Title Participants Abstract "Extensive Reannotation of the Genome of the Model Streptomycete Streptomyces lividans TK24 Based on Transcriptome and Proteome Information" "Kristel Bernaerts, Tassos Economou" "Streptomyces lividans TK24 is a relevant Gram-positive soil inhabiting bacterium and one of the model organisms of the genus Streptomyces. It is known for its potential to produce secondary metabolites, antibiotics, and other industrially relevant products. S. lividans TK24 is the plasmid-free derivative of S. lividans 66 and a close genetic relative of the strain Streptomyces coelicolor A3(2). In this study, we used transcriptome and proteome data to improve the annotation of the S. lividans TK24 genome. The RNA-seq data of primary 5'-ends of transcripts were used to determine transcription start sites (TSS) in the genome. We identified 5,424 TSS, of which 4,664 were assigned to annotated CDS and ncRNAs, 687 to antisense transcripts distributed between 606 CDS and their UTRs, 67 to tRNAs, and 108 to novel transcripts and CDS. Using the TSS data, the promoter regions and their motifs were analyzed in detail, revealing a conserved -10 (TAnnnT) and a weakly conserved -35 region (nTGACn). The analysis of the 5' untranslated region (UTRs) of S. lividans TK24 revealed 17% leaderless transcripts. Several cis-regulatory elements, like riboswitches or attenuator structures could be detected in the 5'-UTRs. The S. lividans TK24 transcriptome contains at least 929 operons. The genome harbors 27 secondary metabolite gene clusters of which 26 could be shown to be transcribed under at least one of the applied conditions. Comparison of the reannotated genome with that of the strain Streptomyces coelicolor A3(2) revealed a high degree of similarity. This study presents an extensive reannotation of the S. lividans TK24 genome based on transcriptome and proteome analyses. The analysis of TSS data revealed insights into the promoter structure, 5'-UTRs, cis-regulatory elements, attenuator structures and novel transcripts, like small RNAs. Finally, the repertoire of secondary metabolite gene clusters was examined. These data provide a basis for future studies regarding gene characterization, transcriptional regulatory networks, and usage as a secondary metabolite producing strain." "Metabolic Characterization and Engineering of Streptomyces lividans Producing Heterologous Proteins (Metabole karakterisering en engineering van Streptomyces lividans voor heterologe eiwitproductie)" "Ivan Lule" "Streptomycetes have become attractive hosts for heterologous protein production due to for instance, their ability to efficiently secrete their proteins in the extracellular environment. Among these microbes, Streptomyces lividans, known to have a reduced restriction-modification barrier, has been used more extensively for heterologous protein production. Hitherto, studies on metabolic characterization and engineering of S. lividans for heterologous protein production are limited, and of these, few are performed in (upscale) controlled fermentation environments. Without metabolomics, the metabolic impact/load that heterologous protein production exerts on the central carbon metabolism of S. lividans remains loosely understood, and can not easily be relieved.Therefore, the general objective of this dissertation was to characterize the growth and metabolism of S. lividans producing heterologous proteins in (upscale) controlled fermentation environments. The study is further extended to assess the effect of overexpressing the gene (pck) encoding phosphoenolpyruvate carboxykinase on heterologous protein production in S. lividans. A genome scale metabolic network model of wild-type S. lividans was extended to include mouse tumor necrosis factor-alpha (mTNF-alpha) production. The resulting model matrix was mathematically analyzed to check for its inherent properties and sensitivity to key measurable fluxes. Results obtained showed that model sensitivity to mTNF-alpha was tenfold higher than biomass production, while the latter was at least tenfold more sensitive than the measurable amino acid fluxes. Metabolic characterization of recombinant S. lividans, in comparison with the wild-type S. lividans, was done to assess metabolic impact based on both extracellular metabolomics and constraint-based modeling. Results obtained showed that the metabolic impact due to heterologous protein production results in lower substrate uptake and slower growth, the former an indication for metabolic channeling. The metabolic impact is widely-distributed in the genome of S. lividans, causing a great shift in its performance. Critical exchange fluxes in the recombinant cells or models performance have been identified as biomass, mTNF-alpha, histidine, valine and alanine. Finally, based on metabolic engineering techniques, it was revealed that pck overexpression in S. lividans TK24 results in up to 1.7-fold increase in hTNF-alpha production. The major findings in this dissertation are an indication of metabolic shifts within the recombinant cell as well as a positive correlation between the explored metabolic engineering approach and heterologous protein production." "Metabolic Modeling of Streptomyces Lividans, a Cell Factory for Heterologous Protein Production (Metabolische modellering van Streptomyces lividans, een celfabriek voor heterologe eiwitproductie)" "Streptomycetes are worldwide used for commercial production of antibiotics and industrial enzymes. Nowadays, several genera of Grampositive bacteria are being tested as cell factories for