High Level of Spinosad Production in the Heterologous Host Saccharopolyspora erythraea

ABSTRACTSpinosad, a highly effective insecticide, has an excellent environmental and mammalian toxicological profile. Global market demand for spinosad is huge and growing. However, after much effort, there has been almost no improvement in the spinosad yield from the original producer,Saccharopolyspora spinosa. Here, we report the heterologous expression of spinosad usingSaccharopolyspora erythraeaas a host. The native erythromycin polyketide synthase (PKS) genes inS. erythraeawere replaced by the assembled spinosad gene cluster through iterative recombination. The production of spinosad could be detected in the recombinant strains containing the whole biosynthesis gene cluster. Both metabolic engineering and UV mutagenesis were applied to further improve the yield of spinosad. The final strain, AT-ES04PS-3007, which could produce spinosad with a titer of 830 mg/liter, has significant potential in industrial applications.IMPORTANCEThis work provides an innovative and promising way to improve the industrial production of spinosad. At the same time, it also describes a successful method of heterologous expression for target metabolites of interest by replacing large gene clusters.

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Overproduction of Ristomycin A by Activation of a Silent Gene Cluster in Amycolatopsis japonicum MG417-CF17

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.03512-14 ◽

2014 ◽

Vol 58 (10) ◽

pp. 6185-6196 ◽

Cited By ~ 51

Author(s):

Marius Spohn ◽

Norbert Kirchner ◽

Andreas Kulik ◽

Angelika Jochim ◽

Felix Wolf ◽

...

Keyword(s):

Mass Spectrometry ◽

Gene Cluster ◽

High Performance ◽

Pathogenic Bacteria ◽

Genome Mining ◽

Gene Clusters ◽

Von Willebrand Disease ◽

Biosynthesis Gene Cluster ◽

Content Type ◽

Bioinformatic Tools

ABSTRACTThe emergence of antibiotic-resistant pathogenic bacteria within the last decades is one reason for the urgent need for new antibacterial agents. A strategy to discover new anti-infective compounds is the evaluation of the genetic capacity of secondary metabolite producers and the activation of cryptic gene clusters (genome mining). One genus known for its potential to synthesize medically important products isAmycolatopsis. However,Amycolatopsis japonicumdoes not produce an antibiotic under standard laboratory conditions. In contrast to mostAmycolatopsisstrains,A. japonicumis genetically tractable with different methods. In order to activate a possible silent glycopeptide cluster, we introduced a gene encoding the transcriptional activator of balhimycin biosynthesis, thebbrgene fromAmycolatopsis balhimycina(bbrAba), intoA. japonicum. This resulted in the production of an antibiotically active compound. Following whole-genome sequencing ofA. japonicum, 29 cryptic gene clusters were identified by genome mining. One of these gene clusters is a putative glycopeptide biosynthesis gene cluster. Using bioinformatic tools, ristomycin (syn. ristocetin), a type III glycopeptide, which has antibacterial activity and which is used for the diagnosis of von Willebrand disease and Bernard-Soulier syndrome, was deduced as a possible product of the gene cluster. Chemical analyses by high-performance liquid chromatography and mass spectrometry (HPLC-MS), tandem mass spectrometry (MS/MS), and nuclear magnetic resonance (NMR) spectroscopy confirmed thein silicoprediction that the recombinantA. japonicum/pRM4-bbrAbasynthesizes ristomycin A.

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The Novel Transcriptional Regulator LmbU Promotes Lincomycin Biosynthesis through Regulating Expression of Its Target Genes in Streptomyces lincolnensis

Journal of Bacteriology ◽

10.1128/jb.00447-17 ◽

2017 ◽

Vol 200 (2) ◽

Cited By ~ 16

Author(s):

Bingbing Hou ◽

Yanwei Lin ◽

Haizhen Wu ◽

Meijin Guo ◽

Hrvoje Petkovic ◽

...

