A new synthetic biology system for investigating the biosynthesis of antibiotics and other secondary metabolites in streptomycetes.

We have created a novel synthetic biology expression system allowing easy refactoring of biosynthetic gene clusters (BGCs) as monocistronic transcriptional units. The system is based on a set of plasmids containing a strong kasOp* promoter, RBS and terminators. It allows the cloning of biosynthetic genes into transcriptional units kasOp*-gene(s)-terminator flanked by several rare restriction cloning sites that can be sequentially combined into the artificial BGC in three compatible Streptomyces integration vectors. They allow a simultaneous integration of these BGCs at three different attB sites in the Streptomyces chromosome. The system was validated with biosynthetic genes from two known BGCs for aromatic polyketides landomycin and mithramycin.

Multiple SigB homologues govern the transcription of the ssgBp promoter in the sporulation-specific ssgB gene in Streptomyces coelicolor A3(2).

Unlike Bacillus subtilis, Streptomyces coelicolor contains nine SigB homologues of the stress-response sigma factor SigB. By using a two-plasmid system, we previously identified promoters recognized by these sigma factors. Almost all promoters were recognized by several SigB homologues. However, no specific sequences of these promoters were found. One of these promoters, ssgBp, was selected to examine this cross-recognition in the native host. It controls the expression of the sporulation-specific gene ssgB. Using a luciferase reporter, the activity of this promoter in S. coelicolor and nine mutant strains lacking individual sigB homologous genes showed that sgBp is dependent on three sigma factors, SigH, SigN, and SigI. To determine which nucleotides in the-10 region are responsible for the selection of a specific SigB homologue, promoters mutated at the last three nucleotide positions were tested in the two-plasmid system. Some mutant promoters were specifically recognized by a distinct set of SigB homologues. Analysis of these mutant promoters in the native host showed the role of these nucleotides. A conserved nucleotide A at position 5 was essential for promoter activity, and two variable nucleotides at positions 4 and 6 were responsible for the partial selectivity of promoter recognition by SigB homologues.

Enhanced protein secretion in reduced genome strains of Streptomyces lividans.

BACKGROUND: S. 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. RESULTS: Two 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. CONCLUSION: The 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.

Investigating the initial steps of auricin biosynthesis using synthetic biology.

Streptomyces lavendulae subsp. lavendulae CCM 3239 (formerly Streptomyces aureofaciens CCM 3239) contains a type II polyketide synthase (PKS) biosynthetic gene cluster (BGC) aur1 whose genes were highly similar to angucycline BGCs. However, its product auricin is structurally different from all known angucyclines. It contains a spiroketal pyranonaphthoquinone aglycone similar to griseusins and is modified with D-forosamine. Here, we describe the characterization of the initial steps in auricin biosynthesis using a synthetic-biology-based approach. We have created a plasmid system based on the strong kasOp* promoter, RBS and phage PhiBT1-based integration vector, where each gene in the artificial operon can be easily replaced by another gene using unique restriction sites surrounding each gene in the operon. The system was validated with the initial landomycin biosynthetic genes lanABCFDLE, leading to the production of rabelomycin after its integration into Streptomyces coelicolor M1146. However, the aur1DEFCGHA homologous genes from the auricin aur1 BGC failed to produce rabelomycin in this system. The cause of this failure was inactive aur1DE genes encoding ketosynthases α and ß (KSα, KSß). Their replacement with homologous aur2AB genes from the adjacent aur2 BGC resulted in rabelomycin production that was even higher after the insertion of two genes from the aur1 BGC, aur1L encoding 4-phosphopantetheinyl transferase (PPTase) and aur1M encoding malonyl-CoA:ACP transacylase (MCAT), suggesting that Aur1L PPTase is essential for the activation of the acyl carrier protein Aur1F. These results suggest an interesting communication of two BGCs, aur1 and aur2, in the biosynthesis of the initial structure of auricin aglycone.