the production of heterologous proteins owing to their ability to efficiently secrete proteins in the culture medium. Among them Streptomyces lividans is the cell factory of choice for the secretory production of heterologous proteins. To acquire a commercially attractive secretion yield of heterologous protein, the availability of suitable nitrogen sources in the medium is often essential. A complex mixture of amino acids, e.g., casamino acids, is usually added to the medium for this purpose. Besides acting as building blocks for biomass production, these amino acids are presumed to play an important role in the biosynthesis of heterologous protein. This dissertation focuses on the qualitative and quantitive characterization of the growth and metabolism of S. lividans TK24 as a cell factory for heterologous protein production. Based on a central set of experimental data, amino acids metabolization is investigated and a framework for the analysis of intracellular fluxes in a complex medium is set up. Experiments are performed with the wild-type and heterologous protein producing S. lividans under controlled bioreactor conditions and in the presence of casamino acids. Mouse Tumor Necrosis Factor alpha is taken as a model heterologous protein. First, the metabolic footprint, also called exometabolome (all extracellular metabolites), is thoroughly analyzed for wild-type and recombinant strain grown in a defined medium supplemented with casamino acids. The metabolite concentration profiles and calculated specific conversion rates reveal amino acid uptake preferences, by-products formation, and the impact and relevance of amino acids on biomass growth and heterologous protein production. In view of optimization of heterologous protein production levels in S. lividans , a deeper understanding of the effects of C- and N-substrates on the (intracellular) metabolism is necessary. Analysis of the intracellular metabolic fluxes via metabolic network modeling techniques is the tool of choice to further unravel this metabolism. To this end, a general metabolic modeling framework is developed to determine metabolic fluxes in S . lividans TK24 grown on a nutrient-rich medium. The plethora of substrates taken up motivates the use of a genome-scale metabolic network model, which makes it also possible to thoroughly study the whole-cell effects (metabolic impact) of heterologous protein production on the host cells metabolism in a further stadium. A genome-scale metabolic network model for wild-type S. lividans TK24 is constructed first. Next, the obtained experimental data are confronted with this genome-scale model using a combination of constraint-based stoichiometric metabolic network modeling techniques. Genome-scale hierarchical flux balance analysis and randomized sampling of the solution space are combined to extract maximum information from the exometabolome profiles. In this dissertation, new insights into the metabolic background of S. lividans as a cell factory forheterologous protein production are presented. The results are based ona systems approach and industrial relevant bioreactor conditions. Besides the exhaustive metabolic footprinting returning qualitative information, a systematic and integrative approach is presented to address the hurdles encountered in genomescale flux analysis by using a combination ofhierarchical flux balance analysis and randomized sampling of the flux solution space. This general approach can easily be adopted in future studies of S. lividans , e.g., as a host for heterologous protein production." "Identification of novel rotihibin analogues in Streptomyces scabies, including discovery of its biosynthetic gene cluster" "Sören Planckaert, Benoit Deflandre, Anne-Mare de Vries, Maarten Ameye, José Martins, Kris Audenaert, Sébastien Rigali, Bart Devreese" "Streptomyces scabies is a phytopathogen associated with common scab disease. This is mainly attributed to its ability to produce the phytotoxin thaxtomin A, the biosynthesis of which is triggered by cellobiose. During a survey of other metabolites released in the presence of cellobiose, we discovered additional compounds in the thaxtomin-containing extract from Streptomyces scabies. Structural analysis by mass spectrometry (MS) and nuclear magnetic resonance (NMR) revealed that these compounds are amino acid sequence variants of the TOR (target of rapamycin) kinase (TORK) pathway-inhibitory lipopeptide rotihibin A, and the main compounds were named rotihibins C and D. In contrast to thaxtomin, the production of rotihibins C and D was also elicited in the presence of glucose, indicating different regulation of their biosynthesis. Through a combination of shotgun and targeted proteomics, the putative rotihibin biosynthetic gene cluster rth was identified in the publicly available genome of S. scabies 87-22. This cluster spans 33 kbp and encodes 2 different nonribosomal peptide synthetases (NRPSs) and 12 additional enzymes. Homologous rth biosynthetic gene clusters were found in other publicly available and complete actinomycete genomes. Rotihibins C and D display herbicidal activity against Lemna minor and Arabidopsis thaliana at low concentrations, shown by monitoring the effects on growth and the maximal photochemistry efficiency of photosystem II. IMPORTANCE Rotihibins A and B are plant growth inhibitors acting on the TORK pathway. We report the isolation and characterization of new sequence analogues of rotihibin from Streptomyces scabies, a major cause of common scab in potato and other tuber and root vegetables. By