Keyword(s):

Gene Cluster ◽

Target Genes ◽

Regulatory Mechanism ◽

Transcriptional Regulator ◽

Gene Clusters ◽

Saccharopolyspora Erythraea ◽

Transcriptional Analysis ◽

Biosynthetic Gene ◽

Content Type ◽

Streptomyces Lincolnensis

ABSTRACT Lincomycin A is a clinically important antimicrobial agent produced by Streptomyces lincolnensis . In this study, a new regulator designated LmbU (GenBank accession no. ABX00623.1) was identified and characterized to regulate lincomycin biosynthesis in S. lincolnensis wild-type strain NRRL 2936. Both inactivation and overexpression of lmbU resulted in significant influences on lincomycin production. Transcriptional analysis and in vivo neomycin resistance (Neo r ) reporter assays demonstrated that LmbU activates expression of the lmbA , lmbC , lmbJ , and lmbW genes and represses expression of the lmbK and lmbU genes. Electrophoretic mobility shift assays (EMSAs) demonstrated that LmbU can bind to the regions upstream of the lmbA and lmbW genes through the consensus and palindromic sequence 5′-CGCCGGCG-3′. However, LmbU cannot bind to the regions upstream of the lmbC , lmbJ , lmbK , and lmbU genes as they lack this motif. These data indicate a complex transcriptional regulatory mechanism of LmbU. LmbU homologues are present in the biosynthetic gene clusters of secondary metabolites of many other actinomycetes. Furthermore, the LmbU homologue from Saccharopolyspora erythraea (GenBank accession no. WP_009944629.1) also binds to the regions upstream of lmbA and lmbW , which suggests widespread activity for this regulator. LmbU homologues have no significant structural similarities to other known cluster-situated regulators (CSRs), which indicates that they belong to a new family of regulatory proteins. In conclusion, the present report identifies LmbU as a novel transcriptional regulator and provides new insights into regulation of lincomycin biosynthesis in S. lincolnensis . IMPORTANCE Although lincomycin biosynthesis has been extensively studied, its regulatory mechanism remains elusive. Here, a novel regulator, LmbU, which regulates transcription of its target genes in the lincomycin biosynthetic gene cluster ( lmb gene cluster) and therefore promotes lincomycin biosynthesis, was identified in S. lincolnensis strain NRRL 2936. Importantly, we show that this new regulatory element is relatively widespread across diverse actinomycetes species. In addition, our findings provide a new strategy for improvement of yield of lincomycin through manipulation of LmbU, and this approach could also be evaluated in other secondary metabolite gene clusters containing this regulatory protein.

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The Swinholide Biosynthesis Gene Cluster from a Terrestrial Cyanobacterium, Nostoc sp. Strain UHCC 0450

Applied and Environmental Microbiology ◽

10.1128/aem.02321-17 ◽

2017 ◽

Vol 84 (3) ◽

Cited By ~ 10

Author(s):

Anu Humisto ◽

Jouni Jokela ◽

Liwei Liu ◽

Matti Wahlsten ◽

Hao Wang ◽

...

Keyword(s):

