RESUMO
Actinobacteria produce numerous antibiotics and other specialized metabolites that have important applications in medicine and agriculture1. Diffusible hormones frequently control the production of such metabolites by binding TetR family transcriptional repressors (TFTRs), but the molecular basis for this remains unclear2. The production of methylenomycin antibiotics in Streptomyces coelicolor A3(2) is initiated by the binding of 2-alkyl-4-hydroxymethylfuran-3-carboxylic acid (AHFCA) hormones to the TFTR MmfR3. Here we report the X-ray crystal structure of an MmfR-AHFCA complex, establishing the structural basis for hormone recognition. We also elucidate the mechanism for DNA release upon hormone binding through the single-particle cryo-electron microscopy structure of an MmfR-operator complex. DNA binding and release assays with MmfR mutants and synthetic AHFCA analogues define the role of individual amino acid residues and hormone functional groups in ligand recognition and DNA release. These findings will facilitate the exploitation of actinobacterial hormones and their associated TFTRs in synthetic biology and in the discovery of new antibiotics.
Assuntos
Antibacterianos/biossíntese , Furanos/metabolismo , Streptomyces coelicolor/metabolismo , Apoproteínas/química , Apoproteínas/metabolismo , Apoproteínas/ultraestrutura , Proteínas de Bactérias/química , Proteínas de Bactérias/classificação , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/ultraestrutura , Microscopia Crioeletrônica , Cristalografia por Raios X , DNA/química , DNA/genética , DNA/metabolismo , DNA/ultraestrutura , Furanos/química , Hormônios/química , Hormônios/classificação , Hormônios/metabolismo , Ligantes , Modelos Moleculares , Peptídeos/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/classificação , Proteínas Repressoras/metabolismo , Proteínas Repressoras/ultraestrutura , Transdução de Sinais , Streptomyces coelicolor/química , Streptomyces coelicolor/genética , Relação Estrutura-AtividadeRESUMO
Advances in DNA sequencing technology and bioinformatics have revealed the enormous potential of microbes to produce structurally complex specialized metabolites with diverse uses in medicine and agriculture. However, these molecules typically require structural modification to optimize them for application, which can be difficult using synthetic chemistry. Bioengineering offers a complementary approach to structural modification but is often hampered by genetic intractability and requires a thorough understanding of biosynthetic gene function. Expression of specialized metabolite biosynthetic gene clusters (BGCs) in heterologous hosts can surmount these problems. However, current approaches to BGC cloning and manipulation are inefficient, lack fidelity, and can be prohibitively expensive. Here, we report a yeast-based platform that exploits transformation-associated recombination (TAR) for high efficiency capture and parallelized manipulation of BGCs. As a proof of concept, we clone, heterologously express and genetically analyze BGCs for the structurally related nonribosomal peptides eponemycin and TMC-86A, clarifying remaining ambiguities in the biosynthesis of these important proteasome inhibitors. Our results show that the eponemycin BGC also directs the production of TMC-86A and reveal contrasting mechanisms for initiating the assembly of these two metabolites. Moreover, our data shed light on the mechanisms for biosynthesis and incorporation of 4,5-dehydro-l-leucine (dhL), an unusual nonproteinogenic amino acid incorporated into both TMC-86A and eponemycin.
