Biosynthesis of plecomacrolide compounds

Abstract

Provided herein are engineered Streptomyces bacteria, and methods of using Streptomyces bacteria to produce Concanamycin A and Concanamycin B. The engineered Streptomyces bacteria overexpress one or more homologous regulator of the Concanamycin biosynthetic gene cluster (BGC), and express one or more heterologous regulator of the Concanamycin BGC. Further provided are optimized culture conditions for inducing Concanamycin A and Concanamycin B production, which can be used to culture Streptomyces bacteria or engineered Streptomyces bacteria.

Description

Docket No. 30275 / 2024-545BIOSYNTHESIS OF PLECOMACROLIDE COMPOUNDSFIELD

[0001] The disclosure relates to methods of increasing Concanamycin production in Streptomyces bacteria, including engineering increased expression of biosynthetic gene cluster regulators in the Streptomyces bacteria and optimizing culture conditions.STATEMENT OF GOVERNMENT INTEREST

[0002] This invention was made with government support under grant no. R01 Al 148383, awarded by the National Institutes of Health. The government has certain rights in the invention.INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

[0003] Incorporation by reference in its entirety is a sequence listing in computer-readable form submitted concurrently herewith and identified as follows: ASCII text file named “2024-545_SeqListing.xml”, 14,115 bytes, created September 17, 2025.BACKGROUND

[0004] Natural products, also referred to as secondary or specialized metabolites, are a valuable source of biologically active molecules and act as important scaffolds to develop new drug leads1. Under laboratory conditions, these compounds are not essential for the growth, development, or reproduction of an organism, but they can often play a vital role in their ecological interactions2. Microorganisms are a significant source of natural products with diverse therapeutic applications, such as antibiotics, anticancer agents, antivirals and immunomodulators. Bacterial survival, resilience, and proliferation across diverse environmental conditions rely on a vast transcriptional regulatory network that triggers metabolic adjustments to adapt rapidly to new environmental conditions. In Streptomyces spp., these processes are coupled with the production of a wide range of pharmaceutically important secondary metabolites. Strains of the genus Streptomyces are among the most prolific microbial sources of bioactive natural products with complex chemical signatures. Even though these organisms are often challenging to culture and engineer, their biochemical potential remains of significant academic and industrial interest34.

[0005] Streptomyces are ubiquitous gram-positive soil bacteria belonging to the order Actinomycetales. One of the most striking features of genus Streptomyces is their ability to produce a wide variety of natural products. Over 50,000 Streptomyces-pmduced natural products have been described, and of more than 12,000 antibiotics used in medicine and agriculture, approximately 55% are derived from Streptomyces species. Traditional methods for increasing production titers of secondary metabolites via fermentation have involved "random mutation and screening” procedures, which requires a considerable amount of time and resources.

[0006] Plecomacrolides are natural products produced by Streptomyces, a class of macrolactone antibiotics that inhibit the evolutionarily conserved vacuolar (V)-ATPase. Concanamycins are a type of 18-memberedDocket No. 30275 / 2024-545 plecomacrolides that have shown great potential as treatments for multiple diseases, including HIV, cancer, and osteoporosis. Due to the complexity of their total synthesis, production of this class of natural products is commercially achieved through microbial fermentation. Yet, the low titers produced by the native isolates are a significant bottleneck for robust structure-activity relationship studies and drug development. Thus, there is a need for the development of metabolic engineering approaches that manipulate secondary metabolite production in bacteria, such as Streptomyces, and thereby improve fermentation titers of natural products.SUMMARY

[0007] Disclosed herein are materials and methods for enhancing Concanamycin production, such as Concanamycin A and Concanamycin B production, in Streptomyces bacteria. The disclosure provides for engineered Streptomyces bacteria that are to be used in the methods for enhanced Concanamycin production. The present disclosure demonstrates that heterologous overexpression of the target regulatory genes in Streptomyces strains which harbor the concanamycin biosynthetic gene cluster (BGC), significantly improved Concanamycin A production and may be used in the disclosed methods. In some embodiments, homologous regulators of the Concanamycin biosynthetic gene cluster (BGC) are overexpressed in an engineered Streptomyces bacteria, heterologous regulators of the Concanamycin BGC are expressed in an engineered Streptomyces bacteria, or a combination thereof. In some embodiments, bacterial culture conditions are optimized to improve Concanamycin production by a Streptomyces bacteria or an engineered Streptomyces bacteria.

[0008] Disclosed herein are various designs of high Concanamycin A- and Concanamycin B-producing Streptomyces strains through rational metabolic engineering approaches. Strain optimization included overexpression of two cms pathway specific regulators, one heterologous regulator, and media optimization, which significantly enhanced the production of Concanamycin A and Concanamycin B (>1000 fold, compared to wild-type under non-optimized cultivation conditions). For example, an engineered strain referred to as DHS10676 (see Table 1) produced the highest concanamycin A titer reported to date (909.8±64.7 mg / L). Supplementation with 0.6% (w / v) of sodium propionate at 48h boosted concanamycin B production to 306.5±42.05 mg / L, resulted in a 7.8-fold improvement compared to non-supplemented cultivation conditions. Subsequent efforts to improve the isolation and purification of concanamycin A and B from crude extracts enabled the isolation of 483.1±25.8 mg / L and 159.4±41.8 mg / L (>90% purity), respectively. Proteomics anaiysis demonstrated a significant increase in abundance of biosynthetic proteins from the cms BGC in the engineered mutant DHS10676 compared to wiid-type. Untargeted metaboiomics anaiysis confirmed the metabolic shift between the wild-type and engineered DHS10676 strains. These improvements were demonstrated in strains other than S. eitanensis, such as S. griseiscabiei, S. steiiiscabiei, and S. neyagawaensis, by heterologous expression of S. lohii bafR regulator and the S. eitanensis cms BGC situated-regulators (see, e.g. Fig. 7D in Example 4).Docket No. 30275 / 2024-545

[0009] Bafilomycin cluster-situated HTH-DNA binding (BafR) protein a 16-membered ring plecomacrolide produced by Streptomyces lohii that is structurally related to concanamycin A, but bears a smaller macrolactone ring, and lacks the carbamoylated deoxyrhamnose sugar. Bafilomycin Ai and concanamycin A directly bind and inhibit the eukaryotic V-ATPase5. In some embodiments, the disclosure provides for an engineered Streptomyces bacteria having increased expression of the BafR protein, wherein the wild-type variant of the Streptomyces bacteria does not express the BafR protein. In various embodiments, the engineered Streptomyces bacteria expresses a BafR protein comprising: I) an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 1, ii) the amino acid sequence of SEQ ID NO: 1, ill) an amino acid sequence that is encoded by a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identity to SEQ ID NO: 4, or, iv) an amino acid sequence that is encoded by a nucleotide sequence of SEQ ID NO: 4. In various embodiments, the engineered Streptomyces bacteria comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 4, or comprises the nucleotide sequence of SEQ ID NO: 4.

[0010] In some embodiments, the engineered Streptomyces bacteria having increased expression of the BafR protein disclosed herein also has increased expression of the CmsG protein. In various embodiments, any of the disclosed engineered Streptomyces bacteria express a CmsG protein comprising: I) an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 2, ii) the amino acid sequence of SEQ ID NO: 2, ill) an amino acid sequence that is encoded by a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 5, or, iv) an amino acid sequence that is encoded by a nucleotide sequence of SEQ ID NO: 5. In various embodiments, the engineered Streptomyces bacteria further comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 5, or comprises the nucleotide sequence of SEQ ID NO: 5.

[0011] In some embodiments, the engineered Streptomyces bacteria having increased expression of the BafR protein and the CmsG protein also has increased expression of the CmsR protein. In various embodiments, any of the disclosed engineered Streptomyces bacteria express a the CmsR protein comprising: I) an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95%% identical to SEQ ID NO: 3, ii) the amino acid sequence of SEQ ID NO: 3, ill) an amino acid sequence that is encoded by a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 6, or, iv) an amino acid sequence that is encoded by a nucleotide sequence of SEQ ID NO: 6. In various embodiments, the engineered Streptomyces bacteria further comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 6, or comprises the nucleotide sequence of SEQ ID NO: 6.

[0012] In some embodiments, the engineered Streptomyces bacteria has increased expression of the BafR protein, the CmsG protein, and the CmsR protein. In various embodiments, the BafR protein comprises the amino acid sequence of SEQ ID NO: 1, the CmsG protein comprises the amino acid sequence of SEQ ID NO: 2, and the amino acid sequence of CmsR comprises the amino acid sequence of SEQ ID NO: 3. In various embodiments, the BafR protein is encoded by the nucleotide sequence of SEQ ID NO: 4, the CmsG protein isDocket No. 30275 / 2024-545 encoded by the nucleotide sequence of SEQ ID NO: 5, and the CmsR protein is encoded by the nucleotide sequence of SEQ ID NO: 6.

[0013] In some embodiments, any of the aforementioned engineered Streptomyces bacteria produce high levels of Concanamycin A. In addition, the disclosure provides for a culture comprising any of the aforementioned engineered Streptomyces bacteria disclosed herein.

[0014] In some embodiments, the engineered Streptomyces bacteria comprises any bacteria belonging to the Streptomyces genus. In some embodiments, the engineered Streptomyces bacteria is Streptomyces eitanensis, Streptomyces stelliscabiei, Streptomyces griseiscabiei, Streptomyces scabiei, or Streptomyces neyagawensis.

[0015] In addition, the disclosure provides for plasmids that are used to engineer any of the Streptomyces bacteria disclosed herein. In some embodiments, the disclosure provides for a plasmid comprising a nucleotide sequence encoding the BafR protein operably linked to a constitutive promoter. In various embodiments, the nucleotide sequence encoding the BafR protein comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 4, or comprises the sequence of SEQ ID NO: 4. The constitutive promoter may be any constitutive promoter known in the art. For example, in some embodiments, the promoter is KasO*p. In various embodiments, the promoter comprises SEQ ID NO: 7.

[0016] In some embodiments, the disclosure provides for a plasmid further comprising a nucleotide sequence encoding the BafR protein operably linked to a constitutive promoter and a nucleotide sequence encoding the CmsG protein operably linked to a constitutive promoter. In various embodiments, the disclosure provides for a plasmid comprising the nucleotide sequence encoding the CmsG protein comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 5, or comprises the sequence of SEQ ID NO: 5. The constitutive promoter may be any constitutive promoter known in the art. For example, in various embodiments, the promoter is KasO*p. In various embodiments, the promoter comprises SEQ ID NO: 7.

[0017] In some embodiments, disclosed herein is a nucleotide sequence encoding the BafR protein, a nucleotide sequence encoding the CmsG protein further comprising a nucleotide sequence encoding the CmsR protein operably linked to a constitutive promoter. In various embodiments, the nucleotide sequence encoding the CmsR protein comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 6, or comprises the sequence of SEQ ID NO: 6. The constitutive promoter may be any constitutive promoter known in the art. For example, in various embodiments, the promoter is KasO*p. In various embodiments, the promoter comprises SEQ ID NO: 7.

[0018] Disclosed herein is a method of engineering a bacteria, wherein the method comprises contacting a Streptomyces bacteria with any of the aforementioned plasmids.

[0019] In some embodiments, disclosed herein is a method of producing Concanamycin A, wherein the method comprises culturing any of the engineered Streptomyces bacteria in media, and wherein the culture comprises: I) peanut meal as an organic nitrogen source, ii) glucose; and ill) soluble starch as a carbon source;Docket No. 30275 / 2024-545 and wherein the culture is incubated at a temperature between about 20°C and 27°C. In various embodiments, the nitrogen source is soybean meal, corn gluten, peanut meal, or a combination thereof. In other embodiments, the carbon source is maltodextrin, potato starch, inulin, soluble starch, or a combination thereof. In some aspects, the Concanamycin A titers are at least about 107 mg / L. In some embodiments, the engineered Streptomyces bacteria is Streptomyces eitanensis, Streptomyces stelliscabiei, or Streptomyces neyagawensis, and the Concanamycin A titers yielded are at least about 107 mg / L. In some embodiments, wherein the engineered Streptomyces bacteria comprises a plasmid comprising BafR, CmsG, and CmsR, all operably linked to a constitutive promoter, the Concanamycin A titers are at least about 843 mg / L.

[0020] Further disclosed herein are methods of producing Concanamycin A, and in some embodiments methods of producing Concanamycin A and Concanamycin B. In some embodiments, methods of producing Concanamycin A comprise culturing any of the aforementioned bacteria. In some embodiments, methods of producing Concanamycin A and B comprise culturing engineered Streptomyces bacteria wherein the culture is supplemented with 0.2-1.0% w / v sodium propionate. For example, this method of producing Concanamycin A yield Concanamycin A titers of about 100 mg / L to about 750 mg / L, about 200 mg / L to about 650 mg / L, about 300 mg / L to about 700mg / L, about 500 mg / L to about 700 mg / L, about 550 mg / L to about 650 mg / L. For example, this method yields Concanamycin A titers of at least about 600 mg / L, about 610 mg / L, about 613 mg / L, about 620 mg / L, about 630 mg / L, or about 650 mg / L. In addition, this method of producing Concanamycin B yield Concanamycin B titers of about 50 mg / L to about 250 mg / L, about 75 mg / L to about 200 mg / L, about 100 mg / L to about 180 mg / L, about 150 Mg / L to about 200 mg / L. For example, this method yields Concanamycin B titers of at least about 100 mg / L, at least about 150 mg / L, at least about 175 mg / L or at least about 200 mg / L.

