A novel isolated streptomyces strain and uses thereof

Abstract

The present invention relates to a novel isolated Streptomyces strain, Streptomyces sungeiensis SD3 (Accession number DSM34917), its engineered version and their uses in producing secondary metabolites

Description

[0001] A NOVEL ISOLATED STREPTOMYCES STRAIN AND USES THEREOF

[0002] CROSS-REFERENCE TO RELATED APPLICATION

[0003] This application claims priority of Singapore Patent Application No. 10202400722U filed on 15 March 2024, the contents of which are incorporated herein in their entirety by reference.

[0004] FIELD OF THE INVENTION

[0005] The present invention relates to a novel isolated Streptomyces strain, Streptomyces sungeiensis SD3 (Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures International Depositary Authority Accession number DSM 34917), and its use in producing secondary metabolites.

[0006] BACKGROUND OF THE INVENTION

[0007] Microbial natural products constitute a significant proportion of pharmaceutical agents utilized in the treatment of a wide spectrum of human afflictions (Newman et al., 2020). Among the microbial producers, actinomycetes stand out as highly prolific sources, producing many clinically important antibacterial, anti-fungal, anti-parasitic, anticancer, and immunosuppressive therapeutics (Krause et al., 2020; Liu et al., 2018). Microbial natural products are secondary metabolites synthesized by the enzymes encoded by biosynthetic gene clusters (BGCs) (Lee et al., 2020). Over the past three decades, genome sequencing has unveiled the presence of multiple BGCs in many actinomycete strains. Many of these BGCs remain dormant or silent, concealing the enigmatic products that are yet to be identified (Xia et al., 2020). The profusion of BGCs within each strain, coupled with the widespread distribution of actinomycete strains in terrestrial and marine environments, bestows a still under tapped reserve for discovering potential drug candidates (Lacey et al., 2022; Nepal et al., 2019).

[0008] Many cryptic BGCs remain dormant under typical laboratory culture conditions due to repressed gene expression, limited precursor availability, or other inhibitory factors (Liu et al., 2021 ). Activating silent BGCs to disclose their cryptic secondary metabolites is often unfruitful, with many reported successes owing to serendipitous discovery of elicitors or fermentation conditions. Rational genetic approaches such as BGC refactoring suffer from low success rate, mainly due to the lack of understanding of the multi-layered genetic regulatory networks governing gene expression (Myronovskyi et al., 2019). BGC activation in the native microbial producers is further complicated because of the slow growth rates and limited genetic manipulability that afflict many non-domesticated actinomycete strains (Myronovskyi et al., 2019). The commonly employed genetic tools and vectors developed for model actinomycetes, primarily Streptomyces species, are often incompatible with wild-type actinomycetes (Xu et al., 2022).

[0009] An alternative approach that has been exploited to activate silent cryptic BGCs involves the expression of cryptic BGCs in a genetically manipulable, fast-growing heterologous chassis (Ahmed et al., 2020). Microbial heterologous expression strains have thus far been developed, most commonly utilising the well-characterized Escherichia coli, Bacillus subtilis and Saccharomyces cerevisiae species as chassis (Hwang et al., 2021 ). However, although these chassis have been more extensively developed and explored for heterologous expression, a large number of Streptomyces BGCs could not be expressed in these strains (Liu et al., 2016; Myronovskyi et al., 2019). This is in part due to the large size and high guaninecytosine (GC) content of Streptomyces BGCs, and the shortage of precursor and cellular energy required for producing secondary metabolites (Liu et al., 2018). In comparison, Streptomyces strains already possess numerous metabolic pathways able to supply necessary precursors, and relevant post-translational modifications needed for the biosynthesis of a diversity of structurally complex secondary metabolites (Kang and Kim, 2021 ).

[0010] Several model Streptomyces strains have been subjected to genome engineering and strain optimization, encompassing S. coelicolor, S. lividans, S. avermitilis, S. albus, S. chattanoogensis, S. griseofuscus, S. atratus, and S. venezuelae (Ahmed et al., 2020; Gomez- Escribano et al., 2011 ; Hwang et al., 2021 ; Liu et al., 2016; Myronovskyi et al., 2018; Whitford et al., 2021 ; Yang et al., 2022). Initially, there was optimism regarding the prospect of identifying a universal Streptomyces heterologous chassis capable of expressing the BGCs from phylogenetically diverse actinomycetes (Liu et al., 2018; Ahmed et aL, 2020; Hwang et al., 2021 ; Yang et al., 2022). However, this aspiration turned out to be insurmountable, likely because of the underestimated differences in regulatory mechanism and cellular environment between the host chassis and native producers. At present, whether a BGC can be successfully expressed in a heterologous Streptomyces host to produce an anticipated secondary metabolite remains largely unpredictable (Myronovskyi et al., 2019; Hwang et aL, 2021 ). Nevertheless, it has been recognized that the likelihood of successful production of secondary metabolites in a heterologous host would increase if the heterologous chassis shares a closer phylogenetic relationship with the native producer (Yue et aL, 2023). Consequently, the strategy of identifying and developing a panel of Streptomyces hosts spanning the phylogenetic spectrum has emerged as a more pragmatic approach, as opposed to the pursuit of a single universal heterologous Streptomyces host (Myronovskyi et al., 2019; Hwang et aL, 2021 ; Liu et al., 2016). Thus, there is a need for improved Streptomyces strains for engineering and enhanced heterologous production of secondary metabolites.

[0011] SUMMARY OF THE INVENTION

[0012] In the present disclosure, the Streptomyces sungeiensis SD3 strain that displays the desired properties of a microbial heterologous chassis, including rapid growth and sporulation on widely employed solid and liquid culture media, was identified. The strain displays susceptibility to antibiotics commonly employed in molecular biology laboratories, rendering it amenable to genetic manipulation. S. sungeiensis SD3 can be readily transformed with genetic material, including large BGC-containing artificial chromosomes using readily available E. coli-Streptomyces shuttling vectors. The genetic tractability of S. sungeiensis SD3 makes it well-suited for optimization and progression through the Design-Build-Test-Learn (DBTL) cycle. One notable feature of S. sungeiensis SD3 is its unique placement within a distinct clade of the phylogenetic tree. It stands apart from other commonly used Streptomyces chassis strains developed by other researchers. This distinctive phylogenetic placement positions S. sungeiensis SD3 as the preferred chassis for the expression of BGCs derived from Streptomyces strains within the same or neighbouring clades.

[0013] In a first aspect, there is provided a novel isolated S. sungeiensis SD3 strain having accession number DSM34917, the said strain being deposited at the Leibniz-lnstitut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), InhoffenstraBe 7B, 38124 Braunschweig, Germany, an International Depositary Authority recognized under the Budapest Treaty.

[0014] In a second aspect, there is provided an engineered S. sungeiensis SD3 strain comprising a heterologous gene encoding a secondary metabolite or a biosynthetic gene cluster (BGC) for producing a secondary metabolite, wherein the engineered S. sungeiensis SD3 strain is derived from the isolated S. sungeiensis SD3 strain deposited at the Leibniz-lnstitut DSMZ- Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) under accession number DSM34917.

[0015] In some embodiments, the secondary metabolite is selected from a group consisting of an antibiotic, antifungal, antitumor agent, immunosuppressant, enzyme inhibitor, herbicide, insecticide, non-ribosomal peptide and combinations thereof. In some embodiments, the secondary metabolite is selected from a group consisting of polyketide, terpene, terpenoid, alkaloid, alkylresorcinol, and combinations thereof. Preferably, the secondary metabolite is selected from a group consisting of streptomycin, tetracycline, erythromycin, nystatin, doxorubicin, mitomycin, tacrolimus, rapamycin, clavulanic acid, obscurolide A2, chartreusin, strptazone B1 , adipostatin A, adipostatin B, furaquinocin M, tasikamide D, tasikamide E, tasikamide F, tasikamide G, chlorostrptazone B1 , and combinations thereof.

[0016] In some embodiments, the engineered S. sungeiensis SD3 strain further comprises a promoter selected from a group consisting of sf14p, KasOp*, gapdhp(EL), and gapdhp(KR), wherein the promoter is operably linked to the heterologous gene.

[0017] In some embodiments, the engineered S. sungeiensis SD3 strain further comprises a heterologous sfp gene.

[0018] In some embodiments, the engineered S. sungeiensis SD3 strain does not comprise a BGC for producing chartreusin. In another embodiment, the beta-ketoacyl synthase genes chaA and chaB within the chartreusin BGC are deleted. Advantageously, deletion of chaA and chaB activity not only eliminates the chartreusin-associated HPLC peaks to facilitate the detection of metabolites produced by heterologous biosynthetic pathways but also frees up malonyl- CoA and NADPH to boost fermentation titer.

[0019] In some embodiments, the engineered S. sungeiensis SD3 strain is AchaAB::sfp.

[0020] In a third aspect, there is provided a method for producing one or more secondary metabolites, comprising culturing the isolated S. sungeiensis SD3 strain or the engineered S. sungeiensis SD3 strain of the present invention in a culture medium under suitable conditions for a period of time, and recovering the secondary metabolites from the culture medium.