Assuntos
Inibidores de Proteassoma , Saccharomyces cerevisiae , Inibidores de Proteassoma/química , Inibidores de Proteassoma/metabolismo , Sequência de Bases , Saccharomyces cerevisiae/genética , Família MultigênicaRESUMO
MOTIVATION: A widely applicable strategy to create cell factories is to knockout (KO) genes or reactions to redirect cell metabolism so that chemical synthesis is made obligatory when the cell grows at its maximum rate. Synthesis is thus growth-coupled, and the stronger the coupling the more deleterious any impediments in synthesis are to cell growth, making high producer phenotypes evolutionarily robust. Additionally, we desire that these strains grow and synthesize at high rates. Genome-scale metabolic models can be used to explore and identify KOs that growth-couple synthesis, but these are rare in an immense design space, making the search difficult and slow. RESULTS: To address this multi-objective optimization problem, we developed a software tool named gcFront-using a genetic algorithm it explores KOs that maximize cell growth, product synthesis and coupling strength. Moreover, our measure of coupling strength facilitates the search so that gcFront not only finds a growth-coupled design in minutes but also outputs many alternative Pareto optimal designs from a single run-granting users flexibility in selecting designs to take to the lab. AVAILABILITY AND IMPLEMENTATION: gcFront, with documentation and a workable tutorial, is freely available at GitHub: https://github.com/lLegon/gcFront and archived at Zenodo, DOI: 10.5281/zenodo.5557755. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Genoma , Software , Fenótipo , Ciclo CelularRESUMO
Mycosporine-like amino acids are a prevalent form of photoprotection in micro- and macro-organisms. Using a combination of natural product extraction/purification and femtosecond transient absorption spectroscopy, we studied the relaxation pathway for a common mycosporine-like amino acid pair, usujirene and its geometric isomer palythene, in the first few nanoseconds following photoexcitation. Our studies show that the electronic excited state lifetimes of these molecules persist for only a few hundred femtoseconds before the excited state population is funneled through an energetically accessible conical intersection with subsequent vibrational energy transfer to the solvent. We found that a minor portion of the isomer pair did not recover to their original state within 3 ns after photoexcitation. We investigated the long-term photostability using continuous irradiation at a single wavelength and with a solar simulator to mimic a more real-life environment; high levels of photostability were observed in both experiments. Finally, we employed computational methods to elucidate the photochemical and photophysical properties of usujirene and palythene as well as to reconcile the photoprotective mechanism.
Assuntos
Aminoácidos , Aminoácidos/química , IsomerismoRESUMO
Plants, as with humans, require photoprotection against the potentially damaging effects of overexposure to ultraviolet (UV) radiation. Previously, sinapoyl malate (SM) was identified as the photoprotective agent in thale cress. Here, we seek to identify the photoprotective agent in a similar plant, garden cress, which is currently used in the skincare product Detoxophane nc. To achieve this, we explore the photodynamics of both the garden cress sprout extract and Detoxophane nc with femtosecond transient electronic absorption spectroscopy. With the assistance of liquid chromatography-mass spectrometry, we determine that the main UV-absorbing compound in garden cress sprout extract is SM. Importantly, our studies reveal that the photoprotection properties of the SM in the garden cress sprout extract present in Detoxophane nc are not compromised by the formulation environment. The result suggests that Detoxophane nc containing the garden cress sprout extract may offer additional photoprotection to the end user in the form of a UV filter booster.
Assuntos
Lepidium sativum/química , Extratos Vegetais/química , Plântula/química , Protetores Solares/químicaRESUMO
Covering: up to December 2018 This article aims to highlight advantages, drawbacks and issues that users should consider when implementing the use of CRISPR/Cas9-tools for genome editing in streptomycetes, the most prolific source of antimicrobial natural products to date. Here, we examine four toolkits that have so far been made available for streptomycete in vivo-engineering and one for in vitro-editing, and review how they have been applied over the last three years. Our critical evaluation of these toolkits intends to support potential users in determining what they could achieve, what they should consider and what system they should select/optimise for their application.
Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Edição de Genes , Streptomyces/genética , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Genoma Bacteriano/genéticaRESUMO
The homologous transcriptional regulators ScbR and ScbR2 have previously been identified as γ-butyrolactone (GBL) and antibiotic receptors, respectively. They regulate diverse physiological processes in Streptomyces coelicolor in response to GBL and antibiotic signals. In this study, ScbR and ScbR2 proteins were shown to interact using a bacterial two-hybrid system where adenylate cyclase activity was reconstituted in Escherichia coli BTH101. These ScbR/ScbR2 interactions in S. coelicolor were then demonstrated by co-immunoprecipitation. The ScbR/ScbR2 heterodimer was shown to co-exist with their ScbR and ScbR2 respective homodimers. When potential operator targets in S. coelicolor were investigated, the heterodimer was found to bind in the promoter region of sco5158, which however was not a target for ScbR or ScbR2 homodimers. These results revealed a new mechanism of regulation by ScbR and ScbR2 in S. coelicolor.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Regulação Bacteriana da Expressão Gênica , Streptomyces coelicolor/genética , Antibacterianos/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Imunoprecipitação , Regiões Promotoras Genéticas , Ligação Proteica , Multimerização ProteicaRESUMO
Gamma-butyrolactones (GBLs) are signalling molecules that control antibiotic production in Streptomyces bacteria. The genetically engineered strain S. coelicolor M1152 was found to overproduce GBLs SCB1-3 as well as five novel GBLs named SCB4-8. Incorporation experiments using isotopically-labelled precursors confirmed the chemical structures of SCB1-3 and established those of SCB4-8.