[0021] In some embodiments, disclosed herein is a method of producing Concanamycin A, the method comprising culturing wild-type Streptomyces eitanensis bacteria in media, wherein the culture comprises: I) corn gluten as an organic nitrogen source; ii) glucose; and ill) inulin as a carbon source; and wherein the culture is incubated at a temperature of between about 20°C to about 27°C. In various embodiments, the nitrogen source is peanut meal, corn gluten, or a combination thereof. In other embodiments, the carbon source is soluble starch, inulin, or a combination thereof. This method of producing Concanamycin A yields titers of about 50 mg / L to about 100 mg / L. For example, this method yields at least about 77mg / L.

[0022] Constitutive heterologous expression of regulators presents a great strategy to screen for novel producing strains, thus accelerating natural product discovery and the design of resilient natural overproducing strains. Disclosed herein are various implementations of a rational metabolic engineering approach for scale-up production of Concanamycin A and Concanamycin B molecules that are potential leads for medicinal chemistry studies against HIV Nef and other disease targets.BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 provides structural formulas of concanamycin A, B, and C.Docket No. 30275 / 2024-545

[0024] Figures 2A-2D show genome assembly, annotation, and analysis of Streptomyces eitanensis. (A) Genome assembled with Unycicler, visualized with Proseek (B) Phylogenetic analysis of 49 core gene (Clusters of Orthologous Groups) families of 33 public RefSeq genomes and Streptomyces eitanensis using SpeciesTree (Kbase) and ITOL. (C) Predicted BGC content in Streptomyces eitanensis strain predicted by AntiSMASH and Gecco. (D) Comparative analysis of Concanamycin A BGC from Streptomyces eitanensis with characterized BGCs from Streptomyces neyagawensis and Streptomyces scabiei using clinker.

[0025] Figures 3A-3B show production of concanamycin A by Streptomyces eitanensis. (A) Architecture and composition of concanamycin A BGC in Streptomyces eitanensis. Cluster representation to scale (bp) with DNAViewer python package. Arrows labeled with “+” indicate the two genes encoding transcription regulators. Genes encoding the biosynthesis of methoxymalonyl-ACP and ethylmalonyl-CoA are represented by arrows labeled with Arrows labeled with "#” show the sugar biosynthetic pathway, glycosyltransferase and carbamoyltransferase. Modular type-1 PKS genes, a Type II thioesterase are indicated by arrows labeled with "I”. Architecture of modular type-1 PKS. Acyl transferase domain AT 11 (dark AT circle) accepts ethylmalonyl-CoA and methylmalonyl-CoA yielding concanamycin A and B, respectively. (B) Optimization of media composition and fermentation temperature for concanamycin A production. Concanamycin A was quantified by AUG (HPLC) with a standard curve of known concentrations. ND - not detected. Symbols represent three independent experiments. Error bars indicate standard deviations (SD). Significant differences between cultivation conditions were determined by Welch's t-test.

[0026] Figures 4A-4D provide a phylogenetic analysis of Streptomyces eitanensis cluster-situated regulators CmsR (A) and CmsG (B) homologs. CmsR (A) and CmsG (B) protein homologues identified in multiple actinomycetes species. Tree designed by ete3, analyzed by ITOL. Uniprot ID is shown for all proteins. Conserved domain analysis of Streptomyces eitanensis cluster-situated regulators CmsR (C) and CmsG (D) using NCBI ‘Conserved Domain Database' and ‘Conserved Domain Architecture Retrieval Tool'. Shown ‘Specific hits' annotated for both proteins.

[0027] Figures 5A-5C show the contribution of overexpression of cluster-situated regulators on concanamycin A production. (A) Design of pSET152 integrative vectors for overexpression of target regulators under the strong constitutive synthetic promoter KasO*. (B) Construction of cmsG, cmsR and cmsG-cmsR overexpression strains DHS10671, DHS10672, and DHS10672, respectively. (C) Concanamycin A production, quantified by AUG (HPLC) with a standard curve of known concentrations. Symbols represent three independent experiments. Error bars indicate standard deviations (SD).

[0028] Figures 6A-6B present a phylogenetic tree of S. lohii Bafilomycin cluster-situated regulator BafR homologs. (A) Protein homologues identified in multiple actinomycetes species. List of S. lohii bafR homologues was identified by BLAST (top-score 12 proteins) using UniProtKB reference proteomes identified in multiple actinomycetes species. Tree designed by ete3 and visualized by ITOL. Uniprot ID is shown for all proteins. (B) Conserved domain analysis using NCBI ‘Conserved Domain Database' and ‘Conserved Domain ArchitectureDocket No. 30275 / 2024-545Retrieval Tool'. Shown ‘Specific hits' annotated for both proteins. Helix-turn-helix transcription regulator, domain architecture ID 10006622.

[0029] Figures 7A-7D show the contribution of BafR and cms cluster-situated regulators from Streptomyces eitanensis in concanamycin A production. (A) Concanamycin A production, at day 7, in engineered Streptomyces eitanensis strains overexpressing bafR alone or in combination with native CMA cluster-situated regulators, cmsG and cmsR, cultivated with diverse organic nitrogen sources and with Zinc supplementation (ZnSO4). (C) Concanamycin A production over time (days, X axis) in DHS10676 and wild-type. (B) Cell dry weight (DCW) of DHS101676 and wild-type cultivated in corn gluten at day 4, 7 and 10. (D) Concanamycin A production in engineered Streptomyces stelliscabiei (NRRL B-2795), Streptomyces neyagawensis (NRRL ISP-5588) and Streptomyces griseiscabiei (NRRL B-2447), and wild-type cultivated with corn gluten and inulin. Symbols represent three independent experiments. Error bars indicate standard deviations (SD). Significant differences between strains and cultivated conditions were calculated by Welch's t-test.

[0030] Figures 8A-8C show the integration of bafR, cmsG, and cmsR in the Streptomyces eitanensis genome.(A) Plasmid pSET'\52-bafR-cmsG-cmsR designed for integration at att site in Streptomyces eitanensis genome.(B) Comparative analysis of wild-type and DHS10676 genomes, and pSET152-baff?-cmsG-cmsR plasmid at att integration site using clinker. (C) Comparative analysis of concanamycin A BGC of the 4 different species engineered in this work generated by clinker.

[0031] Figures 9A-9B provide proteomics and untargeted metabolomics analysis of DHS10676 and wild-type Streptomyces eitanensis. (A) Significant changes in protein abundances from concanamycin BGC, between the engineered DHS10676 strain compared to wild-type at day 4 and 7 Proteins were considered a hit if Iog2fold- change > 1 (X axis) and with adjusted P-value moderated t-test with Benjamini-Hochberg false discovery rate (FDR) adjustment <0.05 (dot size). (B) Feature based analysis of untargeted metabolomics of DHS10676 (darker) and wild-type (lighter) at day 7 of strain cultivation under CMA producing conditions.

[0032] Figures 10A-10D show the impact of sodium propionate supplementation on concanamycin A and B titers. Quantified production of concanamycin A (A) and concanamycin B (B) in wild-type, and engineered Streptomyces eitanensis strains DHS10675 {bafR, cmsG) and DHS10676 {bafR, cmsG, cmsR) with and without 0.6% w / v sodium propionate added at 48h. Concanamycin A and B were quantified by AUG (HPLC) using standard curves with known concentrations of Concanamycin A or B. Symbols represent three independent experiments. Error bars indicate standard deviations (SD). Significant differences between engineered strains and wild-type under the same culture conditions were confirmed by Welch's t-test. (C) Principal component analysis of untargeted metabolome of wild-type (lighter) and engineered DHS10676 (darker) cultivated with sodium propionate (circles) and without (squares). Each symbol represents a biological replicate. (D) Isolated titers of pure concanamycin A and B across wild-type, DHS10675 and DHS10676 quantified in A and B. Symbols represent three independent experiments. Error bars indicate standard deviations (SD).Docket No. 30275 / 2024-545

[0033] Figures 11 A-11D show the metabolomics and proteomics analysis of DHS10676 and wild-type under concanamycin production conditions. (A) Hierarchical clustering of proteins (rows) over each replicate (column). Proteins with unique peptides <2 and present in only 1 biological replicate were excluded. (B) Principal component analysis of label-free proteomics of wild-type (lighter) and engineered DHS10676 (darker) cultivated for 4 (circles) and 7 (squares) days. Each symbol represents a biological replicate. (C) Fold-change (Log2) abundance of BafR (circle) and CmsG (squares) between wild-type and DHS10676 cultivated for 4 (light grey) or 7 (dark grey) days. (D) Hierarchical clustering of metabolite features (rows) over each replicate (column). Features present in less than 2 biological replicates were excluded. Each column represents a biological replicate of Engineered DHS10676 (darker) or Wild-type (lighter) grown under producing conditions for 7 days.

[0034] Figures 12A-12D show the impact of sodium propionate supplementation in concanamycin A and B titers. (A) Concanamycin A and B production by engineered Streptomyces eitanensis strain DHS10676 (pSET152-bafR-cmsG-cmsR) without (square) and with sodium propionate supplementation (0.1, 0.6, 0.74 and 1 % w / v, saturation gradient) at three different times (0, 48 and 72h, size). Each symbol represents an individual growth condition, each combination was tested in triplicate. (B) Concanamycin B production in wild-type (WT) and engineered Streptomyces eitanensis strains DHS10674 (pSET152k-bafR), DHS10673 (pSET152-cmsG- cmsR), DHS10675 (pSET152k-bafR-cmsG) and DHS10676 (pSET152-bafR-cmsG-cmsR) with and without 0.6 (% w / v) sodium propionate (SP) added at 48h. 0% SP is the left column and 0.6% SP is the right column for each strain. Each symbol represents a biological replicate (n). (C) Relative intensity levels of concanamycin A (CMA), B (CMB) and C (CMC) identified by untargeted LC-MS. Concanamycin A, B and C relative levels in wild-type and engineered Streptomyces eitanensis strain DHS10676 (pSET152-bafR-cmsG-cmsR) without and with sodium propionate supplementation (0.6 % w / v). Each condition and strain was tested in triplicate. Concanamycin A, B and C was identified by feature-based untargeted metabolomics in GNPS (level 2). (D) HPLC chromatogram of concanamycin A and B standards, wild-type Streptomyces eitanensis, and engineered DHS10675 and DHS10676 strains cultivated in corn gluten + inulin with / without sodium propionate supplementation.DETAILED DESCRIPTION

[0035] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0036] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0037] The term "a” and "an” as used herein mean "one or more” and include the plural unless the context is appropriate.Docket No. 30275 / 2024-545

[0038] As used herein, the term "bacteria” includes both singular and plural forms, such as a bacterium, a single bacterial cell and bacteria (plural), and genetically modified (recombinant) bacterial cells, bacteria and bacterial strains thereof.

[0039] As used herein, the term "operably linked” refers to a functional linkage between one or more nucleic acid sequences, such as a linkage between a regulatory or promoter sequence and a coding region sequence in which regulatory or promoter and the coding sequence are positioned and oriented for transcription of the coding region sequence to be positively or negatively regulated by the linked regulatory sequence.

[0040] Disclosed herein are materials and methods for sustainable overproduction of plecomacrolide compounds, e.g. concanamycins and bafilomycins. For example, Concanamycin A and B overproduction can be achieved by media and culture optimization, increased expression of target regulatory genes, or a combination thereof.

[0041] Concanamycins are comprised of an 18-membered macrolactone and include a 6-membered hemiketal ring that belong to the polyketide-derived plecomacrolide family of bioactive natural products. Concanamycin A, B, and C were first isolated from Streptomyces diastatochromogenes S-455. These molecules are known to be potent vacuolar-type adenosine triphosphatase (V-ATPase) inhibitors with diverse biological activities, including antifungal, antitumor, anti-viral, and immunomodular activities. As such, these molecules have potential as therapeutics for multiple diseases, including HIV, cancer, and osteoporosis. Concanamycin A in particular has exhibited sub-nanomolar potency against Nef-dependent MHC-I downmodulation activity in HIV- infected T cellsl

[0042] Due to their complex chemical structure, the total synthesis of concanamycins remains challenging and is not a sustainable source of the compounds14. Commercially available concanamycin A and B are produced by large-scale fermentation of Streptomyces strains at low titers, and thus production of these natural products is consequently costly. Despite the four decades since the isolation and characterization of concanamycins was first reported, the highest titers of both concanamycin A and B (Fig. 1) in batch fermentations are 60 and 20 mg / L, respectively1516These low titers produced by native isolates are a significant bottleneck for robust structure-activity studies and drug development.

[0043] Pleiotropic and cluster-situated mechanisms tightly regulate secondary metabolite production2021. Therefore, engineering new regulatory states that modulate natural product biosynthesis can enable controlled production of low-abundant metabolites22’23. Disclosed herein is a rational and systematic approach to increase production of concanamycin A and B. First, optimization of Streptomyces eitanensis cultivation conditions led to a >270-fold increased production of concanamycin A (95.9±17.2 mg / L) in the wild-type strain. Further engineering of native and heterologous regulatory gene overexpression, production of 909.8±64.7 mg / L of concanamycin A was achieved, a 10-fold improvement compared to wild-type. Sodium propionate supplementation boosted production of concanamycin B 306.5±42.1 mg / L, without significantly decreasing concanamycin A yield.Moreover, efforts to optimize extraction and isolation enabled the purification of 483.1 ±25.8 mg / L andDocket No. 30275 / 2024-545159.4±41.8 mg / L concanamycin A and B, respectively, under shake-flask cultivation conditions. Proteomics and metabolomics analysis uncovered a metabolic shift for CMA in the engineered versus wild-type strains. The methods disclosed herein highlight that rational metabolic engineering design principles are broadly applicable for the rapid development of wild-type Streptomyces species for sustainable production of high value natural products.Engineered Bacteria

[0044] Described herein are bacteria of the Streptomyces genus engineered to express one or more proteins that induce increased production of concanamycins. In various embodiments, the Streptomyces bacteria are engineered to express one or more heterologous regulator of the Concanamycin BGC, the Streptomyces bacteria are engineered to have increased expression of one or more homologous regulators of the Concanamycin BGC, or a combination thereof.

