The Impact of Heterologous Regulatory Genes from Lipodepsipeptide Biosynthetic Gene Clusters on the Production of Teicoplanin and A40926.

StrR-like pathway-specific transcriptional regulators (PSRs) function as activators in the biosynthesis of various antibiotics, including glycopeptides (GPAs), aminoglycosides, aminocoumarins, and ramoplanin-like lipodepsipeptides (LDPs). In particular, the roles of StrR-like PSRs have been previously investigated in the biosynthesis of streptomycin, novobiocin, GPAs like balhimycin, teicoplanin, and A40926, as well as LDP enduracidin. In the current study, we focused on StrR-like PSRs from the ramoplanin biosynthetic gene cluster (BGC) in Actinoplanes ramoplaninifer ATCC 33076 (Ramo5) and the chersinamycin BGC in Micromonospora chersina DSM 44151 (Chers28). Through the analysis of the amino acid sequences of Ramo5 and Chers28, we discovered that these proteins are phylogenetically distant from other experimentally investigated StrR PSRs, although all StrR-like PSRs found in BGCs for different antibiotics share a conserved secondary structure. To investigate whether Ramo5 and Chers28, given their phylogenetic positions, might influence the biosynthesis of other antibiotic pathways governed by StrR-like PSRs, the corresponding genes (ramo5 and chers28) were heterologously expressed in Actinoplanes teichomyceticus NRRL B-16726 and Nonomuraea gerenzanensis ATCC 39727, which produce the clinically-relevant GPAs teicoplanin and A40926, respectively. Recombinant strains of NRRL B-16726 and ATCC 39727 expressing chers28 exhibited improved antibiotic production, although the expression of ramo5 did not yield the same effect. These results demonstrate that some StrR-like PSRs can "cross-talk" between distant biosynthetic pathways and might be utilized as tools for the activation of silent BGCs regulated by StrR-like PSRs.

Bacterial community and culturable actinomycetes of Phyllostachys viridiglaucescens rhizosphere.

During the course of development plants form tight interactions with microorganisms inhabiting their root zone. In turn, rhizosphere bacteria, in particular members of the phylum Actinomycetota, positively influence the host plant by increasing access to essential nutrients and controlling the pathogenic microorganism's population. Herein, we report the characterisation of the rhizosphere associated actinobacteria community of Phyllostachys viridiglaucescens growing in the Nikitsky Botanical Garden (Crimean Peninsula, Ukraine). The overall composition of the bacterial community was elucidated by 16S rRNA gene amplicon sequencing followed by isolation of culturable microorganisms with the focus on actinomycetes. The metagenomic approach revealed that the representatives of phylum Actinomycetota (57.1%), Pseudomonadota (20.0%), and Acidobacteriota (12.2%) were dominating in the studied microbiome with Ilumatobacter (phylum Actinomycetota) (13.1%) being the dominant genus. Furthermore, a total of 159 actinomycete isolates, belonging to eight genera of Streptomyces, Micromonospora, Nonomuraea, Arthrobacter, Actinomadura, Kribbella, Cellulosimicrobium, and Mumia, were recovered from P. viridiglaucescens rhizosphere. The isolated species were tested for antimicrobial activity. 64% of isolates were active against at least one bacterial test-culture and 7.5% against fungal test culture. In overall, the rhizosphere bacterial communities act as a great source of actinobacterial diversity with the high potential for production of new bioactive compounds.

Actinoplanes oblitus sp. nov., producing the glycopeptide antibiotic A477.