Assuntos
4-Butirolactona/química , 4-Butirolactona/metabolismo , Antibacterianos/biossíntese , Streptomyces coelicolor/metabolismo , Engenharia Genética , Espectrometria de Massas , Streptomyces coelicolor/genéticaRESUMO
There is a constant need for new and improved drugs to combat infectious diseases, cancer, and other major life-threatening conditions. The recent development of genomics-guided approaches for novel natural product discovery has stimulated renewed interest in the search for natural product-based drugs. Genome sequence analysis of Streptomyces ambofaciens ATCC23877 has revealed numerous secondary metabolite biosynthetic gene clusters, including a giant type I modular polyketide synthase (PKS) gene cluster, which is composed of 25 genes (nine of which encode PKSs) and spans almost 150 kb, making it one of the largest polyketide biosynthetic gene clusters described to date. The metabolic product(s) of this gene cluster are unknown, and transcriptional analyses showed that it is not expressed under laboratory growth conditions. The constitutive expression of a regulatory gene within the cluster, encoding a protein that is similar to Large ATP binding of the LuxR (LAL) family proteins, triggered the expression of the biosynthetic genes. This led to the identification of four 51-membered glycosylated macrolides, named stambomycins A-D as metabolic products of the gene cluster. The structures of these compounds imply several interesting biosynthetic features, including incorporation of unusual extender units into the polyketide chain and in trans hydroxylation of the growing polyketide chain to provide the hydroxyl group for macrolide formation. Interestingly, the stambomycins possess promising antiproliferative activity against human cancer cell lines. Database searches identify genes encoding LAL regulators within numerous cryptic biosynthetic gene clusters in actinomycete genomes, suggesting that constitutive expression of such pathway-specific activators represents a powerful approach for novel bioactive natural product discovery.
Assuntos
Macrolídeos/química , Macrolídeos/farmacologia , Policetídeo Sintases/genética , Streptomyces/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Inativação Gênica , Humanos , Macrolídeos/metabolismo , Família Multigênica , Streptomyces/enzimologia , Ativação TranscricionalRESUMO
Mycosporine glycine (MyG) was produced by the fermentation of a purposely engineered bacterial strain and isolated from this sustainable source. The ultrafast spectroscopy of MyG was then investigated in its native, zwitterionic form (MyGzwitter), via femtosecond transient electronic absorption spectroscopy. Complementary nonadiabatic (NAD) simulations suggest that, upon photoexcitation to the lowest excited singlet state (S1), MyGzwitter undergoes efficient nonradiative decay to repopulate the electronic ground state (S0). We propose an initial ultrafast ring-twisting mechanism toward an S1/S0 conical intersection, followed by internal conversion to S0 and subsequent vibrational cooling. This study illuminates the workings of the archetype mycosporine, providing photoprotection, in the UV-B range, to organisms such as corals, macroalgae, and cyanobacteria. This study also contributes to our growing understanding of the photoprotection mechanisms of life.