Cloning and Expression of Metagenomic DNA in Streptomyces lividans and Its Subsequent Fermentation for Optimized Production.

The choice of an expression system for the metagenomic DNA of interest is of vital importance for the detection of any particular gene or gene cluster. Most of the screens to date have used the Gram-negative bacterium Escherichia coli as a host for metagenomic gene libraries. However, the use of E. coli introduces a potential host bias since only 40% of the enzymatic activities may be readily recovered by random cloning in E. coli. To recover some of the remaining 60%, alternative cloning hosts such as Streptomyces spp. have been used. Streptomycetes are high-GC Gram-positive bacteria belonging to the Actinomycetales and they have been studied extensively for more than 25 years as an alternative expression system. They are extremely well suited for the expression of DNA from other actinomycetes and genomes of high GC content. Furthermore, due to its high innate, extracellular secretion capacity, Streptomyces can be a better system than E. coli for the production of many extracellular proteins. In this article, an overview is given about the materials and methods for growth and successful expression and secretion of heterologous proteins from diverse origin using Streptomyces lividans as a host. More in detail, an overview is given about the protocols of transformation, type of plasmids used and of vectors useful for integration of DNA into the host chromosome, and accompanying cloning strategies. In addition, various control elements for gene expression including synthetic promoters are discussed, and methods to compare their strength are described. Stable and efficient marker-less integration of the gene of interest under the control of the promoter of choice into S. lividans chromosome via homologous recombination using pAMR23A-based system will be explained. Finally, a basic protocol for bench-top bioreactor experiments which can form the start in the production process optimization and up-scaling will be provided.

A stable vector for efficient production of heterologous proteins and secondary metabolites in streptomycetes.

The bacteria of the genus Streptomyces are important producers of a large number of biologically active natural products. Examination of their genomes has revealed great biosynthetic potential for the production of new products, but many of them are silent under laboratory conditions. One of the promising avenues for harnessing this biosynthetic potential is the refactoring and heterologous expression of relevant biosynthetic gene clusters (BGCs) in suitable optimized chassis strains. Although several Streptomyces strains have been used for this purpose, the efficacy is relatively low, and some BGCs have not been expressed. In this study, we optimized our long-term genetically studied Streptomyces lavendulae subsp. lavendulae CCM 3239 strain as a potential host for heterologous expression along with its stable large linear plasmid pSA3239 as a vector system. Two reporter genes, mCherry and gusA under the control of ermEp* promoter, were successfully integrated into pSA3239. The activity of GUS reporter was four-fold higher in pSA3239 than in a single site in S. lavendulae subsp. lavendulae CCM 3239 chromosome, consistent with a higher copy number of pSA3239 (4 copies per chromosome). In addition, the two Att/Int systems (based on PhiC31 and pSAM2) were able to integrate into the corresponding individual attB sites in the chromosome. The BGC for actinorhodin was successfully integrated into pSA3239. However, the resulting strain produced very low amounts of actinorhodin. Its level increased dramatically after integration of the actII-ORF4 gene for the positive regulator under the control of the kasOp* promoter into this strain using the PhiC31 phage integration system. KEY POINTS: • New Streptomyces chassis for heterologous expression of genes and BGCs • Optimized strategy for insertion of heterologous genes into linear plasmid pSA3239 • Efficient heterologous production of actinorhodin after induction of its regulator.

Bacterial Regulatory Proteins.

The regulation of gene expression in bacteria occurs predominantly at the level of transcription, which is controlled by RNA polymerase [...].

A New Family of Transcriptional Regulators Activating Biosynthetic Gene Clusters for Secondary Metabolites.