[0045] The genetic information required for the biosynthesis of secondary metabolites is spatially organized as BGCs that often include cluster-situated regulatory genes. The Concanamycin BGC was first characterized in S. neyagawensis, containing 28 ORFs in a total of -100 Kb19. The aglycone core of concanamycins is derived from the condensation of acyl-CoA building blocks by Type I Polyketide Synthases (Type I PKS). Biosynthetic genes for methoxymalonyl-ACP and ethylmalonyl-CoA production were also identified in this BGC. Concanamycin A differs from concanamycin B (Fig. 1) by the condensation of ethylmalonyl-CoA over methylmalonyl-CoA leading to an ethyl or methyl branch on the macrolactone C8 position. The concanamycin aglycones are modified by the addition of the 4'-O-carbamoyl-2'-deoxyrhamnose (encoded by a six gene deoxysugar subcluster, a carbamoyltransferase, and a glycosyltransferase) at the hemiketal ring C23 hydroxyl group19. Within the gene cluster, two putative pathway specific regulators show high homology to members of the LuxR and SARP protein families19.

[0046] As used herein, the terms "engineered” or "recombinant” can generally refer to a non-naturally occurring organism, nucleic acid, nucleic acid construct, or polypeptide. Such non-naturally occurring nucleic acids may include natural nucleic acids that have been modified, for example that have deletions, substitutions, inversions, insertions, etc., and / or combinations of nucleic acid sequences of different origin that are joined using molecular biology technologies (e.g., a nucleic acid sequences encoding a fusion protein) (e.g., a protein or polypeptide formed from the combination of two different proteins or protein fragments), the combination of a nucleic acid encoding a polypeptide to a promoter sequence, where the coding sequence and promoter sequence are from different sources or otherwise do not typically occur together naturally (e.g., a nucleic acid and a constitutive promoter), etc. Recombinant or engineered can also refer to the polypeptide encoded by the recombinant nucleic acid. Non-naturally occurring nucleic acids or polypeptides include nucleic acids and polypeptides modified by man.

[0047] Engineering of a bacteria involves the transfer of a nucleotide into the bacterial cell. The nucleotide can be a recombinant nucleotide, wherein the transfer of the recombinant nucleotide into the bacterial cell results inDocket No. 30275 / 2024-545 the expression of a homologous nucleotide or a heterologous nucleotide by the bacterial cell. Transferring a recombinant nucleotide into the bacterial cell can result in the integration of the nucleotide into the bacterial genome, or the stable expression of the nucleotide outside of the bacterial genome. Transferring a nucleotide into the bacterial cell can also delete a homologous nucleotide from the bacterial genome. The introduction of the nucleotide into the bacterial cell can be accomplished by any method known in the art, including, but not limited to, transformation, transfection, transduction, conjugation, and the like.

[0048] The introduction of the nucleotide into a host cell may be for instance, but not limited thereto, affected or carried out by protoplast transformation (see, e.g., Chang and Cohen, 1979, Molecular General Genetics 168: 111-115), by electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or by conjugation (see, e.g., Koehler and Thorne, 1987, Journal of Bacteriology 169: 5271-5278). Specific transformation protocols are known in the art for various types of host cells (see, e.g., for Streptomyces conjugation, Matsushima and Baltz, 1996, Microbiology 142: 261-267; see also Bierman et al., 1992, Gene 116(1): 43-49).

[0049] The engineered Streptomyces disclosed herein are genetically modified to have increased expression of at least one protein involved in regulation of the Concanamycin BGC. As used herein, "increased expression” means that the engineered Streptomyces has an improved or increased production of a BGC regulator relative to a control cell (e.g., an unmodified cell).

[0050] As used herein "increased expression” is used to refer to an increased expression of a gene, or gene product thereof (e.g., the encoded protein) in a sample as compared to the expression of said gene or gene product in a suitable control. The term "increased expression” preferably refers to about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%,380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%,540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%,700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%,860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, 1000%,1010%, 1020%, 1030%, 1040%, 1050%, 1060%, 1070%, 1080%, 1090%, 1100%, 1110%, 1120%, 1130%,1140%, 1150%, 1160%, 1170%, 1180%, 1190%, 1200%, 1210%, 1220%, 1230%, 1240%, 1250%, 1260%,1270%, 1280%, 1290%, 1300%, 1310%, 1320%, 1330%, 1340%, 1350%, 1360%, 1370%, 1380%, 1390%,1400%, 1410%, 1420%, 1430%, 1440%, 1450%, 1460%, 1470%, 1480%, 1490%, or / to 1500% or more increased expression relative to a suitable control. For example, "increased expression” refers to about 10% to about 100% increase in expression, or about 50% to about 200% increase in expression, or about 100% to about 250% increase in expression, or about 250% to 500% increase in expression or about 300% to 700% increase in expression, or about 500% to about 1000% increase in expression or about 50% to about 250% increase in expression or about 100% to 500% increase in expression or about 100% to about 1000% increase in expression.Docket No. 30275 / 2024-545

[0051] As used herein, "increased expression” includes when a nucleotide sequence or protein is native to the host cell, whose expression is quantitatively altered or whose expression is directed from a genomic location different from the native host cell as a result of manipulation of the DNA of the host cell by recombinant DNA techniques, including, e.g., a stronger promoter. "Increased expression” also includes when a nucleotide sequence or protein that is not native to the host cell is expressed in the host cell.

[0052] In some embodiments, engineered Streptomyces is genetically modified to express a heterologous (e.g., non-native, exogenous, foreign, or recombinant) target regulatory protein. As used herein, "heterologous” refers to a protein that is not native (or homologous, or endogenous) to the host organism into which it is introduced. As used herein, "increased expression” can also refer to a heterologous gene and corresponding protein. As used herein, engineered Streptomyces bacteria will have higher gene and protein levels of the heterologous protein than a reference bacteria, as the heterologous protein will be absent in the reference bacteria. "Increased expression” may refer to any levels of non-native protein expressed in the engineered Streptomyces.

[0053] In the context of gene expression, "expression” means the transcription of a specific gene or specific genes or a specific nucleotide construct; the transcription of a gene or genes or genetic construct into structural RNA (e.g., rRNA, tRNA) or mRNA with or without subsequent translation of the latter into a protein. The process includes transcription of DNA and processing of the resulting mRNA construct.

[0054] In the present disclosure, an endogenous protein, e.g. native to the host cell, in which structural modifications, e.g., deletions, substitutions, and / or insertions, have been made by recombinant DNA techniques to alter the native polypeptide may be referred to as a protein with "increased expression” in the host cell, if the modified protein levels are compared to levels of the corresponding endogenous protein.

[0055] Protein variants may be defined by their sequence identity when compared to a reference protein. Sequence identity usually is provided as "% sequence identity” or "% identity”. To determine the percent identity between two amino acid sequences in a first step a pairwise sequence alignment is generated between those two sequences, wherein the two sequences are aligned over their complete length (i.e., a pairwise global alignment). The alignment is generated with a program implementing the Needleman and Wunsch algorithm (J. Mol. Biol. (1979) 48, p. 443-453), preferably by using the program "NEEDLE” (The European Molecular Biology Open Software Suite (EMBOSS)) with the programs default parameters (gapopen=10.0, gapextend=0.5 and matrix=EBLOSUM62). The preferred alignment for the purpose of this invention is that alignment, from which the highest sequence identity can be determined.

[0056] After aligning the two sequences, in a second step, an identity value shall be determined from the alignment. Therefore, according to the present invention the following calculation of percent identity applies: % identity = (identical residues I length of the alignment region which is showing the respective sequence of this invention over its complete length) *100. Thus, sequence identity in relation to comparison of two amino acid sequences according to this embodiment is calculated by dividing the number of identical residues by the lengthDocket No. 30275 / 2024-545 of the alignment region which is showing the respective sequence of this invention over its complete length. This value is multiplied with 100 to give "% identity”.

[0057] For calculating the percent identity of two DNA sequences the same applies as for the calculation of percent identity of two amino acid sequences with some specifications. For DNA sequences encoding for a protein the pairwise alignment shall be made over the complete length of the coding region from start to stop codon excluding introns. For non-protein-coding DNA sequences the pairwise alignment shall be made over the complete length of the sequence of this invention, so the complete sequence of this invention is compared to another sequence, or regions out of another sequence. Moreover, the preferred alignment program implementing the Needleman and Wunsch algorithm (J. Mol. Biol. (1979) 48, p. 443-453) is "NEEDLE” (The European Molecular Biology Open Software Suite (EMBOSS)) with the programs default parameters (gapopen=10.0, gapextend=0.5 and matrix=EDNAFULL).Increased expression of Biosynthetic Gene Cluster regulators

[0058] Bafilomycin A1 is a 16-membered ring plecomacrolide produced by Streptomyces lohii that is structurally related to concanamycin A, but bears a smaller macrolactone ring, and lacks the carbamoylated deoxyrhamnose sugar. Bafilomycin A1 and concanamycin A directly bind and inhibit the eukaryotic V-ATPase5. Two cluster-situated regulators, bafG and bafR, are encoded within baf BGC in Streptomyces lohii. BafG (609 amino acids) is a close homologue of the concanamycin cluster-situated gene CmsG from Streptomyces eitanensis. BafR encodes a small 117 amino acid protein, characterized as a LuxR family regulator containing only the conserved helix-turn-helix (HTH-) motif with an inducer-independent type activation31. While BafR homologues containing the LuxR-type HTH-domain exist in multiple actinomycetes species, no such homologue exists in Streptomyces eitanensis.

[0059] In some embodiments, an engineered Streptomyces bacteria expresses a heterologous BafR protein, wherein the wild-type variant of said Streptomyces bacteria does not express endogenous BafR. In some embodiments, an engineered Streptomyces bacteria expresses a heterologous BafR protein, wherein the amino acid sequence of the BafR protein is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 1, or wherein the amino acid sequence of the BafR protein is SEQ ID NO: 1. In some embodiments, an engineered Streptomyces bacteria expresses a heterologous BafR protein, wherein the amino acid sequence is encoded by a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 4, or wherein the amino acid sequence is encoded by the nucleotide sequence of SEQ ID NO: 4.

[0060] In some embodiments, an engineered Streptomyces comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 4, or comprises the nucleotide sequence of SEQ ID NO: 4.

[0061] The structure and gene composition of the concanamycin A BGC in Streptomyces eitanensis is highly conserved between CMA-producing strains (see Fig. 2D, Fig. 3A). The Concanamycin BGC has two cluster- situated proteins with a putative regulatory role, Orf3 (923 amino acids) and Orf17 (701 amino acids)19. The firstDocket No. 30275 / 2024-545Concanamycin BGC cluster-situated protein, Orf17, resembles proteins from the Streptomyces antibiotic regulatory protein (SARP) family, characterized by the presence of OmpR-like DNA-binding and bacterial transcriptional activator (BTA) domains35(see Fig. 4D). The Streptomyces eitanensis protein homolog to Orf17 is CmsG. CmsG shows high homology to BafG, described as a positive AsfR family regulator that activates production of the plecomacrolide bafilomycin Ai in Streptomyces lohiP](see Fig. 4C, 4D).

[0062] In some embodiments, an engineered Streptomyces bacteria exhibits increased expression of a CmsG protein, as compared to endogenous CmsG protein levels. In some embodiments, an engineered Streptomyces bacteria expresses a heterologous BafR protein and exhibits increased expression of a CmsG protein. In some embodiments, an engineered Streptomyces bacteria exhibits increased expression of the CmsG protein, wherein the amino acid sequence of the CmsG protein is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 2, or wherein the amino acid sequence of the CmsG protein is SEQ ID NO: 2. In some embodiments, an engineered Streptomyces bacteria has increased expression of the CmsG protein, wherein the amino acid sequence is encoded by a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 5, or wherein the amino acid sequence is encoded by the nucleotide sequence of SEQ ID NO: 5.

[0063] In some embodiments, an engineered Streptomyces comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 5, or comprises the nucleotide sequence of SEQ ID NO: 5.

[0064] The second Concanamycin BGC cluster-situated protein, Orf3, encodes a protein that shows high sequence similarity to larger ATP-binding regulators of the LuxR family, commonly situated in biosynthetic gene clusters for type I poly ketides34. This class of regulators is characterized by a polypeptide length of >900 amino acids, an ATP-binding motif at the N-terminus, and a helix-turn-helix domain at the C-terminus of the protein (see Fig. 4). The Streptomyces eitanensis protein homolog to Orf3 is CmsR. CmsR is a close homologue of PikD, an ATP-binding protein of the LuxR family that has been characterized as a pathway-specific positive regulatory of pikromycin biosynthesis in Streptomyces venezualae2335(see Fig. 4A).

[0065] In some embodiments, an engineered Streptomyces bacteria exhibits increased expression of a CmsR protein, as compared to endogenous CmsR protein levels. In some embodiments, an engineered Streptomyces bacteria expresses a heterologous BafR protein and exhibits increased expression of a CmsR protein. In some embodiments, an engineered Streptomyces bacteria exhibits increased expression of a CmsG protein and exhibits increased expression of a CmsR protein. In some embodiments, an engineered Streptomyces bacteria expresses a heterologous BafR protein, exhibits increased expression of a CmsG protein, and exhibits increased expression of a CmsR protein. In some embodiments, an engineered Streptomyces bacteria exhibits increased expression of the CmsR protein, wherein the amino acid sequence of the CmsR protein is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 3, or wherein the amino acid sequence of the CmsG protein is SEQ ID NO: 3. In some embodiments, an engineered Streptomyces bacteria has increased expression of the CmsR protein, wherein the amino acid sequence is encoded by a nucleotide sequence that is at least 70%, 75%,Docket No. 30275 / 2024-54580%, 85%, 90%, or 95% identical to SEQ ID NO: 6, or wherein the amino acid sequence is encoded by the nucleotide sequence of SEQ ID NO: 6.

