application for such organisms and may narrow the technological gap towards their non-filamentous competitors