WO2017151059A1 - Activation multiplexable de groupes biosynthétiques silencieux dans des hôtes actinomycètes natifs pour la découverte de produits naturels - Google Patents

Activation multiplexable de groupes biosynthétiques silencieux dans des hôtes actinomycètes natifs pour la découverte de produits naturels Download PDF

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WO2017151059A1
WO2017151059A1 PCT/SG2017/050092 SG2017050092W WO2017151059A1 WO 2017151059 A1 WO2017151059 A1 WO 2017151059A1 SG 2017050092 W SG2017050092 W SG 2017050092W WO 2017151059 A1 WO2017151059 A1 WO 2017151059A1
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streptomyces
cluster
promoter
gene
biosynthetic
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Mingzi Zhang
Fong Tian Wong
Huimin Zhao
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Agency For Science, Technology And Research
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    • C12N15/09—Recombinant DNA-technology
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/76—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Actinomyces; for Streptomyces

Definitions

  • the present invention relates generally to the field of molecular biology.
  • the present invention relates to recombinant genomic editing methods and protein expression.
  • the present invention refers to a recombinant method of activating expression of one or more biosynthetic gene cluster(s), or one or more target gene(s) in a biosynthetic gene cluster comprising more than one gene, the method comprising inserting one or more promoter(s) at one or more transcriptionally functional location(s) relative to the biosynthetic gene cluster(s) or the target gene(s) in the biosynthetic gene cluster(s), whereby the insertion of the promoter(s) results in increased expression of the biosynthetic gene cluster(s) or target gene(s) compared to the expression level of an unmodified biosynthetic gene cluster(s) or target gene(s), wherein the promoter(s) is/are inserted using CRISPR technology.
  • the present invention refers to a recombinant expression plasmid for activating expression of a biosynthetic gene cluster(s), the plasmid comprising one or more promoter(s) as disclosed herein, a biosynthetic gene cluster(s) or one or more target gene(s) as disclosed herein.
  • Fig. 1 shows that the use of CRISPR-Cas9 technology improves genome engineering of Streptomyces.
  • A Conventional gene replacement and genetic knock-in by homologous recombination in Streptomyces involve two steps. In the first step, integration of the suicide plasmid by single crossover is selected for by positive selection. Crossover can occur at either of the homologous regions marked in different shades. Subsequent negative selection yields double crossover clones with either wild type sequence or desired genomic modification.
  • B The gRNA- guided Cas9 nuclease encoded on a replicative plasmid creates a double stranded break at the target genomic locus adjacent to the PAM sequence. Cells that carry out homology-directed repair in the presence of an editing template with homologous flanks survive. For this method, only a single selection step for the plasmid is needed.
  • Fig. 2 shows the activation of the silent indigiodine biosynthetic gene cluster and introduction of heterologous gene in 5. albus.
  • A Part of the indigiodine cluster in 5. albus (not to scale). Indicated is the target site of CRISPR -Cas9 and the introduction site of the kasO*p or tsr- kasO*p for activation of the cluster depending on the donor DNA used, tsr encodes for a thiostrepton resistant gene product.
  • B Knock-in efficiencies of kasO*p or tsr-kasO*p in 5. albus with and without (no protopsacer) targeted double-stranded breaks by CRISPR-Cas9.
  • Fig. 3 shows the results of the activation of the silent undecyprodigionine (RED) biosynthetic gene cluster in 5. lividans.
  • A Part of the RED cluster in 5.
  • lividans (not to scale). Indicated is the target site of CRISPR-Cas9 and the introduction site of the kasO*p by homologous recombination with donor DNA harbouring homologous ends.
  • Arrowheads indicate the relative positions and identities of the primers used for diagnostic PCR to determine knock -in efficiencies.
  • Orange arrowheads indicate kasO*p-specific primers not present in 5. lividans genome.
  • B Knock-in efficiencies of 5.
  • C Diagnostic PCR from genomic DNA isolated from wild type (wt) and exconjugants using the indicated primers to determine kasO*p knock -in at the designated genomic locus. For control PCR of the left and right flanks, primer pairs 1+2 and 3+4 were used respectively. For detection of kasO*p knock-in, primer pairs 1+5 and 3+6 were used.
  • D Wild type and engineered 5. lividans strains on ISP2 plates.
  • E Liquid ISP2 cultures of wild type and engineered 5. lividans strains.
  • Fig. 4 shows the results of the activation of the silent actinorhodin (ACT) biosynthetic gene cluster in 5.
  • ACT silent actinorhodin
  • A Part of the RED cluster in 5.
  • lividans (not to scale). Indicated is the target site of CRISPR-Cas9 and the introduction site of the kasO*p by homologous recombination with donor DNA harbouring homologous ends. The red arrowheads represent primers beyond the homologous regions used for diagnostic PCR of successful knock-in.
  • B Efficiency of kasO*p knock-in for the ACT cluster in 5. lividans.
  • Fig. 5 shows the activation of a silent phosphonate biosynthetic gene cluster in 5. roseosporus.
  • A Uncharacterized phosphonate cluster in 5. roseosporus with homology to the known FR-900098 cluster in S. rubellomurinus in addition to genes predicted to encode for NRPSs and phosphonate transporters.
  • Inset shows a more detailed view of the FR-900098 biosynthetic gene homologs. Promoters are inserted by targeting CRISPR-Cas9 to a region between pepM and frbC homologs indicated by the diverging ORFs in red.
  • Fig. 6 shows the results of multiplex activation and engineering of silent and/or cryptic biosynthetic gene clusters in actinomycetes for natural product discovery. Strategic insertion of constitutive promoters is sufficient to activate expression of relevant genes within the biosynthetic gene cluster and production of the cognate natural product. The same technology can also be used to perform in situ engineering of the gene cluster for the generation of natural product derivatives. Altogether, these approaches will increase the chemical diversity of existing actinomycete strain collections for bioactivity screening and accelerate the natural product discovery process.
  • FIG. 7 presents data showing the relative promoter strengths in different actinomycetes.
  • a copy of the xylE gene driven by the indicated promoters were integarated into the genomes of (A) 5. roseosporus, (B) Micromonospora sp. and (C) 5. erythraea. 1, 2 and 3 day old cultures were harvested and specific xylE activity of the cell lysates was determined.
  • Fig. 8 shows a representation of the biosynthetic pathway for FR-900098.
  • A Part of the phosphonate biosynthetic gene cluster in 5. roseosporus with homology to FR-900098 cluster in 5. rubellomurinus. Genes are labelled according to their homologs in 5. rubellomurinus. Site of promoter(s) insertion is indicated by the red arrow.
  • B FR-900098 cluster in 5. rubellomurinus and the proposed biosynthetic pathway of FR-900098.
  • Figure is obtained from Metcalf et al l
  • C Table showing % identity between homologous genes in the phosphonate cluster of 5. roseosporus and those from the FR-900098 cluster in 5. rubellomurinus.
  • Fig. 9 shows the nucleic acid sequences of the constitutive promoters used for cluster activation. ATG start codons of genes to be activated are underlined.
  • Fig. 10 shows a scheme and sequences of adapters introduced into pCRISPomyces at the Xbal site for making promoter (single and bidirectional) knock-in constructs.
  • promoters such as ermE*p and rcfp
  • the adapter sequences used are the same.
  • Fig. 11 shows a scheme of different cloning schemes for CRISPR/Cas9 editing plasmids for cluster activation.
  • Three cloning schemes were tested over the course of this study to assembly the final genome editing plasmids.
  • Scheme 1 was used to generate editing plasmids for 5. albus.
  • Scheme 2 was used to assemble S. lividans plasmids and a fraction of the 5. roseosporus constructs.
  • Scheme 3 involving modified pCM2 plasmids proved to be the most efficient and were used to make majority of the plasmids in the study. The advantages and limitations of each scheme are listed.
  • Fig. 12 shows a schematic and the results of CRISPR-Cas9-based promoter knock-in strategy to activate silent biosynthetic gene clusters in streptomycetes.
  • Fig. 13 shows graphs depicting the result of the activation of biosynthetic gene clusters in multiple streptomycetes.
  • Fig. 14 shows the results of large scale purification and structural identification of major products from activated polycyclic tetramate macrolactam cluster in 5. roseosporus.
  • A HPLC analysis of crude and fractionated ethyl acetate extracts from 100 ISP2 plates. Extracted ion chromatograms of the 100% methanol fraction contains the major ions m/z 511 and 513 that were produced with activation of the cryptic polycyclic tetramate macrolactam cluster.
  • B The two major products were identified to be photocyclized alteramide A and HSAF. Minor products are likely to be alteramide A and its derivative.
  • Fig. 15 depicts the results of the activation of type II PKS biosynthetic gene cluster in 5.
  • viridochromogenes which yields a novel pigmented compound, (a) Production of brown pigment by the engineered strain but not wild type (wt) 5. viridochromogenes on MGY medium, (b) HPLC analysis of extracts from an engineered 5. viridochromogenes strain harbouring a kasO*p knock-in in front of SSQG_RS26895 (gray) and the parent wild type strain (black). Indicated is the major metabolite 4 that is uniquely produced by the engineered strain. Here the focus is on the major distinct metabolite produced by the engineered strain but it is noted that there are additional differences between the engineered and wild type strain (Fig. 34). (c) Chemical structure of 4. The five rings are labelled A to E.
  • Fig. 16 shows the results of a CRISPR-Cas9 mediated promoter knock-in for activation of pigment biosynthetic gene clusters (BCGs).
