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This work was supported by grants BIO2009-09820 and LE046A11-2 from the Spanish Ministry of Economy and Competitivity and the Junta de Castilla y León, respectively. R. Álvarez-Álvarez and Y. Martínez-Burgo received PFU fellowships from the Spanish Ministry of Education, Culture and Sports. We appreciate the collaboration of Dr. T. López-García in the design of oligonucleotides and RT-qPCR experiments and the reception of plasmid pFL1272 from Dr. F. Lombo.

A salt-tolerant alkaliphilic actinomycete, Mit-1 was isolated from Mithapur, coastal region of Gujarat, India. The strain was identified as Streptomyces clavuligerus and based on 16S rRNA gene sequence (EU146061) homology; it was related to Streptomyces sp. (AY641538.1). The organism could grow with up to 15% salt and pH 11, optimally at 5% and pH 9. It was able to tolerate and secrete alkaline protease in the presence of a number of organic solvents including xylene, ethanol, acetone, butanol, benzene and chloroform. Besides, it could also utilize these solvents as the sole source of carbon with significant enzyme production. However, the organism produced spongy cell mass with all solvents and an orange brown soluble pigment was evident with benzene and xylene. Further, the enzyme secretion increased by 50-fold in the presence of butanol. With acetone and ethanol; the enzyme was highly active at 60-80°C and displayed optimum activity at 70°C. The protease was significantly stable and catalyzed the reaction in the presence of xylene, acetone and butanol. However, ethanol and benzene affected the catalysis of the enzyme adversely. Crude enzyme preparation was more stable at 37°C in solvents as compared to partially purified and purified enzymes. The study holds significance as only few salt-tolerant alkaliphilic actinomycetes are explored and information on their enzymatic potential is still scares. To the best of our knowledge this is the first report on organic solvent tolerant protease from salt-tolerant alkaliphilic actinomycetes.

Streptomyces clavuligerus is a Gram‐positive bacterium that is a high producer of secondary metabolites with industrial applications. The production of antibiotics such as clavulanic acid or cephamycin has been extensively studied in this species; nevertheless, other aspects, such as evolution or ecology, have received less attention. Furthermore, genes that arise from ancient events of lateral transfer have been demonstrated to be implicated in important functions of host species. This approximation discovered relevant genes that genomic analyses overlooked. Thus, we studied the impact of horizontal gene transfer in the S. clavuligerus genome. To perform this task, we applied whole‐genome analysis to identify a laterally transferred sequence from different domains. The most relevant result was a putative antimicrobial peptide (AMP) with a clear origin in the Hymenoptera order of insects. Next, we determined that two copies of these genes were present in the megaplasmid pSCL4 but absent in the S. clavuligerus ATCC 27064 chromosome. Additionally, we found that these sequences were exclusive to the ATCC 27064 strain (and so were not present in any other bacteria) and we also verified the expression of the genes using RNAseq data. Next, we used several AMP predictors to validate the original annotation extracted from Hymenoptera sequences and explored the possibility that these proteins had post‐translational modifications using peptidase cleavage prediction. We suggest that Hymenoptera AMP‐like proteins of S. clavuligerus ATCC 27064 may be useful for both species adaptation and as an antimicrobial molecule with industrial applications. We describe an Hymenoptera antimicrobial peptide horizontally transferred to the antibiotic producer bacteria Streptomyces clavuligerus ATCC 27064

