The Discovery of Weddellamycin, a Tricyclic Polyene Macrolactam Antibiotic from an Antarctic Deep-Sea-Derived Streptomyces sp. DSS69, by Heterologous Expression.

Polyene macrolactams are a special group of natural products with great diversity, unique structural features, and a wide range of biological activities. Herein, a cryptic gene cluster for the biosynthesis of putative macrolactams was disclosed from a sponge-associated bacterium, Streptomyces sp. DSS69, by genome mining. Cloning and heterologous expression of the whole biosynthetic gene cluster led to the discovery of weddellamycin, a polyene macrolactam bearing a 23/5/6 ring skeleton. A negative regulator, WdlO, and two positive regulators, WdlA and WdlB, involved in the regulation of weddellamycin production were unraveled. The fermentation titer of weddellamycin was significantly improved by overexpression of wdlA and wdlB and deletion of wdlO. Notably, weddellamycin showed remarkable antibacterial activity against various Gram-positive bacteria including MRSA, with MIC values of 0.10-0.83 µg/mL, and antifungal activity against Candida albicans, with an MIC value of 3.33 µg/mL. Weddellamycin also displayed cytotoxicity against several cancer cell lines, with IC50 values ranging from 2.07 to 11.50 µM.

Streptomyces-based whole-cell biosensors for detecting diverse cell envelope-targeting antibiotics.

Cell envelope-targeting antibiotics are potent therapeutic agents against various bacterial infections. The emergence of multiple antibiotic-resistant strains underscores the significance of identifying potent antimicrobials specifically targeting the cell envelope. However, current drug screening approaches are tedious and lack sufficient specificity and sensitivity, warranting the development of more efficient methods. Genetic circuit-based whole-cell biosensors hold great promise for targeted drug discovery from natural products. Here, we performed comparative transcriptomic analysis of Streptomyces coelicolor M1146 exposed to diverse cell envelope-targeting antibiotics, aiming to identify regulatory elements involved in perceiving and responding to these compounds. Differential gene expression analysis revealed significant activation of VanS/R two-component system in response to the glycopeptide class of cell envelope-acting antibiotics. Therefore, we engineered a pair of VanS/R-based biosensors that exhibit functional complementarity and possess exceptional sensitivity and specificity for glycopeptides detection. Additionally, through promoter screening and characterization, we expanded the biosensor's detection range to include various cell envelope-acting antibiotics beyond glycopeptides. Our genetically engineered biosensor exhibits superior performance, including a dynamic range of up to 887-fold for detecting subtle antibiotic concentration changes in a rapid 2-h response time, enabling high-throughput screening of natural product libraries for antimicrobial agents targeting the bacterial cell envelope.

Enhancing tylosin production by combinatorial overexpression of efflux, SAM biosynthesis, and regulatory genes in hyperproducing Streptomyces xinghaiensis strain.

Tylosin is a 16-membered macrolide antibiotic widely used in veterinary medicine to control infections caused by Gram-positive pathogens and mycoplasmas. To improve the fermentation titer of tylosin in the hyperproducing Streptomyces xinghaiensis strain TL01, we sequenced its whole genome and identified the biosynthetic gene cluster therein. Overexpression of the tylosin efflux gene tlrC, the cluster-situated S-adenosyl methionine (SAM) synthetase gene metKcs, the SAM biosynthetic genes adoKcs-metFcs, or the pathway-specific activator gene tylR enhanced tylosin production by 18%, 12%, 11%, and 11% in the respective engineered strains TLPH08-2, TLPH09, TLPH10, and TLPH12. Co-overexpression of metKcs and adoKcs-metFcs as two transcripts increased tylosin production by 22% in the resultant strain TLPH11 compared to that in TL01. Furthermore, combinational overexpression of tlrC, metKcs, adoKcs-metFcs, and tylR as four transcripts increased tylosin production by 23% (10.93g/L) in the resultant strain TLPH17 compared to that in TL01. However, a negligible additive effect was displayed upon combinational overexpression in TLPH17 as suggested by the limited increment of fermentation titer compared to that in TLPH08-2. Transcription analyses indicated that the expression of tlrC and three SAM biosynthetic genes in TLPH17 was considerably lower than that of TLPH08-2 and TLPH11. Based on this observation, the five genes were rearranged into one or two operons to coordinate their overexpression, yielding two engineered strains TLPH23 and TLPH24, and leading to further enhancement of tylosin production over TLPH17. In particular, the production of TLPH23 reached 11.35 g/L. These findings indicated that the combinatorial strategy is a promising approach for enhancing tylosin production in high-yielding industrial strains.