[0066] In various embodiments, the engineered Streptomyces bacteria exhibits increased expression of the BafR protein, increased expression of the CmsG protein, increased expression of the CmsR protein, or a combination thereof. In various aspects, the BafR protein comprises the amino acid sequence of SEQ ID NO: 1, the CmsG protein comprises the amino acid sequence of SEQ ID NO: 2, the CmsR protein comprises the amino acid sequence of SEQ ID NO: 3, or a combination thereof. In various aspects, the BafR protein is encoded by the nucleotide sequence of SEQ ID NO: 4, the CmsG protein is encoded by the nucleotide sequence of SEQ ID NO: 5, the CmsR protein is encoded by the nucleotide sequence of SEQ ID NO: 6, or a combination thereof.

[0067] The present disclosure further contemplates a culture comprising any of the Streptomyces bacteria disclosed herein. Exemplary Streptomyces bacteria include, but are not limited to, Streptomyces eitanensis, Streptomyces stelliscabiei, Streptomyces griseiscabiei, Streptomyces scabiei, and Streptomyces neyagawensis.

[0068] In some embodiments, the engineered Streptomyces described above produce high levels of Concanamycin A. As used herein, "high levels” refer to Concanamycin A titers higher than those previously reported in the art by wild-type Streptomyces strains. To date, the highest titers of Concanamycin A produced in batch fermentations are 60 mg / L15.Plasmids

[0069] The term "plasmid”, "vector”, or "construct” as used herein refers to a circular double-stranded (ds) DNA construct used as a cloning vector, and which forms an extrachromosomal self-replicating genetic element in a microorganism such as a bacteria, or integrates into the host chromosome or a microorganism such as a bacteria. The plasmid can be part of an expression system. The plasmid is useful for creating an engineered bacterial cell, for example, that express increased levels of homologous regulators of the Concanamycin BGC, or that expresses heterologous regulators of the Concanamycin BGC.

[0070] The terms "expression” or "express” refers to the production of mRNA from the polynucleotide sequence of a gene or portion of a gene. The production of any polypeptide which is encoded by the mRNA, gene, or portion of the gene is also included within the scope of the terms. The term "encoding” refers to the property of polynucleotide sequences to behave as templates for the production of other macromolecules such as mRNA and proteins.

[0071] When used herein the term "coding sequence" refer to a nucleotide sequence which directly encodes the amino acid sequence of its protein product. The boundaries of the coding sequence are generally determined by an open reading frame, which usually begins with the ATG start codon or alternative start codons such as GTG, CTG or TTG and ends with a stop codon such as TAA, TAG, or TGA. The coding sequence may be a DNA, cDNA, synthetic, or recombinant nucleotide sequence. The start codon can also be named herein asDocket No. 30275 / 2024-545"translational start signal” or "translational start site”. The stop codon can also be named herein as "translational stop signal” or "translational stop site”.

[0072] As used interchangeably herein, "operably linked” and "operatively linked” in the context of recombinant or engineered polynucleotide molecules (e.g., DNA and RNA) vectors, and the like refers to the regulatory and other sequences useful for expression, stabilization, replication, and the like of the coding and transcribed noncoding sequences of a nucleic acid that are placed in the nucleic acid molecule in the appropriate positions and / or orientation relative to the coding sequence so as to positively or negatively affect expression or another characteristic of the coding sequence or transcribed non-coding sequence. This same term can be applied to the position and / or orientation of coding sequences, non-coding and / or transcription control elements (e.g., promoters, enhancers, and termination elements), and / or selectable markers in an expression vector. "Operably linked” can also refer to an indirect attachment (i.e., not a direct fusion) of two or more polynucleotide sequences or polypeptides to each other via a linking molecule (also referred to herein as a "linker”).

[0073] Further disclosed herein are polynucleotides encoding the amino acid sequences disclosed herein, and plasmids comprising the polynucleotides of the disclosure. In various embodiments, a plasmid of the disclosure comprises a nucleotide sequence encoding a BafR protein operably linked to a constitutive promoter. In some embodiments, the plasmid of the disclosure comprises a nucleotide sequence encoding a BafR protein operably linked to a constitutive promoter, wherein the nucleotide sequence encoding the BafR protein comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 4, or comprises the sequence of SEQ ID NO: 4. In some embodiments, the plasmid of the disclosure comprises a nucleotide sequence encoding a BafR protein operably linked to a constitutive promoter, wherein the BafR protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 1, or comprises the amino acid sequence of SEQ ID NO: 1.

[0074] As used herein, "promoter” includes all sequences capable of driving transcription of a coding or a noncoding sequence. In particular, the term "promoter” as used herein refers to a DNA sequence generally described as the 5' regulator region of a gene, located proximal to the start codon. The transcription of an adjacent coding sequence(s) is initiated at the promoter region. The term "promoter” also includes fragments of a promoter that are functional in initiating transcription of the gene. As used herein, the term "constitutive promoter” describes sequences capable of continuously driving transcription of a coding or non-coding sequence. Any plasmids disclosed herein can comprise any constitutive promoter. In some embodiments, the plasmid's constitutive promoter is KasO*p. In some embodiments, the promoter comprises SEQ ID NO: 7. Exemplary constitutive promoters include, but are not limited to, KasO*p, ermEp*,SF14p, SP44, stnYp, SCO5768p, and hrdBp.

[0075] In various embodiments the plasmid of the disclosure comprises a nucleotide sequence encoding a CmsG protein operably linked to a constitutive promoter. In various embodiments, the plasmid of the disclosure comprises a nucleotide sequence encoding a BafR protein and a CmsG protein, wherein both sequences areDocket No. 30275 / 2024-545 operably linked to a constitutive promoter. The disclosed plasmids can comprise any constitutive promoter. In some embodiments, the nucleotide sequence encoding the CmsG protein comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 5, or comprises the sequence of SEQ ID NO: 5. In some embodiments, the nucleotide sequence encodes a CmsG protein comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 2, or comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the plasmid's constitutive promoter is KasO*p. In some embodiments, the promoter comprises SEQ ID NO: 7.

[0076] In various embodiments, the plasmid of the disclosure comprises a nucleotide sequence encoding a CmsR protein operably linked to a constitutive promoter. In various embodiments, the plasmid of the disclosure comprises a nucleotide sequence encoding a CmsR protein and a CmsG protein, wherein both sequences are operably linked to a constitutive promoter. In various embodiments, the plasmid of the disclosure comprises a nucleotide sequence encoding a CmsR protein and a BafR protein, wherein both sequences are operably linked to a constitutive promoter. These disclosed plasmids can comprise any constitutive promoter. In various embodiments, the plasmid of the disclosure comprises a nucleotide sequence encoding a BafR protein, a CmsG protein, and a CmsR protein, wherein each sequence is operably linked to a constitutive promoter. In some embodiments, the nucleotide sequence encoding the CmsR protein comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 6, or comprises the sequence of SEQ ID NO: 6. In some embodiments, the nucleotide sequence encodes a CmsR protein comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 3, or comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the plasmid's constitutive promoter is KasO*p. In some embodiments, the promoter comprises SEQ ID NO: 7.

[0077] Further contemplated herein is a method of engineering a Streptomyces bacteria, wherein the method comprises contacting the Streptomyces bacteria with any of the plasmids disclosed herein.Optimizing Culture Conditions

[0078] Further contemplated herein is a method of producing Concanamycin A, wherein the method comprises culturing any of the Streptomyces bacteria disclosed herein, and wherein in some embodiments the Streptomyces culture yields high levels (or increased levels) of Concanamycin A.

[0079] Disclosed herein are methods of optimizing bacterial culture conditions to improve Concanamycin A yields, wherein the method comprises culturing any of the engineered Streptomyces bacteria disclosed herein in media supplemented with glucose, a carbon, and a nitrogen source, wherein the culture is incubated at a temperature of between about 20°C to about 27°C. As used herein "media” refers to a liquid or solid preparation to support growth of bacteria in culture. In some embodiments, engineered Streptomyces bacteria are cultured in TSB media, T2YE media, YEME media, R2YE media, or any other media acceptable for bacterial culture. In some embodiments, engineered Streptomyces bacteria are cultured in media comprising production media base (such as a base media comprising 5g Bacto Peptone, 5g Yeast extract, 2 g / L CaCOa, pH =7.0) supplementedDocket No. 30275 / 2024-545 with glucose, wherein the carbon source is soluble starch and the nitrogen source is peanut meal. In some embodiments, the carbon source comprises soluble starch, potato starch, inulin, maltodextrin, or a combination thereof. In some embodiments, the nitrogen source comprises peanut meal, corn gluten, soybean meal, or a combination thereof. In some embodiments, the engineered Streptomyces bacteria are cultured in GMSYE media comprising production media base supplemented with glucose, wherein the carbon source is maltodextrin and the nitrogen source is soybean meal. In some embodiments, engineered Streptomyces bacteria are cultured in GSSY media comprising production media base supplemented with glucose, wherein the carbon source is soluble starch and the nitrogen source is soybean meal. In various embodiments, the engineered Streptomyces bacteria are cultured in GICYE media comprising production media base supplemented with glucose, wherein the carbon source is inulin and the nitrogen source is corn gluten. In some embodiments, the engineered Streptomyces bacteria are cultured in GSPYE media comprising production media base supplemented with glucose, wherein the carbon source is soluble starch and the nitrogen source is peanut meal.

[0080] In some embodiments, a method of producing Concanamycin A comprises culturing an engineered Streptomyces bacteria in media. In various embodiments, the Streptomyces bacteria is a Streptomyces eitanensis bacteria. In some embodiments, the S. eitanensis bacteria is engineered to express a plasmid encoding BafR, CmsG, and CmsR, and the culture comprises: I) peanut meal as a nitrogen source, ii) glucose; and ill) soluble starch as a carbon source, wherein the culture is incubated at a temperature between about 20°C to about 27°C, and wherein Concanamycin A titers yielded are least about 600 mg / L, 650 mg / L, 700 mg / L, 750 mg / L, 800 mg / L, 850 mg / L, 900 mg / L, 950 mg / L, or 1000 mg / L. In various embodiments, the Concanamycin A titers yielded are about 600 mg / L to about 1000 mg / L, about 600 mg / L to about 900 mg / L, about 600 mg / L to about 800 mg / L, about 600 mg / L to about 700 mg / L, about 700 mg / L to about 1000 mg / L, about 700 mg / L to about 900 mg / L, about 700 mg / L to about 800 mg / L, about 800 mg / L to about 1000 mg / L, about 800 mg / L to about 900 mg / L, or about 900 mg / L to about 1000 mg / L. In some embodiments, the Concanamycin A titers yielded are at least about 843 mg / L.

[0081] In various embodiments, a method of producing Concanamycin A comprises culturing an engineered Streptomyces bacteria in media, wherein the engineered Streptomyces bacteria is S. griseiscabiei, S. neyagawaensis, or S. stelliscabiei, and wherein the culture comprises: I) peanut meal as a nitrogen source, ii) glucose; and ill) soluble starch as a carbon source, wherein the culture is incubated at a temperature between about 20°C to about 27°C . In some aspects, the S. griseiscabiei bacteria is engineered to express a plasmid encoding BafR, CmsG, and CmsR, and the Concanamycin A titers yielded are at least about 50 mg / L, 60 mg / L 70 mg / L, 80 mg / L, 90 mg / L, or 100 mg / L. In various aspects, the Concanamycin A titers yielded are at least about 50 mg / L to about 100 mg / L, about 50 mg / L to about 90 mg / L, about 50 mg / L to about 80 mg / L, about 50 mg / L to about 70 mg / L, about 50 mg / L to about 60 mg / L, about 60 mg / L to about 100 mg / L, about 60 mg / L to about 90 mg / L, about 60 mg / L to about 80 mg / L, about 60 mg / L to about 70 mg / L, about 70 mg / L to about 100 mg / L, about 70 mg / L to about 90 mg / L, about 70 mg / L to about 80 mg / L, about 80 mg / L to about 100 mg / L, about 80 mg / L to about 90 mg / L, or about 90 mg / L to about 100 mg / L. In some aspects, the S. neyagawaensis bacteriaDocket No. 30275 / 2024-545 is engineered to express a plasmid encoding BafR, CmsG, and CmsR, and the Concanamycin A titers yielded are at least about 80 mg / L, 90 mg / L, 100 mg / L, 110 mg / L, or 120 mg / L. In various aspects, the Concanamycin A titers yielded are at least about 80 mg / L to 120 mg / L, about 80 mg / L to about 110 mg / L, about 80 mg / L to about 100 mg / L, about 80 mg / L to about 90 mg / L, about 90 mg / L to about 120 mg / L, about 90 mg / L to about 110 mg / L, about 90 mg / L to about 100 mg / L, about 100 mg / L to about 120 mg / L, about 90 mg / L to about 100 mg / L, about 90 mg / L to about 100 mg / L, about 100 mg / L to about 120 mg / L, about 100 mg / L to about 110 mg / L, or about 110 mg / L to about 120 mg / L. In some aspects, the Concanamycin A titers yielded are at least about 107 mg / L. In some embodiments, the engineered Streptomyces bacteria is Streptomyces eitanensis, Streptomyces stelliscabiei, or Streptomyces neyagawensis, and the Concanamycin A titers yielded are at least about 107 mg / L. In various aspects, the S. stelliscabiei bacteria is engineered to express a plasmid encoding BafR, CmsG, and CmsR, and the Concanamycin A titers yielded are at least about 400 mg / L, 425 mg / L, 450 mg / L, 475 mg / L, 500 mg / L, or 525 mg / L. In some aspects, the Concanamycin A titers yielded are at least about 400 mg / L to about 525 mg / L, about 400 mg / L to about 500 mg / L, about 400 mg / L to about 475 mg / L, about 400 mg / L to about 450 mg / L, about 400 mg / L to about 425 mg / L, about 425 mg / L to about 525 mg / L, about 425 mg / L to about 500 mg / L, about 425 mg / L to about 475 mg / L, about 425 mg / L to about 450 mg / L, about 450 mg / L to about 525 mg / L, about 450 mg / L to about 500 mg / L, about 450 mg / L to about 475 mg / L, about 475 mg / L to about 525 mg / L, about 475 mg / L to about 500 mgl / L, or about 500 mg / L to about 525 mg / L. In various aspects, the Concanamycin A titers yielded are at least about 442 mg / L.

