Cytokinetic engineering enhances the secretory production of recombinant human lysozyme in Komagataella phaffii.

BACKGROUND: Human lysozyme (hLYZ) is a natural antibacterial protein with broad applications in food and pharmaceutical industries. Recombinant production of hLYZ in Komagataella phaffii (K. phaffii) has attracted considerable attention, but there are very limited strategies for its hyper-production in yeast. RESULTS: Here through Atmospheric and Room Temperature Plasma (ARTP)-based mutagenesis and transcriptomic analysis, the expression of two genes MYO1 and IQG1 encoding the cytokinesis core proteins was identified downregulated along with higher hLYZ production. Deletion of either gene caused severe cytokinesis defects, but significantly enhanced hLYZ production. The highest hLYZ yield of 1,052,444 ± 23,667 U/mL bioactivity and 4.12 ± 0.11 g/L total protein concentration were obtained after high-density fed-batch fermentation in the Δmyo1 mutant, representing the best production of hLYZ in yeast. Furthermore, O-linked mannose glycans were characterized on this recombinant hLYZ. CONCLUSIONS: Our work suggests that cytokinesis-based morphology engineering is an effective way to enhance the production of hLYZ in K. phaffii.

Coordination of Polyketide Release and Multiple Detoxification Pathways for Tolerable Production of Fungal Mycotoxins.

Efficient biosynthesis of microbial bioactive natural products (NPs) is beneficial for the survival of producers, while self-protection is necessary to avoid self-harm resulting from over-accumulation of NPs. The underlying mechanisms for the effective but tolerable production of bioactive NPs are not well understood. Herein, in the biosynthesis of two fungal polyketide mycotoxins aurovertin E (1) and asteltoxin, we show that the cyclases in the gene clusters promote the release of the polyketide backbone, and reveal that a signal peptide is crucial for their subcellular localization and full activity. Meanwhile, the fungus adopts enzymatic acetylation as the major detoxification pathway of 1. If intermediates are over-produced, the non-enzymatic shunt pathways work as salvage pathways to avoid excessive accumulation of the toxic metabolites for self-protection. These findings provided new insight into the interplay of efficient backbone release and multiple detoxification strategies for the production of fungal bioactive NPs.

Genomic and transcriptomic survey of an endophytic fungus Calcarisporium arbuscula NRRL 3705 and potential overview of its secondary metabolites.

BACKGROUND: Secondary metabolites as natural products from endophytic fungi are important sources of pharmaceuticals. However, there is currently little understanding of endophytic fungi at the omics levels about their potential in secondary metabolites. Calcarisporium arbuscula, an endophytic fungus from the fruit bodies of Russulaceae, produces a variety of secondary metabolites with anti-cancer, anti-nematode and antibiotic activities. A comprehensive survey of the genome and transcriptome of this endophytic fungus will help to understand its capacity to biosynthesize secondary metabolites and will lay the foundation for the development of this precious resource. RESULTS: In this study, we reported the high-quality genome sequence of C. arbuscula NRRL 3705 based on Single Molecule Real-Time sequencing technology. The genome of this fungus is over 45 Mb in size, larger than other typical filamentous fungi, and comprises 10,001 predicted genes, encoding at least 762 secretory-proteins, 386 carbohydrate-active enzymes and 177 P450 enzymes. 398 virulence factors and 228 genes related to pathogen-host interactions were also predicted in this fungus. Moreover, 65 secondary metabolite biosynthetic gene clusters were revealed, including the gene cluster for the mycotoxin aurovertins. In addition, several gene clusters were predicted to produce mycotoxins, including aflatoxin, alternariol, destruxin, citrinin and isoflavipucine. Notably, two independent gene clusters were shown that are potentially involved in the biosynthesis of alternariol. Furthermore, RNA-Seq assays showed that only expression of the aurovertin gene cluster is much stronger than expression of the housekeeping genes under laboratory conditions, consistent with the observation that aurovertins are the predominant metabolites. Gene expression of the remaining 64 gene clusters for compound backbone biosynthesis was all lower than expression of the housekeeping genes, which partially explained poor production of other secondary metabolites in this fungus. CONCLUSIONS: Our omics data, along with bioinformatics analysis, indicated that C. arbuscula NRRL 3705 contains a large number of biosynthetic gene clusters and has a huge potential to produce a profound number of secondary metabolites. This work also provides the basis for development of endophytic fungi as a new resource of natural products with promising biological activities.

