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EP3994154A1 - Streptomyces clavuligerus - Google Patents

Streptomyces clavuligerus

Info

Publication number
EP3994154A1
EP3994154A1 EP20737125.3A EP20737125A EP3994154A1 EP 3994154 A1 EP3994154 A1 EP 3994154A1 EP 20737125 A EP20737125 A EP 20737125A EP 3994154 A1 EP3994154 A1 EP 3994154A1
Authority
EP
European Patent Office
Prior art keywords
ccar
clavuligerus
clavulanic acid
streptomyces clavuligerus
fermentation
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP20737125.3A
Other languages
German (de)
English (en)
Inventor
Andrew John Collis
Nicola CROWHURST
Katherine Joyce HONICKER
Steven Gary Kendrew
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
GlaxoSmithKline Intellectual Property No 2 Ltd
Original Assignee
GlaxoSmithKline Intellectual Property No 2 Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GB1909698.1A external-priority patent/GB2585246B/en
Priority claimed from GBGB2005327.8A external-priority patent/GB202005327D0/en
Application filed by GlaxoSmithKline Intellectual Property No 2 Ltd filed Critical GlaxoSmithKline Intellectual Property No 2 Ltd
Publication of EP3994154A1 publication Critical patent/EP3994154A1/fr
Pending legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/42—Oxazoles
    • A61K31/424—Oxazoles condensed with heterocyclic ring systems, e.g. clavulanic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/429—Thiazoles condensed with heterocyclic ring systems
    • A61K31/43—Compounds containing 4-thia-1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula, e.g. penicillins, penems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/36—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Actinomyces; from Streptomyces (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C12P17/188—Heterocyclic compound containing in the condensed system at least one hetero ring having nitrogen atoms and oxygen atoms as the only ring heteroatoms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/465—Streptomyces

Definitions

  • the invention generally relates to the improvement of clavulanic acid production in Streptomyces clavuligerus (S. clavuligerus ).
  • the invention provides a S. clavuligerus comprising point mutations in the ccaR promoter region and gene, a vector comprising a ccaR promoter region and gene comprising said mutations for making said S. clavuligerus and a method for producing clavulanic acid using said S. clavuligerus, and pharmaceutical formulations prepared using clavulanic acid produced using said S. clavuligerus.
  • b-lactam antibiotics such as penicillins
  • penicillins are the most widely used class of antibiotics (Bush & Bradford (2016) Cold Spring Harb Perspect Med).
  • certain pathogens have developed resistance to b-lactam antibiotics by producing b-lactamase, thereby reducing the effectiveness of b- lactam antibiotics.
  • Clavulanic acid is a b-lactam compound structurally related to the penicillins that is produced as a fermentation product by S. clavuligerus. Clavulanic acid was isolated from S. clavuligerus and characterised as a novel b-lactamase inhibitor by Reading and Cole in 1976 (Reading & Cole, (1977) Antimicrob. Agents Chemother.). The addition of clavulanic acid was shown to enhance the antibacterial activity of b-lactamase-labile antibiotics.
  • clavulanic acid has been widely used in the pharmaceutical industry in combination with b-lactam antibiotics to treat infections caused by b-lactamase producing pathogens that would otherwise be resistant to b-lactam antibiotics. It is understood that the b-lactam structure of clavulanic acid binds to the active site of b-lactamase produced by the pathogen, in place of the b-lactam antibiotic, causing irreversible inhibition of the enzyme (Labia and Peduzzi, (1985) Drugs Exp. Clin. Res, ⁇ Georgopapadkou (2004) Exp. Opin. Investig. Drugs).
  • S. c/a vu//gerus strains comprising mutations in the ccaR promoter region and the ccaR gene produces a higher titre of clavulanic acid and at a faster rate as compared to a wild-type (WT) S. clavuligerus.
  • WT wild-type
  • the present disclosure will allow an improved process for clavulanic acid production, particularly at an industrial scale, wherein higher titres of clavulanic acid are produced at lower costs as compared to WT S. clavuligerus.
  • a S. clavuligerus comprising two point mutations in a ccaR promoter region and a substitution mutation in a ccaR gene, wherein the point mutations in the ccaR promoter region are a C (cytosine) to T (thymine) mutation at a site corresponding to position 48 of SEQ ID NO: l and a G (guanine) to A (adenine) point mutation at a site corresponding to position 143 of SEQ ID NO: l; and wherein the mutation in the ccaR gene is an arginine to tryptophan substitution at a site corresponding to position 32 of SEQ ID NO: 2.