In 1973, Eli Lilly and Company described the filamentous actinomycete producing the glycopeptide antibiotic A477 as an Actinoplanes species on the basis of its morphological and physiological features and deposited it as NRRL 3884T. In this paper, we report that the phylogenetic analysis based on the 16S rRNA gene sequence and the whole genome phylogenomic study indicate that NRRL 3884T forms a distinct monophyletic line within the genus Actinoplanes, being most closely related to Actinoplanes octamycinicus NBRC 14524T [99.6 % 16S rRNA gene similarity, 89.4 % average nucleotide identity (ANI), 46.0 % digital DNA-DNA hybridization (dDDH)] and Actinoplanes ianthinogenes NBRC 13996T (98.8 % 16S rRNA gene similarity, 89.0 % ANI, 47.0 % dDDH). NRRL 3884T forms an extensively branched, non-fragmented vegetative mycelium; either sterile aerial hyphae or regular subglobose sporangia are formed depending on cultivation conditions. The cell wall contains meso-2,6-diaminopimelic acid and 2,6-diamino-3-hydroxypimelic acid and the diagnostic sugars are glucose, mannose and ribose with a minor amount of rhamnose. The predominant menaquinone (MK) is MK-9(H4), with minor amounts of MK-9(H2), MK-9(H6) and MK-9(H8). Mycolic acids are absent. The diagnostic phospholipids are diphosphatidylglycerol and phosphatidylethanolamine. The major cellular fatty acids are anteiso-C17 : 0, iso-C16 : 0 and iso-C15 : 0, with moderate amounts of anteiso-C15 : 0 and iso-C17 : 0. The genomic G+C content is 71.5 mol%. Significant differences in the genomic, morphological, chemotaxonomic and biochemical data between NRRL 3884T and the two most closely related Actinoplanes type strains clearly demonstrate that NRRL 3884T represents a novel species of the genus Actinoplanes, for which the name Actinoplanes oblitus sp. nov. is proposed. The type strain is NRRL 3884T (=DSM 116196T).

Properties of Multidrug-Resistant Mutants Derived from Heterologous Expression Chassis Strain Streptomyces albidoflavus J1074.

Streptomyces albidoflavus J1074 is a popular platform to discover novel natural products via the expression of heterologous biosynthetic gene clusters (BGCs). There is keen interest in improving the ability of this platform to overexpress BGCs and, consequently, enable the purification of specialized metabolites. Mutations within gene rpoB for the ß-subunit of RNA polymerase are known to increase rifampicin resistance and augment the metabolic capabilities of streptomycetes. Yet, the effects of rpoB mutations on J1074 remained unstudied, and we decided to address this issue. A target collection of strains that we studied carried spontaneous rpoB mutations introduced in the background of the other drug resistance mutations. The antibiotic resistance spectra, growth, and specialized metabolism of the resulting mutants were interrogated using a set of microbiological and analytical approaches. We isolated 14 different rpoB mutants showing various degrees of rifampicin resistance; one of them (S433W) was isolated for the first time in actinomycetes. The rpoB mutations had a major effect on antibiotic production by J1074, as evident from bioassays and LC-MS data. Our data support the idea that rpoB mutations are useful tools to enhance the ability of J1074 to produce specialized metabolites.

Diversity and bioactive potential of Actinomycetia from the rhizosphere soil of Juniperus excelsa.

Microbial natural products are among the main sources of compounds used in the medical biotechnology field for the purpose of drug development. However, as antibiotic resistance in pathogenic microorganisms is known to be increasing dramatically, there exists a need to develop new antibiotics. Actinomycetia have proven to be a good source of biologically active compounds, although the rediscovery of previously known compounds significantly slows down the introduction of new antibiotics. As a consequence, increasing attention is being paid to the isolation of actinomycete strains from previously unexplored sources, which can significantly increase the likelihood of discovering new biologically active compounds. This study investigated the diversity and bioactive potential of 372 actinomycete strains isolated from the rhizosphere soil of Juniperus excelsa M. Bieb. The examined actinomycete strains belonged to 11 genera, namely, Actinoplanes, Actinorectispora, Amycolatopsis, Kribbella, Micrococcus, Micromonospora, Nocardia, Promicromonospora, Rhodococcus, Saccharopolyspora and Streptomyces. The bioactive potential of each isolated actinomycete strain was determined on the basis of its ability to produce antimicrobial metabolites against Gram-positive and Gram-negative bacteria and yeast. Some 159 strains (42.74%) exhibited antimicrobial activity against at least one of the tested microbial strains. The dereplication analysis of the extract of the Streptomyces sp. Je 1-651 strain, which exhibited strong antimicrobial activity, led to the annotation of spiramycins and stambomycins. Moreover, the phylogenetic analysis based on the 16S rRNA gene sequence of the Je 1-651 strain revealed it to be close to the S. ambofaciens.

Mutations within gene XNR_2147 for TetR-like protein enhance lincomycin resistance and endogenous specialized metabolism of Streptomyces albus J1074.

