Abstract The global rise of antibiotic resistance poses a public health challenge, emphasizing the urgent need for novel antibiotics featuring unique chemical scaffolds and mechanisms of action. Here, we report the discovery and characterization of darumycins, guanidine-containing pentacyclic sesterterpene antibiotics that exhibit potent activity against high-priority Gram-positive pathogens and mycobacteria. The darumycin biosynthetic gene cluster was identified through genome mining in the Actinobacterium Micromonospora rubida and heterologously expressed in Streptomyces chassis strains. Gene cluster engineering facilitated the discovery of novel darumycin derivatives, thereby expanding the chemical diversity within the sesterterpene class of natural products and revealing nuanced variations in their bioactivity. Targeted gene deletions, along with LC–MS and NMR analyses, enabled us to propose a darumycin biosynthetic pathway, further complemented by in vitro biochemical characterization of two O-methyltransferase tailoring enzymes, DarM and DarG. The high-resolution crystal structure of DarM in complex with SAH provided valuable insights into the enzymatic mechanism and revealed a distinct architecture compared to other methyltransferases acting on terpene scaffolds.

Focus on 2004 to 2024The rediscovery of natural products (NPs) as a critical source of new therapeutics has been greatly advanced by the development of heterologous expression platforms for biosynthetic gene clusters (BGCs). Among these, Streptomyces species have emerged as the most widely used and versatile chassis for expressing complex BGCs from diverse microbial origins. In this review, we provide a comprehensive analysis of over 450 peer-reviewed studies published between 2004 and 2024 that describe the heterologous expression of BGCs in Streptomyces hosts. We present a data-driven overview of expression trends across time, BGC types, donor species, and host strain preferences, offering the first quantitative perspective on how this field has evolved over two decades. Our review discusses the key factors influencing successful BGC expression in Streptomyces, including genomic integration strategies, regulatory elements, codon optimization, and precursor supply. We also examine the impact of synthetic biology tools, genome engineering, and host strain tailoring in overcoming common expression barriers. Special emphasis is placed on the role of heterologous expression in accessing silent or cryptic BGCs, elucidating biosynthetic pathways, and generating new-to-nature analogues through combinatorial biosynthesis. By integrating technological advances with practical case studies, we highlight how Streptomyces-based heterologous expression is enabling not only the efficient production of known compounds but also the discovery of structurally novel and biologically potent metabolites. This review aims to serve as a resource for researchers in natural products, synthetic biology, and drug discovery who seek to harness the full potential of microbial biosynthetic diversity.

Streptomyces viridosporus (formerly ghanaensis) ATCC14672 is the best-studied producer of moenomycins, a family of phosphoglycolipid natural products directly inhibiting peptidoglycan glycosyltransferases. The uniqueness of moenomycins, in terms of structure and mode of action, and their extreme potency against several Gram-positive pathogens (including vancomycin- and methicillin-resistant cocci), are their advantages as a drug candidate. However, they are not orally bioavailable; in the bloodstream, moenomycins are characterized by an exceedingly long half-life. These shortcomings are thought to be caused by the long C25 lipid chain (moenocinol) of moenomycins. Here we report the generation and initial studies of two ATCC 14672 mutants to access a greater diversity of moenomycins around its lipid chain. The dO5 mutant cannot produce any moenomycins due to the deletion of the moeO5 gene for the first step in moenomycin assembly. The M12 mutant of ATCC 14672 (knockout of the prenyltransferase gene moeN5) accumulates moenomycins with a C15 lipid chain instead of moenocinol. We demonstrate and discuss the potential applications of the dO5 and M12 mutants for the discovery of novel moenomycins and their genetic determinants.