[0021] In some embodiments, the culture medium is a solid medium. In other embodiments, the culture medium is a liquid medium. Any known solid medium or liquid medium for growing Streptomyces strains may be suitable for culturing SD3 strain. Examples of culture medium include but are not limited to tryptic soy broth (TSB) (30 g / L TSB powder), Mannitol Soya Flour medium (per liter: 20 g Mannitol, 20 g Soya flour, 20 g agar), GYMose medium (per liter: 20 g Glucose, 10 g Maltose, 5 g Yeast Extract), Oatmeal medium (per liter: 25 g Oatmeal Quaker), Media A (per liter: 5 g Mannitol, 5 g soya bean flour, 10 mL Glycerol (86%), 9 g yeast extract, 1 mL trace element solution), Media B (per liter: 10 g Glucose, 1 g asparagine, 1 g K2HPO4, 10 mg MnCl2*4H2O, 1 mL ZnSO4 solution, 1 mL FeSO4 solution), Media C (per liter: 80 g Sucrose, 10 g soybean powder, 2 g yeast extract, 1 g meat extract (beef), 0.3 g K2HPO4, 0.3 g MgSO4’7H2O, 5 mL FeSO4 solution, 1 g CaCOs), Media D (per liter: 20 g Soluble starch, 1 g KNO3, 0.5 g NaCI, 0.5 g MgSO4-7H2O, 0.5 g K2HPO4, 1 mL FeSO4 solution), Media E (per liter: 5 g Glucose, 5 g corn steep powder, 10 g oatmeal, 10 mL oil, 1 g K2HPO4, 1 g MgSO4*7H2O, 1 mL trace element solution), GYM medium (per liter: 10 g Malt extract, 10 g agar, 4 g yeast extract, 4 g glucose, 2 g CaCO3), ISP4 medium (per liter: 10 g soluble potato starch, 1 g MgSO / *7H20, 1 g NaCI, 2 g (NH^SO^ 2 g CaCOs, 20 g agar, 1 mL Trace salts solution), ISP5 medium (per liter: 1 g L-asparagine ,10 mL Glycerol, 1 g K2HPO4, 1 mL Trace salts solution, 20 g Agar), ISP6 medium (per liter: 5 g proteose-peptone, 0.5 g Ammonium iron (II) citrate, 1 g K2HPO4, 15 g agar, 1 g yeast extract), ISP7 medium (per liter: 15 g Glycerol, 0.5 g L-tyrosine, 1 g L-asparagine, 0.5 g K2HPO4, 0.5 g MgSO4-7H20, 0.5 g NaCI, 1 mL FeSO4solution, 20 g agar).

[0022] In some embodiments, the SD strain is cultured at a temperature between 25BC and 35BC. Preferably, the SD3 strain is cultured at 30BC.

[0023] In some embodiments, the secondary metabolite is selected from a group consisting of an antibiotic, antifungal, antitumor agent, immunosuppressant, enzyme inhibitor, herbicide, insecticide, non-ribosomal peptide and combinations thereof. In some embodiments, the secondary metabolite is selected from a group consisting of polyketide, terpene, terpenoid, alkaloid, alkylresorcinol, and combinations thereof. Preferably, the secondary metabolite is selected from a group consisting of streptomycin, tetracycline, erythromycin, nystatin, doxorubicin, mitomycin, tacrolimus, rapamycin, clavulanic acid, obscurolide A2, chartreusin, strptazone B1 , adipostatin A, adipostatin B, furaquinocin M, tasikamide D, tasikamide E, tasikamide F, tasikamide G, chlorostrptazone B1 , and combinations thereof.

[0024] BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Certain embodiments of the present disclosure are described more fully hereinafter with reference to the accompanying drawings.

[0026] Figure 1 : shows (A) Chemical structures for furaquinocins M (6) and D; (B) Key 1 H-1 H COSY and 1 H-13C HMBC correlations of furaquinocin M; (C) Chemical structure for streptazone B1 and chlorostreptazone B1 (11 ); (D) Key 1 H-1 H COSY and 1 H-13C HMBC correlations of chlorostreptazone B1 (1 1 ).

[0027] Figure 2: shows the growth profile of S. sungeiensis SD3 compared with other Streptomyces hosts.

[0028] Figure 3: shows a schematic of a chromosomal genome map and genomic features of S. sungeiensis SD3. Tracks from outermost circle to the center: (i) Marker for genome size, (ii) Predicted coding DNA sequence (CDS) on forward and reverse stands . (iii) Biosynthetic gene clusters predicted by antiSMASH 7.0.0 . (iv) tRNA genes . (v) rRNA genes . (vi) GC content, the outward portion indicating higher than genome-wide average GC content, whilst the inward portion indicating lower than average GC content, (vii) GC skew, the outward the outward portion indicating higher than average GC skew, and the inward portion indicating below average GC skew. The genome figure was constructed using DNAPIotter v 18.1 .0. 12.

[0029] Figure 4: shows a schematic of the phylogenetic relationship between S. sungeiensis SD3 and commonly used Streptomyces chassis. The phylogenetic tree was constructed using TYGS 13 to calculate a Genome BLAST Distance Phylogeny (GBDP) and visualized using Iterative Tree Of Life iTOL (Letunic et aL, 2021 ). The most commonly used Streptomyces chassis are labelled.

[0030] Figure 5: shows a Venn diagram of unique and shared orthologous gene clusters amongst S. sungeiensis SD3, S. avermitilis NBRC 14893 (NC 003155), S. coelicolor A3(2) (CP042324), S. venezuelae ATCC 21782 (CP029193), and S. albus DSM 41398 (CP010519).

[0031] Figure 6: shows chemical structures of secondary metabolites isolated from the fermentation culture of S. sungeiensis SD3 or predicted by AntiSMASH analysis but not detected likely due to repressed gene expression. The structure of streptazone E is included here for comparison.

[0032] Figure 7: shows1H and13C NMR spectra of known abscurolide A2 (1 ) (in DMSO-cfc).

[0033] Figure 8: shows1H and13C NMR spectra of known chartreusin (2) (in DMSO-d6).

[0034] Figure 9: shows1H NMR spectrum of known streptazone B1 (3) (in MeOD-c / 4).

[0035] Figure 10: shows a schematic of a comparison between the streptazone B1 -synthesizing BGC24 from S. sungeiensis SD3 and the streptazone E-synthesizing stz BGC and other similar BGCs that produce polyketide-derived piperidine alkaloids.

[0036] Figure 11 : shows results of heterologous production of secondary metabolites using the S. sungeiensis SD3 host. (A) HPLC chromato-gram and the compounds produced by the heterologously expressed BGCzs and BGC23 from S. tasikensis P46. Note that adipostatin A (4) and B (5) are the products of endogenous SD3_BGCs7b but not P46_BGC23. (B) HPLC chromatogram showing the production of streptazone B1 (3) and chlorostreptazone B1 (1 1 ). Figure 12: shows1H and13C NMR spectra of known adipostatin A (4) (in MeOD-c / 4).

[0037] Figure 13: shows1H NMR spectrum of known adipostatin B (5) (in MeOD-ck).

[0038] Figure 14: shows the core biosynthetic genes of each BGC in SD3 displayed with their protein-encoding gene (PEG) number as annotated by RAST server. The log2FC values of these biosynthetic genes comparing 96 hrs to 12 hrs as analyzed and normalized by DESeq2 are used to generate a heat map with the higher intensity indicating a higher fold change value.

[0039] Figure 15: shows BGCs2b, BGC33, BGC37a and HrdB housekeeping gene read count profiles from 96 hrs mapped onto the S. sungeiensis SD3 genome using HISAT2 v 2.2.1 and visualized using Integrative genomics viewer (IGV) v 2.16.2 (Kim et al., 2019; Robinson et al., 2011 ). The biosynthetic genes of the BGCs as annotated by antiSMASH 7.0 are shown using arrows in the schematic (dark arrows for core biosynthetic genes, grey arrows for accessory biosynthetic genes).

[0040] Figure 16: shows the primary metabolic pathways that underpin chartreusin biosynthesis. The enzyme commission (EC) numbers of relevant metabolic reactions of each metabolic pathway are displayed with the upregulated genes (square) and downregulated genes (diamond) from the pathways highlighted.

[0041] Figure 17: shows a comparison of the strength of constitutively active promoters in S. sungeiensis SD3. (A) The normalized fluorescence intensities are shown for the different S. sungeiensis SD3 strains with the expression of the EGFP gene under exogenous promoter control. The values are means with standard deviation (SD) from three independent experiments. (B) qRT-PCR analysis of the mRNA level of EGFP for the four exogenous promoters. All qRT-PCR values were normalized to endogenous control hrdB.

[0042] Figure 18: shows gene deletion and insertion via CRISPR-Cas9 enabled genome editing in S. sungeiensis. (A-C) Experimental designs to insert the constitutive promoter KasOp* in front of the first T1 PKS module of BGC32b, delete the first AT domain of BGCs2b, and delete the chaA and chaB biosynthetic genes of BGC33 by using CRISPR-Cas9.

[0043] Figure 19: shows PCR results to confirm the promoter insertion or AT domain deletion in BGCs2b or loss of chaA gene in the S. sungeiensis SD3_AchaAB:.sfp strain. The primer regions are denoted by the arrows. Figure 20: shows HPLC analysis confirming the mutant strains have lost the ability to produce chartreusin. The chartreusin peak is denoted by the solid triangle.

[0044] Figure 21 : shows1H NMR spectrum of new furaquinocin M (6) (400 MHz, in DMSO-cfc)-

[0045] Figure 22: shows13C NMR spectrum of new furaquinocin M (6) (100 MHz, in DMSO-cfc)-

[0046] Figure 23: shows1H-1H COSY NMR spectrum of new furaquinocin M (6) (400 MHz, in DMSO- d&).

[0047] Figure 24: shows1H-13C HSQC NMR spectrum of new furaquinocin M (6) (400 MHz, in DMSO-d6).

[0048] Figure 25: shows1H-13C HMBC NMR spectrum of new furaquinocin M (6) (400 MHz, in DMSO-d6).

[0049] Figure 26: shows NOESY NMR spectrum of new furaquinocin M (6) (400 MHz, in DMSO-d6).

[0050] Figure 27: shows HRESIMS of furaquinocin M (6).

[0051] Figure 28: shows1H and13C NMR spectra of new chlorostreptazone B1 (11) (in DMSO-d6).

[0052] Figure 29: shows1H-1H COSY NMR spectrum of new chlorostreptazone B1 (11 ) (in DMSO-d6).

[0053] Figure 30: shows1H-13C HSQC NMR spectrum of new chlorostreptazone B1 (11) (in DMSO- d&)-

[0054] Figure 31 : shows1H-13C HMBC NMR spectrum of new chlorostreptazone B1 (11) (in DMSO- d6).

[0055] Figure 32: shows ESIMS spectrum of chlorostreptazone B1 (11).

[0056] Figure 33: shows heterologously expressed halogenase converts streptazone B1 (3) produced by an endogenous biosynthetic pathway to chlorostreptazone B1 (11) in S. sungeiensis SD3. Figure 34: shows comparison of the fermentation yields of tasikamides D-G for S. tasikensis P46 (solid medium) and SD3::BGC23 (liquid medium).

[0057] DETAILED DESCRIPTION OF THE INVENTION

[0058] Further details of the invention will now be described with reference to the following nonlimiting examples. Unless otherwise defined herein, technical and scientific terms used in the present description have the meanings that are commonly understood by those of ordinary skill in the art.