Insertion sequence transposition inactivates CRISPR-Cas immunity.

CRISPR-Cas immunity systems safeguard prokaryotic genomes by inhibiting the invasion of mobile genetic elements. Here, we screened prokaryotic genomic sequences and identified multiple natural transpositions of insertion sequences (ISs) into cas genes, thus inactivating CRISPR-Cas defenses. We then generated an IS-trapping system, using Escherichia coli strains with various ISs and an inducible cas nuclease, to monitor IS insertions into cas genes following the induction of double-strand DNA breakage as a physiological host stress. We identified multiple events mediated by different ISs, especially IS1 and IS10, displaying substantial relaxed target specificity. IS transposition into cas was maintained in the presence of DNA repair machinery, and transposition into other host defense systems was also detected. Our findings highlight the potential of ISs to counter CRISPR activity, thus increasing bacterial susceptibility to foreign DNA invasion.

The regulatory gene wblA is a target of the orphan response regulator OrrA in Streptomyces coelicolor.

Our previous study using transposon mutagenesis indicated that disruption of the putative response regulator gene orrA impacted antibiotic production in Streptomyces coelicolor. In this study, the role of OrrA was further characterized by comparing the phenotypes and transcriptomic profiles of the wild-type S. coelicolor strain M145 and ΔorrA, a strain with an inactivated orrA gene. Chromatin immunoprecipitation using a strain expressing OrrA fused with FLAG showed that OrrA binds the promoter of wblA, whose expression was downregulated in ΔorrA. The interaction of OrrA with the wblA promoter was further validated by a pull-down assay. Similar to ΔorrA, the deletion mutant of wblA (ΔwblA) was defective in development, and developmental genes were expressed at similar levels in ΔorrA and ΔwblA. Although both OrrA and WblA downregulated actinorhodin and undecylprodigiosin, their roles in regulation of the calcium-dependent antibiotic and yellow-pigmented type I polyketide differed. sco1375, a gene of unknown function, was identified as another OrrA target, and overexpression of either sco1375 or wblA in ΔorrA partially restored the wild-type phenotype, indicating that these genes mediate some of the effects of OrrA. This study revealed targets of OrrA and provided more insights into the role of the orphan response regulator OrrA in Streptomyces.

Unexpected Role of a Short-Chain Dehydrogenase/Reductase Family Protein in Type II Polyketide Biosynthesis.

We investigated the biosynthetic pathway of type II polyketide murayaquinone. The murayaquinone biosynthetic cluster contains genes for three putative short-chain dehydrogenase/reductase family enzymes including MrqF and MrqH with known functions and MrqM with unclear function. We report the functional characterization of MrqM for its role in murayaquinone biosynthesis. Our gene deletion experiment and structural elucidation of the accumulated intermediates revealed that MrqM is related with the second polyketide ring cyclization, because the inactivation of mrqM resulted in the accumulation of an off-pathway intermediate SEK43 and disrupted the second and third ring cyclization. Site-directed mutagenesis studies showed that two conserved residues in MrqM and homologous proteins Y151 and K155 are essential for its activity. The previously proposed second/third ring cyclase, MrqD, might instead play another important role in the chain releasing step of the murayaquinone biosynthesis.