Natural Products ◽

Actin Cytoskeleton ◽

Gene Cluster ◽

Marine Sponge ◽

Gene Clusters ◽

Fold Axis ◽

Biosynthesis Gene Cluster ◽

Content Type ◽

Biosynthesis Gene ◽

Axis Of Symmetry

ABSTRACT Swinholides are 42-carbon ring polyketides with a 2-fold axis of symmetry. They are potent cytotoxins that disrupt the actin cytoskeleton. Swinholides were discovered from the marine sponge Theonella sp. and were long suspected to be produced by symbiotic bacteria. Misakinolide, a structural variant of swinholide, was recently demonstrated to be the product of a symbiotic heterotrophic proteobacterium. Here, we report the production of swinholide A by an axenic strain of the terrestrial cyanobacterium Nostoc sp. strain UHCC 0450. We located the 85-kb trans -AT polyketide synthase (PKS) swinholide biosynthesis gene cluster from a draft genome of Nostoc sp. UHCC 0450. The swinholide and misakinolide biosynthesis gene clusters share an almost identical order of catalytic domains, with 85% nucleotide sequence identity, and they group together in phylogenetic analysis. Our results resolve speculation around the true producer of swinholides and demonstrate that bacteria belonging to two distantly related phyla both produce structural variants of the same natural product. In addition, we described a biosynthesis cluster from Anabaena sp. strain UHCC 0451 for the synthesis of the cytotoxic and antifungal scytophycin. All of these biosynthesis gene clusters were closely related to each other and created a group of cytotoxic macrolide compounds produced by trans -AT PKSs of cyanobacteria and proteobacteria. IMPORTANCE Many of the drugs in use today originate from natural products. New candidate compounds for drug development are needed due to increased drug resistance. An increased knowledge of the biosynthesis of bioactive compounds can be used to aid chemical synthesis to produce novel drugs. Here, we show that a terrestrial axenic culture of Nostoc cyanobacterium produces swinholides, which have been previously found only from marine sponge or samples related to them. Swinholides are polyketides with a 2-fold axis of symmetry, and they are potent cytotoxins that disrupt the actin cytoskeleton. We describe the biosynthesis gene clusters of swinholide from Nostoc cyanobacteria, as well as the related cytotoxic and antifungal scytophycin from Anabaena cyanobacteria, and we study the evolution of their trans -AT polyketide synthases. Interestingly, swinholide is closely related to misakinolide produced by a symbiotic heterotrophic proteobacterium, demonstrating that bacteria belonging to two distantly related phyla and different habitats can produce similar natural products.

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Identification of a Polyketide Synthase Gene Responsible for Ascochitine Biosynthesis in Ascochyta fabae and Its Abrogation in Sister Taxa

mSphere ◽

10.1128/msphere.00622-19 ◽

2019 ◽

Vol 4 (5) ◽

Cited By ~ 1

Author(s):

Wonyong Kim ◽

Judith Lichtenzveig ◽

Robert A. Syme ◽

Angela H. Williams ◽

Tobin L. Peever ◽

...

Keyword(s):

Gene Cluster ◽

Secondary Metabolite ◽

Polyketide Synthase ◽

Animal Health ◽

Gene Clusters ◽

Biosynthesis Gene Cluster ◽

Content Type ◽

Pathogenicity Factor ◽

Biosynthesis Gene ◽

Ascochyta Fabae

ABSTRACT The polyketide-derived secondary metabolite ascochitine is produced by species in the Didymellaceae family, including but not restricted to Ascochyta species pathogens of cool-season food legumes. Ascochitine is structurally similar to the well-known mycotoxin citrinin and exhibits broad-spectrum phytotoxicity and antimicrobial activities. Here, we identified a polyketide synthase (PKS) gene (denoted pksAC) responsible for ascochitine production in the filamentous fungus Ascochyta fabae. Deletion of the pksAC prevented production of ascochitine and its derivative ascochital in A. fabae. The putative ascochitine biosynthesis gene cluster comprises 11 genes that have undergone rearrangement and gain-and-loss events relative to the citrinin biosynthesis gene cluster in Monascus ruber. Interestingly, we also identified pksAC homologs in two recently diverged species, A. lentis and A. lentis var. lathyri, that are sister taxa closely related to ascochitine producers such as A. fabae and A. viciae-villosae. However, nonsense mutations have been independently introduced in coding sequences of the pksAC homologs of A. lentis and A. lentis var. lathyri that resulted in loss of ascochitine production. Despite its reported phytotoxicity, ascochitine was not a pathogenicity factor in A. fabae infection and colonization of faba bean (Vicia faba L.). Ascochitine was mainly produced from mature hyphae at the site of pycnidial formation, suggesting a possible protective role of the compound against other microbial competitors in nature. This report highlights the evolution of gene clusters harnessing the structural diversity of polyketides and a mechanism with the potential to alter secondary metabolite profiles via single nucleotide polymorphisms in closely related fungal species. IMPORTANCE Fungi produce a diverse array of secondary metabolites, many of which are of pharmacological importance whereas many others are noted for mycotoxins, such as aflatoxin and citrinin, that can threaten human and animal health. The polyketide-derived compound ascochitine, which is structurally similar to citrinin mycotoxin, has been considered to be important for pathogenicity of legume-associated Ascochyta species. Here, we identified the ascochitine polyketide synthase (PKS) gene in Ascochyta fabae and its neighboring genes that may be involved in ascochitine biosynthesis. Interestingly, the ascochitine PKS genes in other legume-associated Ascochyta species have been mutated, encoding truncated PKSs. This indicated that point mutations may have contributed to genetic diversity for secondary metabolite production in these fungi. We also demonstrated that ascochitine is not a pathogenicity factor in A. fabae. The antifungal activities and production of ascochitine during sporulation suggested that it may play a role in competition with other saprobic fungi in nature.