  • a S. clavuligerus of the invention for use in the production of clavulanic acid.
  • a vector comprising nucleic acid sequences comprising a ccaR promoter region comprising two point mutations and a ccaR gene comprising a substitution mutation, wherein the point mutations in the ccaR promoter region are a C to T mutation at a site corresponding to position 48 of SEQ ID NO: l and a G to A mutation at a site corresponding to position 143 of SEQ ID NO: l; and wherein the substitution mutation in the ccaR gene is an arginine to tryptophan substitution at a site corresponding to position 32 of SEQ ID NO: 2.
  • a method for producing clavulanic acid comprising the steps of growing a S. clavuligerus of the invention and recovering clavulanic acid produced by said S. clavuligerus, or progeny thereof.
  • a method for producing a pharmaceutical formulation comprising a b-lactam antibiotic and clavulanic acid, the method comprising the steps of (a) growing a S. clavuligerus of the invention; (b) recovering clavulanic acid produced by said S. clavuligerus, or progeny thereof; and (c) preparing the pharmaceutical formulation by combining the clavulanic acid recovered in step (b) with the b-lactam antibiotic.
  • FIG. 1 Effect on fermentation of ccaR mutations Ml, M2 and M3 combined: Clavulanic acid titre profiles
  • FIG: 2 Effect of ccaR mutation: Fermentation titre at 65 hours
  • FIG. 3 Effect of ccaR mutations on fermentation: Clavulanic acid titre profiles
  • FIG. 4 Effect of combined ccaR mutations Ml, M2 and M3 on fermentation viscosity profiles
  • FIG. 5 Effect of ccaR mutations on fermentation viscosity profiles DETAILED DESCRIPTION OF THE INVENTION
  • the present invention provides significant improvements to the process of clavulanic acid production using S. clavuligerus by introducing mutations in the ccaR promoter region and gene.
  • Clavulanic acid produced by S. clavuligerus strains of the present invention may be combined with a b-lactam antibiotic, such as amoxicillin to produce AUGMENTIN.
  • Biosynthesis of clavulanic acid can be divided into early and late stage synthesis and, accordingly, depending on their function, genes involved in clavulanic acid synthesis may also be categorised into early and late stage genes.
  • the S. clavuligerus ccaR gene is located within the cephamycin C gene cluster and is upstream of the blp gene (Perez-Llarena et ai. (1997) J. Bacteriol.).
  • the nucleotide sequence of the ccaR promoter region, ccaR gene and the linked blp gene is available on GenBank (accession no. Z81324).
  • the ccaR gene encodes a positive-acting transcriptional regulator, CcaR, that controls the production of both clavulanic acid and cephamycin C (Alexander & Jensen (1998) J. Bacteriol., ⁇ Santamarta et al. (2011) Mol. Microbiol.). Clavulanic acid and cephamycin C production was abolished in S. clavuligerus ccaR knock-out mutants and restored on re-introduction of wild-type ccaR, showing that CcaR positively regulates the production of both proteins.
  • CcaR regulates clavulanic acid production both directly and indirectly.
  • CcaR regulates the expression of the ceaS2-bls-pah-cas2 polycistronic transcript, which products are involved in the 'early' reaction pathway leading to the formation of clavaminic acid.
  • claR which in turn regulates expression of genes involved in the 'late' reaction pathway of converting clavaminic acid to clavulanic acid.
  • CcaR was shown to bind upstream of ccaR and autoregulate its own expression.
  • the inventors have identified three mutations within the ccaR promoter region and gene, outlined below, for developing an improved S. clavuligerus strain that produces a higher titre of clavulanic acid over a shorter fermentation period as compared to a WT S. clavuligerus strain.
  • Mutation 1 is a point mutation from C to T at a site corresponding position 48 of SEQ ID NO: 1 (48C>T).
  • Mutation 2 is a point mutation from G to A at a site corresponding to position 143 of SEQ ID NO: 1 (143G>A).
  • Mutation 3 is an arginine to tryptophan amino acid substitution at a site corresponding to position 32 of SEQ ID NO:2 (R32W).