Enhancing Ristomycin A Production by Overexpression of ParB-Like StrR Family Regulators Controlling the Biosynthesis Genes.

Amycolatopsis sp. strain TNS106 harbors a ristomycin-biosynthetic gene cluster (asr) in its genome and produces ristomycin A. Deletion of the sole cluster-situated StrR family regulatory gene, asrR, abolished ristomycin A production and the transcription of the asr genes orf5 to orf39. The ristomycin A fermentation titer in Amycolatopsis sp. strain TNS106 was dramatically improved by overexpression of asrR and a heterologous StrR family regulatory gene, bbr, from the balhimycin-biosynthetic gene cluster (BGC) utilizing strong promoters and multiple gene copies. Ristomycin A production was improved by approximately 60-fold, resulting in a fermentation titer of 4.01 g/liter in flask culture, in one of the engineered strains. Overexpression of AsrR and Bbr upregulated transcription of tested asr biosynthetic genes, indicating that these asr genes were positively regulated by AsrR and Bbr. However, only the promoter region of the asrR operon and the intergenic region upstream of orf12 were bound by AsrR and Bbr in gel retardation assays, suggesting that AsrR and Bbr directly regulated the asrR operon and probably orf12 to orf14 but no other asr biosynthetic genes. Further assays with synthetic short probes showed that AsrR and Bbr specifically bound not only probes containing the canonical inverted repeats but also a probe with only one 7-bp element of the inverted repeats in its native context. AsrR and Bbr have an N-terminal ParB-like domain and a central winged helix-turn-helix DNA-binding domain. Site-directed mutations indicated that the N-terminal ParB-like domain was involved in activation of ristomycin A biosynthesis and did not affect the DNA-binding activity of AsrR and Bbr. IMPORTANCE This study showed that overexpression of either a native StrR family regulator (AsrR) or a heterologous StrR family regulator (Bbr) dramatically improved ristomycin A production by increasing the transcription of biosynthetic genes directly or indirectly. The conserved ParB-like domain of AsrR and Bbr was demonstrated to be involved in the regulation of asr BGC expression. These findings provide new insights into the mechanism of StrR family regulators in the regulation of glycopeptide antibiotic biosynthesis. Furthermore, the regulator overexpression plasmids constructed in this study could serve as valuable tools for strain improvement and genome mining for new glycopeptide antibiotics. In addition, ristomycin A is a type III glycopeptide antibiotic clinically used as a diagnostic reagent due to its side effects. The overproduction strains engineered in this study are ideal materials for industrial production of ristomycin A.

Adaptive Optimization Boosted the Production of Moenomycin A in the Microbial Chassis Streptomyces albus J1074.

Great efforts have been made to improve Streptomyces chassis for efficient production of targeted natural products. Moenomycin family antibiotics, represented by moenomycin (Moe) and nosokomycin, are phosphoglycolipid antibiotics that display extraordinary inhibition against Gram-positive bacteria. Herein, we assembled a completed 34 kb hybrid biosynthetic gene cluster (BGC) of moenomycin A (moe-BGC) based on a 24 kb nosokomycin analogue biosynthetic gene cluster (noso-BGC). The heterologous expression of the hybrid moe-BGC in Streptomyces albus J1074 achieved the production of moenomycin A in the recombinant strain LX01 with a yield of 12.1 ± 2 mg/L. Further strong promoter refactoring to improve the transcriptional levels of all of the functional genes in strain LX02 enhanced the production of moenomycin A by 58%. However, the yield improvement of moenomycin A resulted in a dramatic 38% decrease in the chassis biomass compared with the control strain. To improve the weak physiological tolerance to moenomycin A of the chassis, another copy of the gene salb-PBP2 (P238N&F200D), encoding peptidoglycan biosynthetic protein PBP2, was introduced into the chassis strain, producing strain LX03. Cell growth was restored, and the fermentation titer of moenomycin A was 130% higher than that of LX01. Additionally, the production of moenomycin A in strain LX03 was further elevated by 45% to 40.0 ± 3 mg/L after media optimization. These results suggested that the adaptive optimization strategy of strong promoter refactoring in the BGC plus physiological tolerance in the chassis was an efficient approach for obtaining the desired natural products with high titers.