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Identification and Heterologous Expression of the Chaxamycin Biosynthesis Gene Cluster from Streptomyces leeuwenhoekii

Applied and Environmental Microbiology ◽

10.1128/aem.01039-15 ◽

2015 ◽

Vol 81 (17) ◽

pp. 5820-5831 ◽

Cited By ~ 26

Author(s):

Jean Franco Castro ◽

Valeria Razmilic ◽

Juan Pablo Gomez-Escribano ◽

Barbara Andrews ◽

Juan A. Asenjo ◽

...

Keyword(s):

Heterologous Expression ◽

Gene Cluster ◽

Mutational Analysis ◽

Streptomyces Coelicolor ◽

Atacama Desert ◽

Biosynthesis Gene Cluster ◽

Content Type ◽

Biosynthesis Gene ◽

Viable Approach

ABSTRACTStreptomyces leeuwenhoekii, isolated from the hyperarid Atacama Desert, produces the new ansamycin-like compounds chaxamycins A to D, which possess potent antibacterial activity and moderate antiproliferative activity. We report the development of genetic tools to manipulateS. leeuwenhoekiiand the identification and partial characterization of the 80.2-kb chaxamycin biosynthesis gene cluster, which was achieved by both mutational analysis in the natural producer and heterologous expression inStreptomyces coelicolorA3(2) strain M1152. Restoration of chaxamycin production in a nonproducing ΔcxmKmutant (cxmKencodes 3-amino-5-hydroxybenzoic acid [AHBA] synthase) was achieved by supplementing the growth medium with AHBA, suggesting that mutasynthesis may be a viable approach for the generation of novel chaxamycin derivatives.

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Identification of the kinanthraquinone biosynthetic gene cluster by expression of an atypical response regulator

Bioscience Biotechnology and Biochemistry ◽

10.1093/bbb/zbaa082 ◽

2021 ◽

Vol 85 (3) ◽

pp. 714-721

Author(s):

Risa Takao ◽

Katsuyuki Sakai ◽

Hiroyuki Koshino ◽

Hiroyuki Osada ◽

Shunji Takahashi

Keyword(s):

Heterologous Expression ◽

Gene Cluster ◽

Secondary Metabolite ◽

Response Regulator ◽

Bac Library ◽

Gene Clusters ◽

Biosynthetic Gene Cluster ◽

Gene Organization ◽

Biosynthetic Gene ◽

Streptomyces Sp

ABSTRACT Recent advances in genome sequencing have revealed a variety of secondary metabolite biosynthetic gene clusters in actinomycetes. Understanding the biosynthetic mechanism controlling secondary metabolite production is important for utilizing these gene clusters. In this study, we focused on the kinanthraquinone biosynthetic gene cluster, which has not been identified yet in Streptomyces sp. SN-593. Based on chemical structure, 5 type II polyketide synthase gene clusters were listed from the genome sequence of Streptomyces sp. SN-593. Among them, a candidate gene cluster was selected by comparing the gene organization with grincamycin, which is synthesized through an intermediate similar to kinanthraquinone. We initially utilized a BAC library for subcloning the kiq gene cluster, performed heterologous expression in Streptomyces lividans TK23, and identified the production of kinanthraquinone and kinanthraquinone B. We also found that heterologous expression of kiqA, which belongs to the DNA-binding response regulator OmpR family, dramatically enhanced the production of kinanthraquinones.