  • BCGs pigment biosynthetic gene clusters
  • primer pairs 1+2 and 3+4 were used respectively.
  • primer pairs 1+5 and 3+6 were used,
  • PCR product from genomic DNA isolated from wild type (wt) and exconjugants were subjected to BstBI-digestion to determine kasO*p knock- in at the designated genomic locus within the ACT cluster in 5.
  • lividans. M refers to molecular weight ladder. Arrow heads refer to location of primers used for polymerase chain reactions (PCR).
  • Fig. 17 shows graphs depicting the results of liquid chromatography-mass spectrometry (LCMS) analysis of 5.
  • albus strain with activated indigoidine biosynthetic gene cluster (a) HPLC analysis (UV detection at 600 nm) of acidic methanol from wild type (WT) S. albus and the indicated engineered strain (Indigoidine) in which kasO*p was introduced into indigoidine cluster in front of the indC-like ORF.l (b) The masses of the two new major metabolites at 5 min and 5.3 min, indicated by (*), are consistent with indigoidine -related metabolites (m/z 249, 250) and their adducts (m/z 308, 292).
  • Fig. 18 shows graphs depicting the results of liquid chromatography-mass spectrometry (LCMS) analysis of 5.
  • LCMS liquid chromatography-mass spectrometry
  • Fig. 19 shows images of the production of pH-sensitive pigments by engineered 5.
  • lividans strain Wild type (wt) and engineered 5.
  • lividans strains with activated ACT cluster were streaked onto MGY medium.
  • the plate left panel
  • Fig. 20 shows graphs depicting results of liquid chromatography-mass spectrometry (LCMS) analysis of 5.
  • LCMS liquid chromatography-mass spectrometry
  • coelicolor is known to produce different actinorhodin-related metabolites, including gamma-actinorhodin.
  • Fig. 21 show the results of RT-qPCR analysis of 5. roseosporus polycyclic tetramate macrolactam cluster 24.
  • SSGG_ RS02310 is located within the gene cluster and was used as a negative control (NC) for RT-qPCR assay as an example of a gene whose expression is unaffected by knock-in of the kasO*p promoter cassette. Site of kasO*p knock-in is indicated by the arrowhead.
  • Fig. 22 shows the results of LCMS analyses of polycyclic tetramate macrolactam compounds produced by 5. roseosporus.
  • HPLC analysis UV detection at 320 nm
  • ethyl acetate extracts from wild type 5. roseosporus and the indicated engineered strain in which kasO*p is introduced into cluster 24.
  • Fig. 23 shows the results of RT-qPCR analysis of 5. roseosporus phosphonate cluster 10.
  • SSGG_RS 16990 and SSGG_RS 16985 are plotted separately due to differences in scale. Error bars represent the standard deviation of biological triplicates, n.d. indicates undetectable transcript levels,
  • SSGG_RS 16955 is located near the FR-900098 cluster and was used as a negative control (NC) for the RT-qPCR assay as an example of a gene whose expression is unaffected by knock -in of the kasO*p-P8 promoter cassette.
  • Site of kasO*p-P8 promoter cassette knock-in is indicated by the arrowhead.
  • Fig. 24 shows the introduction of kasO*p-P8 promoter cassette for activation of the phosphonate biosynthetic gene cluster in 5. roseosporus. Shown from bottom to top are 1) the native genomic locus with the location of chosen PAM and protospacer sequences, 2) the edited genome locus with the inserted kasO*p-P8 promoter cassette and 3) the sequence traces of the two junctions flanking the promoter cassette. Biosynthetic genes needed for FR-900009 are highlighted in dark and middle gray.
  • Fig. 25 shows the schematic locations of promoter knock-in for 5. roseosporus clusters. Dark gray genes are putative biosynthetic genes while middle gray genes are transport-related and regulation-related genes, respectively. Sites of single or bidirectional promoter cassette knock -in are indicated by the arrowheads. [0032] Fig. 26 shows the schematic location of promoter knock-in for S. venezuelae cluster 16. Indicated in dark gray are putative biosynthetic genes while middle gray genes are transport -related and regulation-related genes, respectively. Site of bidirectional kasO*p-P8 cassette knock-in is indicated by the arrowhead.
  • Fig. 27 shows graphs depicting the results of LCMS analysis of 5. roseosporus strain with an engineered cluster 3.
  • HPLC analysis UV detection at 254 nm
  • ethyl acetate extracts from wild type 5. roseosporus and the indicated engineered strain in which kasO*p is introduced into cluster 3. The major unique product produced by the engineered strain is indicated by (*).
  • Fig. 28 shows graphs depicting the results of LCMS analysis of 5. roseosporus strain with an engineered cluster 18.
  • HPLC analysis UV detection at 254 nm
  • ethyl acetate extracts from wild type 5. roseosporus and the indicated engineered strain in which kasO*p is introduced into cluster 18. The major unique ion detected for the engineered strain is indicated by (*).
  • m/z 380 is the doubly charged species of m/z 780.
  • Extracted ion chromatograms m/z 780 of engineered (top) and wild type (bottom) strains.
  • Fig. 29 shows the results of LCMS analysis of 5. venezuelae strain with an engineered cluster 16.
  • WT Wild type
  • engineered strain in which kasO*p is introduced into cluster 16 on MGY plates HPLC analysis (UV detection at 320 nm) of ethyl acetate extracts from wild type 5. venezuelae and the indicated engineered strain. The major unique ion detected for the engineered strain is indicated by (*).
  • Fig. 30 depicts that data showing that a distinct type II polyketide is produced by 5.
  • viridochromogenes with promoter knock-in (a) Partial schematic of NZ_GG657757 containing majority of biosynthetic genes and the position of kasO*p knock-in.
  • This operon contains contained the minimal set of type II PKS enzymes, including a ketosynthase (SSQG_RS26900), chain-length factor (SSQG_RS26905) and an acyl carrier protein (SSQG_RS26910), together with a polyketide cyclase (SSQG_RS26915), monooxygenase (SSQG_RS26930) and cytochrome P450 (SSQG_RS26935). Except for an additional cytochrome P450, NZ_GG657757 has high homology and similar gene arrangement as a spore pigment biosynthetic gene cluster in 5. avermitilis (Accession number: AB070937.1).
  • Fig. 31 shows data pertaining to constitutive promoters used for cluster activation, (a) Sequences of constitutive promoters used. ATG start codons of genes to be activated are underlined, (b, c) Scheme and sequences of adapters introduced into pCRISPomyces at the Xbal site for making (b) mono-directional and (c) bi-directional promoter knock-in constructs. Restriction sites of selected enzymes are indicated in the sequence maps.
  • Fig. 32 shows the schematic workflow for constructing genome editing plasmid for promoter knock-in.
  • Helper pCRISPomyces-2 plasmids e.g. pCRISPomyces-2-kasO*p
  • promoter knock-in constructs were made by ligating adapter sequences, containing restriction sites flanking the promoter of choice to facilitate insertion of homology arms into pCRISPomyces -2.7
  • the protospacer of a target cluster was first inserted via Bbsl-mediated Golden Gate Assembly.
  • the final editing plasmid was achieved by sequential insertion of the first and second homology arms by Gibson assembly.
  • Fig. 33 shows graphs showing the results of the chemical characterisation of compound 1. NMR analyses of 1. (a) *H NMR (CD 3 OD). (b) COSY (CD 3 OD) (c) HSQC (CD 3 OD). (d) HMBC (CD 3 OD).
  • Fig. 34 shows graphs showing the results of the chemical characterisation of compound 2. NMR analyses of 2 (a) *H NMR of 2 (CD 3 OD). (b) COSY of 2 (CD 3 OD).
  • Fig. 35 shows graphs showing the results of the chemical characterisation of compound 3. 31 P HMBC of authentic FR -900098 sample and 3 produced by the engineered 5. roseosporus strain upon activation of phosphonate biosynthetic gene cluster (cluster 10).
  • Fig. 36 shows graphs showing the results of the chemical characterisation of compound 4.
  • (f) HMBC of 4 (DMSO- e) (g) table showing the NMR peak assignment for 4.
  • Fig. 37 shows the results of the simultaneous introduction of kasO*p and a frameshift mutation into the 5. roseosporus FR-900098 biosynthetic gene cluster.
  • the top panel shows the results of sequence alignments of engineered strains with kasO*p (top 3 sequences) or kasO*p + frameshift mutation (next 4 sequences) introduced into the indicated open reading frame in a single knock-in step.
  • Bottom right panel shows the representative sequence traces of each group of engineered strains in the region containing the frameshift mutation.
  • Bottom left panel shows the knock-in efficiencies for the indicated genomic edits.
  • biosynthetic gene cluster refers to a physically clustered group of two or more genes in a particular genome that together encode a biosynthetic pathway for the production of one or more specialised metabolites, including chemical variants thereof.
  • a wide variety of enzymatic pathways that produce specialized metabolites in bacteria, fungi and plants are known to be encoded in biosynthetic gene clusters.
  • biosynthetic gene cluster and “gene cluster” per se, the latter of which is defined as being a group of two or more genes found within an organism's DNA that encode for similar polypeptides, or proteins, which collectively share a generalized function and are often located within a few thousand base pairs of each other.
  • a biosynthetic gene cluster need not necessarily encode for similar proteins and therefore can encode for proteins that do not have any functional relation.