[0082] In various embodiments, a method of producing Concanamycin A comprises culturing an engineered Streptomyces bacteria in media. In some embodiments, the S. eitanensis bacteria is engineered to express a plasmid encoding CmsG, and wherein the Concanamycin A titers yielded are at least about 250 mg / L, 275 mg / L, 300 mg / L, 325 mg / L, 350 mg / L, or 375 mg / L. In various aspects, the Concanamycin A titers yielded are at least about 250 mg / L to about 375 mg / L, about 250 mg / L to about 350 mg / L, about 250 mg / L to about 325 mg / L, about 250 mg / L to about 300 mg / L, about 250 mg / L to about 275 mg / L, about 275 mg / L to about 350 mg / L, about 275 mg / L to about 325 mg / L, about 275 mg / L to about 300 mg / L, about 300 mg / L to about 350 mg / L, about 300 mg / L to about 325 mg / L, or about 325 mg / L to about 350 mg / L. In some aspects, culturing the engineered S. eitanensis bacteria engineered to express a plasmid encoding CmsG yields Concanamycin A titers of at least about 275 mg / L. In some embodiments, the Streptomyces eitanensis bacteria is engineered to express a plasmid encoding CmsR, and wherein the Concanamycin A titers yielded are at least about 175 mg / L, 200 mg / L, 225 mg / L, 250 mg / L, 275 mg / L, or 300 mg / L. In various aspects, the Concanamycin A titers yielded are at least about 175 mg / L to about 300 mg / L, about 175 mg / L to about 275 mg / L, about 175 mg / L to about 250 mg / L, about 175 mg / L to about 225 mg / L, about 175 mg / L to about 225 mg / L, about 200 mg / L to about 300 mg / L, about 200 mg / L to about 275 mg / L, about 200 mg / L to about 250 mg / L, about 200 mg / L to about 225 mg / L, about 225 mg / L to about 300 mg / L, about 225 mg / L to about 275 mg / L, about 225 mg / L to about 250 mg / L, about 250 mg / L to about 300 mg / L, about 250 mg / L to about 275 mg / L, or about 275 mg / L to about 300 mg / L. In some aspects, culturing the engineered S. eitanensis bacteria engineered to express a plasmid encoding CmsR yields Concanamycin ADocket No. 30275 / 2024-545 titers of at least about 210 mg / L. In some embodiments, the S. eitanensis bacteria is engineered to express a plasmid encoding CmsR and CmsG, and wherein the Concanamycin A titers yielded are at least about 250 mg / L, 275 mg / L, 300 mg / L, 325 mg / L, 350 mg / L, or 375 mg / L. In various aspects, the Concanamycin A titers yielded are at least about 250 mg / L to about 375 mg / L, about 250 mg / L to about 350 mg / L, about 250 mg / L to about 325 mg / L, about 250 mg / L to about 300 mg / L, about 250 mg / L to about 275 mg / L, about 275 mg / L to about 350 mg / L, about 275 mg / L to about 325 mg / L, about 275 mg / L to about 300 mg / L, about 300 mg / L to about 350 mg / L, about 300 mg / L to about 325 mg / L, or about 325 mg / L to about 350 mg / L. In some aspects, culturing the engineered S. eitanensis bacteria engineered to express a plasmid encoding CmsR and CmsG yields Concanamycin A titers of at least about 275 mg / L.

[0083] Further contemplated herein is a method of producing Concanamycin A and Concanamycin B. In some embodiments, the method of producing Concanamycin A and Concanamycin B comprises culturing the aforementioned Streptomyces bacteria, wherein the culture is supplemented with 0.2-1 .0% sodium propionate. In some embodiments, the method of producing Concanamycin A and Concanamycin B yields high levels of Concanamycin A and Concanamycin B, wherein high levels of Concanamycin A are titers higher than those previously reported in the art (60 mg / L15) produced by the same or related strain of Streptomyces bacteria, and similarly high levels of Concanamycin B are titers higher than those previously reported in the art (20 mg / L16) produced by the same or related strain of Streptomyces bacteria. In various embodiments, the Streptomyces bacterial culture supplemented with sodium propionate comprises engineered Streptomyces bacteria. In some embodiments, the Concanamycin A titers yielded by the methods of the disclosure are at least about 500 mg / L, 550mg / L, 600 mg / L, 650 mg / L, 700 mg / L, 750 mg / L, 800 mg / L, or 850 mg / L. In various embodiments, the Concanamycin A titers yielded are at least abut 500 mg / L to about 850 mg / L, about 500 mg / L to about 800 mg / L, about 500 mg / L to about 750 mg / L, about 500 mg / L to about 700 mg / L, about 500 mg / L to about 650 mg / L, about 500 mg / L to about 600 mg / L, about 500 mg / L to about 550 mg / L, about 550 mg / L to about 850 mg / L, about 550 mg / L to about 800 mg / L, about 550 mg / L to about 750 mg / L, about 550 mg / L to about 700 mg / L, about 550 mg / L to about 650 mg / L, about 550 mg / L to about 600 mg / L, about 600 mg / L to about 850 mg / L, about 600 mg / L to about 800 mg / L, about 600 mg / L to about 750 mg / L, about 600 mg / L to about 700 mg / L, about 600 mg / L to about 650 mg / L, about 650 mg / L to about 850 mg / L, about 650 mg / L to about 800 mg / L, about 650 mg / L to about 750 mg / L, about 650 mg / L to about 700 mg / L, about 700 mg / L to about 850 mg / L, about 700 mg / L to about 800 mg / L, about 700 mg / L to about 750 mg / L, about 750 mg / L to about 850 mg / L, about 750 mg / L to about 800 mg / L, or about 800 mg / L to about 850 mg / L. In some embodiments, the Concanamycin A titers yielded by the methods of the disclosure are at least about 613 mg / L. In various embodiments, the Concanamycin B titers yielded by the methods of the disclosure are at least about 150 mg / L, 200 mg / L, 250 mg / L, 300 mg / L, 350 mg / L, or 400 mg / L. In some embodiments, the Concanamycin B titers yielded are at least about 150 mg / L to about 400 mg / L, about 150 mg / L to about 350 mg / L, about 150 mg / L to about 300 mg / L, about 150 mg / L to about 250 mg / L, about 150 mg / L to about 200 mg / L, about 200 mg / L to about 400 mg / L, about 200 mg / L to about 350 mg / L, about 200 mg / L to about 300 mg / L, about 200 mg / L to about 250 mg / L, about 250 mg / L to about 400 mg / L, about 250 mg / L toDocket No. 30275 / 2024-545 about 350 mg / L, about 250 mg / L to about 300 mg / L, about 300 mg / L to about 400 mg / L, about 200 mg / L to about 350 mg / L, or about 350 mg / L to about 400 mg / L. In various embodiments, the Concanamycin B titers yielded by the methods of the disclosure are at least about 175 mg / L.

[0084] In some embodiments, disclosed herein is a method of producing Concanamycin A, the method comprising culturing wild-type Streptomyces eitanensis bacteria in liquid production media base, wherein the culture comprises i) corn gluten replaces soybean meal as a nitrogen source; ii) glucose; and iii) inulin replaces maltodextrin as a carbon source; and wherein the culture is incubated at a temperature of between about 20°C to about 27°C. In some embodiments, a soybean meal nitrogen source is replaced with corn gluten, peanut meal, or a combination thereof. In some embodiments, a maltodextrin carbon source is replaced with soluble starch, inulin, or a combination thereof. In some embodiments, the method comprising culturing Streptomyces eitanensis bacteria in media yields Concanamycin A titers of at least 65 mg / L, 70mg / L, 75 mg / L, 80 mg / L, 85 mg / L, 90 mg / L, 95mg / L, or 100 mg / L. In various embodiments, the Concanamycin A titers yielded are at least about 65 mg / L to about 100 mg / L, about 65 mg / L to about 95 mg / L, about 65 mg / L to about 90 mg / L, about 65 mg / L to about 85 mg / L, about 65 mg / L to about 80 mg / L, about 65 mg / L to about 75 mg / L, about 65 mg / L to about 70 mg / L, about 70 mg / L to about 100 mg / L, about 70 mg / L to about 95 mg / L, about 70 mg / L to about 90 mg / L, about 70 mg / L to about 85 mg / L, about 70 mg / L to about 80 mg / L, about 70 mg / L to about 75 mg / L, about 75 mg / L to about 100 mg / L, about 75 mg / L to about 95 mg / L, about 75 mg / L to about 90 mg / L, about 75 mg / L to about 85 mg / L, about 75 mg / L to about 80 mg / L, about 80 mg / L to about 100 mg / L, about 80 mg / L to about 95 mg / L, about 80 mg / L to about 90 mg / L, about 80 mg / L to about 85 mg / L, about 85 mg / L to about 100 mg / L, about 85 mg / L to about 95 mg / L, about 85 mg / L to about 90 mg / L, about 90 mg / L to about 100 mg / L, about 90 mg / L to about 95 mg / L, or about 95 mg / L to about 100 mg / L. In some embodiments, the method comprising culturing Streptomyces eitanensis bacteria in media yields Concanamycin A titers of at least 77 mg / L.ExamplesExample 1 : Materials and MethodsStrains and culture conditions

[0085] Escherichia coli DH5o (NEB) was used as host for plasmid assembly, replication, and preservation. E. coli S171 strain was used for interspecies conjugation. All E. coli strains were cultivated in LB medium (10g Tryptone, 10g NaCI and 5g of Yeast Extract per liter) at 37 °C. For plasmid maintenance and selection LB media was supplemented with appropriate antibiotics. Concanamycin producing strain (wild-type Streptomyces eitanensis) was a gift from Fermentek. All Streptromyces strains were cultivated in 2xYT (16 g tryptone, 10 g yeast extract, and 5 g NaCI per liter) at 28 °C for seed culture and for genomic DNA extraction. Strains were sporulated in OPAH (1 g Oatmeal, 1 g Pharmamedia, 1 g Arabinose, 0.5 g Humic acid, 0.5 mM KH2PO4, 0.5 mM CaCl2, 0.5 mM MgSO4, 1.9 mg Na2-EDTA'2H20, 1.4 mg FeSO4'7H2O, 0.2 mg H3BO3, 0.05 mg MnSO4'H2O, 0.01 mg ZnSO4'7H2O, 0.01 mg Na2MoO4'2H2O, 0.01 mg CUSO4, 0.01 mg C0CI2, per liter). When appropriate,Docket No. 30275 / 2024-545 media was supplemented with antibiotics: apramycin (50 ug / L), kanamycin (50 ug / mL) and nalidixic acid (25 ug / L).Genome extraction, sequencing, and assembly

[0086] High-quality genomic DNA was prepared using Lucigen Masterpure DNA extraction kit (epicenter) with few modifications. Wild-type and engineered Streptomyces strains were cultivated in 2xYT at 28 °C for 3 days. Cells were pelleted by centrifugation and washed twice with PBS (8g NaCI, 0.1g KCI, 1.44g Na2PO4, 0.22g KH2PO4 per liter, pH=7.4). Biomass was resuspended in 480 .L of EDTA and 120 pL of Lysozyme (10mg / ml) and incubated at 37°C for 45min. Cells were centrifuged for 1min at ~5,000xg, supernatant was discarded, and pellet was resuspended in 200 pL of ‘Tissue and Cell Lysis solution' supplemented with 1 pL of Proteinase K. Samples were incubated at 65 °C for 15 min, and allowed to cooldown at RT for 5 min. Next, samples were incubated at 95 °C for 10 min cooldown at RT, add 30 pL of RNAse A (10mg / ml) and incubate at 37 °C for 1 hr. Genomic DNA was further extracted following kit protocol. Quality of genomic DNA was evaluated by gel electrophoresis.

