Secondary metabolites produced by the fermentation of Streptomyces avermitilis bacterium are powerful antiparasitic agents used in animal health, agriculture and human infection treatments. Avermectin is a macrocyclic lactone with four structural components (A1, A2, B1, B2), each of them containing a major and a minor subcomponent, out of which avermectin B1a is the most effective parasitic control compound. Avermectin B1a produces two homologue avermectins (B1 and B2) that have been used in agriculture as pesticides and antiparasitic agents, since 1985. It has a great affinity with the Cl-channels of the glutamate receptor, allowing the constant flow of Cl- ions into the nerve cells, causing a phenomenon of hyperpolarization causing death by flaccid paralysis. The purpose of this work was to gather information on the production of avermectins and their biocidal effects, with special emphasis on their role in the control of pests and phytopathogenic diseases. The literature showed that S. avermitilis is an important producer of macrocyclic lactones with biocidal properties. In addition, avermectin contributes to the control of ectoparasites and endoparasites in human health care, veterinary medicine and agriculture. Importantly, avermectin is a compound that is harmless to the host (no side effects), non-target organisms and the environment.

Crosstalk regulation is widespread in Streptomyces species. Elucidating the influence of a specific regulator on target biosynthetic gene clusters (BGCs) and cell metabolism is crucial for strain improvement through regulatory protein engineering. PteF and PteR are two regulators that control the biosynthesis of filipin, which competes for building blocks with avermectins in Streptomyces avermitilis. However, little is known about the effects of PteF and PteR on avermectin biosynthesis. In this study, we investigated their impact on avermectin biosynthesis and global cell metabolism. The deletion of pteF resulted in a 55.49% avermectin titer improvement, which was 23.08% higher than that observed from pteR deletion, suggesting that PteF plays a more significant role in regulating avermectin biosynthesis, while PteF hardly influences the transcription level of genes in avermectin and other polyketide BGCs. Transcriptome data revealed that PteF exhibited a global regulatory effect. Avermectin production enhancement could be attributed to the repression of the tricarboxylic acid cycle and fatty acid biosynthetic pathway, as well as the enhancement of pathways supplying acyl-CoA precursors. These findings provide new insights into the role of PteF on avermectin biosynthesis and cell metabolism, offering important clues for designing and building efficient metabolic pathways to develop high-yield avermectin-producing strains.

Avermectins (AVMs), a family of 16-membered macrocyclic macrolides produced by Streptomyces avermitilis, have been the most successful microbial natural antiparasitic agents in recent decades. Doramectin, an AVM derivative produced by S. avermitilis bkd- mutants through cyclohexanecarboxylic acid (CHC) feeding, was commercialized as a veterinary antiparasitic drug by Pfizer Inc. Our previous results show that the production of avermectin and actinorhodin was affected by several other polyketide biosynthetic gene clusters in S. avermitilis and Streptomyces coelicolor, respectively. Thus, here, we propose a rational strategy to improve doramectin production via the termination of competing polyketide biosynthetic pathways combined with the overexpression of CoA ligase, providing precursors for polyketide biosynthesis. fadD17, an annotated putative cyclohex-1-ene-1-carboxylate:CoA ligase-encoding gene, was proven to be involved in the biosynthesis of doramectin. By sequentially removing three PKS (polyketide synthase) gene clusters and overexpressing FadD17 in the strain DM203, the resulting strain DM223 produced approximately 723 mg/L of doramectin in flasks, which was approximately 260% that of the original strain DM203 (approximately 280 mg/L). To summarize, our work demonstrates a novel viable approach to engineer doramectin overproducers, which might contribute to the reduction in the cost of this valuable compound in the future.

The genome of Streptomyces avermitilis contains 33 cytochrome P450 genes. Among the P450 gene products of S. avermitilis, we characterized the biochemical function and structural aspects of CYP184A1. Ultra-performance liquid chromatography-tandem mass spectrometry analysis showed that CYP184A1 induced an epoxidation reaction to produce 9,10-epoxystearic acid. Steady-state kinetic analysis yielded a kcat value of 0.0067 min-1 and a Km value 10 μM. The analysis of its crystal structures illustrated that the overall CYP184A1 structure adopts the canonical scaffold of cytochrome P450 and possesses a narrow and deep substrate pocket architecture that is required for binding to linear chain fatty acids. In the structure of the CYP184A1 oleic acid complex (CYP184A1-OA), C9-C10 of oleic acid was bound to heme for the productive epoxidation reaction. This study elucidates the roles of P450 enzymes in the oxidative metabolism of fatty acids in Streptomyces species.