  • biosynthetic gene cluster implies that 2 or more genes are present, the present invention also allows for targeting of only one of the genes present in a biosynthetic gene cluster.
  • the method can either be used to activate only one or both genes.
  • the method can be used to activate only one or two of the three or all three genes.
  • silent when used in reference to genes, refers to a gene that has no phenotypical effect on the host. This non-effect of the silent gene can be due to the either low or non-existent expression of the silent gene.
  • silent gene may also refer to a transcriptionally inactive gene.
  • orphan when used in reference to genes, refers to genes that lack detectable similarity to genes in other species and, therefore, do not allow for the inference of common descent (i.e., homology). Orphans are an enigmatic portion of the genome because their origin and function are mostly unknown and they typically can represent up to 10% to 30% of all genes in a genome. Several case studies demonstrated that orphans can contribute to lineage-specific adaptation. Without being bound by theory, it is postulated that orphan genes arise from duplication and rearrangement processes followed by fast divergence; however, de novo evolution out of non- coding genomic regions is emerging as an important additional mechanism for the creation of orphan genes.
  • orphans are a subset of taxonomically-restricted genes (TRGs), which are unique to a specific taxonomic level (for example, plant -specific).
  • TRGs taxonomically-restricted genes
  • orphans are usually considered unique to a very narrow taxon, generally a species.
  • the classic model of evolution is based on duplication, rearrangement, and mutation of genes with the idea of common descent. If no orthologous proteins can be found in nearby species, then a gene may be tentatively termed an orphan.
  • Orphan genes differ in that they are lineage- specific and do not show any known history of shared duplication and rearrangement outside of their specific species or clade.
  • Orphan genes may arise through a variety of mechanisms, such as horizontal gene transfer, duplication and rapid divergence, and de novo origination, and may act at different rates in insects, primates, and plants. Despite their relatively recent origin, orphan genes may encode functionally important proteins.
  • the method of preparing or assembling exogenous, homologous and/or heterologous DNA for expression within a host organism is called molecular cloning.
  • the DNA to be cloned is obtained from an organism of interest, and subsequently treated with enzymes in the reaction tube to generate smaller DNA fragments. Subsequently, these fragments are then combined with vector DNA to generate recombinant DNA molecules.
  • the recombinant DNA is then introduced into a host organism (typically an easy-to-grow, benign, laboratory strain of E. coli bacteria). This will generate a population of organisms in which recombinant DNA molecules are replicated along with the host DNA.
  • GMO transgenic or genetically modified microorganisms
  • the method of molecular cloning can also be used to regulate gene expression.
  • regulation of gene expression comprises and includes a wide range of mechanisms that are used by cells to increase or decrease the production of specific gene products (protein or RNA).
  • Sophisticated programs of gene expression are widely observed and know in the art, for example as a mechanism to trigger developmental pathways, respond to environmental stimuli, or adapt to new food sources.
  • Virtually any step of gene expression can be modulated, from transcriptional initiation, to RNA processing, and to the post-translational modification of a protein. Often, one gene regulator controls another, and so on, resulting in a complex gene regulatory network.
  • the process of gene expression itself can be divided into two major processes, transcription and translation.
  • Transcriptional regulation is the means by which a cell regulates the conversion of DNA to RNA (transcription), thereby orchestrating gene activity.
  • a single gene can be regulated in a range of ways, from altering the number of copies of RNA that are transcribed, to the temporal control of when the gene is transcribed.
  • Transcriptional regulation also influences when which proteins are ultimately expressed. This control allows the cell or organism to respond to a variety of intra- and extracellular signals and thus mount a response. Some examples of this include producing the mRNA that encode enzymes to adapt to a change in a food source, producing the gene products, including proteins, involved in cell cycle specific activities, and producing the gene products, including proteins, responsible for cellular differentiation in higher eukaryotes
  • CRISPR-Cas9 refers to genome editing technology based on the capability of clustered regularly interspaced palindromic repeats (CRISPR) and the CRISPR-associated protein-9 nuclease (Cas9) from, for example, Streptococcus pyogenes to induce, for example, double-strand (ds) DNA breaks in a specific location that is complementary to the synthetic guide RNA (sgRNA) sequence integrated into the CRISPR-Cas9 complex, thereby allowing the deletion, addition, and/or modification of genes and/or other genomic elements, such as transcription elements, promoters, promoter enhancers, transcription enhancers, restriction sites, mutations, selection markers, for example antibiotic selection cassettes, and the like.
  • an antibiotic selection cassette is also added to the genome, preceding, simultaneously with, or following insertion of genetic material using the CRISPR technology.
  • deletion of promoter regions, site -directed mutations, mutations and gene deletion is/are performed before, simultaneously or after the addition of the one or more promoter(s) as disclosed herein.
  • deletion of promoter regions, site -directed mutations, mutations and gene deletion is/are performed before performing CRISP- mediated knock-in of the promoter(s).
  • deletion of promoter regions, site-directed mutations, mutations and gene deletion is/are performed after performing CRISP- mediated knock-in of the promoter(s).
  • deletion of promoter regions, site -directed mutations, mutations and gene deletion is/are performed at the same time CRISP- mediated knock-in of the promoter(s) is/are being performed.
  • CRISP- mediated knock-in of the promoter(s) is/are being performed.
  • one or more promoter regions were concurrently deleted.
  • site mutations were concurrently introduced into selected genes.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • Cas CRISPR-associated genes
  • CRISPR-Cas9 mediated defence is as follows: invading DNA from viruses or plasmids is cut into small fragments and incorporated into a CRISPR locus amidst a series of short repeats (around 20 bps).
  • RNAs small RNAs
  • crRNA - CRISPR RNA also referred to as synthetic guide RNA (sgRNA) in an in vitro setting
  • sgRNA synthetic guide RNA
  • the CRISPR - Cas9 works according to the same principle, with the sgRNA guiding the effector nucleases to the desired sections of the DNA, in which the excision is to be made.
  • the term "protospacer” refers to part of the so-called “sgRNA” and is a user defined, 17 to 23 nucleotide long base-pairing region for specific DNA binding.
  • the term “sgRNA” refers to "single guide RNA” or “synthetic guide R A” and is, in the context of CRISPR technology, a chimera of CRISPR RNAs (crRNA) and trans -activating crRNA (tracrRNA), which is typically about 100 nucleotides in length and consists of three regions: a user defined, 17 to 23 nucleotide long base-pairing region for specific DNA binding (which is called a protospacer), a roughly 40 nucleotide long Cas9 handle hairpin for Cas9 protein binding; and a roughly 40 nucleotide long transcription terminator derived from 5.
  • pyogenes that contains hairpin structures that provide stability to the RNA molecule.
  • cassette refers to a nucleic acid sequence that is introduced into the target genome, for example during the knock-in process.
  • examples of a cassette are, but are not limited to mono- or bidirectional promoter sequence, and may also include other elements, such as, for example, an antibiotic resistance marker.
  • promoter refers to a region of a nucleic acid sequence that initiates transcription of a particular gene. Promoters are usually located near the transcription start sites of genes, on the same strand and are usually found upstream on the nucleic acid sequence (towards the 5' region of the sense strand). Promoters can vary in length, from about 100 to 1000 base pairs. Promoters are understood as binding and initiation sites of, for example, RNA polymerases, enzymes which have transcriptional activity, thereby initiating transcription of, for example, DNA to RNA. Different promoters can give genes different expression patterns within a host cell and can also cause simultaneous expression of different genes.
  • Some promoters are active in all cells at all times, while others are specific to different organisms, tissue types (spatial control) or even specific times during the host's development (temporal control). Others promoters are sensitive to external signals, such as changes in temperature or the presence or absence of a certain chemical. Such promoters are known as controllable or inducible on/off switches for genes.
  • the term "bidirectional promoter” refers to regulatory regions that are shared between two genes, when those two genes are transcribed away from one another.
  • the genes are said to be in a head-to-head arrangement, with their transcription start sites (TSSs) positioned nearby one another.
  • TSSs transcription start sites
  • the intergenic distance between these genes can be no greater than 1000 base pairs. This distance is measured from the TSS of the gene on the left of the promoter to the TSS of the gene on the right of the promoter. Head -to-head genes are spaced at this distance more frequently than expected in, for example, the human genome, suggesting a regulatory theme in gene expression.
  • other promoters are termed mono- directional promoters.
  • kasO*p refers to an engineered version of the kasO promoter region (also known as kasOp) from Streptomyces coelicolor.
  • kasO also known as cpkO or SCO6280
  • ermE*p refers to an engineered version of the ermE promoter (aka ermEp) from Saccharopolyspora erythraea. ermEp is the promoter of the erythromycin resistance gene.
  • P2 As used herein, the terms "P2”, “P3”; “P6”, “P8”, “P25” and “rcfP” refer to different promoter regions of housekeeping genes in Streptomyces albus.
  • activation refers to an upregulation of gene expression or transcriptional activation of a gene that was previously not expressed or only expressed in small amounts.
  • suppression refers to a downregulation of gene expression or transcriptional activity of a gene.
  • the term "gene or genome editing” refers to modifying the genetic sequence of an organism, virus, or any other genetic element, to add, delete and/or modify the genetic sequence compare to the sequence as it is present in nature. These alterations are also called mutations (permanent alterations to the nucleotide sequence of an organism, virus, or any other genetic elements) and can also occur naturally.
  • the term "knock-in”, as used in molecular cloning and biology, refers to a type of targeted mutation in which a gene function is produced (also known as a gain of function mutation).