Saccharopolyspora erythraea is an industrially important actinobacteria and a model in polyketide biosynthesis studies. However, its genetic toolbox remains limited. Here, we evaluated the properties of integrative and replicative vectors in Sacch. erythraea DSM40517. Three actinophage-based integrative vectors (φC31, φBT1, VWB) were efficiently transferred into Sacch. erythraea. The attB sites for these vector systems were identified. In case of φBT1- and VWB-based vectors, unique integration sites exists within the chromosome of the strain. In case of pSET152 (φC31), at least four different loci were identified with different efficiency of recombination. Our data revealed that all three integrative vectors are excised from the chromosome of the strain with varying frequencies, highlighting the need for selective pressures for their stable maintenance. Commonly used replicative vectors with Streptomyces replicons (pIJ101 and pSG5) have low transfer efficiency in Sacch. erythraea. To solve this problem, we constructed a shuttle vector pYS191 based on the replicon of plasmid pJV1. This vector was efficiently transferred into Sacch. erythraea and other actinobacteria including Streptomyces, Saccharothrix and Couchioplanes species. pYS191 is maintained at a low copy number under selective conditions, and is rapidly lost without antibiotic pressure. Neither integrative nor replicative vectors significantly affected erythromycin production. This study provides a comprehensive assessment of vector system in Sacch. erythraea and reports a new replicative vector based on rarely exploited pJV1 replicon for transient gene expression and genome engineering. These results expand the genetic toolbox for Saccharopolyspora and facilitate its use in natural products research and industrial biotechnology. • Actinobacterial integrative vectors are suited for gene cloning in Sacch. erythraea. • attB sites for three integrative vectors identified within Sacch. erythraea genome. • A new replicative vector pYS191 for use in Sacch. erythraea was constructed.

The growing threat of multidrug-resistant bacterial infections highlights the urgent need for antibiotics with novel mechanisms of action. Gromomycins, a newly identified class of triterpene antibiotics, exhibit potent activity against Gram-positive bacteria, including drug-resistant species, through a previously uncharacterized mode of action. Here, we report the discovery of a gromomycin-like biosynthetic gene cluster in the Actinoplanes genus through a genome mining approach, leading to the isolation and characterization of new bioactive derivatives that overcome resistance to clinically used drugs in vancomycin-resistant enterococci. Mechanistic studies revealed that gromomycins induce rapid potassium ion leakage and depolarization of the bacterial membrane, resulting in bactericidal activity against Staphylococcus aureus. Gromomycins disrupt the integrity of the cytoplasmic membrane, as evidenced by large pore formation, leakage of intracellular contents, and subsequent cell lysis. Supplementation with membrane lipids and fatty acids neutralized their antibacterial activity, suggesting a direct membrane-targeting mechanism, further supported by the inability to raise gromomycin resistance and their toxic effects on eukaryotic cells. Collectively, these findings deepen our understanding of gromomycin activity and demonstrate the utility of genome mining to uncover structurally novel and biologically active natural products.

Streptomyces albus J1074 (now S. albidoflavus J1074) is a widely used heterologous host for natural product discovery due to its capacity to express biosynthetic gene clusters (BGCs) from diverse organisms. A derivative of this strain, S. albus Del14, enhances heterologous expression by reducing background metabolite production enabling the identification of the previously hidden BGC responsible for producing mansouramycins. In this study, we demonstrate the biosynthetic crosstalk between the native mansouramycin BGC in S. albus Del14 and introduced BGCs from three different organisms results in the production of novel compounds, some featuring rare and complex chemical scaffolds. These include malevonin, which combines NRPS- and mansouramycin-derived building blocks forming a fluorene scaffold, as well as 5'-chloromansouramycin D, a halogenated derivative of mansouramycin D. Additionally, we identified mansevorone, a compound structurally similar to mansouramycin D but utilizing a different tryptophan-derived C7 precursor. This precursor likely arises from the activation of native genes in the host S. albus Del14, triggered by SARP regulators present on the introduced BGC. These findings highlight the evolutionary significance of BGC interactions and underscore their potential as a powerful tool for discovering novel natural products, providing insights that could inform innovative strategies in biosynthetic engineering and the guided evolution of new bioactive compounds.