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Characterization of the Role of para-Aminobenzoic Acid Biosynthesis in Folate Production by Lactococcus lactis

Applied and Environmental Microbiology ◽

10.1128/aem.02174-06 ◽

2007 ◽

Vol 73 (8) ◽

pp. 2673-2681 ◽

Cited By ~ 77

Author(s):

Arno Wegkamp ◽

Wietske van Oorschot ◽

Willem M. de Vos ◽

Eddy J. Smid

Keyword(s):

Gene Cluster ◽

Lactococcus Lactis ◽

Gene Clusters ◽

Defined Medium ◽

Biosynthesis Pathway ◽

Chemically Defined Medium ◽

Aminobenzoic Acid ◽

Biosynthesis Gene Cluster ◽

Gene Coding ◽

Aba Biosynthesis

ABSTRACT The pab genes for para-aminobenzoic acid (pABA) biosynthesis in Lactococcus lactis were identified and characterized. In L. lactis NZ9000, only two of the three genes needed for pABA production were initially found. No gene coding for 4-amino-4-deoxychorismate lyase (pabC) was initially annotated, but detailed analysis revealed that pabC was fused with the 3′ end of the gene coding for chorismate synthetase component II (pabB). Therefore, we hypothesize that all three enzyme activities needed for pABA production are present in L. lactis, allowing for the production of pABA. Indeed, the overexpression of the pABA gene cluster in L. lactis resulted in elevated pABA pools, demonstrating that the genes are involved in the biosynthesis of pABA. Moreover, a pABA knockout (KO) strain lacking pabA and pabB C was constructed and shown to be unable to produce folate when cultivated in the absence of pABA. This KO strain was unable to grow in chemically defined medium lacking glycine, serine, nucleobases/nucleosides, and pABA. The addition of the purine guanine, adenine, xanthine, or inosine restored growth but not the production of folate. This suggests that, in the presence of purines, folate is not essential for the growth of L. lactis. It also shows that folate is not strictly required for the pyrimidine biosynthesis pathway. L. lactis strain NZ7024, overexpressing both the folate and pABA gene clusters, was found to produce 2.7 mg of folate/liter per optical density unit at 600 nm when the strain was grown on chemically defined medium without pABA. This is in sharp contrast to L. lactis strains overexpressing only one of the two gene clusters. Therefore, we conclude that elevated folate levels can be obtained only by the overexpression of folate combined with the overexpression of the pABA biosynthesis gene cluster, suggesting the need for a balanced carbon flux through the folate and pABA biosynthesis pathway in the wild-type strain.

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Plasmid-Borne Type E Neurotoxin Gene Clusters in Clostridium botulinum Strains

Applied and Environmental Microbiology ◽

10.1128/aem.00080-13 ◽

2013 ◽

Vol 79 (12) ◽

pp. 3856-3859 ◽

Cited By ~ 17

Author(s):

Zhen Zhang ◽

Hannamari Hintsa ◽

Ying Chen ◽

Hannu Korkeala ◽

Miia Lindström

Keyword(s):

Gene Cluster ◽

Gel Electrophoresis ◽

Clostridium Botulinum ◽

Southern Hybridization ◽

Gene Clusters ◽

Pulsed Field Gel Electrophoresis ◽

Content Type ◽

Pulsed Field ◽

Large Plasmids

ABSTRACTA collection of 36Clostridium botulinumtype E strains was examined by pulsed-field gel electrophoresis (PFGE) and Southern hybridization with probes targeted tobotEandorfX1in the neurotoxin gene cluster. Three strains were found to contain neurotoxin subtype E1 gene clusters in large plasmids of about 146 kb in size.

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Fur-Dam Regulatory Interplay at an Internal Promoter of the Enteroaggregative Escherichia coli Type VI Secretion sci1 Gene Cluster

Journal of Bacteriology ◽

10.1128/jb.00075-20 ◽

2020 ◽

Vol 202 (10) ◽

Cited By ~ 3

Author(s):

Yannick R. Brunet ◽

Christophe S. Bernard ◽

Eric Cascales

Keyword(s):

