Reconstruction of a Genome-Scale Metabolic Model of Streptomyces albus J1074: Improved Engineering Strategies in Natural Product Synthesis.
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SUBMITTER: Kittikunapong C
PROVIDER: S-EPMC8150979 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundGenome sequencing revealed that Streptomyces sp. can dedicate up to ~ 10% of their genomes for the biosynthesis of bioactive secondary metabolites. However, the majority of these biosynthetic gene clusters are only weakly expressed or not at all. Indeed, the biosynthesis of natural products is highly regulated through integrating multiple nutritional and environmental signals perceived by pleiotropic and pathway-specific transcriptional regulators. Although pathway-specific refactoring has been a proved, productive approach for the activation of individual gene clusters, the construction of a global super host strain by targeting pleiotropic-specific genes for the expression of multiple diverse gene clusters is an attractive approach.ResultsStreptomyces albus J1074 is a gifted heterologous host. To further improve its secondary metabolite expression capability, we rationally engineered the host by targeting genes affecting NADPH availability, precursor flux, cell growth and biosynthetic gene transcriptional activation. These studies led to the activation of the native paulomycin pathway in engineered S. albus strains and importantly the upregulated expression of the heterologous actinorhodin gene cluster.ConclusionsRational engineering of Streptomyces albus J1074 yielded a series of mutants with improved capabilities for native and heterologous expression of secondary metabolite gene clusters.
| S-EPMC5816538 | biostudies-literature
Project description:BACKGROUND: The Streptomyces albus J1074 strain is one of the most widely used chassis for the heterologous production of bioactive natural products. The fast growth and an efficient genetic system make this strain an attractive model for expressing cryptic biosynthetic pathways to aid drug discovery. RESULTS: To improve its capabilities for the heterologous expression of biosynthetic gene clusters, the complete genomic sequence of S. albus J1074 was obtained. With a size of 6,841,649 bp, coding for 5,832 genes, its genome is the smallest within the genus streptomycetes. Genome analysis revealed a strong tendency to reduce the number of genetic duplicates. The whole transcriptomes were sequenced at different time points to identify the early metabolic switch from the exponential to the stationary phase in S. albus J1074. CONCLUSIONS: S. albus J1074 carries the smallest genome among the completely sequenced species of the genus Streptomyces. The detailed genome and transcriptome analysis discloses its capability to serve as a premium host for the heterologous production of natural products. Moreover, the genome revealed 22 additional putative secondary metabolite gene clusters that reinforce the strain's potential for natural product synthesis.
| S-EPMC3937824 | biostudies-literature
Project description:Four novel paulomycin derivatives have been isolated from S. albus J1074 grown in MFE culture medium. These compounds are structural analogs of antibiotics 273a2α and 273a2β containing a thiazole moiety, probably originated through an intramolecular Michael addition. The novel, thiazole, moiety-containing paulomycins show a lower antibiotic activity than paulomycins A and B against Gram-positive bacteria. However, two of them show an improved activity against Gram-negative bacteria. In addition, the four novel compounds are more stable in culture than paulomycins A and B. Thus, the presence of an N-acetyl-l-cysteine moiety linked to the carbon atom of the paulic acid isothiocyanate moiety, via a thioester bond, and the subsequent intramolecular cyclization of the paulic acid to generate a thiazole heterocycle confer to paulomycins a higher structural stability that otherwise will conduce to paulomycin degradation and into inactive paulomenols.
| S-EPMC6151807 | biostudies-literature
Project description:BackgroundNaringenin is an industrially relevant compound due to its multiple pharmaceutical properties as well as its central role in flavonoid biosynthesis.ResultsOn our way to develop Streptomyces albidoflavus J1074 as a microbial cell factory for naringenin production, we have significantly increased the yields of this flavanone by combining various metabolic engineering strategies, fermentation strategies and genome editing approaches in a stepwise manner. Specifically, we have screened different cultivation media to identify the optimal production conditions and have investigated how the additive feeding of naringenin precursors influences the production. Furthermore, we have employed genome editing strategies to remove biosynthetic gene clusters (BGCs) associated with pathways that might compete with naringenin biosynthesis for malonyl-CoA precursors. Moreover, we have expressed MatBC, coding for a malonate transporter and an enzyme responsible for the conversion of malonate into malonyl-CoA, respectively, and have duplicated the naringenin BGC, further contributing to the production improvement. By combining all of these strategies, we were able to achieve a remarkable 375-fold increase (from 0.06 mg/L to 22.47 mg/L) in naringenin titers.ConclusionThis work demonstrates the influence that fermentation conditions have over the final yield of a bioactive compound of interest and highlights various bottlenecks that affect production. Once such bottlenecks are identified, different strategies can be applied to overcome them, although the efficiencies of such strategies may vary and are difficult to predict.