[0087] Streptomyces eitanensis (wild-type) was sequenced using an hybrid approach combining Nanopore technology and Illumina reads (Plasmidosaurus). Genome assembly, annotation and phylogenetic analysis were performed using KBase50platform. Briefly, read quality was checked with FastQC51, and long-reads were cleaned with Filtong before assembly. Genome was assembled with ‘Unicycler - vO.4.8'24, ‘MaSuRCA Assembler - v3.2.9'52and ‘HybridSPAdes - v3.15.3'53. The quality of genome assemblies was accessed with ‘QUAST v4.4‘54. Genome completeness and contamination was assessed with CheckM55. Genome annotation was performed with RASTtk - v1.07356, and eggnog-mapper57.Tested producing conditions

[0088] Production ‘media base' (Bacto Peptone 5g, Yeast extract 5g, CaCO3 2g per liter, pH=7.0) was supplemented with different carbon and complex nitrogen sources to access concanamycin production (Fig. 2, Fig. 5). Several combinations were tested, where maltodextrin was replaced by soluble starch, potato starch or inulin (30g / L), while Soybean meal was replaced by peanut meal or corn gluten inulin (1 Og / L). GMSYE: ‘media base' plus glucose 10g / L, Maltodextrin 30g / L, Soybean meal 10g / L), GSSY: ‘media base' plus glucose 10g / L, Soluble Starch 30g / L, Soybean meal 10g / L), GICYE: media base' plus glucose 10g / L, Inulin 30g / L, Corn Gluten meal 10g / L), GSPYE: ‘media base' plus glucose 10g / L, Soluble Starch 30g / L, Peanut meal 10g / L), GMSoilYE: ‘media base' plus glucose 10g / L, Maltodextrin 30g / L, Soybean meal 10g / L, Soybean oil (6%)). All organic nitrogen sources, with exception of corn gluten, tested in this work were obtained from animal feed and fertilizers suppliers. Therefore, their oil and purity content might vary from batch to batch. Corn gluten meal (C4773-2) was acquired from Sigma-Aldrich.Docket No. 30275 / 2024-545Regulators homology- phylogenetic analysis

[0089] Phylogenetic analysis of Streptomyces eitanensis concanamycin cluster-situated regulators CmsR and CmsG, and S. lohii bafilomycin cluster-situated regulator BafR was performed as follows. Protein homologues identified in multiple actinomycetes species (top-score BLAST homologues using UniProtKB reference proteomes). Uniprot ID is shown for all proteins. Alignment and phylogenetics analysis of cluster-situated regulators (CmsR and CmsG) and BafR was performed by ETE375, with alignment performed by MAFFT and tree constructed using FastTree225. Phylogenetic tree was visualized in ITOL59. Conserved domain analysis was performed with NCBI ‘Conserved Domain Database' and ‘Conserved Domain Architecture Retrieval Tool' (Fig. 4, Fig. 6).Genome-wide phylogenetic analysis

[0090] Followed by genome assembly and annotation, phylogenomic analysis was performed using SpeciesTree in KBase suit. A set of closely related genomes (n=28) from public databases was constructed using 49 core, universal genes defined by Clusters of Orthologous Groups gene families. Phylogeny was inferred using maximum-likelihood analysis of the selected 49 core genes, and the phylogenetic tree is then reconstructed using FastTree225. FastANI58was also used to estimate whole-genome Average Nucleotide Identity (ANI) between Streptomyces eitanensis assembly and the closest genomes in public database. ANI estimations between Streptomyces eitanensis, S. scabiei 87.22 (GCF_000091305.1), S. stelliscabiei(GCA_001008135.1), S. griseiscabiei (GCA_020010925.1) and S. neyagawaensis (GCF_028863365.1) were 89.8%, 89.9%, 90.5% and 87.9%, respectively. Phylogenetic tree generated by SpeciesTree was then exported to ITOL59for visualization and figure design (Fig. 1 B).Plasmid design and genome editing

[0091] The integrative system, mediated by the attP site of the Streptomyces phage cpC3160, was used for the conjugal transfer of DNA from E. coli S171 to Streptomyces eitanensis. To design plasmids for increased expression, the strong synthetic constitutive promoter asO*p61and fd terminator62was synthetized as gene fragment and assembled into the integrative plasmid pSET15260and pSET152- an63via Gibson Assembly, originating plasmid pSET152- asO*p and pSET152k- asO*p respectively. CmsG and cmsR were amplified from gDNA of Streptromyces eitanensis strain, while bafR was amplified from gDNA of Streptomyces lohiP. Selected regulator genes were amplified and assembled, via Gibson Assembly, in pSET152- asO*p and pSET152k- kasO*p between BamHI and EcoRI restriction sites. Synthetic ribosomal binding sites65were used as overlapping regions for multi-gene vector design. Primers were designed using pyDNA66. The integrative plasmids were first introduced into wild-type Streptomyces eitanensis (this work), S. stelliscabiei (NRRL B-2447), S. neyagawaensis (NRRL ISP-5588), and S. griseiscabiei (NRRL B-2795) via interspecies conjugation from E. coli S171. Upon an incubation at 28 °C for 12 h, each plate was overlaid with 1 mL sterilized water containing 1.25 mg apramycin or kanamycin and 0.5 mg nalidixic acid. After additional 3-5 days, the recombinants were transferred to OPAH plates with 25 pg / mL nalidixic acid and 50 pg / mL apramycin or kanamycin. The resultantDocket No. 30275 / 2024-545 antibiotic resistant strains were PCR confirmed and whole-genome sequencing (Nanopore) using their gDNA as template.Concanamycin A production conditions

[0092] Spores from fresh OPAH plates were inoculated into 50 mL of Peanut meal & starch media (Glucose 10g, Starch 30g, Bacto Peptone 5g, Peanut meal 10g, Yeast extract 5g, CaCO3 2g per liter, pH=7.0) and incubated for 3 days at 28°C. Subsequently, 10 mL of pre-culture was used to inoculate 1L of producing media (GICYE: Corn Gluten & Inulin: Glucose 10g, Insulin 30g, Bacto Peptone 5g, Corn Gluten meal 10g, Yeast extract 5g, CaCO3 2g per liter, pH=7.0). Cultures were incubated at 7 days at 22°C 170 rpm. When appropriated, 20mL of a 29.9% (w / v) solution of sodium propionate was added at 48 h.Sample preparation and quantification of concanamycin A and B

[0093] Quantification of concanamycin A and B, reported in the main manuscript, was performed in triplicate from ImL samples from biomass cultivated in 1L shake-flasks. Samples were collected every day for 10 days (Fig. 5B). For all the remaining quantification data samples were collected on day 7. From each 1L cultures, 1 mL was collected, and biomass was pelleted by centrifugation for 5 min at 20000 rpm. Supernatant was mixed with methanol 50 / 50 and stored at -20 °C until further analysis. Cell pellet was resuspended in 1 mL of methanol and 100 pL of glass beads. Tubes were vortexed for 3h at 4 °C, followed by centrifugation for 5 min at 20000 rpm. Routine quantification of concanamycin A and B production was performed by HPLC (Shimadzu) equipped with a PDA detector analyzed with a C18 column (Luna 5 pm C18(2) - Phenomenex, kept at 40 °C),Water(A) / Acetonitrile(B) (10% to 100%B) was used as mobile-phase at 2 ml / min. Calibration curves were performed with known concentrations of pure concanamycin A and B, production titers were quantified by comparing the standard curve of known concentrations and sample peak areas (AUG) at 280 nm.Biomass quantification

[0094] Throughout cultivation, 1 ml samples were taken and filtered through a pre-dried membrane filters. Cells were washed twice with water and dried in a microwave oven for 1 min67. Sampling was performed in triplicate. Due to small pellets morphology, sampling was performed using sterile wide-bore pipette tips.Isolation and purification of concanamycin A and B

[0095] Total biomass (from 1 L culture) was separated by vacuum filtration. Cell pellets were broken by coating the cells with 500 mL of a mixture of 20% methanol (MeOH) in dichloromethane (DCM) and shaking overnight. The organics were obtained by further vacuum filtration and concentrated. The dried extract was resuspended in 40 mL of DCM, and a 1 mL aliquot was set aside for analysis. The remaining DCM mixture was concentrated with silica to be dry loaded for the first round of normal phase purification. Both normal phase purifications were performed on a Biotage flash column system with a 40g column. For the first round of purification an ethyl acetate / hexane gradient (15-100%) was utilized where concanamycins A-C eluted at 100%. The second round of normal phase was run with a step wise gradient of isopropanol and hexanes / chloroformDocket No. 30275 / 2024-545(1 :3 parts). The steps were 0%, 4%, 8%, 25%, and 40% isopropanol. Concanamycins A-C start to elute around 25%. The crude concanamycin mixture is further processed through a reverse phase purification on a biotage flash column system with 55% acetonitrile in water. Two main peaks were obtained: 1stmajor peak comprised mainly of CMB mixed with minor amounts of CMA and CMC and, 2ndpeak was pure CMA. The first peak was further purified on a preparative HPLC as previously reported12. Purity assessment of isolated compounds was performed using HPLC, NMR and LC-MS / MS. Quantification of concanamycin A and B in crude extracts was also performed using HPLC. All extractions were performed in triplicate.Protein extraction and proteomics analysis

[0096] Proteomics analysis was performed at two time-points, day 4 and 7. Three biological replicates were collected for each strain, Streptomyces eitanensis wild-type and DHS10676 (see Table 1), cultivated under concanamycin producing conditions for the two time points. 10 mL of culture were centrifuged at 3000 rpm for 5 min and cell pellet was washed twice with PBS solution and stored at -80 °C. Frozen cell pellets were lysed using 800 l of lysis buffer (6 M urea, 2 M thiourea, 5 mM DTT, 0.1 M TRIS-HCL and 40 pl of proteases inhibitor cocktail), followed by three cycles of sonication. Preparation of cell lysate for proteomic analysis was executed by following the protocol for the S-trap mini kit (Protifi). Briefly, 23 pl of the cell lysate was diluted in 23 pl of buffer 1 (10% SDS, 100mM TEAB pH 8.5) and then vortexed for 20 secs. For reduction 2 pl of 120mM TCEP was added and incubated at 55°C for 15 min. 2 pl of alkylator (500 mM MMTS in isopropanol) was added and incubated ta room temp for 10 min. Lastly, the sample was acidified with 5 pl of 12% phosphoric acid (aq) prior to loading onto the S-trap micro column for binding and washing of proteins as stated in the protocol. Proteins were digested on the S-trap column with trypsin (Promega) in 50 mM TEAB with an enzyme to protein ration 1:10 at 37 °C overnight. Peptides were eluted with 80 pl each of three elution buffers in series: (1) 50 mM TEAB, (2) 0.2% formic acid (aq), and (3) 50% acetonitrile (aq). Eluted peptides were pooled and dried down under N2.Samples were store dried at -20 °C until further analysis. Peptide samples were resuspended in 0.1% formic acid in water and analyzed with a reversed-phase nanoflow UPLC (Ultimate 3000, Thermo) coupled to an Orbitrap Fusion Lumos MS via nano-ESI in positive ion mode. Samples were injected to a C18 trap column (Acclaim PepMap 100, 75 urn x 2 cm, nanoviper) by a loading pump with 2% ACN with 0.1% formic acid at 5 uL / min, and further separated on a C18 separation column (Acclaim Pepmap RSLC, 75 urn x 50 cm, nanoviper) over a 90 min gradient with a flow rate of 300 nL / min. LC separation and elution was performed by following a gradient method composed of solvent A (2% ACN with 0.1% formic acid) and Solvent B (80% ACN with 0.1% formic acid): 0-5min, 5% B; 5-75 min, 5 to 40%B; 75-82 min, 40 to 95% B; 82-84 min, 95% B; 84-90 min, 5% B. LC- MS / MS analysis was performed in data-dependent mode by applying dynamic exclusion after one scan for 30 seconds. Ions at >5e4signal abundance in the survey MS scan were selected and subjected to HCD MS / MS at 35% normalized activation energy for 3 seconds before acquiring another MS scan and subsequent MS / MS scans. The HCD spectral resolution was set at 15,000.Docket No. 30275 / 2024-545

[0097] Table 1: Engineered Streptomyces strains

[0098] Data processing and statistical analysis'. The acquired nLC-HCD MS / MS data were searched using Sequest HT search algorithm on Proteome Discoverer 2.2 (Thermo Fisher) against Streptomyces eitanensis reference proteome (annotated with eggNOG-mapper) with reversed peptide decoys and common contaminants. A maximum of three missed tryptic cleavages were allowed with fixed C-carbamidomethylation, variable M- oxidation, variable N-deamidation, and variable N-terminal acetylation. Precursor mass tolerance was set at 10 ppm, MS / MS mass tolerance was 0.02 Da., and peptide length were limited to minimum 5 amino acids. False discovery rate (FDR) of 1% was applied for peptide and protein identification. Raw data from Proteome Discoverer 2.2 were loaded into R (2623 proteins). Only proteins that were quantified with two unique peptides and were identified in, at least, two biological replicates were used in downstream analysis. A total of 1891 proteins were then used for differential protein abundance. The output data was cleaned for potential batch effect using limma68and normalized with vsn69. Missing values were imputed with the impute function ‘QRILC with package ‘ImputeLCMD’. Differential protein abundance was calculated with limma68between wild-type and engineered DHS10676 strains at 4 and 7 days. Proteins were classified as 'hit' if fold-change was >1 and with a FDR < 5%.Untargeted metabolomics

[0099] Sample preparation: Samples of intracellular and extracellular metabolites were used for untargeted metabolomics. Culture medium was also extracted, as controls and run alongside experimental samples to account for sampling and laboratory contamination. As well, reagent blanks (MeOH) were prepared and measured to account for extraneous signals occurring from chemicals and analytical systems. All samples were prepared in three biological replicates to demonstrate reproducibility.