[0059] A. Definitions

[0060] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0061] As used herein, the term “comprising” may include the embodiments “consisting of’ and “consisting essentially of”. The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases that require the presence of the named features / steps and permit the presence of other features / steps. However, such description should be construed as also describing compositions, mixtures, or processes as “consisting of” and “consisting essentially of” the enumerated features / steps, which allows the presence of only the named features / steps, along with any impurities that might result therefrom, and excludes other features / steps.

[0062] The term “operably linked” as used herein refers to a first molecule that can be joined to a second molecule, wherein the molecules are so arranged that the first molecule affects the function of the second molecule. The term “operably linked” includes the juxtaposition of two or more components (e.g., a promoter and another sequence) such that both components function normally and allow for the possibility that at least one of the components can mediate a function that is exerted upon at least one of the other components. The two molecules may or may not be part of a single contiguous molecule and may or may not be adjacent. For example, a promoter is operably linked to a polynucleotide molecule if the promoter modulates transcription of the polynucleotide molecule of interest in a cell. In additional embodiments, two portions of a transcription regulatory element are operably linked to one another if they are joined such that the transcription-activating functionality of one portion is not adversely affected by the presence of the other portion. Two transcription regulatory elements may be operably linked to one another by way of a linker nucleic acid (e.g., an intervening non-coding nucleic acid) or may be operably linked to one another with no intervening nucleotides present. As used herein, the term “secondary metabolites” refer to organic compounds produced by microorganisms that are not directly involved in the normal growth, development, or reproduction of the microorganisms. Unlike primary metabolites, which are essential for basic cellular functions, secondary metabolites often serve ecological functions such as defence against predators, competition with other organisms, or facilitating reproduction. Examples of secondary metabolites include but not limited to antibiotics, antifungals, antitumor agents, immunosuppressants, enzyme inhibitors, herbicides, insecticides, non-ribosomal peptides, polyketides, terpenes, terpenoids, alkaloids, alkylresorcinols. Preferably, the secondary metabolite is selected from a group consisting of streptomycin, tetracycline, erythromycin, nystatin, doxorubicin, mitomycin, tacrolimus, rapamycin, clavulanic acid, obscurolide A2, chartreusin, strptazone B1 , adipostatin A, adipostatin B, furaquinocin M, tasikamide D, tasikamide E, tasikamide F, tasikamide G, chlorostrptazone B1 , and combinations

[0063] As used herein, the term “biosynthetic gene clusters” or “BCGs” refers to groups of genes that work together to produce a specific biochemical compound. These clusters typically include genes encoding enzymes, regulatory proteins, and transport proteins necessary for the biosynthesis, regulation, and export of the compound. BGCs are diverse and can be categorized based on the types of compounds they produce. One prominent class is the polyketide synthase (PKS) clusters, responsible for the biosynthesis of polyketides. These compounds are known for their structural complexity and include important pharmaceuticals like erythromycin and tetracycline. PKS clusters are further divided into three types: Type I, II, and III, each with distinct enzymatic architectures and biosynthetic pathways. Another significant category is the non-ribosomal peptide synthetase (NRPS) clusters. These clusters synthesize non-ribosomal peptides, which are notable for their diverse biological activities, including antimicrobial and immunosuppressive properties. Unlike ribosomal peptides, NRPS products are assembled by large enzyme complexes that incorporate a wide variety of amino acids, often including non-standard ones. This flexibility allows for the production of compounds like penicillin and vancomycin, which have been instrumental in medical treatments. Terpenoid biosynthetic clusters produce terpenes and terpenoids, the largest class of natural products. These compounds have roles ranging from ecological interactions to pharmaceutical applications. Terpenoid clusters often involve enzymes like terpene synthases and cytochrome P450s, contributing to the structural diversity of these molecules.

[0064] B. Biological deposit Information The bacterial strain Streptomyces sungeiensis SD3 was deposited at the Leibniz- Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), InhoffenstraBe 7B, 38124 Braunschweig, Germany on 23 January 2024 and has been accorded the Accession number DSM34917.

[0065] It should be understood that any and all embodiments of the present disclosure can be combined with technical features in any other embodiment or multiple other embodiments to obtain additional embodiments under the premise of no conflict. The invention includes such combinations resulting in further embodiments.

[0066] EXAMPLES

[0067] The following examples are intended to exemplify the present disclosures and are not limitations of the claimed invention. All molecules, compositions, methods, assays, and results disclosed in the examples and other sections of the specification, figures, and claims form part of the disclosure of the invention.

[0068] Example 1. Methods

[0069] 1.1 General

[0070] NMR data were collected on an Avance NEO 400 MHz or Avance III 600 MHz spectrometer at 298 K. Chemical shifts are expressed in 5 (ppm) and referenced to the residual solvent signals. ESI-MS spectra were acquired on a Thermo LTQ XL spectrometer with ESI source. Flash column chromatography (CC) was performed using silica gel (230-400 mesh, Merck, Darmstadt, Germany). HPLC seperation and purficaiton of the metabolites was conducted using an Agilent 1200 series HPLC-DAD system with an ODS column (Pursuit XRs: diphenyl, 250 mm x 10 mm, 5 pm; Cosmosil: cholester, 250 mm x 10 mm, 5 pm) and a Shimadzu liquid chromatography system equipped with an ODS column (ACE: C18-HL, 250 mm x 10 mm, 5 pm). UV-Vis spectra were collected on a Denovix spectrophotometer. All reagents and materials were purchased from Sigma-Aldrich unless otherwise indicated.

[0071] 1.2 Bacterial strains and culture conditions

[0072] The E. co / / strains Top10, DH10B (Thermo Fisher Scientific) and ET12567 were cultured at 37 °C in LB liquid medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCI, pH 7.2). Selective antibiotics were added when necessary and were used at 50 pg / mL apramycin; 25 pg / mL chloramphenicol; 50 pg / mL kanamycin; 25 pg / mL nalidixic acid (Sigma-Aldrich). Routine culturing of Streptomyces strains on solid media was carried out on mannitol soy (MS) agar (20 g / L soya flour, 20 g / L mannitol and 20 g / L BactoTM agar), while culturing in liquid media was done in tryptic soy broth (TSB) (30 g / L TSB powder) (Merck Millipore, Germany). Incubation of Streptomyces strains on solid media was done at 30 °C incubators whilst for liquid culture 30 °C shaking incubators at 180 rpm was used. Media supplementation used in this study were described: Trace element solution (per liter: 40 mg ZnCI2, 200 mg FeCI3*6H2O, 10 mg CuCI2’2H2O, 10 mg MnCI2«4H2O, 10 mg Na2B4O7*10H2O, 10 mg (NH4)6Mo7O24*4H2O), and Trace salt solution (per liter: 1 g FeSC>4’7H2O, 1 g MnCI2*4H2O, 1 g ZnSO4*7H2O), FeSCU solution (0.01 g FeSC>4 in 1 mL H20), ZnSO4 solution (0.01 g ZnSO4 in 1 mL H20). Media used in this study for fermentation were as described: Mannitol Soya Flour medium (per liter: 20 g Mannitol, 20 g Soya flour, 20 g agar), GYMose medium (per liter: 20 g Glucose, 10 g Maltose, 5 g Yeast Extract), Oatmeal medium (per liter: 25 g Oatmeal Quaker), Media A (per liter: 5 g Mannitol, 5 g soya bean flour, 10 mL Glycerol (86%), 9 g yeast extract, 1 mL trace element solution), Media B (per liter: 10 g Glucose, 1 g asparagine, 1 g K2HPO4, 10 mg MnCI2«4H2O,

[0073] 1 mL ZnSO4solution, 1 mL FeSO4solution), Media C (per liter: 80 g Sucrose, 10 g soybean powder, 2 g yeast extract, 1 g meat extract (beef), 0.3 g K2HPO4, 0.3 g MgSO4*7H2O, 5 mL FeSO4 solution, 1 g CaCO3), Media D (per liter: 20 g Soluble starch, 1 g KNO3, 0.5 g NaCI, 0.5 g MgSO4'7H2O, 0.5 g K2HPO4, 1 mL FeSO4 solution), Media E (per liter: 5 g Glucose, 5 g corn steep powder, 10 g oatmeal, 10 mL oil, 1 g K2HPO4, 1 g MgSO4’7H20, 1 mL trace element solution), GYM medium (per liter: 10 g Malt extract, 10 g agar, 4 g yeast extract, 4 g glucose,

[0074] 2 g CaCOs), ISP4 medium (per liter: 10 g soluble potato starch, 1 g MgSO4*7H20, 1 g NaCI, 2 g (NH4)2SO4, 2 g CaCO3, 20 g agar, 1 mL Trace salts solution), ISP5 medium (per liter: 1 g L- asparagine ,10 mL Glycerol, 1 g K2HPO4, 1 mL Trace salts solution, 20 g Agar), ISP6 medium (per liter: 5 g proteose-peptone, 0.5 g Ammonium iron (II) citrate, 1 g K2HPO4, 15 g agar, 1 g yeast extract), ISP7 medium (per liter: 15 g Glycerol, 0.5 g L-tyrosine, 1 g L-asparagine, 0.5 g K2HPO4, 0.5 g MgSO4-7H20, 0.5 g NaCI, 1 mL FeSO4 solution, 20 g agar).

[0075] 1.3 Biomass measurement of S. sunaeiensis SD3

[0076] Wild type S. sungeiensis SD3 and other Streptomyces strains were grown in 20 mL of TSB for 2 days. 100 mg of biomass of each pre-culture was used for inoculating 50 mL of TSB and cultured at 30 °C and 180 rpm shaking for 3 days. 1 mL of each sample was taken every 24 hrs and centrifuged for 10 min at 14,000 rpm. The supernatant was discarded, and the dry biomass weight was measured.

[0077] 1.4 Transformation of S. sunaeiensis SD3 with BGC-containing PACs pESAC13A containing BGCs from S. spp. P46 was obtained from BioS&T. Three sets of primers were used to confirm the correct insertion of BGCs into the pESAC13A vectors, corresponding to the first, last and middle genes of each cluster delineated by antiSMASH prediction. To integrate the BGC containing pESAC13A vectors into S. sungeiensis SD3, triparental conjugation was first carried out utilizing donor DH10B containing pESAC13A::BGC, DH10B / pR9604 helper, and ET12567 E. coli recipient strains. ET12567 cells harboring the correct pESAC13A::BGC was then conjugated with S. sungeiensis SD3. For conjugation of vectors into S. sungeiensis SD3, spores were germinated by heat-shock at 45 °C for 10 min and mixed with E. coli ET1 567 / pUZ8002 harboring the correct vector at a ratio of 1 :5. The mixture was spread on MS agar supplemented with 20 mM CaCk and 20 mM MgCF and incubated for 20 hours at 30 °C. Following which, the plates were overlaid with 50 pg / mL apramycin and 25 pg / mL nalidixic acid.