Comprehensive dissection of dispensable genomic regions in Streptomyces based on comparative analysis approach.

BACKGROUND: Large-scale genome reduction has been performed to significantly improve the performance of microbial chassis. Identification of the essential or dispensable genes is pivotal for genome reduction to avoid synthetic lethality. Here, taking Streptomyces as an example, we developed a combinatorial strategy for systematic identification of large and dispensable genomic regions in Streptomyces based on multi-omics approaches. RESULTS: Phylogenetic tree analysis revealed that the model strains including S. coelicolor A3(2), S. albus J1074 and S. avermitilis MA-4680 were preferred reference for comparative analysis of candidate genomes. Multiple genome alignment suggested that the Streptomyces genomes embodied highly conserved core region and variable sub-telomeric regions, and may present symmetric or asymmetric structure. Pan-genome and functional genome analyses showed that most conserved genes responsible for the fundamental functions of cell viability were concentrated in the core region and the vast majority of abundant genes were dispersed in the sub-telomeric regions. These results suggested that large-scale deletion can be performed in sub-telomeric regions to greatly streamline the Streptomyces genomes for developing versatile chassis. CONCLUSIONS: The integrative approach of comparative genomics, functional genomics and pan-genomics can not only be applied to perform a multi-tiered dissection for Streptomyces genomes, but also work as a universal method for systematic analysis of removable regions in other microbial hosts in order to generate more miscellaneous and versatile chassis with minimized genome for drug discovery.

The regulatory cascades of antibiotic production in Streptomyces.

Streptomyces is famous for its capability to produce the most abundant antibiotics in all kingdoms. All Streptomyces antibiotics are natural products, whose biosynthesis from the so-called gene clusters are elaborately regulated by pyramidal transcriptional regulatory cascades. In the past decades, scientists have striven to unveil the regulatory mechanisms involved in antibiotic production in Streptomyces. Here we mainly focus on three aspects of the regulation on antibiotic production. 1. The onset of antibiotic production triggered by hormones and their coupled receptors as regulators; 2. The cascades of global and pathway-specific regulators governing antibiotic production; 3. The feedback regulation of antibiotics and/or intermediates on the gene cluster expression for their coordinated production. This review will summarize how the antibiotic production is stringently regulated in Streptomyces based on the signaling, and lay a theoretical foundation for improvement of antibiotic production and potentially drug discovery.

Identification of a secondary metabolism-responsive promoter by proteomics for over-production of natamycin in Streptomyces.

Streptomyces is currently the main producer of microbial pharmaceuticals from its secondary metabolites as natural products. It will be more beneficial if the promoters, which are particularly strong during the secondary metabolism of Streptomyces, are used to drive the efficient production of desired natural products with the coordination of bacterial growth. Here, in an industrial natamycin producer Streptomyces chattanoogensis L10, a strong promoter groESp was identified for this purpose based on the comparative proteomic analysis of the primary and secondary metabolism. With a constitutive promoter ermEp* as a control, the activity of groESp was weak in the primary metabolism, but about sixfold higher than ermEp* in the secondary metabolism, when the representative antibiotic natamycin was highly produced. Furthermore, when ScnRII, a pathway-specific positive regulator in natamycin biosynthesis, was expressed under groESp, the productivity of natamycin was about 20% higher in the secondary metabolism than that from ermEp*, but had no discrimination in the early 2 days. Thus, we showed that proteomics is an effective alternative way to identify promoters for the high yield of natamycin in S. chattanoogensis, and this strategy can be widely adaptable to other Streptomyces species for the full development of secondary metabolites with promising bioactivities.

Rational construction of genome-reduced and high-efficient industrial Streptomyces chassis based on multiple comparative genomic approaches.