[0100] UHPLC-QTOF-MS / MS profiling of crude extracts: Metabolite extracts were analyzed via ultra-high- performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-LCMS) using an Agilent 1290 Infinity II UHPLC coupled to an Agilent 6545 ESI-Q-TOF-MS. The method for data collection is as follows: samples were injected and data acquisition occurred via auto-MS / MS in positive mode. Chromatography was performed using a Phenomenex Kinetex phenyl-hexyl (1.7uM, 2.1 x 50 mm) column and compounds were eluted with an isocratic elution of 90% solvent A (100% H2O + 0.1% formic acid) for 1 minute followed by a 9 minute linear gradient elution to 100% solvent B (95% MeCN + 5% H2O + 0.1% formic acid). The capillaryDocket No. 30275 / 2024-545 temperature was set to 320°C with a source voltage of 3.5kV and a sheath gas flow rate of 11 L / min-1 for electrospray ionization (ESI). Ion fragmentation occurred using ramped collision energy (5 x m / z / 100 + 10 eV) of a maximum of 9 selected precursors per cycle. The positive mode internal lock standards used were Purine C5H4N4 [M + Hb (m / z 121.0508) and hexakis (1 H, 1 H,3H-tetrafluoropropoxy)-phosphazeneC18H18F24N3O6P3 [M + H]+(m / z 922.0098) and ions were included in the static exclusion list. A full static exclusion list was optimized based on high-intensity contaminant ions (>1000 counts) found in blank solvent samples.

[0101] Data processing: Raw mass spectrometry data was converted from vendor-specific instrument files to mzML format using standard peak detection parameters in the msConvert software from ProteoWizard70. Following this, data processing was performed in MZmine3 following an untargeted LC-MS data pre-processing workflow71. Output feature quantification table, peak list, and metadata table was run via feature-based molecular networking on the Global Natural Products Social Molecular Networking (GNPS) platform72. The resultant molecular network was visualized using Cytoscape73.

[0102] Statistical analysis of mass spectrometry data: Statistical analysis of features was performed in R after running in GNPS to pair metadata to features and obtain preliminary annotations74. First, features were filtered to keep only those that were present in two out of the three biological replicates. Next, features that were present in the process and reagent blank samples at greater than 10% intensity were removed from sample feature lists. Subsequently, features were normalized using vsn and missing values were imputed using QRILC imputation.Example 2: Genome analysis of Streptomyces eitanensis

[0103] Streptomyces eitanensis genome was sequenced using Illumina short-read and Oxford Nanopore long-reads, and assembled with Unicycler24. Complete genome assembly consisting of three 'contigs comprising a total of 9,794,182 bp (71.09% GC content) was selected for downstream analysis (Fig. 2A). Assembled genome was annotated with RASP and 8844 coding genes were identified. Next, a phylogenetic analysis using 49 core universal genes was performed with FastTree 225using 31 closely related genomes to estimate the evolutionary relationships between S. eitanensis and reference Streptomyces spp. (Fig. 2B).

[0104] To estimate species demarcation, whole-genome Average Nucleotide Identity (ANI) was also performed between Streptomyces eitanensis genome and the three closest reference genomes, including Streptomyces scabiei 87.22 a known CMA-producing strain26. The estimated ANI between these genomes ranged from 89.8% to 90.5%, suggesting that Streptomyces eitanensis belong to a different Streptomyces species. Biosynthetic potential for secondary metabolite production was also analyzed with ‘antiSMASH v5.1.2’27and ‘Gecco’28. Overall, 38 and 44 biosynthetic gene clusters distributed among two contigs were predicted by antiSMASH and Gecco, respectively (Fig. 2C). One of these clusters was identified as a concanamycin A BGC (~102Kb).

[0105] The organization of the cms BGC in S. eitanensis shows a similar organization and strong amino acid sequence similarity when compared to the known Concanamycin BGCs from Streptomyces neyagawensis (Fig. 2D, Fig. 3A) with a gene identity varying between 85 and 97%.Docket No. 30275 / 2024-545Example 3: Optimization of fermentation conditions for concanamycin A production.

[0106] Concanamycin A (1 in Fig. 1; CMA) production was detected (< 1 mg / L) in wild-type Streptomyces eitanensis strain cultivated in GMSYE at 28°C. GMSYE media is production base media (Bacto Peptone 5g, Yeast extract 5g, CaCO32g per liter, pH=7.0), additionally supplemented with 10g / L glucose, 30 g / L maltodextrin, and 10 g / L soybean meal. Microbial secondary metabolite biosynthesis is regulated by environmental cues such as light, pH, phosphate concentration, oxygenation, temperature, and the identity and quantity of carbon and nitrogen sources29’30. Therefore, the contribution of carbon sources, crude protein (N) sources, and fermentation temperature on CMA production was investigated.

[0107] Polyketides and plecomacrolide production in Streptomyces was shown to be enhanced by soybean oil supplementation31’32. To investigate if CMA yields could also be improved, 6% soybean oil was added to the fermentation medium composition, which completely abrogated production (not detected) (Fig. 3B). GMSYE media uses soybean meal as the crude nitrogen and minerals source. As soybean oil supplementation completely repressed CMA production (Fig. 3B), peanut meal was investigated as an alternative crude protein source. Carbon catabolite repression tightly controls secondary metabolite production, and can be alleviated by cultivation in a media containing a mixture of simple (D-glucose) and complex carbon sources (such as oils, polysaccharides)2933.

[0108] Maltodextrin, initially used as a complex carbon source, consists of 2 to 20 monomers of glucose. Thus, it was reasoned that replacing maltodextrin with a larger polysaccharide, such as soluble starch, could relieve glucose repression increasing secondary metabolite production. Indeed, the new fermentation medium, containing soluble starch and peanut meal, enhanced CMA production by 31 -fold (Fig. 3B). Additionally, decreasing the cultivation temperature to 22°C led to a 6.7-fold increase of CMA titer. Finally, replacing starch with a polysaccharide consisting of D-fructose units and reducing the oil content in the crude protein source slightly increased concanamycin A titers (Fig. 3B). Apart from concanamycin A, production of natural analogues concanamycin B (2) and C (3) was also detected by LC-MS / MS in the optimized culture conditions, at titers below the quantification limit. Optimization of medium composition and fermentation temperature boosted concanamycin A titers to 95.9±17.2 mg / L in the wild-type Streptomyces eitanensis strain that, to date, is the highest reported in shake-flask cultivation.Example 4: Overexpression of cluster-situated regulators.

[0109] Phylogenetic analysis was used to investigate the evolutionary relationship of cluster-situated regulators, revealing a high similarity between Orf3 and Orf17* from Streptomyces eitanensis and S. neyagawensis (Fig. 4A-4B). Hereafter, the Streptomyces eitanensis proteins homologous to Orf3 and Orf17* are referred to as CmsR and CmsG, respectively.

[0110] To investigate the role of pathway regulators in concanamycin production, cmsR and cmsG were overexpressed in Streptomyces eitanensis following their amplification from genomic DNA and assembly into pSET152 under the constitutive synthetic promoter KasO* (Fig. 5A). The resulting plasmids were integrated intoDocket No. 30275 / 2024-545Streptomyces eitanensis genome, generating DHS10671 (pSET152- cmsR), DHS10672 (pSET152k- cmsG) and DHS10673 (pSET152k- cmsG-cmsR) strains (Fig. 5B, Table 1). The production of concanamycin A in each engineered strain was assessed under optimized fermentation conditions (described above, Fig. 3C).

[0111] Overexpression of cmsR, improved CMA production by 2.3-fold (236.2±17.4 mg / L) compared to the wild-type (Fig. 5C). Maximum concanamycin A production was obtained in the strain overexpressing cmsG (DHS10672) reaching 308.5±32.6 mg / L (3.8-fold improvement) compared to the wild-type strain (Fig. 50). Interestingly, concanamycin A production was not enhanced in strain DHS10673 (pSET152k-cmsG-cmsR) compared to engineered strain DHS10672, overexpressing cmsG alone. Although atf-site integration was successfully achieved in Streptomyces eitanensis, disruption of cmsG and cmsR by homologous recombination was not feasible. Nevertheless, these results indicate that overexpression of cmsR and cmsG positively impacts concanamycin A biosynthesis in Streptomyces eitanensis.Example 4: Heterologous expression of the bafilomycin cluster-situated HTH-DNA binding protein (BafR) in Streptomyces eitanensis

[0112] Streptomyces lohii BafG is a close homologue of the concanamycin cluster-situated gene CmsG from Streptomyces eitanensis (Fig. 4B). However, a phylogenetic analysis of BafR revealed homologues containing the LuxR-type HTH-domain in multiple actinomycetes species (Fig. 6A-6B), but not in Streptomyces eitanensis. As overexpression of targeted heterologous regulatory proteins has been shown to complement activity of native activators37, whether concanamycin A production could be improved by heterologous expression of bafR in the Streptomyces eitanensis strain was assessed. BafR was amplified from S. lohii genomic DNA and assembled in the integrative expression plasmid pSET152 under the constitutive synthetic promoter asO*p. The resulting plasmid (pSET152k-ba / R) was then integrated into Streptomyces eitanensis genome, generating DHS10674 (see Table 1). Heterologous expression of bafR slightly decreased CMA production compared to the wild-type Streptomyces eitanensis strain (Fig . 7A). Constitutive expression of bafR in combination with the native con cluster-situated regulator cmsG was also investigated by integrating pSET152k-baff?-cmsG plasmid into Streptomyces eitanensis genome, generating DHS10675 strain. Overexpression of native cmsG and the heterologous bafR, slightly improved concanamycin A production compared to overexpression of cmsG alone (Fig. 7A). An integrative plasmid (pSET152-baff?-cmsG-cmsR) was also designed for overexpression of bafR and both CMA cluster-situated regulators cmsG and cmsR (Fig. 8A). Integration of bafR, cmsG, and cmsR at the attP site was confirmed by whole-genome sequencing (Fig. 8B), yielding the engineered DHS10676 strain.

[0113] Concanamycin A titers in DHS10676 was 909.8±64.7 mg / L, a 10-fold improvement compared to wildtype under the same cultivation conditions (Fig. 7A, Table 2). Concanamycin A production was determined over a time course and maximum titers were observed at day 7 for DHS10676 (Fig. 7B). Production of concanamycin B was also improved in the engineered strains DHS10675 and DHS10676, 16.7±12.5 and 38.8±7.4 mg / L, respectively (Fig. 10A, Table 2). Overexpression of three BGC regulators and overproduction of concanamycins, did not significantly decrease biomass (DCW) compared to wild-type levels (Fig. 7C).Docket No. 30275 / 2024-545

[0114] Table 2: Concanamycin A, B and C quantification and isolated titersQuantification of Concanamycin A and B from biomass (Area Under the Curve using calibration curve of known concentrations)Quantification of Concanamycin A and B from crude extracts (Area Under the Curve using a calibration curve of known concentrations)cPurified concanamycin A and B with purity >90%.dPurified conanamycin C with purity <80%.

[0115] Next, the impact of organic nitrogen sources on concanamycin A production was determined in the engineered strain DHS10676. In contrast to what was observed with wild-type (Fig. 3B), overexpression of the three regulators enable the production of concanamycin A across all organic nitrogen sources tested at comparable titers (Fig. 7A).

[0116] Analysis of the cms BGC with antiSMASH (v 7.1.0.) revealed a putative transcription-factor binding site for a zinc-responsive repressor. As zinc supplementation has been shown to affect secondary metabolite production in multiple Streptomyces species38, it was determined whether concanamycin A production could be activated or repressed by this nutrient. Concanamycin A production in DHS10676 was not affected by increasing exogenous Zn2+(Fig. 7A).

[0117] Concanamycin A production has been reported at low titers in a handful of Streptomyces species19’2639.To understand if the designed plasmid for constitutive expression of bafR, cmsG, and cmsR (Fig. 8A) could activate cms BGC expression across species, strains S. stelliscabiei, S. griseiscabiei, and the known concanamycin producer S. neyagawensis were engineered with the aforementioned plasmid (Fig. 1 C, 1D, Fig. 8C). Improved production of concanamycin A was observed in all engineered strains when compared toDocket No. 30275 / 2024-545 respective wild-type strains (Fig. 7D). Production of concanamycins in S. stelliscabiei and S. griseiscabiei are reported for the first time herein. The maximum titer of 442 mg / L was observed in the engineered S. stelliscabiei (Fig. 7D). Disclosed herein, engineered S. neyagawensis produced 107 mg / L of concanamycin A (Fig. 7D), a 5- fold increase compared with the recently reported expression of S. neyagawensis concanamycin cluster in a chassis strain17. Overexpression of Streptomyces eitanensis cluster-situated regulators, cmsG and cmsR, and the bafilomycin inducer-independent regulator bafR, improved concanamycin A and B production across species, enabling ready access to these structurally complex plecomacrolides for semi-synthetic modifications.Example 5: Proteomics and metabolomics identifies a metabolic switch to CMA / B overproduction.

[0118] The next strain characterized was the engineered strain DHS10676 and wild-type Streptomyces eitanensis, using proteomics and metabolomics under concanamycin production conditions (Fig. 11). Relative protein abundances between DHS10676 and wild-type strains at day 4 and 7 were determined using label-free quantification analysis. Overall, protein abundances between samples cluster based on growth / production stage (4 vs 7 days) (Fig. 11A-11 B). Variance between wild-type and engineered strain (DHS10676) was driven by the differential expression of proteins from the cms BGC (Fig. 11 B). Most of these proteins were identified in DHS10676 and in wild-type strains (Fig. 5A). Interestingly, the overexpressed CmsR was not detected in either DHS10676 or the wild-type strain at day 4 or 7. BafR was only identified in the engineered DHS10676 strain on day 4 of cultivation and not in wild-type (Fig. 11C). CmsG was identified at both sampling times (4 and 7 days) but only in DHS10676 (Fig. 110). Overall, the engineered DHS10676 strain showed a significantly increased abundance of proteins from the cms BGC compared to wild-type cultivated under the same conditions (Fig. 5A). The conclusion drawn from this analysis was that overexpression of the three regulators {bafR, cmsG, cmsR) led to increased levels of biosynthetic proteins involved in concanamycins production.