The strain Je 1-4 T was isolated from the coastal rhizosphere soil of Juniperus excelsa M. Bieb. (Crimean Peninsula). Phylogenetic studies based on the 16S rRNA gene sequence revealed that Je 1-4 T is phylogenetically close to Streptomyces libani subsp. libani NBRC 13452 T (JCM 4322) and Streptomyces nigrescens NBRC 12894 T (DSM 40276) with sequence similarities of 99.86 to 99.93%. The genome of strain Je 1-4 T consisted of a linear chromosome with a size of 8.9 Mbp and a G+C content of 70.9mol%. Digital DNA–DNA hybridization (dDDH) analysis showed that Je 1-4 T is distinct from both S. libani subsp. libani and S. nigrescens (dDDH values of 66.6% and 66.7 %, respectively), while the latter two strains likely represent the same species (dDDH value of 92.0%). The predominant fatty acids in the strains were iso-C 16:0 , anteiso-C 17:0 and anteiso-C 15:0 , and the major menaquinones were MK9 H6 and MK9 H8. Based on these genomic and phenotypic data, strain Je 1-4 T represents a novel species of Streptomyces , for which the name Streptomyces explomaris sp. nov. is proposed. The type strain is Je 1-4 T (=DSM 117375 T =LMG 33490 T ). Additionally, we propose that S. libani subsp. libani Baldacci and Grein 1966 (Approved Lists 1980) is a later heterotypic synonym of S. nigrescens (Sveshnikova 1957) Pridham et al. 1958 (Approved Lists 1980).

BACKGROUND:Streptomyces roseochromogenes NRRL 3504 produces clorobiocin, an aminocoumarin antibiotic that inhibits DNA replication. No other natural products have been isolated from this bacterium so far, despite the presence of a rich repertoire of specialized metabolite biosynthesis gene clusters (smBGCs) within its genome. Heterologous expression of smBGCs in suitable chassis speeds up the discovery of the natural products hidden behind these sets of genes. RESULTS:In this work we focus on one intriguing smBGC of NRRL 3504 bearing some similarity to gene clusters involved in production of manumycin family polyketides. Through heterologous expression in Streptomyces chassis strains S. albus Del14 and S. lividans ΔYA9, this smBGC (hereafter referred to as lim BGC) was shown to direct the production of unusual polyketide limocrocin (LIM) known for its ability to interfere with viral reverse transcriptases. The organization of lim BGC, data on the structures of revealed metabolites as well as manipulations of lim genes allowed us to put forward an initial hypothesis about a biosynthetic pathway leading to LIM. We provide initial data on two LIM derivatives as well as updated NMR spectra for the main product. CONCLUSION:This study reveals the genetic control of biosynthesis of LIM that remained hidden for the last 70 years. This, in turn, opens the door to biological routes towards overproduction of LIM as well as generation of its derivatives.

Nybomycin is a reverse antibiotic with selective activity against fluoroquinolone-resistant Gram-positive bacteria, including Staphylococcus aureus, making it a promising candidate to fight against antimicrobial resistance. However, its clinical development has been limited by the low production yields of native producers. To address this, we explored the heterologous expression of the nybomycin biosynthetic gene cluster (nyb) from the natural producer S. albus subsp. chlorinus NRRL B-24,108 in different marine and terrestrial Streptomyces hosts, aiming to boost production through targeted regulatory and metabolic engineering. We also evaluated the use of seaweed-derived hydrolysates as sustainable fermentation substrates. Among several tested hosts, S. explomaris carrying the nyb gene cluster, produced the highest nybomycin titers. Global transcriptomic analysis identified transcriptional repression and precursor limitation as key bottlenecks. Deletion of the repressors nybW and nybX (NYB-1) significantly increased production, while further overexpression of genes boosting precursor supply (zwf2, nybF) led to the creation of NYB-3B, which reached a titer of 57 mg L− 1—fivefold higher than the previous benchmark. When cultivated on hydrolysates from commercial brown seaweed (Himanthalia elongata) without nutrient supplementation, NYB-3B achieved a titer of 14.8 mg L− 1. This study demonstrates the potential of S. explomaris as a chassis for high-level heterologous nybomycin production and its compatibility with renewable marine feedstocks. Regulatory and metabolic engineering effectively relieved key bottlenecks and improved precursor supply. The use of seaweed hydrolysates supports the development of sustainable nybomycin production. Collectively, these findings provide a valuable foundation for future efforts toward improved supply and clinical development of nybomycin.