  • a Streptomyces clavuligerus comprising two point mutations in a ccaR promoter region and a substitution mutation in a ccaR gene, wherein the point mutations in the ccaR promoter region are a C to T mutation at a site corresponding to 48 of SEQ ID NO: l and a G to A mutation at a site corresponding to 143 of SEQ ID NO: l; and wherein the mutation in the ccaR gene is an arginine to tryptophan substitution at a site corresponding to 32 of SEQ ID NO: 2.
  • the arginine to tryptophan amino acid substitution is a result of a point mutation from C to T at a site corresponding to position 344 of SEQ ID NO: 1 (344C>T).
  • the S. clavuligerus comprises a nucleic acid sequence of SEQ ID NO: 3.
  • a S. clavuligerus comprising a nucleic acid sequence having at least 80%, 85%, 90%, 95% or more identity with SEQ ID NO: 3 wherein said nucleic acid sequence comprises, Ml; M2; M3; Ml and M3; Ml and M2; M2 and M3; or Ml, M2 and M3.
  • nucleic acid sequences of the ccaR promoter region and the nucleic acid sequences encoding the ccaR gene comprising one or more of Ml, M2 and M3 are integrated into the chromosomal DNA of the S. clavuligerus strain.
  • nucleic acid sequences of the ccaR promoter region and the nucleic acid sequences encoding the ccaR gene comprising one or more of Ml, M2 and M3 are located extrachromosoma I ly in the S. clavuligerus strain.
  • S. clavuligerus F613-1 is an industrial strain.
  • the genome of F613-1 was sequenced in 2016, providing a complete genome sequence of S. clavuligerus (GenBank Accession no. CP016559.1).
  • the skilled person would readily identify that within this sequence, the locations of Ml, M2 and M3 correspond to positions 2,011,673, 2,011,768 and 2,011,969, respectively.
  • the S. clavuligerus F613-1 is an industrial strain.
  • the genome of F613-1 was sequenced in 2016, providing a complete genome sequence of S. clavuligerus (GenBank Accession no. CP016559.1).
  • the skilled person would readily identify that within this sequence, the locations of Ml, M2 and M3 correspond to positions 2,011,673, 2,011,768 and 2,011,969, respectively.
  • the S. clavuligerus F613-1 is an industrial strain.
  • the genome of F613-1 was sequenced in 2016, providing a complete genome sequence of S.
  • clavuligerus FID-5 strain chromosome (GenBank Accession no. CP032052J,), the locations of Ml, M2 and M3 correspond to positions 2,013,392, 2,013,487 and 2,013,688, respectively.
  • GenBank accession no. AH006362 S. clavuligerus ATCC 27064
  • the locations of Ml, M2 and M3 correspond to positions 14,042, 14,137 and 14,338, respectively.
  • a significant advantage seen in the strains after introduction of the ccaR promoter region and gene mutations was the speed of accretion of clavulanic acid.
  • the S. clavuligerus strains of the invention produce higher titres of clavulanic acid as compared to WT S. clavuligerus strain and the higher titre is reached within a reduced amount of fermentation time.
  • This increased productivity of clavulanic acid by the mutant S. clavuligerus strains of the invention is key in enabling more efficient downstream processes in extraction and purification of clavulanic acid as the ratio of product to impurity is improved. Further, the capacity and speed of an industrial plant for producing pharmaceutical formulations comprising clavulanic acid is increased, thereby reducing cost of production.
  • the S. clavuligerus strain of the present invention produces a higher titre of clavulanic acid as compared to a WT S. clavuligerus strain.
  • the S. clavuligerus is capable of producing a titre of about 0.5 g/L or more, about 0.75 g/L or more, about 1 g/L or more, about 1.25 g/L or more, about 1.5 g/L or more, about 1.75 g/L or more, about 2 g/L or more or about 2.5 g/L or more of clavulanic acid.
  • the S. clavuligerus is capable of producing a titre of about 2.1 g/L or more of clavulanic acid.
  • the S. clavuligerus is capable of producing a titre of about 0.5 g/L or more, about 0.75 g/L or more, about 1 g/L or more, about 1.25 g/L or more, about 1.5 g/L or more, about 1.75 g/L or more, about 2 g/L or more, about 2.1 g/L or more, or about 2.5 g/L of clavulanic acid is produced after 65 hours of fermentation.
  • the S. clavuligerus is capable of producing a titre of about 2.1 mg/mL or more of clavulanic acid is produced after 65 hours of fermentation.