[0119] As a next step, the non-targeted metabolome of S. eitanensis wild-type and engineered DHS10676 was analyzed, under concanamycin-producing conditions. Metabolites were visualized and annotated by featurebased molecular networking40, which revealed a total of 142 nodes, from which 69 are singletons (Fig. 9B). Only 22 features were annotated by GNPS40, using a level 2 analysis. All features from the concanamycins node (Fig. 9B, Fig. 11D) show increased abundance in engineered DHS10676 compared to wild-type, with the exception of the parent ion 697.4266 m / z, which corresponds to concanamycin H, the ‘open hemiketal' analog of CMA (Fig. 12)41’42. Besides concanamycins, molecular network (Fig. 9B) and clustering (Fig. 11 A) analysis revealed a distinct shift in the identity and abundance of intracellular metabolites from the two strains.Example 6: Sodium propionate supplementation improves concanamycin A and concanamycin B production

[0120] Concanamycin B is a less potent inhibitor of V-ATPase compared to its natural analog concanamyin A, however it has shown promise for osteoclastic V-ATPase inhibiting bone resorption and as a suppressor of antigen presentation by MHC-class II molecules43’44. The Streptomyces eitanensis wild-type strain only produces concanamycin B with titers below the limit of detection. The engineered Streptomyces eitanensis strain,Docket No. 30275 / 2024-545DHS10676, produced 38.8±7.5 mg / L of concanamycin B, which was well below levels achieved for concanamycin A (Fig. 10B). In concanamycin B the C-8 ethyl group derived from ethylmalonyl-CoA was replaced by a methyl group that is derived from methylmalonyl-CoA5(Fig. 3A). Elongation of the concanamycin backbone was shown to incorporate up to seven propionate units in 1, and eight in 2 (see Fig. 1)45. Previous studies reported that addition of low concentrations of sodium propionate can favor incorporation of methylmalonyl-CoA extender units in promiscuous type I PKS AT domains46’47. Additionally, supplementation of sodium propionate at later stages of fermentation was shown to further improve secondary metabolite production in select Streptomyces strains4648. Due to the low titers of concanamycin B, it was reasoned that supplementation with sodium propionate could improve methylmalonyl-CoA pools and enhance concanamycin B production. Therefore, the impact of sodium propionate concentration and supplementation time on concanamycin A and concanamycin B production was investigated by cultivating the engineered DHS10676 strain in small-scale cultures (50 mL). The addition of 0.6% (w / v) of sodium propionate at 48h yielded the highest production of concanamycin B in DHS10676 (Fig. 12A).

[0121] Next, cultivation of wild-type, DHS10673, DHS10674, DHS10675 and DH510676 was conducted with or without supplementation of 0.6% (w / v) of sodium propionate at 48h. Addition of sodium propionate improved concanamycin B production in engineered strains DHS10675 and DHS10676 (Fig. 10), while very low improvement was observed in wild-type and the remaining engineered strains cultivated under the same conditions (Fig. 10, Fig. 12C and 12D). Sodium propionate supplementation (0.6%) enhanced concanamycin A and B titers in DHS10675 to 643.1±30.0 mg / L and 208.5±31.7 mg / L, respectively (Fig. 10A-10B). In the engineered strain DHS10676 concanamycin A titers was 763.6±83.3 mg / L, while the maximum titer of concanamycin B was 306.5±42.05 mg / L (Fig. 10A-10B). Principal component analysis of untargeted metabolomics showed that cultivation with sodium propionate changed the metabolic profile of both engineered and wild-type, reducing the variance between wild-type and engineered strain in the first principal component (PC1 , 55% of total variance) (Fig. 10C). Moreover, untargeted metabolomics was in concordance with targeted analysis, capturing the increased relative abundance of concanamycin B in the engineered strain DHS10676 and wild-type cultivated with sodium propionate (Fig. 12C).

[0122] In the current work, strain I culture condition optimization was combined with improved isolation and purification methods for concanamycin A, B and C (Table 2). After cultivation, concanamycins were obtained by solvent extraction followed by multiple rounds of liquid-chromatography (see methods for detailed protocol; Example 1). As expected, isolated yield titers were lower than quantified titers (using AUG) across all tested strains and conditions (Fig. 10D). It was surmised that loss of material occurred at various stages due to incomplete cell disruption, organic extraction of concanamycins and by the multiple rounds of liquidchromatography required to separate concanamycin A, B, and C from the other compounds produced by this strain. Nevertheless, purified concanamycin A from DHS10676, cultivated with sodium propionate, reached 483.1 ±25.8 mg / L, an 86% recovery of the total quantified compound from crude extract (Fig. 10D, Table 2). Supplementation of sodium propionate also boosted concanamycin B to 159,4±41 .8 mg / L from the DHS10676Docket No. 30275 / 2024-545 engineered strain, an 80-fold improvement compared with wild-type. Thus, addition of sodium propionate has not only increased concanamycin A and B titers, but also decreased the production of several pigmented nuisance compounds, thereby facilitating isolation and purification.

[0123] Various embodiments are described hereinafter. It should be noted that the specific embodiments are not intended as an exhaustive description or as a limitation to the broader aspects discussed herein. One aspect described in conjunction with a particular embodiment is not necessarily limited to that embodiment and can be practiced with any other embodiment(s). Reference throughout this specification to "one embodiment”, "an embodiment,” "an example embodiment,” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases "in one embodiment,” "in an embodiment,” or "an example embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some, but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention. For example, in the appended claims, any of the claimed embodiments can be used in any combination.References:(1) Atanasov, A. G.; Zotchev, S. B.; Dirsch, V. M.; Supuran, C. T. Natural Products in Drug Discovery: Advances and Opportunities. Nat Rev Drug Discov 2021 , 20 (3), 200-216.(2) Newman, D. J.; Gragg, G. M. Natural Products as Sources of New Drugs over the Nearly Four Decades from 01 / 1981 to 09 / 2019. J. Nat. Prod. 2020, 83 (3), 770-803.(3) Pham, J. V.; Yilma, M. A.; Feliz, A.; Majid, M. T.; Maffetone, N.; Walker, J. R.; Kim, E.; Cho, H. 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U.; Zeeck, A. The chemistry of unusual macrolides, 2. Spectroscopic and biosynthetic investigations of the V-type ATPase inhibitor concanamycin A. Liebigs Annalen der Chemie 1994, 1994 (3), 305- 312.Table 3: Sequence TableDocket No. 30275 / 2024-545Docket No. 30275 / 2024-545

Claims

Docket No. 30275 / 2024-545What is claimed is:1 . An engineered Streptomyces bacteria having increased expression of the bafilomycin cluster- situated HTH-DNA binding (BafR) protein; wherein the wild-type variant of the Streptomyces bacteria does not express the BafR protein.

2. The engineered Streptomyces bacteria of claim 1 , wherein the BafR protein comprises:I) an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO:1, ii) the amino acid sequence of SEQ ID NO: 1, ill) an amino acid sequence that is encoded by a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 4, or iv) an amino acid sequence that is encoded by a nucleotide sequence of SEQ ID NO: 4.

3. The engineered Streptomyces bacteria of claim 1 wherein the bacteria comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 4, or comprises the nucleotide sequence of SEQ ID NO: 4.

4. The engineered Streptomyces bacteria of any one of claims 1-3, wherein the bacteria has increased expression of the CmsG protein.

5. The engineered Streptomyces bacteria of claim 4, wherein the CmsG protein comprises:I) an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO:2, ii) the amino acid sequence of SEQ ID NO: 2, ill) an amino acid sequence that is encoded by a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 5, or iv) an amino acid sequence that is encoded by a nucleotide sequence of SEQ ID NO: 5.

6. The engineered Streptomyces bacteria of claim 4, wherein the bacteria comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 5, or comprises the nucleotide sequence of SEQ ID NO: 5.

7. The engineered Streptomyces bacteria of any one of claims 1-6, wherein the bacteria has increased expression of the CmsR protein.

8. The engineered Streptomyces bacteria of claim 7, wherein the CmsR protein comprises:I) an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95%% identical to SEQ ID NO:3,Docket No. 30275 / 2024-545 ii) the amino acid sequence of SEQ ID NO: 3, ill) an amino acid sequence that is encoded by a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 6, or iv) an amino acid sequence that is encoded by a nucleotide sequence of SEQ ID NO: 6.

9. The engineered Streptomyces bacteria of claim 7, wherein the bacteria comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 6, or comprises the nucleotide sequence of SEQ ID NO: 6.

10. The engineered Streptomyces bacteria of claims 1-9, wherein the bacteria has increased expression of the BafR protein, the CmsG protein, and the CmsR protein.11 . The engineered Streptomyces bacteria of claim 10, wherein the BafR protein comprises the amino acid sequence of SEQ ID NO: 1, the CmsG protein comprises the amino acid sequence of SEQ ID NO: 2, and the CmsR protein comprises the amino acid sequence of SEQ ID NO: 3.

12. The engineered Streptomyces bacteria of claim 10, wherein the BafR protein is encoded by the nucleotide sequence of SEQ ID NO: 4, the CmsG protein is encoded by the nucleotide sequence of SEQ ID NO: 5, and the CmsR protein is encoded by the nucleotide sequence of SEQ ID NO: 6.

13. The engineered Streptomyces bacteria of any one of claims 1-12, wherein the bacteria produces high levels of Concanamycin A.

14. A culture comprising the engineered Streptomyces bacteria of any one of claims 1-13.

15. The engineered Streptomyces bacteria of any one of claims 1-14, wherein the bacteria Streptomyces eitanensis, Streptomyces stelliscabiei, Streptomyces griseiscabiei, Streptomyces scabiei, or Streptomyces neyagawensis.

16. A plasmid comprising a nucleotide sequence encoding the BafR protein operably linked to a constitutive promoter.

17. The plasmid of claim 16, wherein the nucleotide sequence encoding the BafR protein comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 4, or comprises the sequence of SEQ ID NO: 4.

18. The plasmid of claim 16, wherein the BafR protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 1, or comprises the amino acid sequence of SEQ ID NO: 1.

19. The plasmid of claim 16, wherein the promoter is KasO*p.

20. The plasmid of claim 16, wherein the promoter comprises SEQ ID NO: 7.Docket No. 30275 / 2024-54521 . The plasmid of claim 16, wherein the plasmid further comprises a nucleotide sequence encoding the CmsG protein operably linked to the constitutive promoter.

22. The plasmid of claim 21 , wherein the nucleotide sequence encoding the CmsG protein comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 5, or comprises the sequence of SEQ ID NO: 5.

23. The plasmid of claim 21 , wherein the CmsG protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 2, or comprises the amino acid sequence of SEQ ID NO: 2.

24. The plasmid of claim 21 , wherein the promoter is KasO*p.

25. The plasmid of claim 21, wherein the promoter comprises SEQ ID NO: 7.

26. The plasmid of claim 21 , wherein the plasmid further comprises a nucleotide sequence encoding the CmsR protein operably linked to the constitutive promoter.

27. The plasmid of claim 26, wherein the nucleotide sequence encoding the CmsR protein comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 6, or comprises the nucleotide sequence of SEQ ID NO: 6.

28. The plasmid of claim 26, wherein the CmsR protein comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 3, or comprises the amino acid sequence of SEQ ID NO: 3.

29. The plasmid of claim 26, wherein the promoter is KasO*p.

30. The plasmid of claim 26, wherein the promoter comprises SEQ ID NO: 7.31 . A method of engineering a bacteria comprising contacting a Streptomyces bacteria with the plasmid of any one of claims 16-30.

32. A method of producing Concanamycin A comprising culturing the engineered Streptomyces bacteria of any one of claims 1-15.

33. The method of claim 32, wherein the method further comprises culturing the engineered Streptomyces bacteria, and wherein the culture comprises:I) peanut meal as a nitrogen source; ii) glucose; andII) inulin as a carbon source; and wherein the culture is incubated at a temperature between about 20°C to about 27°C.Docket No. 30275 / 2024-54534. The method of claim 33, wherein the nitrogen source is soybean meal, corn gluten, peanut meal, or a combination thereof.

35. The method of claim 33 or claim 34, wherein the carbon source is maltodextrin, potato starch, inulin, soluble starch, or a combination thereof.

36. The method of any one of claims 32-35, wherein the Concanamycin A titers are at least about 107 mg / L.

37. The method of any one of claims 33-35, wherein the engineered bacteria is Streptomyces eitanensis, wherein the engineered bacteria further comprises the plasmid of claims 26-30 or the bacteria of claim 10-12, and wherein Concanamycin A titers are at least about 843 mg / L.

38. A method of producing Concanamycin A and Concanamycin B comprising culturing the engineered Streptomyces bacteria of claims 4-10, wherein the culture is supplemented with 0.2-1 .0% w / v sodium propionate.

39. The method of claim 38, wherein Concanamycin A titers are at least about 613 mg / L, and Concanamycin B titers are at least about 175 mg / L.

40. A method of producing Concanamycin A, the method comprising culturing wild-type Streptomyces eitanensis bacteria in liquid production media base, wherein the culture comprises :I) corn gluten as a nitrogen source; ii) glucose; and ill) inulin as a carbon source; and wherein the culture is incubated at a temperature between about 20°C to about 27°C.41 . The method of claim 40, wherein the nitrogen source is peanut meal, corn gluten, or a combination thereof.

42. The method of claim 40 or claim 41 , wherein the carbon source is soluble starch, inulin, or a combination thereof.

43. The method of any one of claims 40-42, wherein Concanamycin A titers are at least about 77mg / L.

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