  • This genetic engineering method can involve a one-for-one substitution of DNA sequence information with a wild-type copy in a genetic locus or the insertion of sequence information not found within the locus, and can be performed by inserting, adding or substituting the wild-type genetic material with other, for example exogenous genetic material or genetic material not usually found at that location.
  • knock-in technology involves a gene inserted into a specific locus, and is thus considered to be a "targeted" insertion
  • transgenic techniques involve modification of the target genome by insertion of the modifying nucleic acid sequence (also known as a trans-gene).
  • the inserted nucleic acid sequence stays in a trans position to the modified sequence, so there is a recombination or transposition between two DNA fragments: the naturally occurring DNA sequence and the inserted cell-modifying nucleic acid sequence.
  • an animal with expressing a newly inserted gene is a knock-in animal; and that both knock-in and knock-out animals are transgenic animals, if, and only then, these animals were obtained by introduction of a nucleic acid, which modified the original genomic sequence.
  • the inserted modification must be stable, for example in the germline, meaning that the offspring of such a transgenic animal must also have the inserted modification.
  • Methods for generating gene knock-ins are known in the art, for example transposon-mediated systems (lox-Cre system), homologous recombination or the recent CRISPR - Cas9 technology.
  • conjuggant refers to a protozoan just after the separation following conjugation, during which an exchange of DNA material has taken place.
  • pentagular refers to a structure having five angles and five sides. This refers to any structure that is derived from or based on the shape of a pentagon, or which is pentagonal. DETAILED DESCRIPTION OF THE PRESENT INVENTION
  • Natural products have been a major and indispensable source of pharmaceuticals and bioactive scaffolds.
  • Genome sequencing of privileged natural product producers, for example, such as actinomycetes reveals a vast untapped resource in the form of silent biosynthetic gene clusters, which can be mined to meet the burgeoning demand for natural products with new bioactivities.
  • the present disclosure demonstrates that CRISPR-Cas9 mediates rapid, multiplex knock-in of heterologous genetic parts in multiple actinomycetes, achieving 50% to 100% knock-in efficiency in one step. It is further shown that this general method of promoter knock-in can be used to activate silent, unexplored biosynthetic gene clusters and to induce the production of secondary metabolites belonging to distinct chemical classes in native producers.
  • CRISPR technology has been demonstrated, for example, genetic for generating knock-outs in Streptomyces, it has not been used for generating more challenging genetic knock-ins, and more importantly for the refactoring and activation of silent biosynthetic gene clusters in native hosts.
  • This method and strategy provides a complementary avenue to better explore the biosynthetic capability and chemical diversity of existing actinomycete strain collections for natural product discovery.
  • an efficient CRISPR -Cas9 knock-in strategy to activate silent biosynthetic gene clusters (BGCs) in various representatives of the Streptomycetes species.
  • CRISPR- Cas9 greatly enhanced knock-in efficiency afforded by CRISPR- Cas9, which enables the genetic manipulation of strains that are usually not genetically amendable and how it can be applied towards activating silent biosynthetic gene clusters.
  • 5. roseosporus has 0% knock-in efficiency in the absence of a protospacer compared to 50% with a functional CRISPR-Cas9.
  • This one-step strategy was used to activate multiple biosynthetic gene clusters of different classes in five Streptomyces species and triggered the production of unique metabolites, including a novel pentangular type II polyketide in Streptomyces viridochromo genes.
  • this method can be used in any organism (prokaryotic or eukaryotic) to access biosynthetic gene clusters, silent or otherwise, or any target gene(s).
  • this CRISPR-Cas9 meditated knock-in technology is used in actinomycetes (also known as actinomycetales or actinobacteria) and can be used in other bacteria, for example, but not limited to, cyanobacteria, Streptomyces sp. or Bacillus sp.
  • actinomycetes also known as actinomycetales or actinobacteria
  • cyanobacteria Streptomyces sp.
  • Bacillus sp Bacillus sp.
  • knock-in in Streptomycetes requires long circuitous selection/screens that are traditionally used to sequentially identify single and double crossover events.
  • This increase in knock-in efficiency due to the use of CRISPR technology also allows genetic manipulation of Streptomycetes to be performed using shorter homology arms and also allows for more challenging genetic manipulations, like the introduction of larger genetic elements to be performed. It is of note that knock-in efficiency drops when the knock-in fragment length increases from 100 base pairs to 1 kilo base pairs.
  • the presently disclosed technology also enables the use of shorter homologous arms (1-2 kilo base pairs instead of much longer arms), which would otherwise be highly inefficient without CRISPR - Cas9.
  • Homologous recombination is known to be relatively inefficient in Streptomycetes, with double crossover events being very rare events.
  • Increasing homology lengths of the editing template help to increase HR efficiency, with a >3 kb homology on each side traditionally used to obtain double crossover recombinants.
  • the length of homology for achieving homologous recombination at acceptable efficiency depends on the Streptomyces strains being engineered but is typically multi-kb in length.
  • the biosynthetic gene cluster(s) or target gene(s) is/are isolated from an Actinobacterium (also known as actinomycetes or actinomycetales).
  • the Actinobacterium is of the genus Streptomyces (nomenclature according to Waksman and Henrici, 1943; synonyms of which include Actinopycnidium (Genus) Krasil'nikov 1962, Actinosporangium (Genus) Krasil'nikov & Yuan 1961, Chainia (Genus) Thirumalachar 1955, Elytrosporangium (Genus) Falcao de Morais et al.
  • biosynthetic gene cluster(s) or target gene(s) is/are isolated from one or more representatives of the Streptomyces genus.
  • the Actinobacterium of the Streptomyces genus is, but is not limited to Streptomyces abietis, Streptomyces abikoensi, Streptomyces aburaviensis, Streptomyces achromogenes, Streptomyces acidiscabies, Streptomyces actinomycinicus, Streptomyces acrimycini, Streptomyces actuosus, Streptomyces aculeolatus, Streptomyces abyssalis, Streptomyces afghaniensis, Streptomyces aidingensis, Streptomyces africanus, Streptomyces alanosinicus, Streptomyces albaduncus, Streptomyces albiaxialis, Streptomyces albidochromogenes, Streptomyces albiflavescens, Streptomyces albiflaviniger, Streptomyces albidoflavus
  • the Actinobacterium is, but is not limited to, Streptomyces albus, Streptomyces avermilitis, Streptomyces erythraeus (also known as Saccharopolyspora erythraed), Streptomyces lividans, Streptomyces griseus, Streptomyces rapamycinicus, Streptomyces roseosporus, Streptomyces rubellomurinus, Streptomyces venezuelae, or Streptomyces viridochomogenes.
  • the biosynthetic gene cluster(s) comprise silent or orphan genes.
  • the target gene(s) is/are silent or orphan genes.
  • the biosynthetic gene cluster(s) or target gene(s) is/are, but are not limited to, SEQ ID NO: 193 to 201.
  • a recombinant method of activating expression of one or more biosynthetic gene cluster(s), or one or more target gene(s) in a biosynthetic gene cluster comprising more than one gene comprising inserting one or more promoter(s) at one or more transcriptionally functional location(s) relative to the biosynthetic gene cluster(s) or the target gene(s) in the biosynthetic gene cluster(s), whereby the insertion of the promoter(s) results in increased expression of the biosynthetic gene cluster(s) or target gene(s) compared to the expression level of an unmodified biosynthetic gene cluster(s) or target gene(s).
  • the method claimed herein is used to insert the promoters into the genomes of native producing hosts without cloning the cluster.
  • promoter(s) is/are inserted into the genome using a plasmid. Insertion of the promoter relies on homologous recombination, which is induced by CRISPR/Cas9-mediated double stranded breaks.
  • the CRISPR/Cas9 and the editing template for the promoter knock-in are encoded in the plasmid. But, in the end, the promoter is inserted into the genome and the plasmid is removed.
  • the biosynthetic gene cluster(s) are activated by insertion of a promoter.
  • the biosynthetic gene cluster(s) are activated by insertion of two or more promoters, that is the multiple promoters are used to express the same target gene or multiple target genes.
  • multiple biosynthetic gene clusters are simultaneously or subsequently activated by the insertion of multiple promoters.
  • multiple promoters are inserted into the same biosynthetic gene cluster.
  • multiple promoters are inserted into different biosynthetic gene cluster(s) within the same host genome.
  • Temporal differentiated use can be instigated, for example, by using two different promoters which are under the different transcription regulatory control or by, for example, using inducible promoters, which are promoters which are activated or repressed by the presence and/or absence of key compounds.
  • inducible promoter is a tetracycline (tet) -inducible system, for which the inducer is tetracycline.
  • the promoter is a bidirectional promoter.
  • the promoter is a unidirectional promoter.
  • the promoter is, but is not limited to, kasO*p, ermE*p, P2, P3, P6, P8, P25, or rcfp.
  • the promoter is kasO*p.
  • the promoter is P8-kasO*p.
  • the promoter is rcpf.
  • the promoter is a cloned native promoter of the target biosynthetic gene cluster(s) or the target gene(s). The choice of promoter depends on the characteristics of the gene to be expressed, for example, the host or species in which the gene is naturally present.
  • the promoter(s) and the biosynthetic gene cluster(s) or target gene(s) are of the same species. In another example, the promoter(s) and the biosynthetic gene cluster(s) or target gene(s) are of different species.
  • the location for the insertion of the promoter need not be localised near or within immediate proximity to the target gene(s) or biosynthetic gene cluster(s).