[0078] 1.5 Standard DNA manipulations

[0079] Standard PCR reactions were carried out using Q5 High Fidelity Polymerase (NEB) in accordance with the manufacturer’s instructions. DNA was purified from 1% agarose gels with SYBR safe (ThermoFisher Scientific) staining. The molecular weight standard used for agarose gel electrophoresis was 1 kb Plus DNA Ladder (ThermoFisher Scientific). The purification of plasmids was carried out using Qiagen Spin Miniprep, and gel extractions using Zymo Research Zymoclean Gel DNA Recovery Kit, according to the manufacturer’s instructions. Restriction endonucleases and DNA-modifying enzymes were from New England Biolabs (NEB). Molecular cloning was carried out using restriction cloning or Gibson Assembly (NEB) to transfer PCR amplified fragments to the vector of choice.

[0080] 1 .6 Whole genome sequencing

[0081] S. sungeiensis SD3 was isolated from a sediment sample collected from Sungei Buloh mangrove wetland reserve, Singapore. The complete genome of S. sungeiensis SD3 was obtained as a linear chromosome of 10,292,314 base pairs using Single Molecule Real Time (SMRT) (Pacific Biosciences, California, USA) sequencing and Illumina sequencing and CLC Genomics Workbench (CLC bio, Denmark).

[0082] 1.7 Phylogenetic and Comparative Genomics Analysis

[0083] The phylogenetic relationship of S. sungeiensis SD3 with other Streptomyces species was established via whole genome comparisons using the TYGS server software and visualised by the online software iTOL. The ANI calculator software from EZBioCloud was used to ascertain the OrthoANIu value between S. sungeiensis SD3 and S. chartreusis. The open reading frame prediction and genome annotation was carried out using RAST software (Rapid Annotation using Subsystem Technology). The predicted proteins obtained by RAST was used in OrthoVenn software to search for orthologous protein clusters. Secondary metabolite biosynthetic gene clusters were identified using the antiSMASH database.

[0084] 1.8 Fermentation and metabolite profiling Spores of S. sungeiensis SD3 wild-type or knockout strains were plated on Mannitol Soya Flour medium and incubated for 4 days at 30fiC. Starter cultures were prepared by inoculating the mycelia to 50 mL GYMose liquid medium in 250 mL conical flask and shaken for 5-7 days at 170 rpm until dispersed. 100 pL starter cultures were used for spreading on solid medium or sub-culturing in liquid medium at 10% and incubated at 305C. For solid medium, after two weeks fermentation the resulting agar plates were mashed and extracted with MeOH. The filtered MeOH extract was concentrated under vacuum and re-dissolved in 500 pL MeOH to obtain the crude sample. For liquid medium fermentation, after 4 days the resultant cultures were centrifuged and separated into supernatant and cell pellet, followed by extraction with ethyl acetate (EA, 1 :1 , v / v) and MeOH respectively. HPLC analysis was performed on Agilentl 200 HPLC-DAD system equipped with an ODS column (Pursuit XRs: 250 mm x 4.6 mm, 5 pm) using a linear gradient of CH3CN in H2O with 0.1% formic acid (0-5 min, 10%-20% CH3CN; 5-35 min, 20%-70% CH3CN; 35-50 min, 70%-90% CH3CN; 50-60 min, 90%-100% CH3CN; 60-70 min, 100% CH3CN) at a flow rate of 1 .0 mL / min.

[0085] 1.9 Isolation and characterization of endogenously produced secondary metabolites

[0086] The liquid GYMose medium with better production titer was chosen for the large-scale fermentation of S. sungeiensis SD3 to obtain the main secondary metabolites. Briefly, four 2.0 L baffled flasks, each containing 800 mL production medium, were inoculated with 40 mL seed culture and incubated for 4 days at 30 °C, 180 rpm. The resulting cultures were centrifuged and separated into supernatant and cell pellet, followed by extraction with ethyl acetate (EA, 1 :1 , v / v) and MeOH (3 x 1 L), respectively. Extracts were combined and evaporated under vacuum to yield the total crude residue, which was then subjected to the flash column separation (CO) over silica gel using a stepwise gradient of hexane / EA / MeOH (20\1\0, 10\1\0, 5\1\0, 1\1\0, 0\1\0, 0\20\1 , 0\10\1 , 0\3\1 , 0\0\1 ) to yield nine fractions (Fr.1 -Fr.9). Further purification was guided by HPLC-UV analysis, which showed the products were mainly in Fr.4 and Fr.5. Fr.4 was purified by repeated semi-preparative RP-HPLC with a high-load ACE ODS column [CH3CN-H2O (containing 0.1% formic acid, v / v) 30:70, v / v; flow rate, 4.7 mL / min] to furnish streptazone B1 (3, 2.0 mg, tn = 12.5 min) and obscurolide A2 (1 , 7.2 mg, fR= 16.5 min). Fr.5 that contains chartreusin peaks was applied to the same RP-HPLC system [ACE ODS column, CH3CN-H2O (containing 0.1% formic acid, v / v) 55:45, v / v; flow rate, 4.7 mL / min], and the collected chartreusin-containing fraction was further purified by a Pursuit XRs Diphenyl column [CH3CN-H2O (containing 0.1% formic acid, v / v) 60:40, v / v; flow rate, 3.0 mL / min] to afford pure chartreusin (2, 8.5 mg, fR= 15.2 min). As for the two other metabolites produced by a PAC-transformed S. sungeiensis SD3, the fermentation of SD3 mutant (i.e., SD3::P46_BGC23) and the broth extraction and isolation procedures were conducted with the same protocol as aforementioned, and the two NMR-pure samples (adipostations A (4) and B (5), tR= 14.5 min and 17.0 min) were finally achieved by preparative RP-HPLC with a Cosmosil Cholester column [CH3CN-H2O (containing 0.1% formic acid, v / v) 88:12, v / v; flow rate, 3.0 mL / min], By comparison of the observed spectroscopic data and physicochemical properties with those reported in the literature, the above isolated compounds were identified to be obsurolide A2 (1 ), chartreusin (2), streptazone B1 (3), adipostatins A (4) and B (5), respectively. The MS,1H and13C NMR spectra can be found in the Figures.

[0087] 1.10 RNA Sequencing

[0088] S. sungeiensis SD3 was grown in TSB media for either 12 hrs or 96 hrs in baffled flasks. Cells were collected and homogenized using a pestle. 1 mg / mL working concentration of lysozyme was used for incubation at 37SC for half an hour. Cells were freeze-thawed using liquid nitrogen for 5-10 cycles. TRIzol Reagent was added according to the manufacturer’s instructions (Invitrogen). Finally, total RNA was extracted and purified using the Purelink RNA Mini kit according to manufacturer’s instructions (Thermo Fisher Scientific). DNAse-l (Thermo Fisher Scientific) was added in accordance with the Purelink RNA Mini kit protocol. For RNA sequencing, the resulting total RNA samples were then sent to Novogene Co., Ltd. for Illumina paired-end 150 bp sequencing. Agilent 2100 Bioanalyzer was used to measure the concentration and quality of total RNA, and samples with integrity values >7.0 was used for library construction. Trimmomatic software was used to carry out read trimming to remove low quality bases and adapter sequences. HISAT2 v 2.2.1 software was used for mapping the RNA-seq to the S. sungeiensis SD3 genome. FeatureCounts software was used to quantify reads based on the “CDS” feature characteristics of the annotation file generated by RAST for S. sungeiensis SD3. DESeq2 software was used for the normalization of reads and detection of differentially expressed genes. Integrative genomics viewer (IGV) v 2.16.2 software was used for simultaneously visualizing RNA sequencing data and genomic annotations.

[0089] 1.1 1 Measurement of promoter strength

[0090] Streptomyces spores were inoculated into 50 mL TSB liquid media starter cultures and shaken for two days, before sub-culturing into 50 mL TSB liquid media for another three days with the addition of 30 5-mm glass beads. 10 mL of culture was extracted and washed and resuspended with 1 x PBS. 100 pl of cells from 10 mL of 1 x PBS cell suspension was used for measurement of OD6oo and normalization of cell concentration to 0.5 OD6oo. Fluorescence measurements were carried out using a Tecan Infinite® 200 PRO Plate Reader. For EGFP, the excitation and emission wavelengths used were 485 nm and 515 nm, respectively. Readings were done in triplicates for each sample. For qRT-PCR, total RNA was used to synthesize cDNA using RevertAid H Minus Reverse Transcriptase (Thermo Scientific). Real-time PCR amplification was performed using the BioRad CFX connect real time PCR detection system. The amplification reaction was performed using the QuantiNova SYBR Green PCR Kit (Qiagen). Experiments were performed using three biological replicates. All qRT-PCR values were normalized to the endogenous housekeeping gene hrdB.

[0091] 1.12 CRISPR / Cas9-assisted gene deletion pCRISPR-Cas9 from Tong et al., 2015 and pQS-idgS from Wang et al., 2020 were utilized in the genome modification efforts, with the deletion strain successfully obtained from the use of pQS-idgS. The construction of pQS-idgS containing the sgRNA and homology arms were as described by Wang et al., 2020. In brief, the short oligonucleotide corresponding to the sgRNA was cloned into the pQS-idgS vector after Nco\ and Xbal digestion. 2 kb homologous recombination templates were introduced to this vector via Gibson assembly, upon linearization by Stul. The Gibson mix was then transformed into chemically competent TOP10 E. coh. Correctly-assembled vectors were then selected using colony PCR and DNA sequencing and subsequently transformed into E. coli ET 12567 / pUZ8002 for conjugation with S. sungeiensis SD3 using conjugation conditions as described above. After 5 days of incubation at 30°C, exconjugants were transferred to MS agar supplemented with 5 pg / mL thiostrepton for activation of the tipA promoter controlling the Streptomyces codon-optimized nuclease gene scas9. Curing of plasmid was done by culturing in TSB liquid media and subsequent plating onto MS agar plates, both without selective antibiotics. Correct mutants were then screened with PCR of deletion regions.