  • said titres are produced after about 40 hours, 45, hours, 50 hours, 55 hours, 60 hours, 65 hours, 70 hours, 72 hours, 75 hours, 80 hours, 85 hours, 90 hours, 95 hours, 100 hours, 105 hours, 110 hours, 115 hours, 120 hours, 125 hours, 130 hours, 135 hours or 140 hours of fermentation.
  • the S. clavuligerus is capable of producing a titre of about 50% or more, about 75% or more, about 100% or more, about 125% or more, about 150% or more, about 175% or more, about 200% or more, about 225% or more, about 250% or more, about 275% or more, about 300% or more, about 325% or more, about 350% or more or about 375% or more, about 400% or more, about 425% or more, about 450% or more, about 475% or more, about 500% or more, about 525% or more, about 550% or more, about 575% or more, about 600% or more, about 625% or more, about 650% or more, about 675% or more, about 700% or more, about 725% or more, about 750% or more, about 775% or more, about 800% or more, about 825% or more, about 850% or more, about 875% or more, about 900% or more, about 925% or more, about 950% or more, about 975% or more, or
  • said percentage increases in titres of clavulanic acid as compared to WT S. clavuligerus are produced after 65 hours of fermentation. In some embodiments, said percentage increases in titres of clavulanic acid as compared to WT S. clavuligerus are produced after about 40, 45, 50, 55, 60, 65, 70, 72, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135 or 140 hours of fermentation.
  • the S. clavuligerus of the invention is capable of producing a titre of about 1000% or more clavulanic acid after 65 hours of fermentation as compared to wild-type S. clavuligerus.
  • a further significant advantage conferred by the strains comprising the ccaR promoter region and gene mutations was a reduction in viscosity of the fermentation culture.
  • S. clavuligerus is a filamentous organism and the morphology of the fermented culture is an important parameter affecting the efficiency of gas and heat transfer in a fermentation.
  • Viscosity measurements of cultured samples are used to indicate the difficulty and energy involved in controlling dissolved oxygen and maintaining temperature in a large-scale fermentation.
  • a desirable feature of a culture is high titre without very high viscosity.
  • the Streptomyces clavuligerus of the present invention is capable of reducing viscosity of a fermentation broth, of which it is comprised, by about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, or about 40% or more as compared to WT Streptomyces clavuligerus.
  • the Streptomyces clavuligerus of the present invention is capable of reducing viscosity of a fermentation broth by about 40% or more after 65 hours of fermentation as compared to WT Streptomyces clavuligerus.
  • the fermentation broth comprises the Streptomyces clavuligerus of the present invention
  • the Streptomyces clavuligerus of the present invention is capable of a viscosity of a fermentation broth, of which it is comprised, of about 35 centipoise or less, about 30 centipoise or less, 25 centipoise or less or 20 centipoise or less.
  • the Streptomyces clavuligerus of the present invention is capable of a reduction in viscosity of a fermentation broth of which it is comprised, relative to the viscosity of WT Streptomyces clavuligerus, of about 5 centipoise or more, about 7 or more, about 10 centipoise or more, 13 centipoise or more, 15 centipoise or more, 17 centipoise or more, or 20 centipoise or more.
  • the Streptomyces clavuligerus of the present invention is capable of a reduction in viscosity of a fermentation broth of which it is comprised, relative to the viscosity of WT Streptomyces clavuligerus, of about 6 centipoise or more, 13 centipoise or more, or 17 centripoise or more. In one embodiment, said viscosity, or reduction in viscosity is after 65 hours of fermentation. In one embodiment, the Streptomyces clavuligerus of the present invention is capable of a viscosity of a fermentation broth, of which it is comprised, of about 28 centipoise or less after 65 hours of fermentation. In one embodiment, the Streptomyces clavuligerus of the present invention is capable of a viscosity of a fermentation broth, of which it is comprised, of about 25 centipoise or less after 65 hours of fermentation.
  • S. clavuligerus may comprise one or more mutations selected from Ml, M2 and M3.
  • the S. clavuligerus may comprise two mutations selected from Ml, M2 and M3.
  • the S. clavuligerus may comprise two mutations selected from Ml, M2 and M3, wherein one of the two mutations is M2.
  • the S. clavuligerus may comprise a single mutation selected from Ml, M2 and M3.
  • a S. clavuligerus of the invention for use in the production of clavulanic acid.