The current situation with drug-resistant microbial pathogens is critical, dictating an acute need for novel efficient antibiotics. Herein, we report a new class of antibiotics named gromomycins with significant activity, especially against drug-resistant Gram-positive pathogens, including methicillin- and daptomycin-resistant Staphylococcus aureus. Gromomycins are pentacyclic triterpenes with a cyclic guanidino group forming the fifth six-membered ring. We have used transposon mutagenesis to identify the gromomycin biosynthetic gene cluster, since it could not be assigned by any available bioinformatics tools, highlighting its unique biosynthetic route. Using gene cluster engineering, feeding experiments, and LC-MS and NMR analyses we have proposed the biosynthetic pathway for gromomycins, which are the first bacterial triterpenes synthesized independently of the squalene pathway. They also exhibit a so far unprecedented cyclization route that utilizes a hexaprenylguanidine linear precursor. Leveraging our understanding of their biosynthesis, we have identified additional gromomycin producers, resulting in the isolation of novel bioactive derivatives.

Pyrazinones are a growing family of microbial NRPS-derived natural products showing interesting biological activities. These compounds are characterized by the presence of either a di- or trisubstituted heterocyclic, nonaromatic 2(1 H)-pyrazinone core in their structure. The most commonly occurring disubstituted pyrazinone natural products are synthesized through a dipeptide intermediate, which is further cyclized to yield the pyrazinone moiety. Trisubstituted pyrazinones are seldom found in natural products, with JBIR56 and JBIR57, isolated from marine Streptomyces, being notable examples. In contrast to the simply organized disubstituted pyrazinones, JBIR56 and JBIR57 are syn-thesized as tetrapeptides with unnatural beta-amino acid residue involved in the for-mation of the pyrazinone moiety. Despite interesting structural features, biosynthetic routes leading to the production of these compounds have not been reported yet. Here we report the discovery of new members of trisubstituted pyrazinone family– tetrapeptides ichizinones A-C in Streptomyces sp. LV45-129. Through sequence analysis and heterologous expression, a biosynthetic gene cluster encoding ichizinone production was identified. Based on gene annotation and sequence homology, a biosynthetic model was suggested. The presented results provide insights into the biosynthesis of rare trisubstituted pyrazinone natural products.

Actinosynnema mirum DSM 43827 is a bacterium from the small genus Actinosynnema within the rapidly growing actinomycete family Pseudonocardiaceae (Land M et al. Stand Genomic Sci 1:46–53 2009). Despite its diverse repertoire of specialized metabolite biosynthetic gene clusters (BGCs), the potential of A. mirum for production of bioactive molecules is not fully explored. Here, we used a heterologous expression approach to gain deeper insight into this issue. In this work we report that expression of in silico predicted BGC#4 from A. mirum in S. albus Del14 and S. lividans ΔYA9 led to production of several prenylated derivatives of tyrosine. Their most likely structures, according to MS and MS/MS data, agreed with 4-O-prenyl-(l)-tyrosine and its N-acetyl derivative, previously described in lichen-forming fungi (Iacovelli R et al. J Nat Prod 87:2243–2254 2024). Further experiments confirm the production of the aforementioned compounds is governed by a single structural gene, amir_2071 for prenyltransferase of dimethylallyltryptophan synthase (DMATS) superfamily, whose homologs are abundant in bacterial genomes.