| S-EPMC10466684 | biostudies-literature
Project description:Heterocyclic natural products with various bioactivities play significant roles in pharmaceuticals. Here, we isolated a heterocyclic compound salumycin (1) from a Streptomyces albus J1074 mutant strain. The structure of (1) was elucidated via single-crystal X-ray diffraction, mass spectrometry (MS), fourier transform infrared spectrometer (FTIR), and nuclear magnetic resonance (NMR) data analysis. Salumycin (1) contained a novel pyrazolequinone ring, which had never been previously reported in a natural product. Salumycin (1) exhibited moderate 2,2'-diphenyl-1-picrylhydrazyl (DPPH)-radical scavenging activity (EC50 = 46.3 ± 2.2 μM) compared with tert-butylhydroquinone (EC50 = 4.7 ± 0.3 μM). This study provides a new example of discovering novel natural products from bacteria.
| S-EPMC7570766 | biostudies-literature
Project description:A large majority of genome-encrypted chemical diversity in actinobacteria remains to be discovered, which is related to the low level of secondary metabolism genes expression. Here, we report the application of a reporter-guided screening strategy to activate cryptic polycyclic tetramate macrolactam gene clusters in Streptomyces albus J1074. The analysis of the S. albus transcriptome revealed an overall low level of secondary metabolism genes transcription. Combined with transposon mutagenesis, reporter-guided screening resulted in the selection of two S. albus strains with altered secondary metabolites production. Transposon insertion in the most prominent strain, S. albus ATGSal2P2::TN14, was mapped to the XNR_3174 gene encoding an unclassified transcriptional regulator. The mutant strain was found to produce the avenolide-like compound butenolide 4. The deletion of the gene encoding a putative acyl-CoA oxidase, an orthologue of the Streptomyces avermitilis avenolide biosynthesis enzyme, in the S. albus XNR_3174 mutant caused silencing of secondary metabolism. The homologues of XNR_3174 and the butenolide biosynthesis genes were found in the genomes of multiple Streptomyces species. This result leads us to believe that the discovered regulatory elements comprise a new condition-dependent system that controls secondary metabolism in actinobacteria and can be manipulated to activate cryptic biosynthetic pathways.
| S-EPMC5575351 | biostudies-literature
Project description:In streptomycetes, autoregulators are important signaling compounds that trigger secondary metabolism, and they are regarded as Streptomyces hormones based on their extremely low effective concentrations (nM) and the involvement of specific receptor proteins. Our previous distribution study revealed that butenolide-type Streptomyces hormones, including avenolide, are a general class of signaling molecules in streptomycetes and that Streptomyces albus strain J1074 may produce butenolide-type Streptomyces hormones. Here, we describe metabolite profiling of a disruptant of the S. albusaco gene, which encodes a key biosynthetic enzyme for butenolide-type Streptomyces hormones, and identify four butenolide compounds from S. albus J1074 that show avenolide activity. The compounds structurally resemble avenolide and show different levels of avenolide activity. A dual-culture assay with imaging mass spectrometry (IMS) analysis for in vivo metabolic profiling demonstrated that the butenolide compounds of S. albus J1074 stimulate avermectin production in another Streptomyces species, Streptomyces avermitilis, illustrating the complex chemical interactions through interspecies signals in streptomycetes.IMPORTANCE Microorganisms produce external and internal signaling molecules to control their complex physiological traits. In actinomycetes, Streptomyces hormones are low-molecular-weight signals that are key to our understanding of the regulatory mechanisms of Streptomyces secondary metabolism. This study reveals that acyl coenzyme A (acyl-CoA) oxidase is a common and essential biosynthetic enzyme for butenolide-type Streptomyces hormones. Moreover, the diffusible butenolide compounds from a donor Streptomyces strain were recognized by the recipient Streptomyces strain of a different species, resulting in the initiation of secondary metabolism in the recipient. This is an interesting report on the chemical interaction between two different streptomycetes via Streptomyces hormones. Information on the metabolite network may provide useful hints not only to clarification of the regulatory mechanism of secondary metabolism, but also to understanding of the chemical communication among streptomycetes to control their physiological traits.