We previously identified the aur1 biosynthetic gene cluster (BGC) in Streptomyceslavendulae subsp. lavendulae CCM 3239 (formerly Streptomycesaureofaciens CCM 3239), which is responsible for the production of the unusual angucycline-like antibiotic auricin. Auricin is produced in a narrow interval of the growth phase after entering the stationary phase, after which it is degraded due to its instability at the high pH values reached after the production phase. The complex regulation of auricin BGC is responsible for this specific production by several regulators, including the key activator Aur1P, which belongs to the family of atypical response regulators. The aur1P gene forms an operon with the downstream aur1O gene, which encodes an unknown protein without any conserved domain. Homologous aur1O genes have been found in several BGCs, which are mainly responsible for the production of angucycline antibiotics. Deletion of the aur1O gene led to a dramatic reduction in auricin production. Transcription from the previously characterized Aur1P-dependent biosynthetic aur1Ap promoter was similarly reduced in the S. lavendulaeaur1O mutant strain. The aur1O-specific coactivation of the aur1Ap promoter was demonstrated in a heterologous system using a luciferase reporter gene. In addition, the interaction between Aur1O and Aur1P has been demonstrated by a bacterial two-hybrid system. These results suggest that Aur1O is a specific coactivator of this key auricin-specific positive regulator Aur1P. Bioinformatics analysis of Aur1O and its homologues in other BGCs revealed that they represent a new family of transcriptional coactivators involved in the regulation of secondary metabolite biosynthesis. However, they are divided into two distinct sequence-specific subclasses, each of which is likely to interact with a different family of positive regulators.

The linear plasmid pSA3239 is essential for the replication of the Streptomyces lavendulae subsp. lavendulae CCM 3239 chromosome.

We previously reported the complete genome of Streptomyces lavendulae subsp. lavendulae CCM 3239, containing the linear chromosome and the large linear plasmid pSA3239. Although the chromosome exhibited replication features characteristic for the archetypal end-patching replication, it lacked the tap/tpg gene pair for two proteins essential for this process. However, this archetypal tpgSa-tapSa operon is present in pSA3239. Complete genomic sequence of the S. lavendulae Del-LP strain lacking this plasmid revealed the circularization of its chromosome with a large deletion of both arms. These results suggest an essential role of pSA3239-encoded TapSa/TpgSa in the end-patching replication of the chromosome.

Cross-Recognition of Promoters by the Nine SigB Homologues Present in Streptomyces coelicolor A3(2).

In contrast to Bacillus subtilis, Streptomyces coelicolor A3(2) contains nine homologues of stress response sigma factor SigB with a major role in differentiation and osmotic stress response. The aim of this study was to further characterize these SigB homologues. We previously established a two-plasmid system to identify promoters recognized by sigma factors and used it to identify promoters recognized by the three SigB homologues, SigF, SigG, and SigH from S. coelicolor A3(2). Here, we used this system to identify 14 promoters recognized by SigB. The promoters were verified in vivo in S. coelicolor A3(2) under osmotic stress conditions in sigB and sigH operon mutants, indicating some cross-recognition of these promoters by these two SigB homologues. This two-plasmid system was used to examine the recognition of all identified SigB-, SigF-, SigG-, and SigH-dependent promoters with all nine SigB homologues. The results confirmed this cross-recognition. Almost all 24 investigated promoters were recognized by two or more SigB homologues and data suggested some distinguishing groups of promoters recognized by these sigma factors. However, analysis of the promoters did not reveal any specific sequence characteristics for these recognition groups. All promoters showed high similarity in the -35 and -10 regions. Immunoblot analysis revealed the presence of SigB under osmotic stress conditions and SigH during morphological differentiation. Together with the phenotypic analysis of sigB and sigH operon mutants in S. coelicolor A3(2), the results suggest a dominant role for SigB in the osmotic stress response and a dual role for SigH in the osmotic stress response and morphological differentiation. These data suggest a complex regulation of the osmotic stress response in relation to morphological differentiation in S. coelicolor A3(2).

Screening Systems for Stable Markerless Genomic Deletions/Integrations in Streptomyces Species.