combining proteomics data with genomic analysis, we found a cryptic biosynthetic gene cluster coding for enzyme machinery capable of rotihibin production. This work may lead to the biotechnological production of variants of this lipopeptide to investigate the exact mechanism by which it can target the plant TORK pathway in Arabidopsis thaliana. In addition, bioinformatics revealed the existence of other variants in plant-associated Streptomyces strains, both pathogenic and nonpathogenic species, raising new questions about the actual function of this lipopeptide. The discovery of a module in the nonribosomal peptide synthetase (NRPS) that incorporates the unusual citrulline residue may improve the prediction of peptides encoded by cryptic NRPS gene clusters." "Safety evaluation of the food enzyme protein–glutamine γ-glutamyltransferase from the non-genetically modified Streptomyces mobaraensis strain M2020197." "Lieve Herman" "The food enzyme protein-glutamine γ-glutamyltransferase (protein-glutamine: amine γ-glutamyltransferase; EC 2.3.2.13) is produced with the non-genetically modified Streptomyces mobaraensis strain M2020197 by Taixing Dongsheng Bio-Tech Co. Ltd. The identity of the production strain and the absence of viable cells could not be established. The food enzyme is intended to be used in eight food manufacturing processes: processing of cereals and other grains for the production of (1) baked products, (2) cereal-based products other than baked; processing of dairy products for the production of (3) fermented dairy products, (4) cheese, (5) dairy desserts; processing of plant- and fungal-derived products for the production of (6) meat analogues, (7) plant-based analogues of milk and milk products; processing of meat and fish products for the production of (8) modified meat and fish products. Dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 3.498 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 91 mg TOS/kg bw per day. The calculated margin of exposure for each age group was 36 (infants), 26 (toddlers), 50 (children), 99 (adolescents), 115 (adults) and 133 (the elderly). A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that a risk of allergic reactions upon dietary exposure to this food enzyme cannot be excluded, but the likelihood is low. The safety of the food enzyme could not be established given the derived margins of exposure. Therefore, the Panel concluded that the food enzyme could not be considered safe under the intended conditions of use." "Safety evaluation of the food enzyme xylose isomerase from the genetically modified Streptomyces rubiginosus strain DP-Pzn37" "Lieve Herman" "The food enzyme is a d-xylose aldose-ketose-isomerase (EC 5.3.1.5) produced with the genetically modified Streptomyces rubiginosus strain DP-Pzn37 by Danisco US Inc. Although the production strain contains antibiotic resistance genes, the food enzyme was shown to be free from viable cells of the production organism and its DNA. The food enzyme is intended to be used in an immobilised form for the isomerisation of glucose for the production of high fructose syrups. Residual amounts of total organic solids (TOS) are eliminated by the use of an immobilised food enzyme and further removed by the purification steps applied during the production of high fructose syrups using the immobilised enzyme; consequently, dietary exposure was not calculated. Genotoxicity tests did not raise safety concerns. The systemic toxicity was assessed by a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 85.2 mg TOS/kg body weight (bw) per day, the highest dose tested. Similarity of the amino acid sequence to those of known allergens was searched and no match was found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood is considered to be low. Based on the data provided, the immobilisation process and the removal of total organic solids during the production of high fructose syrups, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use." "Characterization of Sigma Factor Genes in Streptomyces lividans TK24 Using a Genomic Library-Based Approach for Multiple Gene Deletions" "Tassos Economou" "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." "Secretory production of recombinant proteins by Streptomyces" "Kristof Vrancken, Jozef Anné" "Bacterial systems are widely applied as production platforms for proteins of biopharmaceutical or therapeutic interest and industrial enzymes. Among these prokaryotic systems, streptomycetes are attractive host cells because several strains of these Gram-positive bacteria have a high innate secretion capacity and extensive knowledge on their fermentation is available. A survey of the literature and our own experience suggests that several proteins are secreted to commercially acceptable levels. However, many heterologous proteins, most often of eukaryotic origin, are currently only poorly secreted by this host, indicating the need for further optimization of Streptomyces as a production host. In this review, the considerable efforts and strategies made in recent years aimed at improving streptomycetes as a host for the production of recombinant proteins will be discussed." "A panoramic view of the genomic landscape of the genus Streptomyces" "Marios Nikolaidis, Andrew Hesketh, Nikoletta Frangou, Dimitris Mossialos, Yves Van de Peer, Stephen G. Oliver, Grigorios D. Amoutzias" "Disulfide bridges as essential elements for the thermostability of lytic polysaccharide monooxygenase LPMO10C from Streptomyces coelicolor" "Magali Tanghe, Barbara Danneels, Matthias Last"