  • a vector comprising nucleic acid sequences comprising a ccaR promoter region comprising two point mutations and a ccaR gene comprising a substitution mutation, wherein the mutations in the ccaR promoter region are a C to T point mutation at a site corresponding to position 48 of SEQ ID NO: l and a G to A point mutation at a site corresponding to position 143 of SEQ ID NO: l; and wherein the mutation in the ccaR gene is an arginine to tryptophan substitution at a site corresponding to position 32 of SEQ ID NO: 2.
  • the arginine to tryptophan amino acid substitution is a result of a point mutation from C to T at a site corresponding to position 344 of SEQ ID NO: 1.
  • the vector is a plasmid. In another embodiment, the vector is linear DNA. In yet another embodiment, the vector is a bacteriophage. Other vectors able to artificially carry genetic material into another cell, where it can be replicated and/or expressed are well known in the art and may be used.
  • the ccaR promoter region is operably linked to the ccaR gene. However, in one embodiment, the ccaR promoter region is not operably linked to the ccaR gene.
  • the vector comprises a nucleic acid sequence of SEQ ID NO: 3.
  • S. clavuligerus comprising a nucleic acid sequence having at least 80, 85, 90, 95% or more identity with SEQ ID NO: 3, wherein said nucleic acid sequence comprises Ml and M2, M2 and M3, or Ml, M2 and M3.
  • the vector of the invention is used for producing a S. clavuligerus of the invention.
  • the mutated ccaR promoter region and gene of the present invention may be obtained by conventional cloning methods (such as PCR) based on the sequences provided herein.
  • the vectors of the invention are used to prepare a S. clavuligerus of the present invention by way of transformation into an organism capable of subsequent conjugation with S. clavuligerus using methods known in the art.
  • techniques and base vectors used in Streptomyces biology are described in Practical Streptomyces Genetics (T. Keiser, M.J. Bibb, M.J. Buttner, K.F. Chater, D.A. Hopwood (2000)), a manual published by the John Innes Centre.
  • G3P glyceraldehyde-3-phosphate
  • arginine is two essential precursors in the clavulanic acid pathway.
  • G3P may alternatively enter the glycolytic pathway and then the Krebs cycle, channelled by glyceraldehyde-3- phosphate dehydrogenase (gap).
  • gap glyceraldehyde-3- phosphate dehydrogenase
  • cvml encodes an enzyme involved in the conversion of clavaminic acid to 3S, 5S clavams, which, contrary to the 3R, 5R stereochemistry of clavulanic acid, do not possess b-lactamase inhibitory property. Inactivation of cvml led to increased levels of clavulanic acid production (Paradkar et al. (2001) Appl. Environ. Microbiol.).
  • the S. clavuligerus strains of the invention further comprises genetic modifications known in the art.
  • a vector of the invention is used to introduce the ccaR promoter region and gene mutations to an S. clavuligerus strain comprising further genetic mutations (as compared to WT S. clavuligerus).
  • Genetic modifications of the art may be targeted mutations based on a knowledge-based approach or they may be mutations resulting from a random mutagenesis and selection approach. It is envisaged that such genetic modifications known in the art in combination with ccaR promoter region and gene mutations of the invention will provide further improvements to the S. clavuligerus strain, such as further increased clavulanic acid titre as compared to the respective genetic modification known in the art alone or the ccaR promoter and gene mutations of the invention alone.
  • the mutations within the ccaR promoter region and gene disclosed herein may be introduced into a S. clavuligerus strain comprising further mutations but comprising WT ccaR promoter region and gene. This may be performed using the vectors carrying ccaR promoter region and gene mutations disclosed herein.
  • strains of the invention may be introduced into S. c/a vu//gerus strains of the invention. Such strains may be produced using routine genetic engineering methods known in the art.
  • the S. clavuligerus strains of the present invention comprises a copy of the WT ccaR promoter region and gene.
  • ccaR promoter region and gene comprising the mutations described herein does not replace the WT ccaR promoter region and gene, but is added to it.
  • the S. clavuligerus strains of the invention comprising mutations in the ccaR promoter region and gene comprise one or more copies of the WT ccaR promoter region and gene.
  • a method for producing clavulanic acid comprising the steps of growing a Streptomyces clavuligerus of the invention and recovering clavulanic acid produced by said modified Streptomyces clavuligerus, or progeny thereof.