Escherichia Coli ◽

Gene Cluster ◽

Gene Clusters ◽

Secretion System ◽

Target Cells ◽

Type Vi Secretion System ◽

Content Type ◽

Internal Promoter ◽

E Coli ◽

Type Vi Secretion

ABSTRACT The type VI secretion system (T6SS) is a weapon for delivering effectors into target cells that is widespread in Gram-negative bacteria. The T6SS is a highly versatile machine, as it can target both eukaryotic and prokaryotic cells, and it has been proposed that T6SSs are adapted to the specific needs of each bacterium. The expression of T6SS gene clusters and the activation of the secretion apparatus are therefore tightly controlled. In enteroaggregative Escherichia coli (EAEC), the sci1 T6SS gene cluster is subject to a complex regulation involving both the ferric uptake regulator (Fur) and DNA adenine methylase (Dam)-dependent DNA methylation. In this study, an additional, internal, promoter was identified within the sci1 gene cluster using +1 transcriptional mapping. Further analyses demonstrated that this internal promoter is controlled by a mechanism strictly identical to that of the main promoter. The Fur binding box overlaps the −10 transcriptional element and a Dam methylation site, GATC-32. Hence, the expression of the distal sci1 genes is repressed and the GATC-32 site is protected from methylation in iron-rich conditions. The Fur-dependent protection of GATC-32 was confirmed by an in vitro methylation assay. In addition, the methylation of GATC-32 negatively impacted Fur binding. The expression of the sci1 internal promoter is therefore controlled by iron availability through Fur regulation, whereas Dam-dependent methylation maintains a stable ON expression in iron-limited conditions. IMPORTANCE Bacteria use weapons to deliver effectors into target cells. One of these weapons, the type VI secretion system (T6SS), assembles a contractile tail acting as a spring to propel a toxin-loaded needle. Its expression and activation therefore need to be tightly regulated. Here, we identified an internal promoter within the sci1 T6SS gene cluster in enteroaggregative E. coli. We show that this internal promoter is controlled by Fur and Dam-dependent methylation. We further demonstrate that Fur and Dam compete at the −10 transcriptional element to finely tune the expression of T6SS genes. We propose that this elegant regulatory mechanism allows the optimum production of the T6SS in conditions where enteroaggregative E. coli encounters competing species.

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Engineering the Erythromycin-Producing Strain Saccharopolyspora erythraea HOE107 for the Heterologous Production of Polyketide Antibiotics

Frontiers in Microbiology ◽

10.3389/fmicb.2020.593217 ◽

2020 ◽

Vol 11 ◽

Author(s):

Jin Lü ◽

Qingshan Long ◽

Zhilong Zhao ◽

Lu Chen ◽

Weijun He ◽

...

Keyword(s):

Gene Cluster ◽

Polyketide Synthase ◽

Streptomyces Coelicolor ◽

Genome Mining ◽

Bac Library ◽

Gene Clusters ◽

Artificial Chromosome ◽

Saccharopolyspora Erythraea ◽

Heterologous Production ◽

Integration Sites

Bacteria of the genus Saccharopolyspora produce important polyketide antibiotics, including erythromycin A (Sac. erythraea) and spinosad (Sac. spinosa). We herein report the development of an industrial erythromycin-producing strain, Sac. erythraea HOE107, into a host for the heterologous expression of polyketide biosynthetic gene clusters (BGCs) from other Saccharopolyspora species and related actinomycetes. To facilitate the integration of natural product BGCs and auxiliary genes beneficial for the production of natural products, the erythromycin polyketide synthase (ery) genes were replaced with two bacterial attB genomic integration sites associated with bacteriophages ϕC31 and ϕBT1. We also established a highly efficient conjugation protocol for the introduction of large bacterial artificial chromosome (BAC) clones into Sac. erythraea strains. Based on this optimized protocol, an arrayed BAC library was effectively transferred into Sac. erythraea. The large spinosad gene cluster from Sac. spinosa and the actinorhodin gene cluster from Streptomyces coelicolor were successfully expressed in the ery deletion mutant. Deletion of the endogenous giant polyketide synthase genes pkeA1-pkeA4, the product of which is not known, and the flaviolin gene cluster (rpp) from the bacterium increased the heterologous production of spinosad and actinorhodin. Furthermore, integration of pJTU6728 carrying additional beneficial genes dramatically improved the yield of actinorhodin in the engineered Sac. erythraea strains. Our study demonstrated that the engineered Sac. erythraea strains SLQ185, LJ161, and LJ162 are good hosts for the expression of heterologous antibiotics and should aid in expression-based genome-mining approaches for the discovery of new and cryptic antibiotics from Streptomyces and rare actinomycetes.

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