Clavulanic acid (CA) is a specific metabolite that inhibits β-lactamases, which inactivate β-lactam antibiotics, therefore reinstating the activity of β-lactams against β-lactamase-producing pathogens, and surmounting β-lactam resistance. While extensive efforts have been made to optimize CA production through random mutagenesis, these approaches often introduce deleterious mutations, limiting further yield improvements. In this study, whole-genome sequencing, time-resolved transcriptomics, and independent component analysis (ICA)-based gene co-expression network analysis were employed to elucidate the genetic and transcriptomic factors underlying enhanced CA production in mutant Streptomyces clavuligerus strains developed through ultraviolet-induced random mutagenesis. By analyzing strains with varying CA productivities and correlating genomic and transcriptomic changes, we identified multiple candidate mutations, key transcriptional regulators, and metabolic pathways influencing CA yield. Notable findings include large plasmid deletions, an enrichment of mutations in secondary metabolite biosynthesis and regulatory genes, and metabolic shifts redirecting amino acid and carbon flux toward CA biosynthetic pathways. ICA revealed gene modules directly associated with CA biosynthesis, precursor supply, and transcriptional regulation. This integrative approach generated a comprehensive paired genomic-transcriptomic dataset for CA production. The insights gained offer targeted strategies for rational strain engineering, advancing more efficient and sustainable antibiotic production.

Cephamycin C is an extracellular broad spectrum β-lactam antibiotic produced by Streptomyces clavuligerus, S. cattleya and Nocardia lactamdurans. In the present study, different substrates for solid-state fermentation were screened for maximum cephamycin C production by S. clavuligerus NT4. The fermentation parameters such as substrate concentration, moisture content, potassium dihydrogen phosphate, inoculum size and ammonium oxalate were optimized by response surface methodology (RSM). The optimized conditions yielded 21.68 ± 0.76 mg gds-¹ of cephamycin C as compared to 10.50 ± 1.04 mg gds-¹ before optimization. Effect of various amino acids on cephamycin C production was further studied by using RSM, which resulted in increased yield of 27.41 ± 0.65 mg gds-¹.

Streptomyces clavuligerus is a species used worldwide to industrially produce clavulanic acid (CA), a molecule that enhances antibiotic effectiveness against β-lactamase-producing bacterial strains. Despite its low inherent CA production, hyper-producing strains have been developed. However, genomic analyses specific to S. clavuligerus and CA biosynthesis are limited. Genomic variations that may influence CA yield were explored using S. clavuligerus strain genomes from diverse sources. Despite the slight differences obtained by similarity index calculation, pan-genome estimation revealed that only half of the genes identified were present in all strains. As expected, core genes were associated with primary metabolism, while the remaining genes were linked to secondary metabolism. Differences at the sequence level were more likely to be found in regions close to the tips of the linear chromosome. Wild-type strains preserved larger chromosomal and plasmid regions compared to industrial and/or hyper-producing strains; such a grouping pattern was also found through refined phylogenetic analyses. These results provide essential insights for the development of hyper-producing S. clavuligerus strains, attending to the critical demand for this antibiotic enhancer and contributing to future strategies for CA production optimization.

Introduction: Clavulanic acid is the first B-Lactamase inhibitor that has been used clinically since 1981. This inhibitor is the natural product of the fermentation of Streptomyces clavuligerus and is useful for protecting B-lactams against lots of important B-lactamases such as Group A B-lactamases and Group D Cloxacillin lysis enzymes. The aim of this study was to optimize the composition of the fermentation medium in terms of the carbon source in the production of clavulanic acid. Materials and Methods: In this experiment, the influence of different concentrations of glycerol 15 g/L, wheat bran 17 g/L, and glucose 18.1 g/L on the production of clavulanic acid was tested. After passing the levels of sporulation, seeding, and fermentation, the influence of different concentrations of glucose, wheat bran, and glycerol on pH rates, biomass, and morphology of Streptomyces clavuligerus was tested by the light microscope. The concentration of clavulanic acid produced was measured by spectrophotometry. Results: According to the results, the highest production of clavulanic acid in all substrates was on the eighth day. The use of glycerol at a concentration of 15 g/l produced 238.3 mg/l clavulanic acid, which had the highest production rate and also reduced production costs. Discussion and Conclusion: Due to the price, availability, and also increasing the production of glycerol and wheat bran (compared to corn oil), the production of clavulanic acid in environments containing glycerol and wheat bran is easier and more convenient.