  • Methods for growing S. clavuligerus and recovering clavulanic acid are known in the art.
  • suitable conditions for fermentation of S. clavuligerus and extraction of clavulanic acid have been described in WOOl/87891 (SmithKIine Beecham P.L.C.) and by Ser etal. (Ser etal. (2016) Front Microbio.).
  • the method produces a titre of a titre of about 0.5 g/L or more, about 1 g/L or more, about 1.5 g/L or more, about 2 g/L or more or about 2.5 g/L or more of clavulanic acid.
  • said titres of clavulanic acid are produced by the method after 65 hours of fermentation.
  • said titres of clavulanic acid are produced by the method after about 50, 55, 60, 65, 70, 72, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135 or 140 hours of fermentation.
  • the method produces about 50% or more, about 75% or more, about 100% or more, about 125% or more, about 150% or more, about 175% or more, about 200% or more, about 225% or more, about 250% or more, about 275% or more, about 300% or more, about 325% or more, about 350% or more or about 375% or more, about 400% or more, about 425% or more, about 450% or more, about 475% or more, about 500% or more, about 525% or more, about 550% or more, about 575% or more, about 600% or more, about 625% or more, about 650% or more, about 675% or more, about 700% or more, about 725% or more, about 750% or more, about 775% or more, about 800% or more, about 825% or more, about 850% or more, about 875% or more, about 900% or more, about 925% or more, about 950% or more, about 975% or more, or about 1000% or more of clavulanic acid as
  • said percentage increases in titres of clavulanic acid as compared to WT S. clavuligerus are produced after 65 hours of fermentation. In some embodiments, said percentage increases in titres of clavulanic acid as compared to WT S. clavuligerus are produced after about 40, 45, 50, 55, 60, 65, 70, 72, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135 or 140 hours of fermentation.
  • the S. clavuligerus of the invention is capable of producing a titre of about 1000% or more clavulanic acid after 65 hours of fermentation as compared to wild-type S. clavuligerus.
  • the viscosity of the fermentation broth comprising a mutant Streptomyces clavuligerus strain of the present invention is reduced by about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, or about 40% or more as compared to WT Streptomyces clavuligerus.
  • said reduction in viscosity is obtained after 65 hours of fermentation.
  • said reduction in viscosity is obtained after about 40, 45, 50, 55, 60, 65, 70, 72, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, or 140 hours of fermentation.
  • the viscosity of the fermentation broth comprising a mutant Streptomyces clavuligerus strain of the present invention is reduced by about 40% or more after 65 hours of fermentation as compared to WT Streptomyces clavuligerus. In one embodiment, the viscosity of the fermentation broth comprising a mutant Streptomyces clavuligerus strain of the present invention is about 35 centipoise or less, about 30 centipoise or less, 25 centipoise or less or 20 centipoise or less. In one embodiment, said viscosity is after 65 hours of fermentation.
  • the viscosity of the fermentation broth comprising a mutant Streptomyces clavuligerus strain of the present invention is about 28 centipoise or less after 65 hours of fermentation. In one embodiment, the viscosity of the fermentation broth comprising a mutant Streptomyces clavuligerus strain of the present invention is about 25 centipoise or less after 65 hours of fermentation.
  • step (c) preparing the pharmaceutical formulation by combining the clavulanic acid recovered in step (b) with the b-lactam antibiotic.
  • the b-lactam antibiotic is amoxicillin.
  • the product co-amoxiclav is marketed by GlaxoSmithKline as AUGMENTIN for treating bacterial infections. It comprises a combination of the b-lactam antibacterial agent amoxycillin and clavulanic acid.
  • the product is provided in various pharmaceutical formulations, for instance tablets, capsule powders and sachets containing free flowing granules.
  • the pharmaceutical formulation may comprise different ratios of b-lactam antibiotic, such as amoxicillin or ticarcillin, and clavulanic acid.
  • clavulanic acid is combined with a b-lactam antibiotic at ratios ranging from 3: 1 to 30: 1. The specific ratio may be dependent on type of formulation, dosing regime, route of administration and/or target indication.
  • the pharmaceutical formulations of the invention comprise amoxycillin trihydrate and potassium clavulanate.
  • Suitable pharmaceutical formulations comprising amoxicillin and clavulanic acid for use in the present invention have been described in W094/27557 (SmithKIine Beecham Corporation) and W098/35672 (SmithKIine Beecham Laboratoires Pharmaceutica).