Impact of MtrA on phosphate metabolism genes and the response to altered phosphate conditions in Streptomyces.

Phosphate metabolism is known to be regulated by the PhoPR regulatory system in Streptomyces and some other bacteria. In this study, we report that MtrA also regulates phosphate metabolism in Streptomyces. Our data showed that, in Streptomyces coelicolor, MtrA regulates not only phosphate metabolism genes such as phoA but also phoP under different phosphate conditions, including growth on rich complex media without added inorganic phosphate and on defined media with low or high concentrations of inorganic phosphate. Cross-regulation was also observed among mtrA, phoP and glnR under these conditions. We demonstrated both in vitro and in vivo binding of MtrA to the promoter regions of genes associated with phosphate metabolism and to the intergenic region between phoR and phoU, indicating that these phosphate metabolism genes are targets of MtrA. We further showed that MtrA in S. lividans and S. venezuelae has detectable regulatory effects on expression of phosphate metabolism genes. Additionally, the MtrA homologue from Corynebacterium glutamicum bound predicted MtrA sites of multiple phosphate metabolism genes, implying its potential for regulating phosphate metabolism in this species. Overall, our findings support MtrA as a major regulator for phosphate metabolism in Streptomyces and also potentially in other actinobacteria.

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

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.

A novel XRE family regulator that controls antibiotic production and development in Streptomyces coelicolor.

Although the genome of the Streptomyces model strain S. coelicolor was sequenced nearly two decades ago, the function of many annotated genes has not been verified, including that of gene sco1979, which was predicted to encode a transcriptional regulator of the xenobiotic response element (XRE) family. In this study, we showed that SCO1979 represses its own transcription and that deletion of sco1979 from S. coelicolor markedly enhanced production of three antibiotics, which are actinorhodin (ACT), undecylprodigiosin (RED), and calcium-dependent antibiotic (CDA), suggesting that SCO1979 represses their biosynthesis. We demonstrated that transcription of genes in the ACT, RED, and CDA pathways was generally increased in the mutant strain Δ1979 compared with levels in the wild-type strain M145. Additionally, purified recombinant SCO1979 interacted with DNA sequences upstream of sco1979 and actII-orf4, redZ, and cdaR, the pathway-specific regulators for the three pathways, implying that SCO1979 potentially regulates the ACT, RED, and CDA pathways via their specific regulators. In addition, disruption of sco1979 led to the notably delayed formation of aerial mycelium and spores, and consistent with this, transcription of genes associated with aerial hyphae and spore formation, such as chp and rdl, and ram, was reduced in Δ1979, implying the involvement of SCO1979 in cellular development control as well. In summary, our findings demonstrated that SCO1979 is a pleiotropic regulator with roles in both secondary metabolism and morphological development in S. coelicolor. KEY POINTS: • SCO1979 is a novel Streptomyces regulator of the XRE family. • SCO1979 regulates its own transcription. • SCO1979 regulates antibiotic production and cellular development.

Functional Genome Mining Reveals a Class V Lanthipeptide Containing a d-Amino Acid Introduced by an F420 H2 -Dependent Reductase.