Assuntos
Glicina , Glicina/química , Glicina/análogos & derivados , Bioengenharia , Raios Ultravioleta , CicloexanóisRESUMO
Inhibition of histone lysine acetyltransferases (KATs) KAT6A and KAT6B has shown antitumor activity in estrogen receptor-positive (ER+) breast cancer preclinical models. PF-07248144 is a selective catalytic inhibitor of KAT6A and KAT6B. In the present study, we report the safety, pharmacokinetics (PK), pharmacodynamics, efficacy and biomarker results from the first-in-human, phase 1 dose escalation and dose expansion study (n = 107) of PF-07248144 monotherapy and fulvestrant combination in heavily pretreated ER+ human epidermal growth factor receptor-negative (HER2-) metastatic breast cancer (mBC). The primary objectives of assessing the safety and tolerability and determining the recommended dose for expansion of PF-07248144, as monotherapy and in combination with fulvestrant, were met. Secondary endpoints included characterization of PK and evaluation of antitumor activity, including objective response rate (ORR) and progression-free survival (PFS). Common treatment-related adverse events (any grade; grades 3-4) included dysgeusia (83.2%, 0%), neutropenia (59.8%, 35.5%) and anemia (48.6%, 13.1%). Exposure was approximately dose proportional. Antitumor activity was observed as monotherapy. For the PF-07248144-fulvestrant combination (n = 43), the ORR (95% confidence interval (CI)) was 30.2% (95% CI = 17.2-46.1%) and the median PFS was 10.7 (5.3-not evaluable) months. PF-07248144 demonstrated a tolerable safety profile and durable antitumor activity in heavily pretreated ER+HER2- mBC. These findings establish KAT6A and KAT6B as druggable cancer targets, provide clinical proof of concept and reveal a potential avenue to treat mBC. clinicaltrial.gov registration: NCT04606446 .
RESUMO
γ-butyrolactone and related signalling systems are found in Streptomyces and other actinobacteria where they control the production of secondary or specialized metabolites such as antibiotics. Genetic manipulation of these regulatory systems therefore leads to changes in the secondary metabolite profile of a strain and has been used to activate previously silent secondary metabolite gene clusters. However, there is no easy way to assess the presence of γ-butyrolactone-like systems in Streptomyces strains without whole-genome sequencing. We have therefore developed and tested a PCR screen that is able to detect homologues of the commonly co-located butenolide synthase and γ-butyrolactone receptor genes. This PCR screen could be employed for the screening of strain libraries to detect signalling systems without the necessity for whole-genome sequencing.
RESUMO
Biomimicry has become a key player in researching new materials for a whole range of applications. In this study, we have taken a crude extract from the red algae Palmaria palmata containing mycosporine-like amino acids - a photoprotective family of molecules. We have applied the crude extract onto a surface to assess if photoprotection, and more broadly, light-to-heat conversion, is retained; we found it is. Considering sunscreens as a specific application, we have performed transmission and reflection terahertz spectroscopy of the extract and glycerol to demonstrate how one can monitor stability in real-world applications.
RESUMO
The growing problem of antibiotic resistance has led to the exploration of uncultured bacteria as potential sources of new antimicrobials. PCR amplicon analyses and short-read sequencing studies of samples from different environments have reported evidence of high biosynthetic gene cluster (BGC) diversity in metagenomes, indicating their potential for producing novel and useful compounds. However, recovering full-length BGC sequences from uncultivated bacteria remains a challenge due to the technological restraints of short-read sequencing, thus making assessment of BGC diversity difficult. Here, long-read sequencing and genome mining were used to recover >1400 mostly full-length BGCs that demonstrate the rich diversity of BGCs from uncultivated lineages present in soil from Mars Oasis, Antarctica. A large number of highly divergent BGCs were not only found in the phyla Acidobacteriota, Verrucomicrobiota and Gemmatimonadota but also in the actinobacterial classes Acidimicrobiia and Thermoleophilia and the gammaproteobacterial order UBA7966. The latter furthermore contained a potential novel family of RiPPs. Our findings underline the biosynthetic potential of underexplored phyla as well as unexplored lineages within seemingly well-studied producer phyla. They also showcase long-read metagenomic sequencing as a promising way to access the untapped genetic reservoir of specialised metabolite gene clusters of the uncultured majority of microbes.