Bacteria of the genus Streptomyces are one of the most important producers of biologically active natural products. Recent robust genomic sequencing of Streptomyces strains has shown enormous genetic potential for new natural products. However, many biosynthetic gene clusters are silent. Therefore, efficient and stable genome modification methods are needed to induce their production or to manipulate them for the production of new compounds or biotechnologically improved strains. We have recently developed a simple and efficient markerless genome modification system for these bacteria based on the positive selection of double crossovers using the blue pigment indigoidine bpsA gene. This chapter is an attempt to provide methodological details of this strategy for stable markerless genomic engineering (deletions/insertions) to improve their biotechnological properties and to produce biologically active compounds.

An efficient system for stable markerless integration of large biosynthetic gene clusters into Streptomyces chromosomes.

The bacteria of the genus Streptomyces are among the most important producers of biologically active secondary metabolites. Moreover, recent genomic sequence data have shown their enormous genetic potential for new natural products, although many new biosynthetic gene clusters (BGCs) are silent. Therefore, efficient and stable genome modification techniques are needed to activate their production or to manipulate their biosynthesis towards increased production or improved properties. We have recently developed an efficient markerless genome modification system for streptomycetes based on positive blue/white selection of double crossovers using the bpsA gene from indigoidine biosynthesis, which has been successfully applied for markerless deletions of genes and BGCs. In the present study, we optimized this system for markerless insertion of large BGCs. In a pilot test experiment, we successfully inserted a part of the landomycin BGC (lanFABCDL) under the control of the ermEp* promoter in place of the actinorhodin BGC (act) of Streptomyces lividans TK24 and RedStrep 1.3. The resulting strains correctly produced UWM6 and rabelomycin in twice the yield compared to S. lividans strains with the same construct inserted using the PhiBT1 phage-based integration vector system. Moreover, the system was more stable. Subsequently, using the same strategy, we effectively inserted the entire BGC for mithramycin (MTM) in place of the calcium-dependent antibiotic BGC (cda) of S. lividans RedStrep 1.3 without antibiotic-resistant markers. The resulting strain produced similar levels of MTM when compared to the previously described S. lividans RedStrep 1.3 strain with the VWB phage-based integration plasmid pMTMF. The system was also more stable. KEY POINTS: • Optimized genome editing system for markerless insertion of BGCs into Streptomyces genomes • Efficient heterologous production of MTM in the stable engineered S. lividans strain.

The antitumor antibiotic mithramycin: new advanced approaches in modification and production.

The aureolic acid-type polyketide mithramycin (MTM) has a remarkable cytotoxicity against a variety of human tumors and has been used for the treatment of several types of cancer, including chronic and acute myeloid leukemia, testicular carcinoma, hypercalcemia, and Paget's disease. However, its clinical use is quite limited due to its toxicity. Recently, interest in MTM has been renewed after its identification as a top candidate for the inhibition of the aberrant fusion transcription factor EWS-FLI1, associated with malignant transformation and progression of Ewing sarcoma tumor family. The mechanism of MTM inhibition involves its reversible non-intercalative interaction with GC-rich DNA regions. As a result of this binding, MTM blocks binding of transcription factors (such as Sp1) to their GC-rich promoters and inhibits transcription of several proto-oncogenes and thus suppresses various types of cancer. Knowledge of the biosynthesis of MTM and its gene cluster has enabled genetic modifications of the gene cluster and combinatorial biosynthesis to produce new modified MTM molecules ("mithralogues") with improved efficacy and lower toxicity, which has also renewed interest in the clinical development of MTM. However, production yields of MTM and its analogues are low in the natural production strains. Recent developments in genetic engineering approaches have made it possible to increase MTM production through more rational strategies based on genetic manipulations and heterologous expression in optimized chassis. Recent construction of various genetically modified strains of Streptomyces lividans has shown their use for efficient heterologous production of various biologically active secondary metabolites including MTM. KEY POINTS: • Discovery a novel bifunctional glycosyl hydrolase from uncultured microorganism. • Heterologous production of MTM in engineered S. lividans strains is efficient.