Streptomyces albus J1074 is one of the most popular heterologous expression platforms among streptomycetes. Identification of new genes and mutations that influence specialized metabolism in this species is therefore of great applied interest. Here, we describe S. albus KO-1304 that was isolated as a spontaneous lincomycin-resistant variant of double rpsLR94G rsmGR15SG40E mutant KO-1295. Besides altered antibiotic resistance profile, KO-1304 exhibited increased antibiotic activity as compared to its parental strains. KO-1304 genome sequencing revealed mutations within gene XNR_2147 encoding putative TetR-like protein. Gene XNR_2146 for efflux protein is the most likely target of repressing action of Xnr_2147. Our data agree with the scenario where lincomycin resistance phenotype of KO-1304 arose from inability of mutated Xnr_2147 protein to repress XNR_2146. Introduction of additional copy of XNR_2146 into wild type strain increased antibiotic activity of the latter, attesting to the practical value of transporter genes for strain improvement.

Heterologous Expression Reveals Ancient Properties of Tei3­A VanS Ortholog from the Teicoplanin Producer Actinoplanes teichomyceticus

Glycopeptide antibiotics (GPAs) are among the most clinically successful antimicrobials. GPAs inhibit cell-wall biosynthesis in Gram-positive bacteria via binding to lipid II. Natural GPAs are produced by various actinobacteria. Being themselves Gram-positives, the GPA producers evolved sophisticated mechanisms of self-resistance to avoid suicide during antibiotic production. These self-resistance genes are considered the primary source of GPA resistance genes actually spreading among pathogenic enterococci and staphylococci. The GPA-resistance mechanism in Actinoplanes teichomyceticus­the producer of the last-resort-drug teicoplanin­has been intensively studied in recent years, posing relevant questions about the role of Tei3 sensor histidine kinase. In the current work, the molecular properties of Tei3 were investigated. The setup of a GPA-responsive assay system in the model Streptomyces coelicolor allowed us to demonstrate that Tei3 functions as a non-inducible kinase, conferring high levels of GPA resistance in A. teichomyceticus. The expression of different truncated versions of tei3 in S. coelicolor indicated that both the transmembrane helices of Tei3 are crucial for proper functioning. Finally, a hybrid gene was constructed, coding for a chimera protein combining the Tei3 sensor domain with the kinase domain of VanS, with the latter being the inducible Tei3 ortholog from S. coelicolor. Surprisingly, such a chimera did not respond to teicoplanin, but indeed to the related GPA A40926. Coupling these experimental results with a further in silico analysis, a novel scenario on GPA-resistance and biosynthetic genes co-evolution in A. teichomyceticus was hereby proposed.

Occurrence of vanHAX and Related Genes beyond the Actinobacteria Phylum.

Clinically relevant glycopeptide antibiotics remain among the most successful classes of natural antibacterials. This success, however, is endangered by the spread of glycopeptide resistance genes, also known as van genes. Thus, it is important to trace and comprehend possible routes of van gene dissemination. In the current work, we present a comprehensive bioinformatic analysis aimed at mapping the occurrence of van genes beyond the Actinobacteria phylum-the most likely natural reservoir of van genes. We show that two additional classes of Gram-positive bacteria, Erysipelotrichia and Ktedonobacteria, as well as one class of Gram-negative bacteria, Anaerolineae, carry van genes. Additionally, we demonstrate that various new genera belonging to the classes Clostridia and Bacilli also carry van genes. The majority of discovered van loci are co-localized with MGE-related genes of various types. Finally, we propose a phylogeny-based scenario for the spread of van genes, unraveling a network of consequential horizontal gene transfer events linking the phylum Actinobacteria with the five other bacterial classes carrying van genes.

Screening of Thiopeptide-Producing Streptomycetes Isolated From the Rhizosphere Soil of Juniperus excelsa.

The identification of an increasing number of drug-resistant pathogens has stimulated the development of new therapeutic agents to combat them. Microbial natural products are among the most important elements when it comes to drug discovery. Today, thiopeptide antibiotics are receiving increasing research attention due to their potent activity against Gram-positive bacteria. In this study, we demonstrated the successful use of a whole-cell microbial biosensor (Streptomyces lividans TK24 pMO16) for the specific detection of thiopeptide antibiotics among the native actinomycete strains isolated from the rhizosphere soil of Juniperus excelsa (Bieb.). Among the native strains, two strains of Streptomyces, namely sp. Je 1-79 and Je 1-613, were identified that were capable of producing thiopeptide antibiotics. A multilocus sequence analysis of five housekeeping genes (gyrB, atpD, recA, rpoB, and trpB) classified them as representatives of two different species of the genus Streptomyces. The thiopeptide antibiotics berninamycin A and B were identified in the extracts of the two strains by means of a dereplication analysis. The berninamycin biosynthetic gene cluster was also detected in the genome of the Streptomyces sp. Je 1-79 strain and showed a high level of similarity (93%) with the ber cluster from S. bernensis. Thus, the use of this whole-cell biosensor during the first stage of the screening process could serve to accelerate the specific detection of thiopeptide antibiotics.