Dysbiosis, an imbalance within the oral microbiome, is associated with several diseases, particularly periodontitis, which is characterized by a transition towards Gram-negative bacteria. Conventional treatments, including antibiotics and surgery, have limitations, which have led to an exploration of alternative methods. The application of probiotic bacteria to restore a balanced microbiome is such a minimally invasive alternative. In practice, the use of probiotic bacteria is hampered by the insufficient survival rate of the bacteria in the formulations. Using Limosilactobacillus strains as an example, this study addresses these challenges and discusses three concrete measures to extend the duration of the bacterial viability. First, bacterial cultures were exposed to stress inducers during cultivation, such as osmotic stress or acidic pH, to induce protective physiological responses and enhance resilience. Next, the probiotic bacteria were microencapsulated via spray-drying with Eudragit® EPO & RL30D. Besides the protective effects, the aim of microencapsulation is to ensure the gradual release of the bacteria, i.e. Limosilactobacillus reuteri (formerly known as Lactobacillus reuteri). Finally, films comprising mucoadhesive polymers were created with the objective of prolonging the residence of bacteria in the oral cavity through mucoadhesive interactions and rendering the bacteria in a form that is suitable for application. Our research underscores the significance of cultivation conditions in improving bacterial survival in subsequent formulation steps. We confirm the efficacy of microencapsulation of L. reuteri through spray-drying. Its success is evidenced by the controlled release identified during the dissolution process. A suitable method for the production of mucoadhesive polymer films is described. Encountered challenges when embedding microencapsulated bacteria in polymer films are discussed and a set of conditions, including growth phase, pH, and osmotic stress, was evaluated to identify factors influencing survival. In summary, the results enhance the progress of focused measures for preserving dental health, highlighting the capability of mucoadhesive polymer films as delivery vehicles for microencapsulated probiotic bacteria.

Streptomyces cyanogenus S136 is known to produce landomycin family antibiotics, particularly its largest congener, landomycin A. Except for landomycins and polyene lucensomycin, no other specialized metabolites were sourced from S136. Nevertheless, S136 genome sequencing revealed over 40 biosynthetic gene clusters (BGCs), implying underappreciated potential of this strain for the production of novel bioactive natural compounds. We set out to gain deeper insight into the specialized metabolome of this strain. First, trans­criptomic analysis of S136 grown under landomycin production conditions has been carried out, revealing that most of them are expressed at a basal level. This, likely, leads to a phenotypic silence of most of the BGCs. Nevertheless, several notable exceptions have been spotted. First of all, landomycin BGC is expressed at high level (at least 100 Transcripts Per Million mapped reads (TPM); and around 1000 TPM for minimal polyketide synthase genes lanFABC). Similarly, high levels of expression showed BGCs # 2, 4, 7 and 33, of which #2, encoding unknown saccharide, is the most dissimilar to the described precedents. RNAseq data also allowed us to delineate better the borders of several presumed BGCs. In the next phase of the work we singled out a few BGCs within S136 that appeared to be promising. First, these BGCs exhibited low similarity to the other gene clusters directing the production of known natural products. Second, the BGCs harbored cluster-situated regulatory genes that can be employed in the attempts to activate the expression of cryptic pathways. For one such BGC we constructed two plasmids for expression of several such regulatory genes and introduced them into S136 and its derivative deficient in production of landomycin A. Bioassays showed no differences in bioactivity of the recombinant strains as compared to the initial strains. Liquid chromatography coupled to mass spectrometry (LC-MS) analysis of several S. cyanogenus samples revealed the effects of genotype, growth conditions and extraction on specialized metabolome of this species, setting reference point for further stu­dies.