Lantibiotics are a type of ribosomally synthesized and post-translationally modified peptides (termed lanthipeptides) with often potent antimicrobial activity. Herein, we report the discovery of a new lantibiotic, lexapeptide, using the library expression analysis system (LEXAS) approach. Lexapeptide has rare structural modifications, including N-terminal (N,N)-dimethyl phenylalanine, C-terminal (2-aminovinyl)-3-methyl-cysteine, and d-Ala. The characteristic lanthionine moiety in lexapeptide is formed by three proteins (LxmK, LxmX, and LxmY), which are distinct from enzymes known to be involved in lanthipeptide biosynthesis. Furthermore, a novel F420 H2 -dependent reductase (LxmJ) from the lexapeptide biosynthetic gene cluster (BGC) is identified to catalyze the reduction of dehydroalanine to install d-Ala. Our findings suggest that lexapeptide is the founding member of a new class of lanthipeptides that we designate as class V. We also identified further class V lanthipeptide BGCs in actinomycetes and cyanobacteria genomes, implying that other class V lantibiotics await discovery.

Biosynthetic access to the rare antiarose sugar via an unusual reductase-epimerase.

Rubrolones, isatropolones, and rubterolones are recently isolated glycosylated tropolonids with notable biological activity. They share similar aglycone skeletons but differ in their sugar moieties, and rubterolones in particular have a rare deoxysugar antiarose of unknown biosynthetic provenance. During our previously reported biosynthetic elucidation of the tropolone ring and pyridine moiety, gene inactivation experiments revealed that RubS3 is involved in sugar moiety biosynthesis. Here we report the in vitro characterization of RubS3 as a bifunctional reductase/epimerase catalyzing the formation of TDP-d-antiarose by epimerization at C3 and reduction at C4 of the key intermediate TDP-4-keto-6-deoxy-d-glucose. These new findings not only explain the biosynthetic pathway of deoxysugars in rubrolone-like natural products, but also introduce RubS3 as a new family of reductase/epimerase enzymes with potential to supply the rare antiarose unit for expanding the chemical space of glycosylated natural products.

RedH and PigC Catalyze the Biosynthesis of Hybrubins via Phosphorylation of 4'-Methoxy-2,2'-Bipyrrole-5'-Carbaldehyde.

Hybrubins are "unnatural" alkaloids with the same 4'-methoxy-2,2'-bipyrrole-5'-methine moiety found in prodiginines and a different ring derived from tetramic acids. Here, we demonstrated that RedH, a homologue of prodigiosin synthetase PigC, was responsible for the biosynthesis of hybrubins A and B in Streptomyces lividansIn vitro reactions indicated that RedH and PigC catalyzed the intermolecular condensation between 4'-methoxy-2,2'-bipyrrole-5'-carbaldehyde (MBC) and (Z)-5-ethylidenetetramic acid (ETA) to produce hybrubin B. Moreover, we demonstrated that RedH and PigC activated MBC via phosphorylation of the aldehyde group to form an intermediate Pi-MBC and that the subsequent condensation between Pi-MBC and (Z)-5-ethylidenetetramic acid occurs in a nonenzymatic way.IMPORTANCE Hybrubins are an emerging class of prodiginines possessing a new C ring derived from 5'-substituted tetramic acids and the methylene bridge connecting the C ring at a different position. We have supposed that condensation between 4'-methoxy-2,2'-bipyrrole-5'-carbaldehyde (MBC) and 5-ethylidenetetramic acid (ETA) yields the hybrid natural products hybrubins, which was proposed to be catalyzed by the undecylprodigiosin synthetase RedH. However, it is doubted whether RedH is able to catalyze another type of condensation between MBC and tetramic acids. In this study, we have demonstrated that the MBC-ETA condensation proceeds through RedH/PigC-catalyzed enzymatic activation of MBC via phosphorylation and a nonenzymatic condensation of Pi-MBC with ETA. Since MBC analogues have been shown to be accepted by PigC, more hybrubin analogues might be produced by using combinations of MBC analogues and other tetramic acids in future studies.

Genome-Wide Mutagenesis Links Multiple Metabolic Pathways with Actinorhodin Production in Streptomyces coelicolor.