Assuntos
Metagenoma , Solo , Regiões Antárticas , Bactérias/genética , Bactérias/metabolismo , Metagenômica , Família MultigênicaRESUMO
The genome sequence of Streptomyces ambofaciens, a species known to produce the congocidine and spiramycin antibiotics, has revealed the presence of numerous gene clusters predicted to be involved in the biosynthesis of secondary metabolites. Among them, the type II polyketide synthase-encoding alp cluster was shown to be responsible for the biosynthesis of a compound with antibacterial activity. Here, by means of a deregulation approach, we gained access to workable amounts of the antibiotics for structure elucidation. These compounds, previously designated as alpomycin, were shown to be known members of kinamycin family of antibiotics. Indeed, a mutant lacking AlpW, a member of the TetR regulator family, was shown to constitutively produce kinamycins. Comparative transcriptional analyses showed that expression of alpV, the essential regulator gene required for activation of the biosynthetic genes, is strongly maintained during the stationary growth phase in the alpW mutant, a stage at which alpV transcripts and thereby transcripts of the biosynthetic genes normally drop off. Recombinant AlpW displayed DNA binding activity toward specific motifs in the promoter region of its own gene and that of alpV and alpZ. These recognition sequences are also targets for AlpZ, the γ-butyrolactone-like receptor involved in the regulation of the alp cluster. However, unlike that of AlpZ, the AlpW DNA-binding ability seemed to be insensitive to the signaling molecules controlling antibiotic biosynthesis. Together, the results presented in this study reveal S. ambofaciens to be a new producer of kinamycins and AlpW to be a key late repressor of the cellular control of kinamycin biosynthesis.
Assuntos
Antibacterianos/metabolismo , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Quinonas/metabolismo , Streptomyces/metabolismo , Sequência de Aminoácidos , Antibacterianos/química , Proteínas de Bactérias/genética , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Conformação Proteica , Quinonas/química , Streptomyces/genéticaRESUMO
Streptorubin B is a structurally remarkable member of the prodiginine group of antibiotics produced by several actinobacteria, including the model organism Streptomyces coelicolor A3(2). Transannular strain within the pyrrolophane structure of this molecule causes restricted rotation that gives rise to the possibility of (diastereomeric) atropisomers. Neither the relative nor the absolute stereochemistry of streptorubin B is known. NOESY NMR experiments were used to define the relative stereochemistry of the major atropisomer of streptorubin B·HCl in solution as anti. We exploited this finding together with our knowledge of streptorubin B biosynthesis in S. coelicolor to determine the absolute stereochemistry of the anti atropisomer. 2-Undecylpyrrole stereoselectively labeled with deuterium at C-4' was synthesized and fed to a mutant of S. coelicolor, which was unable to produce streptorubin B because it was blocked in 2-undecylpyrrole biosynthesis, and in which the genes responsible for the last two steps of streptorubin B biosynthesis were overexpressed. (1)H and (2)H NMR analysis of the stereoselectively deuterium-labeled streptorubin B·HCl produced by this mutasynthesis strategy allowed us to assign the absolute stereochemistry of the major (anti) atropisomer as 7'S. HPLC analyses of streptorubin B isolated from S. coelicolor on a homochiral stationary phase and comparisons with streptorubin B derived from an enantioselective synthesis showed that the natural product consists of an approximately 88:7:5 mixture of the (7'S, anti), (7'S, syn), and (7'R, anti) stereoisomers.
Assuntos
Antibacterianos/química , Prodigiosina/análogos & derivados , Antibacterianos/biossíntese , Deutério/química , Espectroscopia de Ressonância Magnética , Prodigiosina/biossíntese , Prodigiosina/química , Prótons , Soluções , Estereoisomerismo , Streptomyces coelicolor/metabolismoRESUMO
All of the genetic elements necessary for the production of the antibiotic methylenomycin (Mm) and its regulation are contained within the 22-kb mmy-mmf gene cluster, which is located on the 356-kb linear plasmid SCP1 of Streptomyces coelicolor A3(2). A putative operon of 3 genes within this gene cluster, mmfLHP, was proposed to direct the biosynthesis of an A-factor-like signaling molecule, which could play a role in the regulation of Mm biosynthesis. The mmfLHP operon was expressed under the control of its native promoter in S. coelicolor M512, a host lacking the SCP1 plasmid, and the ability to produce prodiginine and actinorhodin antibiotics. Comparative metabolic profiling led to the identification and structure elucidation of a family of 5 new 2-alkyl-4-hydroxymethylfuran-3-carboxylic acids (AHFCAs), collectively termed Mm furans (MMFs), as the products of the mmfLHP genes. MMFs specifically induce the production of the Mm antibiotics in S. coelicolor. Comparative genomics analyses and searches of the natural product chemistry literature indicated that other streptomycetes may produce AHFCAs, suggesting that they could form a general class of antibiotic biosynthesis inducers in Streptomyces species, with analogous functions to the better known gamma-butyrolactone regulatory molecules.