Small non-coding RNAs play a pivotal role in regulating various metabolic processes in both prokaryotic and eukaryotic organisms. However, knowledge about small RNAs (sRNAs) in Streptomyces clavuligerus (S. clavuligerus) is scarce. This study aimed to use cutting-edge bioinformatics tools and a compendium of RNA-seq data to predict the potential coding of sRNAs that might be present in the genome of S. clavuligerus ATCC 27064. In the genome of S. clavuligerus, 606 intergenic regions (IGRs) are conserved, and 272 possess a highly thermodynamically stable and conserved secondary structure, indicating the presence of non-coding RNA in these regions. The transcriptome assembly of S. clavuligerus showed that the genome is completely functional, as all the annotated genes are expressed under the conditions analyzed. From this assembly, transcripts originating from IGRs were labeled as putative sRNAs, and their differential expression during the growth curve of S. clavuligerus for clavulanic acid (CA) production was established. The interactome of these differentially expressed (DE) RNAs displayed the sRNAs as global regulators, as they can have multiple mRNA targets. The functional annotation of the target genes of DE sRNAs demonstrated that they are directly involved in secondary metabolite production. Specifically, two sRNA have the genes of the biosynthetic gene cluster of CA as targets. Thus, these molecules add an additional layer to the regulatory cascade for CA biosynthesis, and we propose them as targets for metabolic engineering to increase CA production.

Drimane-type sesquiterpenoids (DMTs) are characterized by a distinctive 6/6 bicyclic skeleton comprising the A and B rings. While DMTs are commonly found in fungi and plants, their presence in bacteria has not been reported. Moreover, the biosynthetic pathways for DMTs have been primarily elucidated in fungi, with identified P450s only acting on the B ring. In this study, we isolated and characterized three bacterial DMTs, namely 3β-hydroxydrimenol ( 2 ), 2α-hydroxydrimenol ( 3 ), and 3-ketodrimenol ( 4 ), from Streptomyces clavuligerus . Through genome mining and heterologous expression, we identified a cav biosynthetic gene cluster responsible for the biosynthesis of DMTs 2 – 4 , along with a P450, CavA, responsible for introducing the C-2 and C-3 hydroxy groups. Furthermore, the substrate scope of CavA revealed its ability to hydroxylate drimenol analogs. This discovery not only broadens the known chemical diversity of DMTs from bacteria, but also provides new insights into DMT biosynthesis in bacteria.

Streptomyces clavuligerus is an industrially important producer of clavulanic acid (CA), a β-lactamase inhibitor which is used together with amoxicillin in one of the most widely prescribed antibacterial medicines, the co-amoxiclav. In a mid-eighties ATCC vial of S . clavuligerus ATCC 27064 culture, we have found a new genotype, which was apparently lost from the subsequent ATCC collection stocks, and has remained obscure to the scientific community. Most importantly, this genotype harbors teleocidin (lyngbyatoxin) biosynthetic genes, which are located on an enigmatic 138 kb chromosomal region and support accumulation of significant amounts of these highly toxic, tumor-promoting secondary metabolites in cultures of S . clavuligerus . While this genomic region is completely absent from all published sequences for S . clavuligerus ATCC strain, at least one of the industrial strains for commercial production of CA, originating from ATCC 27064, retained the genetic potential for production of teleocidins. The origin of teleocidin biosynthetic cluster can now be traced back to early S . clavuligerus stocks at the ATCC. Our work provides a genome sequence and a deposited monoisolate of this genotype. Given the scale of industrial use of S . clavuligerus world-wide and toxicity of teleocidins, we also discuss the environmental and safety implications and provide a method of abolishing teleocidin production without affecting productivity of CA. Key points • Early stocks of S. clavuligerus ATCC 27064 produce toxic teleocidins • Teleocidin biosynthetic genes were found within a distinct S. clavuligerus genotype • The genotype has been passed on to some industrial clavulanic acid producer strains