Avermectins (AVEs) are economically potent anthelmintic agents produced by Streptomyces avermitilis. Among eight AVE components, B1a exhibits the highest insecticidal activity. The purpose of this study was to enhance B1a production, particularly in the high-yielding industrial strain A229, by a combination strategy involving the following steps. (i) aveC gene was engineered to increase B1a:B2a ratio. Three aveC variants (aveC2m, aveC5m, and aveC8m, respectively encoding two, five, and eight amino acid mutations) were synthesized by fusion PCR. B1a:B2a ratio in A229 derivative having kasOp*-controlled aveC8m reached 1.33 (B1a and B2a titers were 8120 and 6124 μg/mL). Corresponding values in A229 were 0.99 and 6447 and 6480 μg/mL. (ii) β-oxidation pathway genes fadD and fadAB were overexpressed in wild-type (WT) strain and A229 to increase supply of acyl-CoA precursors for AVE production. The resulting strains all showed increased B1a titer. Co-overexpression of pkn5p-driven fadD and fadAB in A229 led to B1a titer of 8537 μg/mL. (iii) Genes bicA and ecaA involved in cyanobacterial CO2-concentrating mechanism (CCM) were introduced into WT and A229 to enhance carboxylation velocity of acetyl-CoA and propionyl-CoA carboxylases, leading to increased supply of malonyl- and methylmalonyl-CoA precursors and increased B1a titer. Co-expression of bicA and ecaA in A229 led to B1a titer of 8083 μg/mL. (iv) aveC8m, fadD-fadAB, and bicA-ecaA were co-overexpressed in A229, resulting in maximal B1a titer (9613 μg/mL; 49.1% increase relative to A229). Our findings demonstrate that the combination strategy we provided here is an efficient approach for improving B1a production in industrial strains.Key points• aveC mutation increased avermectin B1a:B2a ratio and B1a titer.• Higher levels of acyl-CoA precursors contributed to enhanced B1a production.• B1a titer in an industrial strain was increased by 49.1% via a combination strategy.

BACKGROUND: The gram-positive bacterium, Streptomyces avermitilis, holds industrial importance as the producer of avermectin, a widely used anthelmintic agent, and a heterologous expression host of secondary metabolite-biosynthetic gene clusters. Despite its industrial importance, S. avermitilis\' genome organization and regulation of gene expression remain poorly understood. In this study, four different types of Next-Generation Sequencing techniques, including dRNA-Seq, Term-Seq, RNA-Seq and ribosome profiling, were applied to S. avermitilis to determine transcription units of S. avermitilis at a genome-wide level and elucidate regulatory elements for transcriptional and translational control of individual transcription units.
RESULTS: By applying dRNA-Seq and Term-Seq to S. avermitilis MA-4680, a total of 2361 transcription start sites and 2017 transcript 3\'-end positions were identified, respectively, leading to determination of 1601 transcription units encoded in S. avermitilis\' genome. Cataloguing the transcription units and integrated analysis of multiple high-throughput data types revealed the presence of diverse regulatory elements for gene expression, such as promoters, 5\'-UTRs, terminators, 3\'-UTRs and riboswitches. The conserved promoter motifs were identified from 2361 transcription start sites as 5\'-TANNNT and 5\'-BTGACN for the - 10 and - 35 elements, respectively. The - 35 element and spacer lengths between - 10 and - 35 elements were critical for transcriptional regulation of functionally distinct genes, suggesting the involvement of unique sigma factors. In addition, regulatory sequences recognized by antibiotic regulatory proteins were identified from the transcription start site information. Analysis of the 3\'-end of RNA transcript revealed that stem structure formation is a major determinant for transcription termination of most transcription units.
CONCLUSIONS: The transcription unit architecture elucidated from the transcripts\' boundary information provides insights for unique genetic regulatory mechanisms of S. avermitilis. Our findings will elevate S. avermitilis\' potential as a production host for a diverse set of secondary metabolites.

Ivermectin (IVM) is an FDA approved macrocyclic lactone compound traditionally used to treat parasitic infestations and has shown to have antiviral potential from previous in-vitro studies. Currently, IVM is commercially available as a veterinary drug but have also been applied in humans to treat onchocerciasis (river blindness - a parasitic worm infection) and strongyloidiasis (a roundworm/nematode infection). In light of the recent pandemic, the repurposing of IVM to combat SARS-CoV-2 has acquired significant attention. Recently, IVM has been proven effective in numerous in-silico and molecular biology experiments against the infection in mammalian cells and human cohort studies. One promising study had reported a marked reduction of 93% of released virion and 99.98% unreleased virion levels upon administration of IVM to Vero-hSLAM cells. IVM\'s mode of action centres around the inhibition of the cytoplasmic-nuclear shuttling of viral proteins by disrupting the Importin heterodimer complex (IMPα/β1) and downregulating STAT3, thereby effectively reducing the cytokine storm. Furthermore, the ability of IVM to block the active sites of viral 3CLpro and S protein, disrupts important machinery such as viral replication and attachment. This review compiles all the molecular evidence to date, in review of the antiviral characteristics exhibited by IVM. Thereafter, we discuss IVM\'s mechanism and highlight the clinical advantages that could potentially contribute towards disabling the viral replication of SARS-CoV-2. In summary, the collective review of recent efforts suggests that IVM has a prophylactic effect and would be a strong candidate for clinical trials to treat SARS-CoV-2.