BACKGROUND: Streptomyces chattanoogensis L10 is the industrial producer of natamycin and has been proved a highly efficient host for diverse natural products. It has an enormous potential to be developed as a versatile cell factory for production of heterologous secondary metabolites. Here we developed a genome-reduced industrial Streptomyces chassis by rational 'design-build-test' pipeline. RESULTS: To identify candidate large non-essential genomic regions accurately and design large deletion rationally, we performed genome analyses of S. chattanoogensis L10 by multiple computational approaches, optimized Cre/loxP recombination system for high-efficient large deletion and constructed a series of universal suicide plasmids for rapid loxP or loxP mutant sites inserting into genome. Subsequently, two genome-streamlined mutants, designated S. chattanoogensis L320 and L321, were rationally constructed by depletion of 1.3 Mb and 0.7 Mb non-essential genomic regions, respectively. Furthermore, several biological performances like growth cycle, secondary metabolite profile, hyphae morphological engineering, intracellular energy (ATP) and reducing power (NADPH/NADP+) levels, transformation efficiency, genetic stability, productivity of heterologous proteins and secondary metabolite were systematically evaluated. Finally, our results revealed that L321 could serve as an efficient chassis for the production of polyketides. CONCLUSIONS: Here we developed the combined strategy of multiple computational approaches and site-specific recombination system to rationally construct genome-reduced Streptomyces hosts with high efficiency. Moreover, a genome-reduced industrial Streptomyces chassis S. chattanoogensis L321 was rationally constructed by the strategy, and the chassis exhibited several emergent and excellent performances for heterologous expression of secondary metabolite. The strategy could be widely applied in other Streptomyces to generate miscellaneous and versatile chassis with minimized genome. These chassis can not only serve as cell factories for high-efficient production of valuable polyketides, but also will provide great support for the upgrade of microbial pharmaceutical industry and drug discovery.

FadR1, a pathway-specific activator of fidaxomicin biosynthesis in Actinoplanes deccanensis Yp-1.

Fidaxomicin, an 18-membered macrolide antibiotic, is highly active against Clostridium difficile, the most common cause of diarrhea in hospitalized patients. Though the biosynthetic mechanism of fidaxomicin has been well studied, little is known about its regulatory mechanism. Here, we reported that FadR1, a LAL family transcriptional regulator in the fidaxomicin cluster of Actinoplanes deccanensis Yp-1, acts as an activator for fidaxomicin biosynthesis. The disruption of fadR1 abolished the ability to synthesize fidaxomicin, and production could be restored by reintegrating a single copy of fadR1. Overexpression of fadR1 resulted in an approximately 400 % improvement in fidaxomicin production. Electrophoretic mobility shift assays indicated that fidaxomicin biosynthesis is under the control of FadR1 through its binding to the promoter regions of fadM, fadA1-fadP2, fadS2-fadC, and fadE-fadF, respectively. And the conserved binding sites of FadR1 within the four promoter regions were determined by footprinting experiment. All results indicated that fadR1 encodes a pathway-specific positive regulator of fidaxomicin biosynthesis and upregulates the transcription levels of most of genes by binding to the four above intergenic regions. In summary, we not only clearly elucidate the regulatory mechanism of FadR1 but also provide strategies for the construction of industrial high-yield strain of fidaxomicin.

Transcriptome-Based Identification of a Strong Promoter for Hyper-production of Natamycin in Streptomyces.

Streptomyces are famed producers of secondary metabolites with diverse bioactivities and structures. However, biosynthesis of natural products will consume vast precursors from primary metabolism, and some secondary metabolites are toxic to the hosts. To overcome this circumstance and over-produce secondary metabolites, one of the strategies is to over-express biosynthetic genes under strong promoters specifically expressed during secondary metabolism. For this purpose, here based on Microarray and eGFP reporter assays, we obtained a promoter thlM4p, whose activity was undetectable in the first 2 days of fermentation, but sevenfold higher than the strong promoter ermE*p in the following days. Moreover, when the positive regulator gene scnRII was driven from thlM4p, natamycin yield increased 30% compared to ermE*p. Therefore, we provide a new way to identify promoters, which is silenced during primary metabolism while strongly expressed under secondary metabolism of Streptomyces.

Dual regulation between the two-component system PhoRP and AdpA regulates antibiotic production in Streptomyces.