Pleiotropic anti-anti-sigma factor BldG is phosphorylated by several anti-sigma factor kinases in the process of activating multiple sigma factors in Streptomyces coelicolor A3(2).

The anti-anti-sigma factor BldG has a pleiotropic function in Streptomyces coelicolor A3(2), regulating both morphological and physiological differentiation. Together with the anti-sigma factor UshX, it participates in a partner-switching activation of the sigma factor σH, which has a dual role in the osmotic stress response and morphological differentiation in S. coelicolor A3(2). In addition to UshX, BldG also interacts with the anti-sigma factor ApgA, although no target sigma factor has yet been identified. However, neither UshX nor ApgA phosphorylates BldG. This phosphorylation is provided by the anti-sigma factor RsfA, which is specific for the late developmental sigma factor σF. However, BldG is phosphorylated in the rsfA mutant, suggesting that some other anti-sigma factors containing HATPase_c kinase domain are capable to phosphorylate BldG in vivo. Bacterial two-hybrid system (BACTH) was therefore used to investigate the interactions of all suitable anti-sigma factors of S. coelicolor A3(2) with BldG. At least 15 anti-sigma factors were found to interact with BldG. These interactions were confirmed by native PAGE. In addition to RsfA, BldG is specifically phosphorylated on the conserved phosphorylation Ser57 residue by at least seven additional anti-sigma factors. However, only one of them, SCO7328, has been shown to interact with three sigma factors, σG, σK and σM. A mutant with deleted SCO7328 gene was prepared in S. coelicolor A3(2), however, no specific function of SCO7328 in growth, differentiation or stress response could be attributed to this anti-sigma factor. These results suggest that BldG is specifically phosphorylated by several anti-sigma factors and it plays a role in the regulation of several sigma factors in S. coelicolor A3(2). This suggests a complex regulation of the stress response and differentiation in S. coelicolor A3(2) through this pleiotropic anti-sigma factor.

A Structural Analysis of the Angucycline-Like Antibiotic Auricin from Streptomyces lavendulae Subsp. Lavendulae CCM 3239 Revealed Its High Similarity to Griseusins.

We previously identified the aur1 gene cluster in Streptomyces lavendulae subsp. lavendulae CCM 3239 (formerly Streptomyces aureofaciens CCM 3239), which is responsible for the production of the angucycline-like antibiotic auricin (1). Preliminary characterization of 1 revealed that it possesses an aminodeoxyhexose d-forosamine and is active against Gram-positive bacteria. Here we determined the structure of 1, finding that it possesses intriguing structural features, which distinguish it from other known angucyclines. In addition to d-forosamine, compound 1 also contains a unique, highly oxygenated aglycone similar to those of spiroketal pyranonaphthoquinones griseusins. Like several other griseusins, 1 also undergoes methanolysis and displays modest cytotoxicity against several human tumor cell lines. Moreover, the central core of the aur1 cluster is highly similar to the partial gris gene cluster responsible for the biosynthesis of griseusin A and B in both the nature of the encoded proteins and the gene organization.

Recent achievements in the generation of stable genome alterations/mutations in species of the genus Streptomyces.

The bacteria of the genus Streptomyces are the most valuable source of natural products of industrial and medical importance. A recent explosion of Streptomyces genome sequence data has revealed the enormous genetic potential of new biologically active compounds, although many of them are silent under laboratory conditions. Efficient and stable manipulation of the genome is necessary to induce their production. Comprehensive studies in the past have led to a large and versatile collection of molecular biology tools for gene manipulation of Streptomyces, including various replicative plasmids. However, biotechnological applications of these bacteria require stable genome alterations/mutations. To accomplish such stable genome editing, two major strategies for streptomycetes have been developed: (1) integration into the chromosome through Att/Int site-specific integration systems based on Streptomyces actinophages (ΦC31, ΦBT1, VWB, TG1, SV1, R4, ΦJoe, µ1/6) or pSAM2 integrative plasmid; (2) integration by homologous recombination using suicidal non-replicating vectors. The present review is an attempt to provide a comprehensive summary of both approaches for stable genomic engineering and to outline recent advances in these strategies, such as CRISPR/Cas9, which have successfully manipulated Streptomyces strains to improve their biotechnological properties and increase production of natural or new gene-manipulated biologically active compounds.