A n ovel glycoside hydrolase (GH) family 46 chitosanase (SaCsn46A) from Streptomyces avermitilis was cloned and functionally expressed in Escherichia coli Rosetta (DE3) strains. SaCsn46A consists of 271 amino acids, which includes a 34-amino acid signal peptide. The protein sequence of SaCsn46A shows maximum identity (83.5%) to chitosanase from Streptomyces sp. SirexAA-E. Then, the mature enzyme was purified to homogeneity through Ni-chelating affinity chromatography with a recovery yield of 78% and the molecular mass of purified enzyme was estimated to be 29 kDa by SDS-PAGE. The recombinant enzyme possessed a temperature optimum of 45 °C and a pH optimum of 6.2, and it was stable at pH ranging from 4.0 to 9.0 and below 30 °C. The Km and Vmax values of this enzyme were 1.32 mg/mL, 526.32 U/mg/min, respectively (chitosan as substrate). The enzyme activity can be enhanced by Mg2+ and especially Mn2+, which could enhance the activity about 3.62-fold at a 3-mM concentration. The enzyme can hydrolyze a variety of polysaccharides which are linked by β-1,4-glycosidic bonds such as chitin, xylan, and cellulose, but it could not hydrolyze polysaccharides linked by α-1,4-glycosidic bonds. The results of thin-layer chromatography and HPLC showed that the enzyme exhibited an endo-type cleavage pattern and could hydrolyze chitosan to glucosamine (GlcN) and (GlcN)2. This study demonstrated that SaCsn46A is a promising enzyme to produce glucosamine and chitooligosaccharides (COS) from chitosan.

The heat shock response (HSR) is a universal cellular response that promotes survival following temperature increase. In filamentous Streptomyces, which accounts for ∼70% of commercial antibiotic production, HSR is regulated by transcriptional repressors; in particular, the widespread MerR-family regulator HspR has been identified as a key repressor. However, functions of HspR in other biological processes are unknown. The present study demonstrates that HspR pleiotropically controls avermectin production, morphological development, and heat shock and H2O2 stress responses in the industrially important species Streptomyces avermitilis. HspR directly activated ave structural genes (aveA1 and aveA2) and H2O2 stress-related genes (katA1, catR, katA3, oxyR, ahpC, and ahpD), whereas it directly repressed heat shock genes (HSGs) (the dnaK1-grpE1-dnaJ1-hspR operon, clpB1p, clpB2p, and lonAp) and developmental genes (wblB, ssgY, and ftsH). HspR interacted with PhoP (response regulator of the widespread PhoPR two-component system) at dnaK1p to corepress the important dnaK1-grpE1-dnaJ1-hspR operon. PhoP exclusively repressed target HSGs (htpG, hsp18_1, and hsp18_2) different from those of HspR (clpB1p, clpB2p, and lonAp). A consensus HspR-binding site, 5\'-TTGANBBNNHNNNDSTSHN-3\', was identified within HspR target promoter regions, allowing prediction of the HspR regulon involved in broad cellular functions. Taken together, our findings demonstrate a key role of HspR in the coordination of a variety of important biological processes in Streptomyces species. IMPORTANCE Our findings are significant to clarify the molecular mechanisms underlying HspR function in Streptomyces antibiotic production, development, and H2O2 stress responses through direct control of its target genes associated with these biological processes. HspR homologs described to date function as transcriptional repressors but not as activators. The results of the present study demonstrate that HspR acts as a dual repressor/activator. PhoP cross talks with HspR at dnaK1p to coregulate the heat shock response (HSR), but it also has its own specific target heat shock genes (HSGs). The novel role of PhoP in the HSR further demonstrates the importance of this regulator in Streptomyces. Overexpression of hspR strongly enhanced avermectin production in Streptomyces avermitilis wild-type and industrial strains. These findings provide new insights into the regulatory roles and mechanisms of HspR and PhoP and facilitate methods for antibiotic overproduction in Streptomyces species.

Avermectins are a group of drugs that occurs naturally as a product of fermenting Streptomyces avermitilis, an actinomycetes, isolated from the soil. Eight different structures, including ivermectin, abamectin, doramectin, eprinomectin, moxidectin, and selamectin, were isolated and divided into four major components (A1a, A2a, B1a and B2a) and four minor components (A1b, A2b, B1b, and B2b). Avermectins are generally used as a pesticide for the treatment of pests and parasitic worms as a result of their anthelmintic and insecticidal properties. Additionally, they possess anticancer, anti-diabetic, antiviral, antifungal, and are used for treatment of several metabolic disorders. Avermectin generally works by preventing the transmission of electrical impulse in the muscle and nerves of invertebrates, by amplifying the glutamate effects on the invertebrates-specific gated chloride channel. Avermectin has unwanted effects or reactions, especially when administered indiscriminately, which include respiratory failure, hypotension, and coma. The current review examines the mechanism of actions, biosynthesis, safety, pharmacokinetics, biological toxicity and activities of avermectins.