  • transcription regulatory elements can be found along stretches of the genome that may not appear to be in immediate proximity to the target gene(s) or biosynthetic gene cluster(s).
  • regulatory sequences for example promoters
  • biosynthetic gene cluster(s) are then brought into proximity of the transcription target(s), thereby resulting in functional expression of the target gene(s) or biosynthetic gene cluster(s).
  • the promoters are inserted at transcriptionally functional location(s), which is/are upstream of the biosynthetic gene cluster(s) or target gene(s). In another example, the promoters are inserted at transcriptionally functional location(s), which is/are downstream of the biosynthetic gene cluster(s) or target gene(s). In yet another example, the promoters are inserted at transcriptionally functional location(s), which is/are both upstream and downstream of the biosynthetic gene cluster(s) or target gene(s).
  • Microbial natural products are a rich source of pharmaceutical agents and current advances in genomics have unveiled a vast source of potential unexplored biosynthetic gene clusters. Because majority of encoded metabolites of these biosynthetic gene clusters are undetectable using current analytical methods due to minimal or zero biosynthetic gene cluster expression under laboratory conditions (such biosynthetic gene clusters are commonly defined as silent biosynthetic gene clusters), strategies to activate biosynthetic gene cluster expression and trigger metabolite production are critical to realize the full potential of nature's chemical repertoire. While heterologous expression bypass native regulation networks and can be engineered rationally, entire biosynthetic pathways often spanning large areas of genomes will have to be cloned and refactored. Additionally, heterologous hosts may lack regulatory, enzymatic or metabolic requirements necessary for product biosynthesis. Inducing cluster expression in native hosts circumvents these limitations but may be hindered by low homologous recombination efficiencies.
  • Actinobacteria are traditionally rich sources of natural products but 50-80% of biosynthetic gene clusters in actinomycetes with encoding for pathways to potentially novel bioactive compounds are silent under normal laboratory conditions.
  • CRISPR clustered regularly interspaced palindromic repeat
  • Cas9 nucleases can be directed to any site on the genome simply by transcribing a synthetic guide RNA (sgRNA), requiring only a protospacer adjacent motif (PAM) sequence at the target site.
  • sgRNA synthetic guide RNA
  • PAM protospacer adjacent motif
  • Staphylococcus pyogenes Cas9 PAMs are especially abundant in the GC-rich actinomycete genomes, greatly increasing the number of potential target sites and coverage of CRISPR-Cas9 genome editing in these natural product relevant organisms.
  • the promoter(s) is/are inserted using CRISPR-Cas9 technology.
  • one aim of the disclosed method is to yield and/or increase production of one or more molecules encoded by a biosynthetic gene cluster.
  • the CRISPR-Cas9 has been reconstituted in multiple Streptomyces strains and used to perform precise deletions of individual genes and entire biosynthetic gene clusters of up to 82.2 kb8, at high efficiencies of 60-100% with minimal off-target activity.
  • This unprecedented recovery of desired mutants can be due to the fact that CRISPR-Cas9 selects against wild type sequences in favour of double-crossover recombinants in the presence of double stranded homology-flanked editing templates (Fig. 1).
  • the CRISPR/Cas9 technology reduces the required time for homology-directed recombination by one -half by circumventing the conventional two-step selection/screening method for single and double crossover events (Fig. 1).
  • CRISPR technology has also enabled the genetic manipulation of many genetically recalcitrant organisms.
  • the Streptococcus pyogenes CRISPR-Cas9 system is recently reconstituted in model Streptomycetes to delete genes and entire biosynthetic gene clusters, as well as perform site-directed mutagenesis and gene replacement at significantly improved efficiencies.
  • the CRISPR-Cas9 technology has been extended to perform strategic promoter knock-in for the activation of silent biosynthetic gene clusters in native Streptomyces hosts (Fig. 12a). Shown herein is the use of this technology to perform strategic promoter knock-in and site-directed mutagenesis for efficient activation of silent and uncharacterized biosynthetic gene clusters in multiple actinomycetes.
  • manipulating growth conditions such as media composition to ensure expression of pathway-specific activator(s), presence of physiological and environmental co-inducers, engineering of the translational and transcriptional machineries, suppression of the genes regulated by repressor(s), overexpressing pathway-specific regulator(s), testing a variety of heterologous hosts to express target clusters and silencing major secondary metabolite biosynthetic pathways to relieve competition for key precursors.
  • Screening growth conditions is time and labour intensive while the other strategies can only applied on a case- by-case basis. Furthermore, this strategy does not enable one to identify the biosynthetic gene cluster responsible for a given secondary metabolite, knowledge of which will be valuable towards structure identification and downstream titer improvement.
  • a major consideration of the disclosed activation strategy is the selection of site(s) for promoter knock-in.
  • the activators and repressors can be predicted with certain confidence.
  • SARP Streptomyces antibiotic regulatory protein
  • LAL LuxR
  • the core operon(s) encoding for key biosynthetic enzymes within the biosynthetic gene clusters have been targeted, inserting promoters in front of the first open reading frame of an operon.
  • Multiplex promoter knock-in will be useful for more "fragmented" biosynthetic gene clusters that require the introduction of multiple promoters for activation.
  • CRISPR-Cas9 can be used to efficiently and precisely introduce heterologous promoters into Streptomyces genomes for biosynthetic genetic cluster activation
  • well- characterized pigment biosynthetic genetic clusters were selected, namely the indigoidine cluster in Streptomyces albus, as well as the actinorhodin (ACT) and undecylprodigiosin (RED) clusters in Streptomyces lividans.
  • CRISPR-Cas9 mediated knock-in, upstream promoter regions of main biosynthetic operons or pathway-specific activators were replaced with constitutive promoters that are stronger than the commonly used ermE* promoter and work in multiple Streptomyces species (Fig. 16).
  • CRISPR-Cas9 increased knock-in efficiency of the kasO* promoter upstream of the indC-like indigoidine synthase gene compared to without CRISPR-Cas9 (Fig. 12b). Higher knock-in efficiency observed with 2 kb homologous arms as compared to 1 kb arms is consistent with homology-directed repair of Cas9-induced double stranded breaks.
  • CRISPR-Cas9 can be used to precisely introduce heterologous genetic elements into Streptomyces genomes at relatively high efficiencies for secondary metabolite production from silent biosynthetic gene clusters.
  • the enhanced knock-in efficiencies allowed use of donor DNA with shorter homology flanks as well as the introduction of larger genetic elements, both of which will be challenging without CRISPR-Cas9. While homologous recombination occurs efficiently in model strains like 5. lividans and 5. albus without CRISPR-Cas9, for other strains like Streptomyces roseosporus, the increase in efficiency afforded by CRISPR-Cas9 is critical and allows genetic manipulation of otherwise challenging strains (Fig. 12b).
  • roseosporus also possesses a phosphonate biosynthetic gene cluster with genes showing high homology and synteny to the Streptomyces rubellomurinus FR-900098 biosynthetic gene cluster (Table 8). Intriguingly, BLASTP search within -2000 NCBI-deposited actinobacteria assemblies for FR-900098 biosynthetic enzymes did not uncover similar biosynthetic gene clusters, suggesting that 5. roseosporus has the uncommon biosynthetic potential to synthesize the antimalarial compound, which to date has been attributed to 5. rubellomurinus and 5. lavendulae. To determine if 5.
  • roseosporus can produce FR-900098, a bidirectional P8-kasO*p promoter cassette was introduced to drive expression of the putative frbD operon and frbC homolog (Fig. 23 and 24).
  • the engineered strain produced 3 with 31 P-NMR, HMBC and mass values consistent with FR-900098 (Fig. 13b, and Figs. 33 to 36), validating the inherent ability of 5. roseosporus to make FR-900098.
  • growth e.g. oxygen levels, trace elements, biosynthetic precursors
  • workup extraction or detection methods
  • biosynthetic gene clusters may be extinct and rendered non-functional by mutations occurring beyond the biosynthetic gene clusters.
  • LCMS liquid chromatography-mass spectrometry
  • venezuelae yielded production of unique compounds that were not observed for the parent strains (Fig. 13c to e).
  • roseosporus was predicted to be a nucleoside-type I PKS with biosynthetic enzymes for incorporation of a 3-amino-5-hydroxybenzoic acid starter unit and naphthalene ring formation. Insertion of kasO*p upstream of the main synthase gene encoding a loading domain and three PKS modules triggered the production of a major metabolite with m/z 405 (Fig. 17c). A distinct compound with m/z 780 was observed for another engineered 5.
  • lividans does not produce any pigmented product in this medium.
  • ACT cluster kasO*p was knocked-in in front of ActII-orf4 at 100% efficiency as determined by diagnostic PCR -digestion and sequencing (Fig. 4A, B). Consistent with forced expression of the ACT pathway-specific positive regulator and production of the pH-sensitive actinorhodin antibiotic, engineered 5. lividans strains were pigmented and turned dark blue with ammonia fuming (Fig. 4C).
  • CRISPR-Cas9 system can be used for cluster activation and induction of secondary metabolite production in Streptomyces by mediating efficient promoter knock -in at target genomic loci to drive the expression of biosynthetic or regulatory genes. It was also shown that heterologous genes or functionalities can be introduced in the same step at comparable efficiencies, a property that can be exploited to further improve production titer from the activated biosynthetic gene clusters.
  • This system should provide a powerful means to activate silent biosynthetic gene clusters and induce secondary metabolite production in actinomycetes for natural product discovery.