Genetics Behind the Glycosylation Patterns in the Biosynthesis of Dalbaheptides.

Glycopeptide antibiotics are valuable natural metabolites endowed with different pharmacological properties, among them are dalbaheptides used to treat different infections caused by multidrug-resistant Gram-positive pathogens. Dalbaheptides are produced by soil-dwelling high G-C Gram-positive actinobacteria. Their biosynthetic pathways are encoded within large biosynthetic gene clusters. A non-ribosomally synthesized heptapeptide aglycone is the common scaffold for all dalbaheptides. Different enzymatic tailoring steps, including glycosylation, are further involved in decorating it. Glycosylation of dalbaheptides is a crucial step, conferring them specific biological activities. It is achieved by a plethora of glycosyltransferases, encoded within the corresponding biosynthetic gene clusters, able to install different sugar residues. These sugars might originate from the primary metabolism, or, alternatively, their biosynthesis might be encoded within the biosynthetic gene clusters. Already installed monosaccharides might be further enzymatically modified or work as substrates for additional glycosylation. In the current minireview, we cover recent updates concerning the genetics and enzymology behind the glycosylation of dalbaheptides, building a detailed and consecutive picture of this process and of its biological evolution. A thorough understanding of how glycosyltransferases function in dalbaheptide biosynthesis might open new ways to use them in chemo-enzymatic synthesis and/or in combinatorial biosynthesis for building novel glycosylated antibiotics.

Scandium-microorganism interactions in new biotechnologies.

Scandium (Sc) plays a special role in high-tech industries because of its wide application in green, space, and defense technologies. However, Sc mining and purification are problematic due to political, technological, and environmental difficulties. The deficit of this element limits global technological development. One sustainable solution to this problem is to use microorganisms to extract Sc from ore and waste, as well as to concentrate and separate it from other elements. Sc also demonstrates attractive metabolic effects on microbes that is of great interest in white biotechnology. Sc increases the production of proteins and secondary metabolites and activates poorly expressed genes. This review offers a comprehensive analysis of current knowledge on the application of Sc-microorganism interactions in promising biotechnologies, its perspectives, and future challenges.

Genetic approaches to improve clorobiocin production in Streptomyces roseochromogenes NRRL 3504.

Streptomyces roseochromogenes NRRL 3504 is best known as a producer of clorobiocin, a DNA replication inhibitor from the aminocoumarin family of antibiotics. This natural product currently draws attention as a promising adjuvant for co-application with other antibiotics against Gram-negative multidrug-resistant pathogens. Herein, we expand the genetic toolkit for NRRL 3504 by showing that a set of integrative and replicative vectors, not tested previously for this strain, could be conjugally transferred at high frequency from Escherichia coli to NRRL 3504. Using this approach, we leverage a cumate-inducible expression of cluster-situated regulatory gene novG to increase clorobiocin titers by 30-fold (up to approximately 200 mg/L). To our best knowledge, this is the highest level of clorobiocin production reported so far. Our findings set a working ground for further improvement of clorobiocin production as well as for the application of genetic methods to illuminate the cryptic secondary metabolome of NRRL 3504. Key Points • Efficient system for conjugative transfer of plasmids into NRRL 3504 was developed. • Expression of regulatory genes in NRRL 3504 led to increase in clorobiocin titer. • Secondary metabolome of NRRL 3504 becomes an accessible target for genetic manipulations using the expanded vector set and improved intergeneric conjugation protocol.

Structural diversity, bioactivity, and biosynthesis of phosphoglycolipid family antibiotics: Recent advances.