Nybomycin is an antibiotic compound with proven activity against multi-resistant Staphylococcus aureus, making it an interesting candidate for combating these globally threatening pathogens. For exploring its potential, sufficient amounts of nybomycin and its derivatives must be synthetized to fully study its effectiveness, safety profile, and clinical applications. As native isolates only accumulate low amounts of the compound, superior producers are needed. The heterologous cell factory S. albidoflavus 4N24, previously derived from the cluster-free chassis S. albidoflavus Del14, produced 860 mu g L-1 of nybomycin, mainly in the stationary phase. A first round of strain development modulated expression of genes involved in supply of nybomycin precursors under control of the common Perm* promoter in 4N24, but without any effect. Subsequent studies with mCherry reporter strains revealed that Perm* failed to drive expression during the product synthesis phase but that use of two synthetic promoters (PkasOP* and P41) enabled strong constitutive expression during the entire process. Using PkasOP*, several rounds of metabolic engineering successively streamlined expression of genes involved in the pentose phosphate pathway, the shikimic acid pathway, supply of CoA esters, and nybomycin biosynthesis and export, which more than doubled the nybomycin titer to 1.7 mg L-1 in the sixth-generation strain NYB-6B. In addition, we identified the minimal set of nyb genes needed to synthetize the molecule using single-gene-deletion strains. Subsequently, deletion of the regulator nybW enabled nybomycin production to begin during the growth phase, further boosting the titer and productivity. Based on RNA sequencing along the created strain genealogy, we discovered that the nyb gene cluster was unfavorably downregulated in all advanced producers. This inspired removal of a part and the entire set of the four regulatory genes at the 3 '-end nyb of the cluster. The corre-sponding mutants NYB-8 and NYB-9 exhibited marked further improvement in production, and the deregulated cluster was combined with all beneficial targets from primary metabolism. The best strain, S. albidoflavus NYB-11, accumulated up to 12 mg L-1 nybomycin, fifteenfold more than the basic strain. The absence of native gene clusters in the host and use of a lean minimal medium contributed to a selective production process, providing an important next step toward further development of nybomycin.

Derivatizing natural products (NPs) is essential in structure-activity relationship (SAR) studies, compound optimization, and drug development. Ribosomally synthesized and post-translationally modified peptides (RiPPs) represent one of the major classes of natural products. Thioholgamide represents thioamitide - a recently emerged family of RiPPs with unique structures and great potential in anticancer drug development. Although the method for generating the RiPP library by codon substitutions in the precursor peptide gene is straightforward, the techniques to perform RiPP derivatization in Actinobacteria remain limited and time-consuming. Here, we report a facile system for producing a library of randomized thioholgamide derivatives utilizing an optimized Streptomyces host. This technique enabled us to access all possible amino acid substitutions of the thioholgamide molecule, one position at a time. Out of 152 potential derivatives, 85 were successfully detected, revealing the impact of amino acid substitutions on thioholgamide post-translational modifications (PTMs). Moreover, new PTMs were observed among thioholgamide derivatives: thiazoline heterocycles, which have not yet been reported for thioamitides, and S-methylmethionine, which is very rare in nature. The obtained library was subsequently used for thioholgamide SAR studies and stability assays.

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.

Pamamycins are a family of highly bioactive macrodiolide polyketides produced by Streptomyces alboniger as a complex mixture of derivatives with molecular weights ranging from 579 to 705 Daltons. The large derivatives are produced as a minor fraction, which has prevented their isolation and thus studies of chemical and biological properties. Herein, we describe the transcriptional engineering of the pamamycin biosynthetic gene cluster (pam BGC), which resulted in the shift in production profile toward high molecular weight derivatives. The pam BGC library was constructed by inserting randomized promoter sequences in front of key biosynthetic operons. The library was expressed in Streptomyces albus strain with improved resistance to pamamycins to overcome sensitivity-related host limitations. Clones with modified pamamycin profiles were selected and the properties of engineered pam BGC were studied in detail. The production level and composition of the mixture of pamamycins was found to depend on balance in expression of the corresponding biosynthetic genes. This approach enabled the isolation of known pamamycins and the discovery of three novel derivatives with molecular weights of 663 Da and higher. One of them, homopamamycin 677A, is the largest described representative of this family of natural products with an elucidated structure. The new pamamycin 663A shows extraordinary activity (IC50 2 nM) against hepatocyte cancer cells as well as strong activity (in the one-digit micromolar range) against a range of Gram-positive pathogenic bacteria. By employing transcriptional gene cluster refactoring, we not only enhanced the production of known pamamycins but also discovered novel derivatives exhibiting promising biological activities. This approach has the potential for broader application in various biosynthetic gene clusters, creating a sustainable supply and discovery platform for bioactive natural products.