  • Streptomyces roseosporus is best known and studied for being the native producer of lipopeptide antibiotic daptomycin, which is one of the frontline antibiotics against drug resistant gram positive pathogens. While mass spectrometry studies further identified additional non-ribosomal peptide synthetase products with antimicrobial activities (arylomycin, napsamycin and stenothricin), the relevant biosynthetic genes have yet to be identified, hindering efforts to overproduce these products by microbial fermentation using engineered hosts. Using the CRISPR-Cas9 system to strategically knock in constitutive promoters, the biosynthetic capability of 5.
  • antiSMASH stands for antibiotics & Secondary Metabolite Analysis Shell, a genome-mining software that is capable of analysing the sequenced genome in silico, identifying potential biosynthetic gene clusters and predicting core structures of encoded metabolites. See for example https://antismash.secondarymetabolites.org/.
  • roseosporus is not known to produce phosphonate compounds but its genome encodes a predicted phosphonate biosynthetic gene cluster with the genes showing high homology and synteny (>94% identity) to those within the reported biosynthetic gene cluster of antimalarial FR-900098 in Streptomyces rubellomurinus (Fig. 5 A and Fig. 8). Targeting genes involved in the first two steps in the FR-900098 biosynthetic pathway (Fig.
  • the new phosphonate compound produced by the engineered strain with the bidirectional promoter knock-in was determined to be FR-900098 when spiking of an authentic sample increased the intensity of the signals at -21-22 ppm with no change in spectrum profile (Fig. 5C).
  • the estimated FR-900098 titer of 10-12 mg/L is more than 100-fold higher than the IC 50 against Plasmodium falciparum, demonstrating that this strategy of knocking in promoters can be used to activate production of secondary metabolites from silent biosynthetic gene clusters at sufficient quantities for bioactivity screening.
  • roseosporus can be achieved by knocking in a single 97 base pairs kasO*p in front of the first open reading frame of the cluster that encodes a sterol desaturase.
  • LCMS liquid chromatography-mass spectrometry
  • analysis of wild type and activated strain revealed a series of new peaks at retention times of 21 to 23 minutes with two major products identified by their mass values of m/z 511 and 513 (Fig. 14A). Minor products with m/z 501, 511, 515 and 555 were also detected.
  • alteramide A photocyclization is a spontaneous and efficient reaction with quantitative conversion following 6 hours of light exposure, it was inferred that the photocyclized compound is most likely a by-product of the workup and alteramide A is the original metabolite produced by 5. roseosporus. Whether alteramide A is a biosynthetic precursor to HSAF remains to be determined.
  • PKS-NRPS cluster consisting of one polyketide synthase (PKS) module with a ketosynthase (KS) domain and one NRPS module with two condensation domains.
  • PKS polyketide synthase
  • KS ketosynthase
  • NRPS module with two condensation domains.
  • PKS polyketide synthase
  • KS ketosynthase
  • NRPS module with two condensation domains.
  • Its closest homologous biosynthetic gene cluster is the antifungal ECO-02301 in Streptomyces aizunenesis, of which only 7% of the genes show homology to the 5. roseosporus cluster.
  • Introducing kasO*p before the first open reading frame, which encodes a transporter of the major facilitator superfamily, of the main PKS-NRPS operon corresponded with the production of a new metabolite at 8.6 min retention time with m/z 344 (Fig. 14).
  • PKS nucleoside-type I polyketide synthase
  • AHBA 3-amino-5-hydroxybenzoic acid
  • CAL Coenzyme A-ligase
  • KR-KS-AT-DH ketoreductase-ketosynthase-acyltransferase-dehydratase
  • 1 L of ISP2 medium contains 10 g malt extract broth (Sigma- Aldrich), 4 g Bacto yeast extract (BD Biosciences), 4 g glucose (Sigma -Aldrich) and for ISP2 agar plates an additional 20 g of agar (BD Biosciences). Conjugation experiments involving WM6026 and WM3780 E.
  • coli strains were performed on R2 agar without sucrose: 0.25 g K2S04 (Sigma), 10.12 g MgCl 2 , 6H 2 0, 10 g glucose, 0.1 g Bacto casamino acids (BD Biosciences), 5.73 g TES (Sigma), 20 g agar in 1 L water, autoclaved, after which 1 mL filter-sterilized 50 mg/mL KH 2 P0 4 solution and filter-sterilized 2.94 g CaCl 2 , 2H 2 0 and 3 g L-proline in 5 mL 1 N NaOH were added to the medium. Conjugation experiments involving the ET 12567 E.
  • coli strain was performed on SFM agar with 10 mM MgCl 2 : 20 g/L mannitol, 20 g/L soya flour, 20 g/L agar were stirred at 95 C for 2 to 4 hours prior to autoclaving. After which, 1 M MgCl 2 was added to the medium for a final concentration of 10 mM.
  • pCRISPomyces-2 plasmids for making promoter knock-in constructs were made by inserting adapter sequences with restriction sites flanking the promoter of choice to facilitate insertion of homology arms (Fig.15).
  • Protospacer for a target cluster was first inserted via Bbsl Golden Gate Assembly.
  • the helper plasmid was linearized using Spel and assembled with the downstream homology arm by Gibson assembly (New England Biolab).
  • the second upstream homology arm is also inserted by Gibson assembly using Hindlll or Nhel linearized construct containing the first homology arm.
  • Promoter knock-in constructs are transformed into conjugating E. coli strains and colonies with the appropriate antibiotic resistance (e.g. 50 mg/L apramycin (Sigma)) were picked into Luria-Bertani (LB) medium with antibiotics.
  • LB Luria-Bertani
  • WM6026 requires diaminopimelic acid (Sigma) in LB medium for growth and subsequent wash and re-suspension steps involving LB medium.
  • Overnight cultures were diluted 1 : 100 into fresh LB medium with antibiotics and grown to an optical density (OD600) of 0.4 to 0.6. 400 ⁇ L ⁇ of the culture was pelleted, washed twice and resuspended in LB medium without antibiotics. The washed E.
  • coli cells were then mixed with spores at 1 :5 volume ratio and spotted on R2 plate. After incubation for 16 to 20 hours at 30 C, the plates were flooded with nalidixic acid and apramycin and incubated until exconjugants appear. Exconjugants were streaked into ISP2 plates containing apramycin at 30 °C followed by re -streaking to ISP2 plates at 37 C to cure the CRISPR-Cas9 plasmid containing a temperature sensitive origin of replication. Apramycin-sensitive clones growing at 37 C were then subjected to validation of promoter knock -in and genome editing as described below.
  • Genomic DNA from wild type and exconjugants from the indicated strains were isolated from liquid cultures using the Blood and Tissue DNeasy kit (Qiagen) after pre -treating the cells with 20 mg/mL lysozyme for 0.5 to 1 hour at 30 C.
  • PCR Polymerase chain reaction
  • Table 4 Polymerase chain reaction (PCR) was performed using control primers beyond the homology regions or knock-in specific primers (Table 4) with Taq polymerase (KODXtreme, Millipore) with the following step-down cycling conditions: 94 C for 2 minutes; [98 C for 10 seconds, 68 C for 1 kb/minute] x 5 cycles; [98 C for 10 seconds, 66 C for 10 seconds, 68 C for 1 kb/minute] x 5 cycles; [98 C for 10 seconds, 64 C for 10 seconds, 68 C for 1 kb/minute] x 5 cycles; [98 C for 10 seconds, 62 C for 10 seconds, 68 C for 1 kb/minute] x 15 cycles; hold 4 C.
  • PCR products were subjected to digest with specific restriction enzymes to differentiate between PCR products were further wild type genomic sequences and successful genome editing by knock-ins. Positive samples were purified using Qiaquick PCR purification kit (Qiagen) and validated by Sanger sequencing.
  • HPLC parameters were as follows: solvent A, 0.1% formic acid in water; solvent B, 0.1% formic acid in acetonitrile; gradient at a constant flow rate of 0.3 mL/ minute, 10% B for 5 minutes, 10% to 100% B in 25 minutes, maintain at 100% B for 10 minutes, return to 10% B in 1 minute and finally maintain at 10% B for 9 minutes; detection by ultraviolet spectroscopy at 210, 254 and 280 nm.
  • Liquid seed cultures (2 mL ISP2) of wildtype and engineered 5. roseosporus strains were inoculated from a plate or spore stock in the 14 mL culture tube. Seed cultures were incubated at 30 C with 250 rpm shaking until achieving turbidity or high particle density (typically 2 to 3 days). Seed cultures were diluted 1 : 100 into 50 mL of ISP2 broth in 250 mL baffled flasks containing -30-40 5 mm glass beads (Sigma) and incubated at 30 °C with 250 rpm shaking for 14 days. The cultures harvested by pelleting at maximum speed in an Eppendorf 5810R centrifuge for 10 minutes.
  • the cell pellet was stored at -80 °C while the supernatants were split into two 50 mL falcon tubes, flash frozen liquid nitrogen and lyophilized to dryness. 25 and 10 mL of methanol was added to each tube containing dried supernatant and frozen cell pellets respectively. The methanol mixtures were vortexed for 1 minute each and incubated on a platform shaker at 4 C for 2 hours. Samples were clarified by spinning at maximum speed in an Eppendorf 5810R centrifuge for 10 minutes twice and pooling the methanol extracts from the respective pellets and lyophilized culture supernatants. A generous amount of anhydrous sodium sulfate was added to the extracts and stirred.