Moenomycins, such as moenomycin A, are phosphoglycolipid specialized metabolites produced by a number of actinobacterial species. They are among the most potent antibacterial compounds known to date, which drew numerous studies directed at various aspects of the chemistry and biology of moenomycins. In this review, we outline the advances in moenomycin research over the last decade. We focus on biological aspects, highlighting the contribution of the novel methods of genomics and molecular biology to the deciphering of the biosynthesis and activity of moenomycins. Specifically, we describe the structural diversity of moenomycins as well as the underlying genomic variations in moenomycin biosynthetic gene clusters. We also describe the most recent data on the mechanism of action and assembly of complicated phosphoglycolipid scaffold. We conclude with the description of the genetic control of moenomycin production by Streptomyces bacteria and a brief outlook on future developments.

Genomic Insights into the Distribution and Phylogeny of Glycopeptide Resistance Determinants within the Actinobacteria Phylum.

The spread of antimicrobial resistance (AMR) creates a challenge for global health security, rendering many previously successful classes of antibiotics useless. Unfortunately, this also includes glycopeptide antibiotics (GPAs), such as vancomycin and teicoplanin, which are currently being considered last-resort drugs. Emerging resistance towards GPAs risks limiting the clinical use of this class of antibiotics-our ultimate line of defense against multidrug-resistant (MDR) Gram-positive pathogens. But where does this resistance come from? It is widely recognized that the GPA resistance determinants-van genes-might have originated from GPA producers, such as soil-dwelling Gram-positive actinobacteria, that use them for self-protection. In the current work, we present a comprehensive bioinformatics study on the distribution and phylogeny of GPA resistance determinants within the Actinobacteria phylum. Interestingly, van-like genes (vlgs) were found distributed in different arrangements not only among GPA-producing actinobacteria but also in the non-producers: more than 10% of the screened actinobacterial genomes contained one or multiple vlgs, while less than 1% encoded for a biosynthetic gene cluster (BGC). By phylogenetic reconstructions, our results highlight the co-evolution of the different vlgs, indicating that the most diffused are the ones coding for putative VanY carboxypeptidases, which can be found alone in the genomes or associated with a vanS/R regulatory pair.

Complete genome sequence of Streptomyces cyanogenus S136, producer of anticancer angucycline landomycin A.

Streptomyces cyanogenus S136 is the only known producer of landomycin A (LaA), one of the founding members of angucycline family of aromatic polyketides. LaA displays potent anticancer activities which has made this natural product a target of numerous chemical and cell biological studies. Little is known about the potential of S136 strain to produce other secondary metabolites. Here we report complete genome sequence of LaA producer and how we used this sequence to evaluate for this species its phylogenetic position and diversity of gene clusters for natural product biosynthesis. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02834-4.

Genetically engineered rpsL merodiploidy impacts secondary metabolism and antibiotic resistance in Streptomyces.

Certain point mutations within gene for ribosomal protein S12, rpsL, are known to dramatically change physiological traits of bacteria, most prominently antibiotic resistance and production of various metabolites. The rpsL mutants are usually searched among spontaneous mutants resistant to aminoglycoside antibiotics, such as streptomycin or paromomycin. The shortcomings of traditional selection are as follows: random rpsL mutants may carry undesired genome alterations; many rpsL mutations cannot be isolated because they are either not associated with increased antibiotic resistance or non-viable in the absence of intact rpsLWT gene. Introduction of mutant rpsL alleles in the rpsLWT background can be used to circumvent these obstacles. Here we take the latter approach and report the generation and properties of a set of stable rpsL merodiploids for Streptomyces albus J1074. We identified several rpsL alleles that enhance endogenous and heterologous antibiotic production by this strain and show that rpsLWTrpsLK88E merodiploid displays increased streptomycin resistance. We further tested several promising rpsL alleles in two more strains, Streptomyces cyanogenus S136 and Streptomyces ghanaensis ATCC14672. In S136, plasmid-borne rpsLK88E+P91S and rpsLK88R led to elevated landomycin production; no changes were detected for ATCC14672 merodiploids. Our data outline the prospects for and limitations to rpsL merodiploids as a tool for rapid enhancement of secondary metabolism in Streptomyces.

Eliciting the silent lucensomycin biosynthetic pathway in Streptomyces cyanogenus S136 via manipulation of the global regulatory gene adpA.