Antibiotic production during secondary metabolism in Streptomyces spp. is elaborately controlled by multiple environmental signals and intracellular cascades. These include the two-component system PhoRP responding to phosphate starvation and a conserved signaling pathway mediated by the pleiotropic regulator AdpA. However, little information exists about how these two pathways work together for secondary metabolite production of Streptomyces. Herein, we report the dual regulation from the phosphate starvation-responsive regulator PhoP and AdpA on atrA promoter (atrAp) for the production of daptomycin, an antibiotic produced by Streptomyces roseosporus. We found that PhoP directly binds to atrAp, positively regulates atrA expression and thus daptomycin production. We also observed positive auto-regulation of phoRP expression during fermentation for daptomycin production. Moreover, partial overlap between PhoP- and AdpA-binding sites on atrAp was observed, which results in partial competitive binding between these two regulators. This partial overlapping and competition between PhoP and AdpA was further confirmed by mutations and binding assays. In summary, our findings have revealed dual regulation of PhoP and AdpA on the same promoter for antibiotic production in Streptomyces. This mechanism would be beneficial to further environment-responsive fermentation optimization for antibiotic production.

Multiple transporters are involved in natamycin efflux in Streptomyces chattanoogensis L10.

Antibiotic-producing microorganisms have evolved several self-resistance mechanisms to prevent auto-toxicity. Overexpression of specific transporters to improve the efflux of toxic antibiotics has been found one of the most important and intrinsic resistance strategies used by many Streptomyces strains. In this work, two ATP-binding cassette (ABC) transporter-encoding genes located in the natamycin biosynthetic gene cluster, scnA and scnB, were identified as the primary exporter genes for natamycin efflux in Streptomyces chattanoogensis L10. Two other transporters located outside the cluster, a major facilitator superfamily transporter Mfs1 and an ABC transporter NepI/II were found to play a complementary role in natamycin efflux. ScnA/ScnB and Mfs1 also participate in exporting the immediate precursor of natamycin, 4,5-de-epoxynatamycin, which is more toxic to S. chattanoogensis L10 than natamycin. As the major complementary exporter for natamycin efflux, Mfs1 is up-regulated in response to intracellular accumulation of natamycin and 4,5-de-epoxynatamycin, suggesting a key role in the stress response for self-resistance. This article discusses a novel antibiotic-related efflux and response system in Streptomyces, as well as a self-resistance mechanism in antibiotic-producing strains.

Transposon-based identification of a negative regulator for the antibiotic hyper-production in Streptomyces.

Production of secondary metabolites in Streptomyces is regulated by a complex regulatory network precisely, elaborately, and hierarchically. One of the main reasons for the low yields of some high-value secondary metabolites is the repressed expression of their biosynthetic gene clusters, supposedly by some gene cluster out-situated negative regulators. Identification of these repressors and removal of the inhibitory effects based on the regulatory mechanisms will be an effective way to improve their yields. For proof of the concept, using an antibiotic daptomycin from Streptomyces roseosporus, we introduced Himar1-based random mutagenesis combined with a reporter-guided screening strategy to identify a transcriptional regulator PhaR, whose loss-of-function deletion led to about 2.68-fold increase of the gene cluster expression and approximately 6.14-fold or 43% increased daptomycin production in the flask fermentation or in the fed-batch fermentation, respectively. Further study showed that PhaR negatively regulates the expression of daptomycin biosynthetic gene cluster by direct binding to its promoter (dptEp). Moreover, phaR expression gradually drops down during fermentation, and PhaR is positively auto-regulated by directly binding to its own promoter, which results in positive feedback regulation to persistently reduce phaR expression. Meanwhile, the declining PhaR protein remove its repressive effects during daptomycin production. All these results support that our strategy would be a powerful method for genetic screening and rational engineering for the yield improvement of antibiotics, and could be potentially used widely in other Streptomyces species.

Regulatory and biosynthetic effects of the bkd gene clusters on the production of daptomycin and its analogs A21978C1-3.

Daptomycin is a cyclic lipopeptide antibiotic produced by Streptomyces roseosporus in an acidic peptide complex A21978C. In this complex, A21978C1-3 is most abundant and contains branched-chain fatty acyl groups, while daptomycin has a straight decanoic acyl group. The branched-chain α-keto acid dehydrogenase complex (BCDH complex), encoded by bkd gene clusters in Streptomyces, is responsible for the early step of converting branched-chain amino acids into branched-chain fatty acids. In a daptomycin industrial producer S. roseosporus L30, two alleles of bkd gene clusters, bkdA1B1C1/bkdA2B2C2, and a regulatory gene bkdR located upstream of bkdA2B2C2 are identified. We show that BkdR positively regulated bkdA2B2C2 expression and was negatively auto-regulated, but is not directly involved in regulation of daptomycin gene cluster expression. However, BkdR is required for both daptomycin and A21978C1-3 production. Furthermore, deletion of bkdA2B2C2 only led to partial reduction of A21978C1-3 production, while the ΔbkdA1B1C1 mutant shows very weak production of A21978C1-3, and the double bkd mutant has a similar production profile as the single ΔbkdA1B1C1 mutant, suggesting that bkdA1B1C1 gene cluster plays a dominant role in branched-chain fatty acid biosynthesis. So we reveal a unique regulatory function of BkdR and genetic engineered a bkd null strain for daptomycin production with reduced impurities.