Streptomyces species are important antibiotic-producing organisms that tightly regulate their antibiotic production. Actinorhodin is a typical antibiotic produced by the model actinomycete Streptomyces coelicolor To discover the regulators of actinorhodin production, we constructed a library of 50,000 independent mutants with hyperactive Tn5 transposase-based transposition systems. Five hundred fifty-one genes were found to influence actinorhodin production in 988 individual mutants. Genetic complementation suggested that most of the insertions (76%) were responsible for the changes in antibiotic production. Genes involved in diverse cellular processes such as amino acid biosynthesis, carbohydrate metabolism, cell wall homeostasis, and DNA metabolism affected actinorhodin production. Genome-wide mutagenesis can identify novel genes and pathways that impact antibiotic levels, potentially aiding in engineering strains to optimize the production of antibiotics in StreptomycesIMPORTANCE Previous studies have shown that various genes can influence antibiotic production in Streptomyces and that intercommunication between regulators can complicate antibiotic production. Therefore, to gain a better understanding of antibiotic regulation, a genome-wide perspective on genes that influence antibiotic production was needed. We searched for genes that affected production of the antibiotic actinorhodin using a genome-wide gene disruption system. We identified 551 genes that altered actinorhodin levels, and more than half of these genes were newly identified effectors. Some of these genes may be candidates for engineering Streptomyces strains to improve antibiotic production levels.

Novel Two-Component System MacRS Is a Pleiotropic Regulator That Controls Multiple Morphogenic Membrane Protein Genes in Streptomyces coelicolor.

As with most annotated two-component systems (TCSs) of Streptomyces coelicolor, the function of TCS SCO2120/2121 was unknown. Based on our findings, we have designated this TCS MacRS, for morphogenesis and actinorhodin regulator/sensor. Our study indicated that either single or double mutation of MacRS largely blocked production of actinorhodin but enhanced formation of aerial mycelium. Chromatin immunoprecipitation (ChIP) sequencing, using an S. coelicolor strain expressing MacR-Flag fusion protein, identified in vivo targets of MacR, and DNase I footprinting of these targets revealed a consensus sequence for MacR binding, TGAGTACnnGTACTCA, containing two 7-bp inverted repeats. A genome-wide search revealed sites identical or highly similar to this consensus sequence upstream of six genes encoding putative membrane proteins or lipoproteins. These predicted sites were confirmed as MacR binding sites by DNase I footprinting and electrophoretic mobility shift assays in vitro and by ChIP-quantitative PCR in vivo, and transcriptional analyses demonstrated that MacR significantly impacts expression of these target genes. Disruption of three of these genes, sco6728, sco4924, and sco4011, markedly accelerated aerial mycelium formation, indicating that their gene products are novel morphogenic factors. Two-hybrid assays indicated that these three proteins, which we have named morphogenic membrane protein A (MmpA; SCO6728), MmpB (SCO4924), and MmpC (SCO4011), interact with one another and with the putative membrane protein and MacR target SCO4225. Notably, SAV6081/82 and SVEN1780/81, homologs of MacRS TCS from S. avermitilis and S. venezuelae, respectively, can substitute for MacRS, indicating functional conservation. Our findings reveal a role for MacRS in cellular morphogenesis and secondary metabolism in StreptomycesIMPORTANCE TCSs help bacteria adapt to environmental stresses by altering gene expression. However, the roles and corresponding regulatory mechanisms of most TCSs in the Streptomyces model strain S. coelicolor are unknown. We investigated the previously uncharacterized MacRS TCS and identified the core DNA recognition sequence, two seven-nucleotide inverted repeats, for the DNA-binding protein MacR. We further found that MacR directly controls a group of membrane proteins, including MmpA-C, which are novel morphogenic factors that delay formation of aerial mycelium. We also discovered that these membrane proteins interact with one another and that other Streptomyces species have conserved MacRS homologs. Our findings suggest a conserved role for MacRS in morphogenesis and/or other membrane-associated activities. Additionally, our study showed that MacRS impacts, albeit indirectly, the production of the signature metabolite actinorhodin, further suggesting that MacRS and its homologs function as novel pleiotropic regulatory systems in Streptomyces.