  • the extracts were decanted, was dried using a rotary evaporator and resuspended in 700 ⁇ L ⁇ deuterium oxide (Sigma) added in two 350 ⁇ L ⁇ aliquots.
  • a spatula-full of Chelex 100 resin (Bio-Rad) was added to each sample in a 1.7 mL centrifuge tube, which was incubated for 30 minutes at room temperature with agitation on a Thermo microplate shaker.
  • the samples were clarified twice by centrifuging at maximum speed in an Eppendorf bench-top centrifuge for 1 minute each time.
  • MGY medium contains 10 g malt extract broth, 4 g Bacto yeast extract (BD Biosciences), 4 g glucose (1st Base, Axil Scientific) and for MGY agar plates, an additional 20 g of Bacto agar (BD Biosciences). Conjugation experiments involving WM6026 and WM3780 E.
  • coli strains were performed on R2 agar without sucrose: 0.25 g K 2 S0 4 , 10.12 g MgCl 2 , 6H 2 0, 10 g glucose, 0.1 g Bacto casamino acids (BD Biosciences), 5.73 g TES, 20 g agar in 1 L water, autoclaved, after which 1 mL filter-sterilized 50 mg/mL KH2P04 solution and filter-sterilized 2.94 g CaCl 2 , 2H 2 0 and 3 g L-proline in 5 mL 1 N NaOH were added to the medium.
  • potential promoter knock-in sites were selected based on: a. the presence of a potential transcriptional activator; b. minimal set of core biosynthetic genes; and/or c. if multiple promoters need to be inserted.
  • PAMs and protospacer sequences were designed to be as close as possible to cut sites. Select a 20 nt protospacer of interest.
  • the 3' protospacer adjacent sequence (PAM) must be NGG, where N is any nucleotide. Preference is given to one or more of the following: sequences with purines (A, G) occupying the last four (3') bases of the protospacer; sequences on the non-coding strand; and/or sequences in which the last 12 nt of protospacer + 3 nt PAM (15 nt total) are unique in the genome. The sequences are verified using BLAST.
  • Homology arms are designed according to one or more of the following criteria: a. for robust PCR - optimization performed, primer design (and by extension, homology arm junction) is key; b. removal of Cas9-recutting site while minimizing genetic perturbation (e.g. disruption of genes or remaining sequences). While the latter step is not required for deletion studies, it is required for activation studies.
  • the protospacer of a target cluster was first inserted via Bbsl-mediated Golden Gate Assembly as previously described.
  • the helper plasmid (pCRISPomyces-2-kasO*p, pCRISPomyces-2-P8-kasO*p) was linearized using Spel and assembled with the downstream homology arm, which is 2 kb unless otherwise indicated (data not shown) by Gibson assembly (New England Biolabs).
  • the second upstream homology arm (2 kb, unless otherwise indicated) was subsequently inserted by Gibson assembly using Hindlll or Nhel linearized construct containing the first homology arm. See Fig. 32 for workflow to construct genome editing plasmids. Different workflows for assembling the knock-in plasmids were tried and this workflow was deemed most attractive in terms of ease, efficiency and modularity (see Fig. 11).
  • the 3' protospacer adjacent sequence must be NGG, where N is any nucleotide. Preference is given to one or more of the following: sequences with purines (A, G) occupying the last four (3') bases of the protospacer; sequences on the non-coding strand; sequences in which the last 12 nt of protospacer + 3 nt PAM (15 nt total) are unique in the genome (check by BLAST with all four possible NGG sequences).
  • anneal spacer oligos as follows: re-suspend both oligos to ⁇ in water. Mix 5 ⁇ L ⁇ FOR + 5 REV and 90 ⁇ 30mM HEPES, pH 7.8. Heat to 95 °C for 5 minutes, then ramp to 4 °C at a rate of 0.1 °C/second. Insert annealed spacer by Golden Gate assembly. Perform the chosen assembly method to insert the 2 kb homology arms sequentially in the digested, dephosphorylated backbone (see above.)
  • Promoter knock-in constructs were used to transform conjugating E. coli strains and colonies with the appropriate antibiotic resistance (e.g. 50 mg/L apramycin) were picked into Luria-Bertani (LB) medium with antibiotics.
  • LB Luria-Bertani
  • WM6026 requires diaminopimelic acid in LB medium for growth and it was added to LB medium for subsequent wash and re-suspension steps. Overnight cultures were diluted 1 : 100 into fresh LB medium with antibiotics and grown to an OD600 of 0.4-0.6. 400 ⁇ L ⁇ of the culture was pelleted, washed twice and re- suspended in LB medium without antibiotics. The washed E.
  • coli cells were then mixed with spores at 1 :5 volume ratio and spotted on R2 without sucrose plates. After incubation for 16 to 20 hours at 30 °C, the plates were flooded with nalidixic acid and apramycin and incubated until exconjugants appear. Exconjugants were streaked onto MGY plates containing apramycin at 30 °C followed by restreaking to MGY plates at 37 °C to cure the CRISPR-Cas9 plasmid containing a temperature- sensitive origin of replication. Apramycin-sensitive clones growing at 37 °C were then subjected to validation of promoter knock-in and genome editing as described below.
  • Genomic DNA from wild type and exconjugants from the indicated strains were isolated from liquid cultures using the Blood and Tissue DNeasy kit (Qiagen) after pre -treating the cells with 20 mg/mL lysozyme for 0.5 to 1 hours at 30 °C.
  • PCR was performed using control primers beyond the homology regions or knock-in specific primers (Table 4) with KODXtreme Taq polymerase (Millipore). PCR conditions were optimised for high GC templates.
  • PCR products were subjected to digest with specific restriction enzymes to differentiate between PCR products of wild type genomic sequences and successful genome editing by knock-ins. Positive samples were purified using Qiaquick PCR purification kit (Qiagen) and validated by Sanger sequencing.
  • Qiaquick PCR purification kit Qiagen
  • Sanger sequencing A schematic example of PCR-digest determination of promoter knock-in can be seen in Fig. 16.
  • RNA isolation and Real-time quantitative PCR (RT-qPCR).
  • Seed cultures were diluted 1: 100 into 50 mL of MGY broth in 250 mL baffled flasks containing -30-40 5 mm glass beads and incubated at 30 °C with 250 rpm shaking (10 to 14 days for 5. roseosporus, 5 to 7 days for 5. venezuelae). The cultures were harvested by pelleting at maximum speed in an Eppendorf 5810R centrifuge for 10 minutes. The cell pellet was stored at -80 °C while the supernatants were split into two 50 mL falcon tubes. Culture supernatants were extracted three times with equal volume ethyl acetate. For solid-state cultures, the strains were grown on MGY plates at 30 °C for 10 days.
  • HPLC parameters were as follows: solvent A, 0.1% trifluoroacetic acid in water; solvent B, 0.1% trifluoroacetic acid in acetonitrile; gradient at a constant flow rate of 0.2 mL/min, 10% B for 5 minutes, 10% to 100% B in 35 minutes, maintain at 100% B for 10 minutes, return to 10% B in 1 minutes and finally maintain at 10% B for 10 minutes; detection by ultraviolet spectroscopy at 210 nm, 254 nm, 280 nm, 320 nm.
  • the cell pellet was stored at -80 °C while the supernatants were split into two 50 mL falcon tubes, flash frozen liquid nitrogen and lyophilized to dryness. 25 and 10 mL of methanol was added to each tube containing dried supernatant and frozen cell pellets respectively. The methanol mixtures were vortexed for 1 min each and incubated on a platform shaker at 4 °C for 2 hours. Samples were clarified by spinning at maximum speed in an Eppendorf 5810R centrifuge for 10 minutes twice and pooling the methanol extracts from the respective pellets and lyophilized culture supernatants. A generous amount of anhydrous sodium sulfate was added to the extracts and stirred.
  • the extracts were decanted, concentrated to dryness and resuspended in 700 ⁇ L ⁇ deuterium oxide added in two 350 ⁇ L ⁇ aliquots.
  • a spatula-full of Chelex-100 resin (Bio-Rad) was added to each sample in a 1.7 mL centrifuge tube, which was incubated for 30 minutes at room temperature with agitation on a Thermo microplate shaker.
  • the samples were clarified twice by centrifuging at maximum speed in an Eppendorf bench top centrifuge for 1 minute each time.
  • the supernatants were then filtered using a 10 kDa Vivaspin column (GE Healthcare) and the filtrates were transferred to a 5 mm NMR tube for NMR analysis.
  • 31 P-NMR has been acquired using a Bruker DRX-600 spectrometer equipped with a 5mm BBFO cryoprobe. Proton decoupled 31 P- NMR spectra are referenced to an external H 3 P0 4 (aq) standard ( ⁇ 0.0 ppm). All samples have been acquired for 6000 scans. Identity of FR-900098 was confirmed by 1) spiking with the sample with authentic FR-900098, 2) 31 P HMBC data comparison; 3) HRMS data. Production titers were estimated by spiking in known amounts of FR-900098.
  • PTM was eluted in Fl l according to LCMS analysis.
  • Fl l was subjected to semi-prep HPLC using a C18 column (Phenomenex, 250 x 10 mm) with the following gradient: 5-40 minutes 5%-20% acetonitrile in water with 0.1% formic acid; 40-60 minutes 20%-50% acetonitrile in water with 0.1% formic acid; 60-70 min 50%-60% acetonitrile in water with 0.1% formic acid.