Assuntos
Antibacterianos/biossíntese , Furanos/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Família Multigênica/genética , Streptomyces coelicolor/genética , Cromatografia Líquida de Alta Pressão , Biologia Computacional , Primers do DNA/genética , Furanos/química , Genômica/métodos , Espectrometria de Massas , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Peptídeos/genética , Plasmídeos/genéticaRESUMO
Microorganisms require protection against the potentially damaging effects of ultraviolet radiation exposure. Photoprotection is, in part, provided by mycosporine-like amino acids (MAAs). Previous reports have proposed that nonradiative decay mediates the impressive photoprotection abilities of MAAs. In this letter, we present the first ultrafast dynamics study of two MAAs, shinorine and porphyra-334. We demonstrate that, in aqueous solution, these MAAs relax along their S1 coordinates toward the S1/S0 conical intersection within a few hundred femtoseconds after photoexcitation and then traverse the conical intersection and vibrationally cool in approximately 1 ps through heat transfer to the solvent. This new insight allows a quintessential component of microbial life to be unraveled and informs the development of molecular photon-to-heat converters for a myriad of applications.
Assuntos
Cicloexanonas/química , Cicloexilaminas/química , Glicina/análogos & derivados , Raios Ultravioleta , Glicina/química , Processos FotoquímicosRESUMO
Filamentous members of the phylum Actinobacteria are a remarkable source of natural products with pharmaceutical potential. The discovery of novel molecules from these organisms is, however, hindered because most of the biosynthetic gene clusters (BGCs) encoding these secondary metabolites are cryptic or silent and are referred to as orphan BGCs. While co-culture has proven to be a promising approach to unlock the biosynthetic potential of many microorganisms by activating the expression of these orphan BGCs, it still remains an underexplored technique. The marine actinobacterium Salinispora tropica, for instance, produces valuable compounds such as the anti-cancer molecule salinosporamide but half of its putative BGCs are still orphan. Although previous studies have used marine heterotrophs to induce orphan BGCs in Salinispora, its co-culture with marine phototrophs has yet to be investigated. Following the observation of an antimicrobial activity against a range of phytoplankton by S. tropica, we here report that the photosynthate released by photosynthetic primary producers influences its biosynthetic capacities with production of cryptic molecules and the activation of orphan BGCs. Our work, using an approach combining metabolomics and proteomics, pioneers the use of phototrophs as a promising strategy to accelerate the discovery of novel natural products from marine actinobacteria.
Assuntos
Actinobacteria , Micromonosporaceae , Actinobacteria/genética , Micromonosporaceae/genética , Família Multigênica , FitoplânctonRESUMO
Prokaryotic cell-free coupled transcription-translation (TX-TL) systems are emerging as a powerful tool to examine natural product biosynthetic pathways in a test tube. The key advantages of this approach are the reduced experimental time scales and controlled reaction conditions. To realize this potential, it is essential to develop specialized cell-free systems in organisms enriched for biosynthetic gene clusters. This requires strong protein production and well-characterized synthetic biology tools. The Streptomyces genus is a major source of natural products. To study enzymes and pathways from Streptomyces, we originally developed a homologous Streptomyces cell-free system to provide a native protein folding environment, a high G+C (%) tRNA pool, and an active background metabolism. However, our initial yields were low (36 µg/mL) and showed a high level of batch-to-batch variation. Here, we present an updated high-yield and robust Streptomyces TX-TL protocol, reaching up to yields of 266 µg/mL of expressed recombinant protein. To complement this, we rapidly characterize a range of DNA parts with different reporters, express high G+C (%) biosynthetic genes, and demonstrate an initial proof of concept for combined transcription, translation, and biosynthesis of Streptomyces metabolic pathways in a single "one-pot" reaction.