Biosynthesis of Tropolones in Streptomyces spp.: Interweaving Biosynthesis and Degradation of Phenylacetic Acid and Hydroxylations on the Tropone Ring.

Tropolonoids are important natural products that contain a unique seven-membered aromatic tropolone core and exhibit remarkable biological activities. 3,7-Dihydroxytropolone (DHT) isolated from Streptomyces species is a multiply hydroxylated tropolone exhibiting antimicrobial, anticancer, and antiviral activities. In this study, we determined the DHT biosynthetic pathway by heterologous expression, gene deletion, and biotransformation. Nine trl genes and some of the aerobic phenylacetic acid degradation pathway genes (paa) located outside the trl biosynthetic gene cluster are required for the heterologous production of DHT. The trlA gene encodes a single-domain protein homologous to the C-terminal enoyl coenzyme A (enoyl-CoA) hydratase domain of PaaZ. TrlA truncates the phenylacetic acid catabolic pathway and redirects it toward the formation of heptacyclic intermediates. TrlB is a 3-deoxy-d-arabino-heptulosonic acid-7-phosphate (DAHP) synthase homolog. TrlH is an unusual bifunctional protein bearing an N-terminal prephenate dehydratase domain and a C-terminal chorismate mutase domain. TrlB and TrlH enhanced de novo biosynthesis of phenylpyruvate, thereby providing abundant precursor for the prolific production of DHT in Streptomyces spp. Six seven-membered carbocyclic compounds were identified from the trlC, trlD, trlE, and trlF deletion mutants. Four of these chemicals, including 1,4,6-cycloheptatriene-1-carboxylic acid, tropone, tropolone, and 7-hydroxytropolone, were verified as key biosynthetic intermediates. TrlF is required for the conversion of 1,4,6-cycloheptatriene-1-carboxylic acid into tropone. The monooxygenases TrlE and TrlCD catalyze the regioselective hydroxylations of tropone to produce DHT. This study reveals a natural association of anabolism of chorismate and phenylpyruvate, catabolism of phenylacetic acid, and biosynthesis of tropolones in Streptomyces spp.IMPORTANCE Tropolonoids are promising drug lead compounds because of the versatile bioactivities attributed to their highly oxidized seven-membered aromatic ring scaffolds. Our present study provides clear insight into the biosynthesis of 3,7-dihydroxytropolone (DHT) through the identification of key genes responsible for the formation and modification of the seven-membered aromatic core. We also reveal the intrinsic mechanism of elevated production of DHT and related tropolonoids in Streptomyces spp. The study on DHT biosynthesis in Streptomyces exhibits a good example of antibiotic production in which both anabolic and catabolic pathways of primary metabolism are interwoven into the biosynthesis of secondary metabolites. Furthermore, our study sets the stage for metabolic engineering of the biosynthetic pathway for natural tropolonoid products and provides alternative synthetic biology tools for engineering novel tropolonoids.

Reconstitution of Kinamycin Biosynthesis within the Heterologous Host Streptomyces albus J1074.

Diazofluorene compounds such as kinamycin and lomaiviticin feature unique molecular structures and compelling medicinal bioactivities. However, a complete understanding of the biosynthetic details for this family of natural products has yet to be fully elucidated. In addition, a lack of genetically and technically amenable production hosts has limited access to the full medicinal potential of these compounds. Here, we report the capture of the complete kinamycin gene cluster from Streptomyces galtieri Sgt26 by bacterial artificial chromosome cloning, confirmed by successful production of kinamycin in the heterologous host Streptomyces albus J1074. Sequence analysis and a series of gene deletion experiments revealed the boundary of the cluster, which spans 75 kb DNA. To probe the last step in biosynthesis, acetylation of kinamcyin F to kinamycin D, gene knockout, and complementation experiments identified a single gene product involved with final acetylation conversions. This study provides full genetic information for the kinamycin gene cluster from S. galtieri Sgt26 and establishes heterologous biosynthesis as a production platform for continued mechanistic assessment of compound formation and utilization.