[0092] 1.13 Isolation and characterization of secondary metabolites produced by heterologous pathways

[0093] For heterologous expression of Tsk BGC in SD3: As tasikamides generally feature with similar characteristic UV absorptions and the inventors have tasikamides as authentic samples in hand, the reality of tasikamide production by the Tsk BGC-transformed strain SD3::P46_BGC23 was readily confirmed though the HPLC-UV and MS analysis of the organic extract of the culture broth.

[0094] For heterologous expression of furaquinocin BGC in SD3: A noticeable secondary metabolite was observed when P46_BGC28 was heterologously expressed within SD3 host, and a routine HPLC-guided isolation and purification for the extract of 4L culture broth of SD3::P46_BGC28 leads to the collection of the target compound with bright yellow colour. Based on the fact that BGC28 is predicted to produce a furaquinocin-type product coupled with the observed characteristic UV absorption for the isolated compound (6), this yellow metabolite was deduced to be a furaquinocin analogue. The detail of MS and NMR-based structural characterization is described below: the molecular formula of compound 6 was established as C21H24O6 on the basis of HRESIMS data (m / z, 373.1631 [M+H]+, calcd for C21 H25O6 373.1645 ), which requires 10 degrees of unsaturation. The13C NMR data of 6 displayed resonances for two carbonyls at 5 182.6 and 179.9 ppm and ten olefinic / aromatic carbons between 5 107.9 and 6 162.1 ppm. The1H NMR spectrum accordingly showed resonances attributable to two aromatic proton (6 7.01 , 6.05), one methoxy (5 3.77), four methyl groups {6 1.35, 1.47, 1 .49, 1.62), three methines (5 5.15, 4.51 , 3.67), and one methylene (6 2.12 / 2.17) (Figure 21 ). The above NMR data (Table 5) were highly identical to those of furaquinocin D, with the only difference being that the Me-8 substitution in furaquinocin D was replaced by a proton in compound 6 (Figure 1 ). This realization was further confirmed by an extensive 2D NMR analysis (Figures 21 -27), especially for the key HMBC correlations from H-8 (66.05) to C-6 (6 179.9) / C-9 (6 182.6) / 9a (6 107.9). Given the structure of compound 6, it was determined as a new member of the furaquinocins family and named as furaquinocin M (6, Figure 1 ).

[0095] Table 5.1H (400 MHz) and13C (100 MHz) NMR Data (6 ppm, in DMSO-d6) for furaquinocin

[0096] M (6)a

[0097] For heterologous expression of chlorostreptazone B1 (1 1 ) in SD3: P46 BGC13 encodes a marinopyrrole pathway that contains six putative halogenase genes. Although the S. sungeiensis SD3 host transformed with P46 BGC13 did not produce the anticipated marinopyrroles, the strain produced a new compound 1 1 that features a characteristic UV absorption similar to those of streptazones (Puder et al., 2000). The compound was isolated and characterized to establish its identify as a hitherto unreported compound. The molecular formula of compound 11 was shown to be C10H10CINO by HREIMS analysis. Its1H NMR spectrum (Figure 28) displayed one exchangeable proton (58.05), two olefinic protons (56.36 and 6.29), four aliphatic protons and one methyl protons (5 2.21 ). The13C NMR spectrum (Figure 28) of 11 indicated the presence of one C-methyl, two aliphatic methylenes, one hetero-substituted and two olefinic methines, one carbonyl, and three other quaternary carbons. On the basis of HMBC correlations (Figure 31), biosynthetic considerations, and comparison of its1H and13C NMR data (Table S6) with structurally related known streptazone B1 (Puder et al., 2000). Compound 1 1 was assigned to be a chlorinated derivative of streptazone B1 at carbon C-7 and named as chlorostreptazone B1 (1 1 , Figure 1 ).

[0098] Example 2. Selection of S. sungeiensis SD3 for chassis development

[0099] The use of fast-growing microbial chassis is crucial for efficient genetic modification and culturing work, particularly in the case of Streptomyces strains, which tend to grow slower than microbial chassis like E. coli. To identify potential chassis strains, a collection of actinomycete strains isolated from the soil and aquatic environments of Singapore was screened. The strains were evaluated based on their growth rate and sporulation on common media, including MS and GYM solid agar media, and TSB liquid media. After filtering out slow-growing or non-sporulating strains, twelve strains (including SD3) were identified with growth rates comparable to or higher than the model Streptomyces strains S. coelicolor M1 154 and S. lividans TK24 (Figure 2). The twelve strains exhibited readily observable sporulation on MS solid agar within 3-4 days, which is essential for long-term storage and DNA transformation via E. coli-Streptomyces conjugation.

[0100] For a microbial strain to serve as a reliable heterologous chassis in the production of secondary metabolites, it is imperative for the strain to be amenable to genetic manipulation and proficient in the uptake of exogenous DNA vectors. This ability to accept foreign genetic material is not only essential for introducing BGCs into the chassis but also crucial in strain improvement through targeted gene knock-ins and knock-outs. Considering that most wildtype or undomesticated Streptomyces spp. are inherently genetically recalcitrant and unresponsive to conventional transformation methods, genetic tractability stands out as a pivotal criterion when choosing a suitable heterologous chassis. As many DNA vectors employed for genetic manipulation of Streptomyces employed today harbour Aprr, CnT, or Kanrmarkers, the susceptibility to the three antibiotics is also an important requirement of a chassis. The antibiotic sensitivity of the twelve strains was assessed and it was found that ten of the twelve strains are susceptible to apramycin (50 pg / mL), chloramphenicol (25 pg / mL), and kanamycin (50 pg / mL) on solid agar media. The capability of the strains in accepting DNA vectors carrying large foreign DNA inserts was next evaluated. 14 pESAC13A-derived vectors derived from P1 -derived artificial chromosomes (PACs) obtained from BioS&T (https: / / www.biost.com / pesac_13-libraries) were tested (Sosio et al., 2000). These 14 PACs, which contain long DNA fragments in the range of 80 to 160 Kb, originated from the two genomic libraries constructed from the genomic DNA of Streptomyces tasikensis P46 (pE- SAC13A-P46-BGC1 / 13 / 15 / 23 / 28 / 30) and Streptomyces sp. SD50 (pESAC13A-P46- BGC7 / 18 / 23 / 32 / 38 / 44 / 45) (Table 1 ). Table 1. Plasmids used in this study.

[0101] By introducing the 14 PACs into the strains through E. coli-Streptomyces conjugation, it was found that only four of the ten strains displayed natural competence in accepting the PACs. Notably, S. sungeiensis SD3 exhibited the highest rate of successful conjugation with BGC- containing PACs, with 13 of the 14 PACs resulting in viable exconjugants as confirmed by positive PCR results (data not shown). Based on the number of exconjugants, the transformation efficiency of S. sungeiensis is comparable to the widely used host S. lividans TK24 (Table 2).

[0102] Table 2. Conjugation efficiency of S. sungeiensis SD3 and S. li

[0103] 5x108of ET12567(pR9604) donor cells containing pESAC13A-P46-BGC28 vector was used in intergeneric conjugation with 108of S. sungeiensis SD3 and S. lividans TK24 recipient spores. Exconjugant frequency was calculated by dividing the number of exconjugant colonies by the number of the recipient spores used. Values represent the mean frequencies from three independent experiments.

[0104] S. sungeiensis SD3 was subjected to further transformation experiments using a variety of DNA vectors, including some common plJ101 and pSET152-derived conjugative and integrative plasmids (e.g., plJ12551 , plJ8630, pSEVA28c1 ) used by academic researchers. These plasmids rely on the phiC31 integration system to target the con-served phiC31 -attB site present in many actin omycetes. All the plJ101 and pSET152-derived plasmids tested were successfully conjugated into S. sungeiensis SD3 and their integration into the chromosome was confirmed by PCR (data not shown). The observations suggest that S. sungeiensis SD3 can be readily transformed with the readily available conjugative and / or integrative plasmids, which are often employed for the heterologous expression of individual biosynthetic genes or entire BGCs.

[0105] S. sungeiensis SD3 was initially isolated from a sediment sample obtained from the mangrove swamp of Sungei Buloh Wetland Reserve of Singapore (Table 3). It was confirmed that S. sungeiensis SD3 displayed compatibility with International Streptomyces Project (ISP) media, specifically ISP2-7. No additional media requirements were needed to induce the sporulation of S. sungeiensis SD3, as it readily sporulated on MS media within a short period of 3-4 days. On the other hand, it generally takes a week for a typical model Streptomyces strain (e.g., S. lividans ref: Christian Ruckert et al., 2015) to sporulate. Some environmental strains even take 10-15 days to sporulate (e,g., S. tasikensis P46 ref. Guang-Lei Ma et al., 2022). Based on these promising outcomes, S. sungeiensis SD3 was further characterized and optimized as a heterologous chassis for the expression of exogenous secondary bio-synthetic pathways.

[0106] Table 3. Strains used in this study.

[0107] Example 3. Phylogenetic relationship and genomic features of S. sungeiensis SD3

[0108] To gain a better understanding of the primary and secondary metabolic capability of the strain, the whole genome of S. sungeiensis SD3 was sequenced and assembled using a hybrid approach combining Illumina and PacBio sequencing data. The complete genome had a total length of 10,292,314 bp and a guanine-cytosine content (GC) of 70.88% (Figure 3). To determine its closest phylogenetic neighbours, the whole genome sequence of S. sungeiensis SD3 was used to construct a Genome BLAST Distance Phylogeny (GBDP) tree using the Type Strain Genome Software (TYGS) (Meier-Kolthoff et al., 2019). Notably, S. sungeiensis SD3 seems to belong to a distinct clade separate from other commonly used Streptomyces chassis strains that include S. albus DSM 41398, S. coelicolor A3(2), S. lividans TK24, S. avermitilis NBRC 14893, S. venezuelae ATCC 21782, S. griseofuscus DSM 40191 , S. chattanoogensis, and S. atratus SCSIO ZH16 (Figure 4). S. sungeiensis SD3 forms a monophyletic lineage with S. chartreusis and other closely related strains. The OrthoANIu value obtained from the EzBioCloud database between S. sungeiensis SD3 and S. chartreusis is 84.03%, which meets the ANI threshold value for establishing a novel species or type strain (95%) (Hitch et al., 2021 ). Considering that the success rate of heterologous production of secondary metabolites usually increases with the phylogenetic proximity of the original producer and the heterologous host (Nepal et al., 2019; Myronovskyi et al., 2019), it was reasoned that S. sungeiensis SD3 could be utilized as a heterologous chassis to complement the existing Streptomyces chassis.