Secretome Dynamics in a Gram-Positive Bacterial Model.

Protein secretion is a central biological process in all organisms. Most studies dissecting bacterial secretion mechanisms have focused on Gram-negative cell envelopes such as that of Escherichia coli However, proteomics analyses in Gram negatives is hampered by their outer membrane. Here we studied protein secretion in the Gram-positive bacterium Streptomyces lividans TK24, in which most of the secretome is released in the growth medium. We monitored changes of the secretome as a function of growth phase and medium. We determined distinct protein classes of "house-keeping" secreted proteins that do not change their appearance or abundance in the various media and growth phases. These comprise mainly enzymes involved in cell wall maintenance and basic transport. In addition, we detected significant abundance and content changes to a sub-set of the proteome, as a function of growth in the different media. These did not depend on the media being minimal or rich. Transcriptional regulation but not changes in export machinery components can explain some of these changes. However, additional downstream mechanisms must be important for selective secretome funneling. These observations lay the foundations of using S. lividans as a model organism to study how metabolism is linked to optimal secretion and help develop rational optimization of heterologous protein production.

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.

An efficient blue-white screening system for markerless deletions and stable integrations in Streptomyces chromosomes based on the blue pigment indigoidine biosynthetic gene bpsA.

We previously developed an efficient deletion system for streptomycetes based on the positive selection of double-crossover events using bpsA, a gene for producing the blue pigment indigoidine. Using this system, we removed interfering secondary metabolite clusters from Streptomyces lividans TK24, resulting in RedStrep strains with dramatically increased heterologous production of mithramycin A (up to 3-g/l culture). This system, however, required a time-consuming step to remove the resistance marker genes. In order to simplify markerless deletions, we prepared a new system based on the plasmid pAMR18A. This plasmid contains a large polylinker with many unique restriction sites flanked by apramycin and kanamycin resistance genes and the bpsA gene for selecting a double-crossover event. The utility of this new markerless deletion system was demonstrated by its deletion of a 21-kb actinorhodin gene cluster from Streptomyces lividans TK24 with 30% efficiency. We used this system to efficiently remove the matA and matB genes in selected RedStrep strains, resulting in biotechnologically improved strains with a highly dispersed growth phenotype involving non-pelleting small and open mycelia. No further increase in mithramycin A production was observed in these new RedStrep strains, however. We also used this system for the markerless insertion of a heterologous mCherry gene, an improved variant of the monomeric red fluorescent protein, under the control of the strong secretory signal sequence of the subtilisin inhibitor protein, into the chromosome of S. lividans TK24. The resulting recombinant strains efficiently secreted mCherry into the growth medium in a yield of 30 mg/l.

Rapid identification of Streptomyces tetracycline producers by MALDI-TOF mass spectrometry.

The main objective of this study was using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for assembling of DSM (German Collection of Microorganisms) Streptomyces spectral database and identification of wild Streptomyces cultures, which were clustered by MALDI-TOF Biotyper OC software as well as for teracycline detection by observing of obtained spectra using flexAnalysis software. Production of tetracycline was confirmed by thin-layer chromatography. Presence of tetracycline mass spectrum was verified by several tetracycline producers (Streptomyces aureofaciens LMG 5968, S. aureofaciens 84/25, and S. aureofaciens BMK) and by pure tetracycline mass. Our results showed that it is possible to use MALDI-TOF MS for identification of tetracycline producers within Streptomyces genera by several easy steps. The purpose of this study was to establish cheap and quick detection of tetracycline producers.