Negative regulation of daptomycin production by DepR2, an ArsR-family transcriptional factor.

Daptomycin, a lipopeptide antibiotic potently active against Gram-positive bacterial pathogens, is produced by Streptomyces roseosporus, but the transcriptional regulation on its biosynthesis is not fully understood. Here, we report that DepR2, an ArsR-family transcriptional regulator isolated previously by DNA-affinity purification, interacts directly with dptEp, the major promoter of the daptomycin gene cluster. DepR2 binds to an imperfect palindromic sequence at the very upstream of dptEp. Meanwhile, higher dptEp activities were consistently observed in the ΔdepR2 mutant, correlating with a nearly 2.5-fold increased production of daptomycin and three structurally related secondary metabolites A21978C1-3. Thus, our data suggest that the ArsR-family transcriptional regulator DepR2 negatively regulates production of daptomycin by directly repressing the expression of its gene cluster in S. roseosporus. To the best of our knowledge, this is the first report to show the involvement of an ArsR-family regulator in the direct regulation of secondary metabolite biosynthesis in Streptomyces.

Transcriptional regulation of the daptomycin gene cluster in Streptomyces roseosporus by an autoregulator, AtrA.

Daptomycin is a cyclic lipopeptide antibiotic produced by Streptomyces roseosporus. To reveal the transcriptional regulatory mechanism of daptomycin biosynthesis, we used the biotinylated dptE promoter (dptEp) as a probe to affinity isolate the dptEp-interactive protein AtrA, a TetR family transcriptional regulator, from the proteome of mycelia. AtrA bound directly to dptEp to positively regulate gene cluster expression and daptomycin production. Meanwhile, both ΔatrA and ΔadpA mutants showed bald phenotype and null production of daptomycin. AdpA positively regulated atrA expression by direct interaction with atrA promoter (atrAp), and removal of ArpA in S. roseosporus, a homolog of the A-factor receptor, resulted in accelerated morphological development and increased daptomycin production, suggesting that atrA was the target of AdpA to mediate the A-factor signaling pathway. Furthermore, AtrA was positively autoregulated by binding to its own promoter atrAp. Thus, for the first time at the transcriptional level, we have identified an autoregulator, AtrA, that directly mediates the A-factor signaling pathway to regulate the proper production of daptomycin.

DepR1, a TetR Family Transcriptional Regulator, Positively Regulates Daptomycin Production in an Industrial Producer, Streptomyces roseosporus SW0702.

Daptomycin is a potent cyclic lipopeptide antibiotic. It is widely used against various Gram-positive bacterial pathogens. Historically, a poor understanding of the transcriptional regulation of daptomycin biosynthesis has limited the options for targeted genetic engineering toward titer improvement. Here, we isolated a TetR family transcriptional regulator, DepR1, from the industrial producer Streptomyces roseosporus SW0702 using a biotinylated dptE promoter (dptEp) as a probe. The direct interaction between DepR1 and dptEp then was confirmed by electrophoretic mobility shift assays and DNase I footprinting assays. The deletion of depR1 led to a reduction in dptEp activity and the cessation of daptomycin production. The ΔdepR1 mutant produced less red pigment and failed to sporulate on R5 medium. This suggests that DepR1 plays a positive role in the control of morphological differentiation. Moreover, DepR1 was positively autoregulated by directly binding to its own promoter. This might account for the positive feedback regulation of daptomycin production. Based on these positive effects, genetic engineering by overexpression of depR1 raised daptomycin production and shortened the fermentation period both in flask and in fermentor.

Efficient Biosynthesis of Fungal Polyketides Containing the Dioxabicyclo-octane Ring System.