[0109] Genome annotation of S. sungeiensis SD3 using RAST resulted in the identification of 9,154 protein-coding ORFs and 90 tRNA genes (Aziz et al., 2008). A total of 1 ,589 genes were categorized into subsystems by RAST, with the highest relative abundance observed in genes involved in amino acid metabolism (29.8%) and carbohydrate metabolism (26.8%). To analyze genomic content differences, Ortho-Venn2 was employed to compare the annotated proteins of S. sungeiensis SD3 with those of S. coeli-color, S. avermitilis, S. albus, and S. venezuelae (Xu et al., 2019). A total of 6,754 orthologous putative proteins were identified in S. sungeiensis SD3, with 3,054 shared among the other four strains, representing the core proteins shared by the Streptomyces strains. S. sungeiensis SD3 shared the highest number of orthologous protein clusters with S. avermitilis (545), followed by S. coelicolor (361 ), S. venezuelae (239), and S. albus (1 1 1 ), reflecting their phylogenetic relatedness (Figure 5). 75 protein clusters are unique to S. sungeiensis SD3 and are not found in the other four strains, while only 62, 56, 54, and 49 clusters are unique to S. coelicolor, S. venezue-lae, S. avermitilis, and S. albus, respectively. The 75 unique protein clusters are primarily involved in various metabolic processes, including amino acid and carbohydrate metabolism, with the largest gene ontology group associated with DNA binding and regulation (14.28%). S. sungeiensis SD3 possesses more enzymes involved in the metabolism of aromatic compounds such as benzoate (75) compared to S. coelicolor (42) and S. avermitilis (38). Another unique group of proteins found in S. sungeiensis SD3 are involved in the biosynthesis of pyrroloquinoline quinone (8%), which is an organic cofactor essential for bacterial dehydrogenases, including methanol dehydrogenases (Matsutani et al., 2018).

[0110] Example 4. The biosynthetic capability of S. sungeiensis SD3 To assess the capacity of S. sungeiensis SD3 in producing secondary metabolites, we mined its genome for BGCs using antiSMASH 7.0 (Blin et aL, 2023). The mining yielded at least 37 BGCs, including nine coding for Type I, II, or III polyketide synthases (PKS), and seven coding for non-ribosomal peptide synthetases (NRPS) (Table 4). Among the 37 BGCs, several BGCs bear resemblance to characterized BGCs from other strains in terms of gene composition and sequence homology. These putative homologous BGCs are anticipated to produce secondary metabolites that are either identical or structurally similar to coelichelin, ectoine, ishi-gamide, albaflavenone, focixins, chartreusin, lagunapyrone A, desferrioxamine, streptazone E, and isocomplestatin (Table 4). Interestingly, although BGC24 shares similarity with the BGC for streptazone E biosynthesis in Streptomyces sp. MSC090213JE0829, the modular PKS encoded by the BGC only contain five modules, in contrast to the six modules encoded by the streptazone E BGC, indicating a potential difference in carbon-chain length between the products of the two BGCs. Meanwhile, several BGCs are predicted to synthesize unknown secondary metabolites with novel polyketide or non-ribosomal peptide backbone. The number and diversity of BGCs found in the genome suggests S. sungeiensis SD3 possesses the metabolic networks and pathways to support the synthesis of diverse secondary metabolites.

[0111] Table 4. BGCs predicted by AntiSMASH analysis.

[0112]

[0113] S. sungeiensis SD3 was cultured in 27 different culture media, including 8 liquid and 19 solid media to substantiate the biosynthetic potential and identify the constitutively active BGCs. Following the fermentation, the organic metabolites were extracted from the culture broth and mycelium. The metabolites were subsequently profiled using liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS). Aided by an in-house natural product library and characterization by NMR spectroscopy, two major secondary metabolites produced by S. sungeiensis SD3 that include obscurolide A2 (1 ) ([M + H]+, m / z 276.08) and chartreusin ([M + Na]+, m / z 663.25) (2) were identified (Figures 6, 7 and 8) (Hoff et al., 1992). Obscurolide is a putative butyrolactone derivative that was produced in abundance under several fermentation conditions. The other major product chartreusin is a glycosylated aromatic polyketide known for its dsDNA-binding and cleaving activity (Xu et al., 2005), which is being studied as a DNA-damaging chemotherapy agent. The production of chartreusin agrees with the presence of the Type II PKS-encoding BGC33 that is highly similar to the chartreusin BGC found in S. chartreusis HKI-249.

[0114] S. sungeiensis SD3 also produced streptazone B1 ([M + H]+, m / z 162.08) (3) under several fermentation conditions. Streptazone B1 is a piperidine alkaloid that features a densely packed functionalised scaffold and unknown biosynthetic pathway (Puder et al., 2000) (Figures 6, 9), and is considered a potent anticancer compound. As noted earlier, S. sungeiensis SD3 contains a Type-I modular PKS-encoding BGC (BGC24, Table 4) that shares homology with the BGCs that produces streptazone E and other polyketide-derived piperidine alkaloids (Ohno et al., 2015; Puder et al., 2000; Warmer et al., 2022). However, BGC24 encodes a five- module PKS system, in contrast to the six-module PKS system for streptazone E biosynthesis (Figure 10). With BGC24 assigned as the putative BGC for streptazone B1 biosynthesis, it was surprisingly found that streptazone B1 and streptazone E are produced by similar but different pathways. S. sungeiensis SD3 produced two highly hydrophobic compounds (4 and 5) that share the same mass ([M + H]+, m / z 321.28) after the strain was transformed with a P1 -derived artificial chromosome (PAC) that contains an exogenous DNA fragment (P46_BGC23). The two compounds were found to be adipostatins A (4) and B (5) by HRMS and NMR spectroscopy (Figures 6, 1 1 A, 12, and 13). Adipostatins are alkylresorcinols or Type III PKS-generated phenolic lipids with modest antifilarial activity against the nematode parasite Brugia malayi that causes elephantiasis (Rateb et al., 2015). Considering that the exogenous BGC does not contain any Type III PKS genes, the production of adipostatin A and B is likely due to the inadvertent activation of the native alkylresorcinols BGC37b.

[0115] The results from antiSMASH mining analysis underscore the ability of S. sungeiensis SD3 to synthesize a wide array of secondary metabolites, supporting that the genetic makeup of the chassis encompasses a robust metabolic network conducive to the heterologous production of secondary metabolites. Considering that only a small fraction of the predicted secondary metabolites has been identified by the metabolite profiling, many of the endogenous BGCs are likely to be silent under laboratory fermentation conditions. The discrepancy between the large number of secondary metabolites suggested by antiSMASH analysis and the small number of metabolites detected suggests that the production of secondary metabolites is tightly regulated in S. sungeiensis SD3.

[0116] Example 5. Gene expression profile of S. sungeiensis SD3

[0117] Transcriptomic profiling was performed by RNA sequencing to gain a better understanding of the metabolic activity and gene expression pattern of S. sungeiensis SD3. Based on the timing of the production of chartreusin and streptazone B1 , RNA sequencing was performed at two time points: pre-BGC expression (12 hours) and at the on-set of BGC expression (96 hours). Total RNA was extracted from biological triplicates at these two time points and subjected to RNA-seq analysis. Analysis of the RNA-Seq results revealed that at 96 hours, 22 out of 41 BGCs exhibited upregulation of expression for at least 50% for the core biosynthetic genes (log2FC > 1 , P < 0.05, DESeq2) (Figures 14 and 15). The highest log2FC between 12 and 96 hours was observed for the chartreusin BGC (BGC33, 10.8-1 1.5 for PEG 8216-8217) and BGC37a (1 1.1 -12.3 for PEG 8652-8655). Interestingly, comparison of the 96 hr reads shows comparable transcriptional activity for BGC33 and the BGC37a that is predicted to produce a non-ribosome peptide that resembles isocomplestatin. However, the anticipated product of BGC37a could not be detected by HPLC and LC / MS-aided metabolite profiling, suggesting the BGC remains silent likely because of post-transcriptional regulation. While for the two cryptic BGC32a and 32b that are predicted to produce novel polyketide natural products, the low gene expression levels suggest that they are silent BGCs because of repressed gene expression.

[0118] The main metabolic pathways that support the production of the aromatic polyketide chartreusin were further investigated to understand why the fermentation titer (~ 2.7 mg / L) for chartreusin was high. Glucose and maltose served as the primary carbon sources in the GYM liquid media used for chartreusin production and RNA isolation.

[0119] Analysis of the glycolysis pathway revealed downregulation of the majority of genes associated with the glycolysis pathway and the citric acid cycle (log2FC < 1 , P < 0.05, DESeq2), indicating a slowdown of metabolism and growth at 96hr. In contrast, the majority of genes involved in the conversion of pyruvate to acetate (1 1 out of 19 genes) and the genes that convert acetate to acetyl-CoA and malonyl-CoA were upregulated. Moreover, 34 out of 67 genes from the beta-oxidation pathway produces acetyl-CoA also showed upregulation at 96 hr. In addition to acetyl-CoA and malonyl-CoA, monosaccharides that include D-fucose and D-digitalose are also required as the precursors for chartreusin biosynthesis (Xu et aL, 2005). The majority of associated genes involved in D-glucose-1 P to dTDP-L-rhamnose production were upregulated at 96 hours (Figure 16). The observations suggest that the high fermentation titer (2.7 mg / L) of chartreusin is likely due to the synergized result of the high-level expression of chartreusin biosynthetic genes and the upregulation of the primary metabolic genes for acetyl-CoA and monosaccharide production.