Actinobacteria are among the most prolific sources of medically and agriculturally important compounds, derived from their biosynthetic gene clusters (BGCs) for specialized (secondary) pathways of metabolism. Genomics witnesses that the majority of actinobacterial BGCs are silent, most likely due to their low or zero transcription. Much effort is put into the search for approaches towards activation of silent BGCs, as this is believed to revitalize the discovery of novel natural products. We hypothesized that the global transcriptional factor AdpA, due to its highly degenerate operator sequence, could be used to upregulate the expression of silent BGCs. Using Streptomyces cyanogenus S136 as a test case, we showed that plasmids expressing either full-length adpA or its DNA-binding domain led to significant changes in the metabolome. These were evident as changes in the accumulation of colored compounds, bioactivity, as well as the emergence of a new pattern of secondary metabolites as revealed by HPLC-ESI-mass spectrometry. We further focused on the most abundant secondary metabolite and identified it as the polyene antibiotic lucensomycin. Finally, we uncovered the entire gene cluster for lucensomycin biosynthesis (lcm), that remained elusive for five decades until now, and outlined an evidence-based scenario for its adpA-mediated activation.

Teicoplanin biosynthesis: unraveling the interplay of structural, regulatory, and resistance genes.

Teicoplanin (Tcp) is a clinically relevant glycopeptide antibiotic (GPA) that is produced by the actinobacterium Actinoplanes teichomyceticus. Tcp is a front-line therapy for treating severe infections caused by multidrug-resistant Gram-positive pathogens in adults and infants. In this review, we provide a detailed overview of how Tcp is produced by A. teichomyceticus by describing Tcp biosynthesis, regulation, and resistance. We summarize the knowledge gained from in vivo and in vitro studies to provide an integrated model of teicoplanin biosynthesis. Then, we discuss genetic and nutritional factors that contribute to the regulation of teicoplanin biosynthesis, focusing on those that have been successfully applied for improving teicoplanin production. A current view on teicoplanin self-resistance mechanisms in A. teichomyceticus is given, and we compare the Tcp biosynthetic gene cluster with other glycopeptide gene clusters from actinoplanetes and from unidentified isolates/metagenomics samples. Finally, we provide an outlook for further directions in studying Tcp biosynthesis and regulation.

Development of a gene expression system for the uncommon actinomycete Actinoplanes rectilineatus NRRL B-16090.

The urgent need for discovering new bioactive metabolites prompts exploring novel actinobacterial taxa by developing appropriate tools for their genome mining and rational genetic engineering. One promising source of new bioactive natural products is the genus Actinoplanes, a home to filamentous sporangia-forming actinobacteria producing many important specialized metabolites such as teicoplanin, ramoplanin, and acarbose. Here we describe the development of a gene expression system for a new Actinoplanes species, A. rectilineatus (NRRL B-16090), which is a potential producer of moenomycin-like antibiotics. We have determined the optimal conditions for spore formation in A. rectilineatus and a plasmid transfer procedure for its engineering via intergeneric E. coli-A. rectilineatus conjugation. The φC31- and pSG5-based vectors were successfully transferred into A. rectilineatus, but φBT1- and VWB-based vectors were not transferable. Finally, using the glucuronidase reporter system, we assessed the strength of several heterologous promoters for gene expression in A. rectilineatus.

Genetic insights into the mechanism of teicoplanin self-resistance in Actinoplanes teichomyceticus.

Actinoplanes teichomyceticus NRRL B-16726 is the only known producer of the clinically important glycopeptide antibiotic teicoplanin. The producing strain is highly self-resistant to teicoplanin. Although the biosynthesis of teicoplanin has been investigated, much of our understanding of self-resistance in the producing strain is based on the extrapolation of existing data about glycopeptide resistance (mediated by the expression of vanRS-vanHAX genes) in other actinomycetes and cocci. The organization of the operons carrying putative van orthologues in A. teichomyceticus differs from known precedents, further adding up to the uncertainty about teicoplanin self-resistance mechanisms. Here, we determined operon structure of the teicoplanin resistance genes in A. teichomyceticus. Although Tei15* is necessary to activate teicoplanin biosynthetic genes, the expression of van orthologues was shown to be independent of Tei15*. We further showed that tei7 promoter driving the expression of vanHAX orthologues is dependent on Tei2 (VanR). Finally, we demonstrate the utility of the tei2 promoter as a new tool to achieve strong constitutive expression in A. teichomyceticus.