  • 2 was eluted at 62 minutes. 1 was eluted at 61 minutes.
  • NMR analysis was performed on an Agilent 600 MHz NMR spectrometer.
  • albus seed cultures were diluted 1 : 100 into 50 mL of MGY broth in 250 mL baffled flasks and grown at 25 °C with 250 rpm shaking for 2 to 3 days. Culture supernatants of wild type and engineered 5. albus strains were extracted twice with equal volume ethyl acetate containing 1 % (v/v) formic acid. Extracts were dried and re-suspended in methanol prior to analysis by LCMS using ESI source in positive ion mode (Bruker, Amazon SL Ion Trap) equipped with a Kinetex 2.6 ⁇ XB-C18 100 A (Phenomenex).
  • HPLC parameters were as follows: solvent A, 0.1% trifluoroacetic acid in water; solvent B, 0.1% trifluoroacetic acid in acetonitrile; gradient at a constant flow rate of 1 mL/minute, 5% B for 2 minutes, 5% to 100% B in 15 minutes, maintain at 100% B for 2 minutes, return to 5% B and maintain for 2 minutes; detection by ultraviolet spectroscopy at 500 nm (RED, ACT) or 600 nm (indigoidine). MS/MS was performed in positive auto MS(n) mode with scan range m z 100-1000.
  • Table 1 Bacterial strains and plasmids used in this study. Plasmids with editing templates used for promoter knock-in are included below the respective engineered strains in underlined font.
  • 2k-2 (sgRNA 2) application pCM2-kasO *p-indC- pCM2-kasO*p-indC-2k-l with 1 kb homology arms
  • This lk-1 instead of 2kb application pCM2-kasO *p-indC- pCM2-kasO *p-indC-2k-2 with 1 kb homology arms
  • This lk-2 instead of 2kb application pCM2-kasO *p-indC-
  • NRRL refers to the ARS Culture Collection (NRRL), a culture collection of the Agricultural Research Service (ARS).
  • ARS Agricultural Research Service
  • cagtcctgcacg construct (Scheme 2) npPP269 aaaaaTCTAGActcaggaacggtcggttccggg PCR flank 1 for 5.
  • gcgccggtcagccaaca editing construct npPP271 aaaaaCATATGatgaagcgtttccgcttactcgtcctc PCR flank 2 for 5.
  • cagtcctgcacg construct (Scheme 2) npPP273 aaaaaTCTAGAacgccatcccgatgacggctgc PCR flank 1 for 5.
  • cagtcctgcacg construct (Scheme 2) npPP281 aaaaaTCTAGAtgccacagcagatagtgcggatcaca PCR flank 1 for 5.
  • ttacgagcggaagaacgac editing construct npPP283 aaaaaCATATGatgtctgaagacagActggtcggcgcg PCR flank 2 for 5.
  • npPP612 PCR flank 2 for 5.
  • npPP602 PCR flank 2 for 5.
  • cluster (cluster 24))
  • npPP342 caggcggcgtcgcttttcag Sequencing primers for edited genomic region npPP343 tagacgaaaacgttcaacgccacca (5. roseosporus R32 cluster)
  • npPP360 gatgagcaggtcccagaaggcctcgg PCR of target genomic locus for 5. roseosporus npPP361 gttcgccgtgctcgaagtcctgatcgg
  • npPP344 gccacggacatgcacgacga Sequencing primers for edited genomic region npPP345 gcgagcggttccacggtgt (5. roseosporus R35 cluster)
  • npPP362 cgttcggcgatcgcgttcatcgcc PCR of target genomic locus for 5. roseosporus npPP363 gtcgcgttgattccgaccatcgccc
  • npPP340 agtttgccgggcattctgtcca Sequencing primers for edited genomic region npPP341 gcgtccatgagccgcttgttct (5. roseosporus R22 cluster)
  • npPP286 tccggcgaagtgcacatggcagtc PCR of target genomic locus for 5. roseosporus npPP287 accagcgccatctcgaagacctgga
  • npPP244 gcaactgaatctccaggtcgg Sequencing primers for edited genomic region npPP245 cagcgccacggttccactg (5. roseosporus R26 cluster)
  • npPP674 ggacgggaagatcacaccggtctccgtgg PCR of target genomic locus for 5. roseosporus npPP675 ctgcgaccgcttcgtcaggtcgcattcg
  • roseosporus R3 cluster (cluster 3) npPP677 tcctggaggagaagatccgttcgctgga PCR of target genomic locus for 5. roseosporus npPP678 cgcagcacctcgacggccttgatcagccc
  • R14 cluster npPP679 cggtatcgaccggtccgagggtgattcacg Sequencing primers for edited genomic region npPP701 cccggcccgtcgtctcgtagacgaagagat (5. roseosporus R14 cluster) npPP680 ccgcgactggctgcgcgtgaagacgagag PCR of target genomic locus for 5. roseosporus npPP681 ccggccttccaggagggtcacgtcgagt
  • Sequencing primer for edited genomic region npPP685 acaggaacggaacccgtcggaccggcgt
  • npPP693 gaaggtcggcgaagatctcgccccagtacg PCR of target genomic locus for 5. roseosporus npPP694 cgcttgtcggtcttgccgttcggcgtgagc
  • npPP695 acgtaccccgtgacgaaggcctgttcacc Sequencing primers for edited genomic region npPP696 gtaccggaccgcccgtacatcgatatcggg (5. roseosporus R5 cluster)
  • npPP689 gtcaccatcggctcctacgacggggtgcac PCR of target genomic locus for 5.
  • venezuelae npPP699 ccttcggcatgatctcgcaggcgctgatgg
  • SV16 cluster (cluster 16) npPP700 ccggtcatcttggtgacctgctggtcgagc Sequencing primers for edited genomic region, npPP701 gcttcagggtctcctcgatgggctgcacg 5. venezuelae SV16 cluster (cluster 16) npPP200 gcctccgccgacctgtgaacggta PCR of target genomic locus for 5. lividans npPP201 cggcgagtcagcaggactccgaacggac
  • npPP164 cgtgatcgacgacgaaccgcaga PCR of target genomic locus for 5.
  • npPP178 gcgcctggagggcgttgaggacg RED cluster - control primer pair for left flank
  • npPP355 cataactcccccagtcctgcacg RED cluster - kasO*p- specific primer for left flank, used with npPP164
  • npPP176 cggcaccccatccgctcatgggag PCR of target genomic locus for 5.
  • lividans RED cluster - control npPP227 tggtagaggtcccggtcgaacaactcggccgg
  • Sequencing primers for edited genomic region npPP804 caccacagtgccagtaggtctggtacggta
  • knock-ins were performed with editing templates containing the indicated insert with 2 kb homology flanks.
  • No protospacer refers to the same knock-in constructs for the indicated cluster without a protospacer.
  • b kasO*p and P8-kasO*p cassettes are 97 and 774 bp respectively, tsr refers to a ⁇ 1 kb thiostrepton- resistance cassette.
  • Table 4 AntiSMASH analyses of 5. roseosporus NRRL15998 (NCBI Reference Sequence: NZ_DS999644.1). Previously observed compounds include daptomycin (clusters 1, 2), napsamycin (cluster 9), stenothricin (cluster 5) and arylomycin (cluster 20).
  • Table 6 Sequence homology of 5. roseosporous cluster 10 to FR-900098 biosynthetic gene cluster from S. rubellomurinus.
  • Cluster 27 Melanin 7484949 7495338
  • Cluster 28 Nrps 7706602 7760938
  • Cluster 29 Terpene 7788497 7809951
  • Cluster 30 T3pks 7946146 7987237
  • Cluster 31 Terpene-Nrps 8189935 8226158
  • Table 8 AntiSMASH analyses of 5. viridochr omo genes DSM 40736 (NCBI Reference Sequence: NZ_ACEZ00000000.1). Previously observed product from 5. viridochromogenes include phosphinothricin.

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Abstract

L'invention concerne des procédés par recombinaison d'activation de l'expression d'au moins un groupe de gènes biosynthétiques, ou d'au moins un gène cible dans un groupe de gènes biosynthétiques comprenant plus d'un gène, le procédé consistant à insérer un ou plusieurs promoteurs à l'aide de la technologie CRISPR dans au moins un emplacement fonctionnel sur le plan transcriptionnel par rapport au(x) groupe(s) de gènes biosynthétiques ou du ou des gènes cibles dans le groupe de gènes biosynthétiques, l'introduction du ou des promoteurs entraînant une augmentation de l'expression du groupe ou des groupes de gènes biosynthétiques ou du ou des gènes cibles par comparaison avec le niveau d'expression d'un ou plusieurs groupes de gènes biosynthétiques ou d'un ou plusieurs gènes cibles non modifiés. L'invention concerne également des plasmides d'expression recombinés pour activer l'expression d'un ou plusieurs groupes de gènes biosynthétiques.
PCT/SG2017/050092 2016-02-29 2017-02-28 Activation multiplexable de groupes biosynthétiques silencieux dans des hôtes actinomycètes natifs pour la découverte de produits naturels WO2017151059A1 (fr)

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CN114075522B (zh) * 2021-11-26 2023-10-24 河南省科学院生物研究所有限责任公司 一株具有广谱抗菌活性的放线菌f8及其应用
CN115960791A (zh) * 2023-01-08 2023-04-14 天津大学 一种海洋来源放线菌基因遗传操作体系及构建方法

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