Aurovertins are fungal polyketides that exhibit potent inhibition of adenosine triphosphate synthase. Aurovertins contain a 2,6-dioxabicyclo[3.2.1]octane ring that is proposed to be derived from a polyene precursor through regioselective oxidations and epoxide openings. In this study, we identified only four enzymes required to produce aurovertin E. The core polyketide synthase produces a polyene α-pyrone. Following pyrone O-methylation by a methyltransferase, a flavin-dependent mono-oxygenase and an epoxide hydrolase can iteratively transform the terminal triene portion of the precursor into the dioxabicyclo[3.2.1]octane scaffold. We demonstrate that a tetrahydrofuranyl polyene is the first stable intermediate in the transformation, which can undergo epoxidation and anti-Baldwin 6-endo-tet ring opening to yield the cyclic ether product. Our results further demonstrate the highly concise and efficient ways in which fungal biosynthetic pathways can generate complex natural product scaffolds.

Sigma factor WhiGch positively regulates natamycin production in Streptomyces chattanoogensis L10.

The roles of many sigma factors are unclear in regulatory mechanism of secondary metabolism in Streptomyces. Here, we report the regulation network of a group 3 sigma factor, WhiGch, from a natamycin industrial strain Streptomyces chattanoogensis L10. WhiGch regulates the growth and morphological differentiation of S. chattanoogensis L10. The whiG ch deletion mutant decreased natamycin production by about 30 % and delayed natamycin production more than 24 h by delaying the growth. Overexpression of the whiG ch gene increased natamycin production in large scale production medium by about 26 %. WhiGch upregulated the transcription of natamycin biosynthetic gene cluster and inhibited the expression of migrastatin and jadomycin analog biosynthetic polyketide synthase genes. WhiGch positively regulated natamycin biosynthetic gene cluster by directly binding to the promoters of scnC and scnD, which were involved in natamycin biosynthesis, and these binding sites adjacent to translation start codon were determined. Thus, this paper further elucidates the high natamycin yield mechanisms of industrial strains and demonstrates that a valuable improvement in the yield of the target metabolites can be achieved through manipulating the transcription regulators.

Epigenetic genome mining of an endophytic fungus leads to the pleiotropic biosynthesis of natural products.

The small-molecule biosynthetic potential of most filamentous fungi has remained largely unexplored and represents an attractive source for the discovery of new compounds. Genome sequencing of Calcarisporium arbuscula, a mushroom-endophytic fungus, revealed 68 core genes that are involved in natural product biosynthesis. This is in sharp contrast to the predominant production of the ATPase inhibitors aurovertin B and D in the wild-type fungus. Inactivation of a histone H3 deacetylase led to pleiotropic activation and overexpression of more than 75 % of the biosynthetic genes. Sampling of the overproduced compounds led to the isolation of ten compounds of which four contained new structures, including the cyclic peptides arbumycin and arbumelin, the diterpenoid arbuscullic acid A, and the meroterpenoid arbuscullic acid B. Such epigenetic modifications therefore provide a rapid and global approach to mine the chemical diversity of endophytic fungi.

Dual positive feedback regulation of protein degradation of an extra-cytoplasmic function σ factor for cell differentiation in Streptomyces coelicolor.

Here we report that in Streptomyces coelicolor, the protein stability of an ECF σ factor SigT, which is involved in the negative regulation of cell differentiation, was completely dependent on its cognate anti-σ factor RstA. The degradation of RstA caused a ClpP/SsrA-dependent degradation of SigT during cell differentiation. This was consistent with the delayed morphological development or secondary metabolism in the ΔclpP background after rstA deletion or sigT overexpression. Meanwhile, SigT negatively regulated clpP/ssrA expression by directly binding to the clpP promoter (clpPp). The SigT-clpPp interaction could be disrupted by secondary metabolites, giving rise to the stabilized SigT protein and retarded morphological development in a non-antibiotic-producing mutant. Thus a novel regulatory mechanism was revealed that the protein degradation of the ECF σ factor was initiated by the degradation of its anti-σ factor, and was accelerated in a dual positive feedback manner, through regulation by secondary metabolites, to promote rapid and irreversible development of the secondary metabolism. This ingenious cooperation of intracellular components can ensure economical and exquisite control of the ECF σ factor protein level for the proper cell differentiation in Streptomyces.