Engineered Streptomyces lividans Strains for Optimal Identification and Expression of Cryptic Biosynthetic Gene Clusters.

Streptomyces lividans is a suitable host for the heterologous expression of biosynthetic gene clusters (BGCs) from actinomycetes to discover "cryptic" secondary metabolites. To improve the heterologous expression of BGCs, herein we optimized S. lividans strain SBT5 via the stepwise integration of three global regulatory genes and two codon-optimized multi-drug efflux pump genes and deletion of a negative regulatory gene, yielding four engineered strains. All optimization steps were observed to promote the heterologous production of polyketides, non-ribosomal peptides, and hybrid antibiotics. The production increments of these optimization steps were additional, so that the antibiotic yields were several times or even dozens of times higher than the parent strain SBT5 when the final optimized strain, S. lividans LJ1018, was used as the heterologous expression host. The heterologous production of these antibiotics in S. lividans LJ1018 and GX28 was also much higher than in the strains from which the BGCs were isolated. S. lividans LJ1018 and GX28 markedly promoted the heterologous production of secondary metabolites, without requiring manipulation of gene expression components such as promoters on individual gene clusters. Therefore, these strains are well-suited as heterologous expression hosts for secondary metabolic BGCs. In addition, we successfully conducted high-throughput library expression and functional screening (LEXAS) of one bacterial artificial chromosome library and two cosmid libraries of three Streptomyces genomes using S. lividans GX28 as the library-expression host. The LEXAS experiments identified clones carrying intact BGCs sufficient for the heterologous production of piericidin A1, murayaquinone, actinomycin D, and dehydrorabelomycin. Notably, due to lower antibiotic production, the piericidin A1 BGC had been overlooked in a previous LEXAS screening using S. lividans SBT5 as the expression host. These results demonstrate the feasibility and superiority of S. lividans GX28 as a host for high-throughput screening of genomic libraries to mine cryptic BGCs and bioactive compounds.

Deletion of MtrA Inhibits Cellular Development of Streptomyces coelicolor and Alters Expression of Developmental Regulatory Genes.

The developmental life cycle of Streptomyces species includes aerial hyphae formation and spore maturation, two distinct developmental processes that are controlled, respectively, by two families of developmental regulatory genes, bld and whi. In this study, we show that the response regulator MtrA (SCO3013) is critical for normal development of aerial hyphae in S. coelicolor and related species. ΔmtrA, a deletion mutant of the response regulator gene mtrA, exhibited the bald phenotype typical of bld mutants defective in aerial mycelium formation, with formation either much delayed or absent depending on the culture medium. Transcriptional analysis indicated that MtrA activates multiple genes involved in formation of aerial mycelium, including chp, rdl, and ram genes, as well as developmental regulatory genes of the bld and whi families. However, the major regulatory gene bldD showed enhanced expression in ΔmtrA, suggesting it is repressed by MtrA. electrophoretic mobility shift assays indicated that MtrA binds upstream of several genes with altered expression in ΔmtrA, including bldD and whiI, and sequences similar to the consensus binding sequence for MtrA of another actinomycete, Mycobacterium tuberculosis, were found in the bound sites. A loosely conserved recognition sequence containing two short, direct repeats was identified for MtrA of S. coelicolor and was validated using mutational analysis. MtrA homologs are widely distributed among Streptomyces species, and as with S. coelicolor, deletion of the mtrA homologs sve_2757 from S. venezuelae and sli_3357 from S. lividans resulted in conditional bald morphology. Our study suggests a critical and conserved role for MtrA in Streptomyces development.