[0120] Example 6. Assessment of the strength of orthogonal promoters in S. sungeiensis SD3

[0121] For heterologous production of secondary metabolites, constitutively active promoters are often required to activate gene expression. To identify orthogonal promoters that are functional in S. sungeiensis SD3, we assessed the strength of several widely used promoters that were shown to function in multiple actinomycete strains. The chosen promoters sf14p, KasOp*, gapdhp(EL), gapdhp(KR) were placed upstream of the engineered green fluorescent protein (EGFP) reporter gene in the plJ8630 vector (Shao et al., 2013; Sun et al., 1999; Wang et al., 2013). The promoter-containing plasmids were conjugated into S. sungeiensis SD3 and the EGFP fluorescence intensity of the culture broth was used as a measure of the promoter strength. By measuring the fluorescence intensity of the cells, it was found that KasOp*, gapdhp(EL), gapdhp(KR) induced the highest EGFP expression, whereas SF14p induced moderate EGFP expression (Figure 17A). As the background fluorescence of the Streptomyces may interfere with the measurement of fluorescence intensity, quantitative real time-PCR (qRT-PCR) experiments was also performed to confirm that the mRNA levels of the egfp gene under the control of KasOp*, gapdhp(EL), gapdhp(KR) are significantly elevated; whereas the one under the control of SF14p only showed moderate increase in mRNA level (Figure 17B).

[0122] Example 7. S. sungeiensis SD3 is amenable to CRISPR / Cas9-based genome editing

[0123] Strain optimization is often required to improve the performance of microbial heterologous hosts. Aside from the UV light or chemical induced random mutagenesis employed for the improvement of industrial strains, gene-targeted genome editing has become an increasing attractive approach in strain improvement. For example, the removal of competing biosynthetic pathways by genetic approaches in S. lividans, S. avermitilis, and S. albus has resulted in cleaner metabolite background and improved fermentation yield (Ahmed et al., 2020; Myronovskyi et al., 2018; Hwang et al., 2023. The emergence of CRISPR / Cas9-assisted methods as efficient genome editing tools for actinomycetes continues to advocate the effectiveness of targeted genome editing (Tao et al., 2018).

[0124] Two Cas9-expressing vectors were tested, including the pSG5-based pCRISPR-Cas9, and the plJ101 -based pQS-idgS (Tong et al., 2015; Wang et al., 2020), for gene deletion and insertion in S. sungeiensis SD3. The conjugation of pCRISPR-Cas9 into S. sungeiensis SD3 was not successful after repeated attempts, whereas the conjugation of pQS-idgS into S. sungeiensis SD3 yielded exconjugants. The observations seem to suggest that the temperature sensitive pSG5 replicon in pCRISPR-Cas9 is incompatible with S. sungeiensis SD3. A similar observation was reported earlier for Saccharopolyspora erythraea, and the authors proposed that the pSG5 replicon can cause unpredicted gene recombination and instability (Mo et al., 2019). Wang et al. also observed similar issues, which led to their use of the plJ101 replicon for the construction of the pQS-idgS (Wang et al., 2020).

[0125] The utility of pQS-idgS for gene deletion and promoter insertion in S. sungeiensis SD3 was validated. The first pks gene (ctg1 8216) of the silent cryptic BGC32b was chosen to target by deleting a fragment comprising the first 1 ,323 bp of the pks gene (Figure 18A). The pQS- idgS-based plasmid was also constructed to insert the KasOp* promoter in front of ctg1_8216 to test whether the constitutively active KasOp* can activate the expression of the BGC to produce its unknown metabolite (Figure 18B). Another plasmid was constructed to target the beta-ketoacyl synthase genes chaA and chaB within the chartreusin BGC to disrupt the production of chartreusin while inserting a PhiC31 attB attachment site and a sfp gene (Figure 18C). Disrupting chaA and chaB would abolish chartreusin production, which will not only eliminate the chartreusin-associated HPLC peaks to facilitate the detection of metabolites produced by heterologous biosynthetic pathways but also free up malonyl-CoA and NADPH to boost fermentation titer. The additional attB site is expected to allow the integration of an additional copy of exogenous BGC in the chromosome; whereas the insertion of the sfp gene that codes for the promiscuous Sfp 4’- Phosphopantetheinyl transferase may lead to the activation of silent BGCs (Zhang et al., 2017). Upon the conjugation of the pQS-idgS-based plasmids that harbor the corresponding sgRNA and homology arms into S. sungeiensis SD3, exconjugants were readily observed. After the curing of the plasmid following the procedure described by Zhang and co-workers (Wang et al., 2020), the successful deletion of the pks gene fragment and insertion of the KasOp* promoter were confirmed by PCR and DNA sequencing (Figure 19). The deletion of chaA and chaB genes was successful as well, which was further confirmed by the observation that the mutant strain no longer produced chartreusin (Figure 20).

[0126] While the intended gene deletion and promoter or gene insertions were successful, the whole genome sequencing of one of the mutant strains unearthed an unexpectedly extensive deletion for one of the chaAB deletion clones. The deletion resulted in the loss of a 240.6 kb DNA fragment, stretching 206,425 bp upstream of chaA and 30,843 bp downstream of chaB. As a result, the entirety of BGC33 (chartreusin BGC) was eradicated while half of the upstream BGC32a / b was also lost. It is worth noting that while the large deletion was unexpected, the experiment proved efficacious in disrupting chartreusin production and resulting in a notable genome reduction of over 200 Kb. The genome reduction did not elicit any noticeable variances in growth and sporulation rate or morphology.

[0127] Taken together, the outcomes from the gene deletion and promoter insertion experiments underscored the compatibility of S. sungeiensis SD3 with CRISPR-Cas9-based genome editing techniques. The unexpected large deletion is not an uncommon phenomenon as it has been observed by the inventors as well as other research labs (Hoff et al., 2018). The observations reinforce the view that judicious selection of the PAM and Cas9-cutting sites is needed to avert unintended instances of homologous recombination events in S. sungeiensis SD3 and other Streptomyces strains.

[0128] Example 8. Heterologous production of secondary metabolites using S. sungeiensis SD3 To establish S. sungeiensis SD3 as a useful microbial chassis for expressing heterologous BGCs from closely related phylogenetic strains, the expression of BGCs from the S. tasikensis P46 strain was tested. While sharing a close phylogenetic relationship with S. sungeiensis SD3 (Figure 4), S. tasikensis P46 displays a much slower growth rate, typically requiring more than 14 days to reach maturity on agar plates, in contrast to the rapid five-day growth cycle of S. sungeiensis SD3 (Ma et al., 2022). Based on the inventors’ experience, the production of secondary metabolites, including tasikamides, furaquinocins, and pentalenolactones, are notoriously inconsistent in S. tasikensis P46. S. tasikensis P46 is also difficult to manipulate genetically and not amenable to CRISPR / Cas9 editing (such as pCRISPR-Cas9 and pQS- idgS) due to the toxicity of Cas9 for the strain (Ma et al., 2022; Candra et al., 2022). Hence, if the expression of the BGCs of S. tasikensis P46 in S. sungeiensis SD3 is successful, it will not only enable the production of the secondary metabolites in a less erratic manner but also offer the opportunity to activate the novel cryptic BGCs of S. tasikensis P46 by heterologous expression.

[0129] A genomic DNA library of S. tasikensis P46 constructed using the P1 -derived artificial chromosome (PAC) vector pESAC13A from BioS&T (Canada) was obtained. From the genomic library, six clones that contain the BGCs predicted to produce polyketides, non- ribosomal peptides, terpenoids, and ribosomal synthesized and post-translationally modified peptides (RiPPs) were obtained (i.e., P46_BCG1 , BCG13, BCG15, BCG23, BCG28, BCG30). The six BGC-harbouring pESAC13A plasmids were subsequently integrated into the chromosome of S. sungeiensis SD3 via E. coli-Streptomyces conjugation. The transformed S. sungeiensis SD3 strains were cultivated in various liquid and solid fermentation cultures for the production of secondary metabolites. Among the six transformed strains, S. sungeiensis SD3::P46_BGC23 and S. sungeiensis SD3::P46_BGC28 were found to produce compounds that were not observed for the S. sungeiensis SD3 control strain transformed with empty pESAC13A (Figure 11 A).

[0130] P46_BGC28 encodes a pathway predicted to produce the polyketide-terpene hybrid products known as furaquinocins. This pathway involves a prenyltransferase that introduces an isoprene unit and a Type III PKS that generates the 1 ,3,6,8-tetrahydroxynaphthalene (THN) skeleton through the condensation of five units of malonyl-CoA (Kumano et al., 2010). Analysis of the HPLC chromatogram from the S. sungeiensis SD3::BGC28 strain revealed the presence of a new compound (m / z 372.1573) in the culture broth when cultured in GYMose liquid medium. Subsequent isolation and structural elucidation confirmed that it is a novel furaquinocin and herein named as furaquinocin M (6) (Figures 1 , 11 A, and 21 -27, and Table 5). Generally, furaquinocins are known to display a broad-spectrum antibacterial activity. P46_BGC23 codes for an NPRS pathway that produces the cyclic peptides tasikamides. HPLC analysis of the fermentation products of the S. sungeiensis SD3::BGC23 strain revealed the presence of a group of metabolites that exhibit similar UV-Vis spectra, molecular masses ([M + H]+, m / z 809.38, 841.41 , 855.38, and 857.40), and retention times as tasikamides D-G (7-10) (Ma et al., 2022). Tasikamides D-G represent a unique family of non-ribosomal peptides characterized by the presence of methylated and hydroxylated alpha and beta amino acid building blocks (Ma et aL, 2022). The production of compounds 7-10, in conjunction with the presence of several NRPS-encoding BGCs in the S. sungeiensis SD3 genome, suggests S. sungeiensis SD3 could be exploited as a robust chassis to produce non-ribosomal peptides. It is worth noting that the production of tasikamides in S. sungeiensis SD3 only required five days in GYMose liquid medium (versus 15 days for S. tasikensis P46 on solid medium), and that S. sungeiensis SD3 produces 7-10 in higher fermentation titer (2 to 4-fold by estimation; Figure 34) than S. tasikensis P46, highlighting its advantages for the production of tasikamides and potentially other NRPS peptides.

[0131] Example 9. Application of S. sungeiensis SD3 in enzyme discovery

[0132] The functionality of numerous biosynthetic enzymes continues to elude comprehension, primarily owing to the formidable challenges associated with the production of fully functional recombinant proteins. This predicament is particularly pronounced when dealing with enzymes sourced from actinomycetes, as their expression in conventional microbial cell hosts like E. coli and B. subtilis poses significant hurdles. Recognizing the potential inherent in S. sungeiensis SD3, characterized by its rapid growth and genetic amenability, a novel application for this strain was conceived - the expression of individual enzymes for the purpose of elucidating their substrate specificity through cell-based assays.