| S-EPMC5930338 | biostudies-literature
Project description:Streptomyces albus J1074 is a streptomycete strain widely used as a host for expression of secondary metabolite gene clusters. Bioinformatic analysis of the genome of this organism predicts the presence of 27 gene clusters for secondary metabolites. We have used three different strategies for the activation of some of these silent/cryptic gene clusters in S. albus J1074: two hybrid polyketide-non-ribosomal peptides (PK-NRP) (antimycin and 6-epi-alteramides), a type I PK (candicidin), a non-ribosomal peptides (NRP) (indigoidine) and glycosylated compounds (paulomycins). By insertion of a strong and constitutive promoter in front of selected genes of two clusters, production of the blue pigment indigoidine and of two novel members of the polycyclic tetramate macrolactam family (6-epi-alteramides A and B) was activated. Overexpression of positive regulatory genes from the same organism also activated the biosynthesis of 6-epi-alteramides and heterologous expression of the regulatory gene pimM of the pimaricin cluster activated the simultaneous production of candicidins and antimycins, suggesting some kind of cross-regulation between both clusters. A cluster for glycosylated compounds (paulomycins) was also identified by comparison of the high-performance liquid chromatography profiles of the wild-type strain with that of a mutant in which two key enzymes of the cluster were simultaneously deleted.
| S-EPMC3992020 | biostudies-literature
Project description:Because the majority of environmental bacteria are not easily culturable, access to many bacterially encoded secondary metabolites will be dependent on the development of improved functional metagenomic screening methods. In this study, we examined a collection of diverse Streptomyces species for the best innate ability to heterologously express biosynthetic gene clusters. We then optimized methods for constructing high quality metagenomic cosmid libraries in the best Streptomyces host. An initial screen of a 1.5 million-membered metagenomic library constructed in Streptomyces albus, the species that exhibited the highest propensity for heterologous expression of gene clusters, led to the identification of the novel natural product metatricycloene (1). Metatricycloene is a tricyclic polyene encoded by a reductive, iterative polyketide-like gene cluster. Related gene clusters found in sequenced genomes appear to encode a largely unexplored collection of structurally diverse, polyene-based metabolites.
| S-EPMC5469685 | biostudies-literature
Project description:An important unresolved issue in microbial secondary metabolite production is the abundance of biosynthetic gene clusters that are not expressed under typical laboratory growth conditions. These so-called silent or cryptic gene clusters are sources of new natural products, but how they are silenced, and how they may be rationally activated are areas of ongoing investigation. We recently devised a chemogenetic high-throughput screening approach ("HiTES") to discover small molecule elicitors of silent biosynthetic gene clusters. This method was successfully applied to a Gram-negative bacterium; it has yet to be implemented in the prolific antibiotic-producing streptomycetes. Herein we have developed a high-throughput transcriptional assay format in Streptomyces spp. by leveraging eGFP, inserted both at a neutral site and inside the biosynthetic cluster of interest, as a read-out for secondary metabolite synthesis. Using this approach, we successfully used HiTES to activate a silent gene cluster in Streptomyces albus J1074. Our results revealed the cytotoxins etoposide and ivermectin as potent inducers, allowing us to isolate and structurally characterize 14 novel small molecule products of the chosen cluster. One of these molecules is a novel antifungal, while several others inhibit a cysteine protease implicated in cancer. Studies addressing the mechanism of induction by the two elicitors led to the identification of a pathway-specific transcriptional repressor that silences the gene cluster under standard growth conditions. The successful application of HiTES will allow future interrogations of the biological regulation and chemical output of the countless silent gene clusters in Streptomyces spp.
| S-EPMC5617735 | biostudies-literature