[0133] As a preliminary demonstration of this concept, a transformative experiment wherein S. sungeiensis SD3 was genetically modified using a P1 -derived artificial chromosome (PAC) carrying six distinct halogenase genes sourced from the marinopyrrole pathway of S. tasikensis P46 was initiated. Specifically, P46_BGC13 codes for a putative marinopyrrole pathway that contains six potential flavin monooxygenase-type halogenases. The six putative halogenases share sequence homology with some of the characterized flavo-halogenases from the pyrrolomycin and marinopyrrole pathways (Flatt et al., 2013; Zhang et al., 2007; Yamanaka et aL, 2012; El Gamal et a., 2016). Although the S. sungeiensis SD3 host transformed with P46_BGC13 did not produce the anticipated marinopyrroles, the strain produced a new compound consistently when compared to the control strain when cultured in oatmeal liquid medium (Figure 1 1 B). The compound was isolated and characterized to establish its identify as a hitherto unreported compound, chlorostreptazone B1 (11 , [M + H]+, m / z 196.08). The structure of 11 was unambiguously established by 1 D and 2D NMR spectroscopic studies (Figures 1 and 28-32, and Table 6).

[0134] Table 6.1H (400 MHz) and13C (100 MHz) NMR data (6 in ppm, in DMSO-cfc) for chlorostreptazone B1 (11 )a

[0135] Unlike S. sungeiensis SD3, the S. sungeiensis SD3::P46_BGC13 strain produced negligible amount of streptazone B1 . It was reasoned that one of the six halogenases encoded by P46_BGC13 was expressed in S. sungeiensis SD3, resulting in the cross-chlorination of streptazone B1 produced by one of the endogenous pathways (Figure 33). This unexpected finding suggests that some of the biosynthetic enzymes from the BGC13 were expressed in S. sungeiensis SD3, and that the halogenase encoded by P46 BGC13 can chlorinate the nonnative substrate streptazone B1 likely because of relaxed substrate selectivity. Further, it underscored the capability of S. sungeiensis SD3 as a valuable tool for the systematic screening of enzyme substrates through the utilization of live-cell or cell lysate-based enzymatic assays.

[0136] Summary

[0137] S. sungeiensis SD3 was identified as a promising microbial chassis for the heterologous production of secondary metabolites. This strain possesses several advantageous characteristics that make it well-suited for expressing heterologous biosynthetic pathways.

[0138] (a) S. sungeiensis SD3 occupies a unique position within a distinct branch on the phylogenetic tree, distinguishing it from previously developed Streptomyces chassis strains. This phylogenetic classification designates S. sungeiensis SD3 as the preferred chassis for expressing BGCs originating from Streptomyces strains within the same phylogenetic clade. The use of closely related chassis could enhance the likelihood of successful expression and production of secondary metabolites, owing to the shared cellular environment and metabolic capabilities with the native producer.

[0139] (b) S. sungeiensis SD3 harbours primary metabolic networks that support the synthesis of polyketides and non-ribosomal peptides, two classes of natural products renowned for their diverse biological activities and promising therapeutic applications. Based on the strain's inherent metabolic capabilities, S. sungeiensis SD3 can be exploited as a versatile chassis for the heterologous production of the polyketides and non-ribosomal peptides.

[0140] (c) S. sungeiensis SD3 also demonstrates remarkable genetic tractability, which means it can be easily manipulated and modified genetically. This trait enables researchers to improve the strain and optimize its performance through iterative cycles of Design-Build-Test-Learn (DBTL). By introducing genetic modifications, researchers can enhance the expression of BGCs, increase production yields, and improve the strain's overall performance.

[0141] (d) The fast growth rate of S. sungeiensis SD3 is advantageous for experimental work. Rapid growth allows for shorter cultivation times and faster generation of results. Additionally, the strain's susceptibility to antibiotics can be beneficial in experimental setups where selective pressure or antibiotic-based selection is required.

[0142] The successful production of the polyketide-terpenoid compound 6 and NRPS peptides 7-10 demonstrates the versatility of S. sungeiensis SD3 as a microbial chassis for expressing BGCs from closely related strains. The expedited fermentation and heightened fermentation titer further highlight the advantages associated with employing S. sungeiensis SD3 as a heterologous expression host. It is noteworthy that, among the six tested BGCs, four failed to produce in both the native producer (P46) and the host (SD3), indicating that silent BGCs in the native producer are likely to remain as inactive in a heterologous host. Consequently, the activation of these silent BGCs through genetic or non-genetic means remains imperative for the heterologous production of secondary metabolites. Unlike many undomesticated wild-type strains, the outstanding genetic tractability of S. sungeiensis SD3 will enable the genetic engineering of silent BGCs within the host, facilitating their activation. The availability of genome-editing tools further renders S. sungeiensis SD3 amenable to metabolite engineering, boosting precursor and cofactor supplies to enhance fermentation titers. The accessibility of its comprehensive genome sequence and transcriptomic profile paves the way for further strain enhancement through genome editing. It is envisioned that optimizing S. sungeiensis SD3 via genome editing will result in specialized mutant strains tailored for the efficient production of diverse classes of secondary metabolites. The removal of the chartreusin BGC and the adjacent 200kb region has already yielded the S. sungeiensis SD3_A240kb strain that is characterized by genome reduction and a cleaner HPLC background. Concurrently, the introduction of the sfp gene yielded the S. sungeiensis SD3_AchaAB::sfp strain that may facilitate the activation of silent BGCs reliant on post- translational modification through phosphopantetheinylation (Zhang et al., 2017). These two mutant strains will serve as promising starting points for the development of customized chassis tailored for the production of polyketide natural products.

[0143] In conclusion, these findings underscore the potential of S. sungeiensis SD3 as a promising microbial chassis for heterologous expression of secondary biosynthetic pathways. The capacity to express diverse biosynthetic pathways, coupled with its genetic tractability, unique phylogenetic position, fast growth rate, and susceptibility to antibiotics, positions S. sungeiensis SD3 as a valuable and complementary chassis within the Streptomyces chassis repertoire for uncovering novel natural products and producing commercially valuable compounds. Ongoing optimization endeavours, encompassing both genetic and non-genetic strategies, aim to generate specialized mutant strains tailored for the production of distinct natural product classes. This optimization process also seeks to streamline metabolic networks, ultimately achieving higher fermentation titers essential for industrial applications.

[0144] While the subject matter of this disclosure has been described and shown in considerable detail with reference to certain illustrative aspects, including various combinations and subcombinations of features, those skilled in the art will readily appreciate other aspects and variations and modifications thereof as encompassed within the scope of the present disclosure. Moreover, the descriptions of such aspects, combinations, and sub-combinations is not intended to convey that the claimed subject matter requires features or combinations of features other than those expressly recited in the claims. Accordingly, the scope of this disclosure is intended to include all modifications and variations encompassed within the spirit and scope of the following appended claims. Bibliography

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Claims

Claims1. An isolated S. sungeiensis SD3 strain deposited at the Leibniz-lnstitut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) under accession number DSM34917.

2. An engineered S. sungeiensis SD3 strain comprising a heterologous gene encoding a secondary metabolite or a biosynthetic gene cluster (BGC) for producing a secondary metabolite, wherein the engineered S. sungeiensis SD3 strain is derived from an isolated S. sungeiensis SD3 strain deposited at the Leibniz-lnstitut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) under accession number DSM34917.

3. The engineered S. sungeiensis SD3 strain of claim 2, wherein the secondary metabolite is selected from a group consisting of an antibiotic, antifungal, antitumor agent, immunosuppressant, enzyme inhibitor, herbicide, insecticide, non-ribosomal peptide and combinations thereof.

4. The engineered S. sungeiensis SD3 strain of claim 2, wherein the secondary metabolite is selected from a group consisting of polyketide, terpene, terpenoid, alkaloid, alkylresorcinol, and combinations thereof.

5. The engineered S. sungeiensis SD3 strain of claim 2, wherein the secondary metabolite is selected from a group consisting of streptomycin, tetracycline, erythromycin, nystatin, doxorubicin, mitomycin, tacrolimus, rapamycin, clavulanic acid, obscurolide A2, chartreusin, strptazone B1 , adipostatin A, adipostatin B, furaquinocin M, tasikamide D, tasikamide E, tasikamide F, tasikamide G, chlorostrptazone B1 , and combinations thereof.

6. The engineered S. sungeiensis SD3 strain of any one of claims 2-5, further comprising a promoter selected from a group consisting of sf 14p, KasOp*, gapdhp(EL), and gapdhp(KR), wherein the promoter is operably linked to the heterologous gene.

7. The engineered S. sungeiensis SD3 strain of any one of claims 2-6, wherein said strain further comprises a heterologous sfp gene.

8. The engineered S. sungeiensis SD3 strain of claim 2, wherein said strain does not comprise a BGC for producing chartreusin.

9. A method for producing one or more secondary metabolites, comprising culturing the isolated S. sungeiensis SD3 strain of claim 1 or the engineered S. sungeiensis SD3 strain of any one of claims 2-8 in a culture medium under suitable conditions for a period of time, and recovering the secondary metabolites from the culture medium.

10. The method of claim 9, wherein the culture medium is a solid medium.

11. The method of claim 9, wherein the culture medium is a liquid medium.

12. The method of any one of claims 9-11 , wherein the secondary metabolite is selected from a group consisting of an antibiotic, antifungal, antitumor agent, immunosuppressant, enzyme inhibitor, herbicide, insecticide, non-ribosomal peptide and combinations thereof.

13. The method of any one of claims 9-11 , wherein the secondary metabolite is selected from a group consisting of polyketide, terpene, terpenoid, alkaloid, alkylresorcinol, and combinations thereof.

14. The method of any one of claims 9-11 , wherein the secondary metabolite is selected from a group consisting of streptomycin, tetracycline, erythromycin, nystatin, doxorubicin, mitomycin, tacrolimus, rapamycin, clavulanic acid, obscurolide A2, chartreusin, strptazone B1, adipostatin A, adipostatin B, furaquinocin M, tasikamide D, tasikamide E, tasikamide F, tasikamide G, chlorostrptazone B1 , and combinations thereof.

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