  • a pharmaceutical formulation comprising clavulanic acid produced by the method described herein, further comprising a b-lactam antibiotic.
  • the b-lactam antibiotic is amoxicillin.
  • the pharmaceutical formulation is AUGMENTIN.
  • a genetically engineered microorganism that produces clavulanic acid comprising two point mutations in a ccaR promoter region and a substitution mutation in a ccaR gene, wherein the point mutations in the ccaR promoter region are a C to T point mutation at a site corresponding to position 48 of SEQ ID NO: l and a G to A point mutation at a site corresponding to position 143 of SEQ ID NO: l; and wherein the substitution mutation in the ccaR gene is an arginine to tryptophan substitution at a site corresponding to position 32 of SEQ ID NO: 2.
  • the genetically engineered microorganism is Streptomyces clavuligerus.
  • the ccaR gene is integrated into the chromosomal DNA of said microorganism.
  • the arginine to tryptophan substitution is a result of a C to T point mutation at a site corresponding to position 344 of SEQ ID NO: 1.
  • the genetically engineered microorganism comprises a nucleic acid sequence of SEQ ID NO:3.
  • the genetically engineered microorganism is capable of producing a titre of about 0.5 g/L or more, about 1 g/L or more, about 1.5 g/L or more, about 2 g/L or more or about 2.5 g/L or more of clavulanic acid.
  • the genetically engineered microorganism is capable of producing a titre of about 2.1 g/L or more of clavulanic acid.
  • said titre of clavulanic acid is produced after 65 hours of fermentation.
  • the genetically engineered microorganism is capable of producing a titre of about 100% or more, about 200% or more, about 300% or more, about 400% or more, about 500% or more, about 600% or more, about 700% or more, about 800% or more, about 900% or more, or about 1000% or more of clavulanic acid after 65 hours of fermentation as compared to wild-type (WT) microorganism.
  • WT microorganism refer to the same species of microorganism as the genetically engineered microorganis, that is to say a non-genetically engineered microorganism of the same species.
  • the genetically engineered microorganism is capable of producing a titre of about 1000% or more of clavulanic acid after 65 hours of fermentation as compared to WT microorganism. In one embodiment, the genetically engineered microorganism is capable of reducing viscosity of a fermentation broth by about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, or about 40% or more after 65 hours of fermentation as compared to wild-type (WT) microorganism. In another embodiment, the microorganism is capable of reducing viscosity of a fermentation broth by about 40% or more after 65 hours of fermentation as compared to WT microorganism.
  • a genetically engineered microorganism described herein for use in the production of clavulanic acid.
  • a method for producing clavulanic acid comprising the steps of growing a genetically engineered microorganism described herein; and recovering clavulanic acid produced by the genetically engineered microorganism, or progeny thereof.
  • a titre of about 0.5 g/L or more, about 1 g/L or more, about 1.5 g/L or more, about 2 g/L or more or about 2.5 g/L or more of clavulanic acid is produced.
  • a titre of about 2.1 g/L or more of clavulanic acid is produced.
  • said titre of clavulanic acid is produced after 65 hours of fermentation.
  • a titre of about 100% or more, about 200% or more, about 300% or more, about 400% or more, about 500% or more, about 600% or more, about 700% or more, about 800% or more, about 900% or more, or about 1000% or more of clavulanic acid is produced after 65 hours of fermentation as compared to wild-type (WT) microorganism. In one embodiment, a titre of 1000% or more of clavulanic acid is produced after 65 hours of fermentation as compared to WT microorganism.
  • viscosity of a fermentation broth comprising the genetically engineered microorganism is reduced by about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, or about 40% or more after 65 hours of fermentation as compared to WT microorganism. In one embodiment, viscosity of the fermentation broth comprising the genetically engineered microorganism is reduced by about 40% or more after 65 hours of fermentation as compared to WT microorganism.
  • step (c) preparing the pharmaceutical formulation by combining the clavulanic acid recovered in step (b) with the b-lactam antibiotic.
  • the b-lactam antibiotic is amoxicillin.
  • the pharmaceutical formulation is AUGMENTIN.
  • S. clavuligerus strains described hereinbefore also apply to the genetically engineered microorganism that produces clavulanic acid.
  • aspects and embodiments also include methods for producing clavulanic acid using the S. clavuligerus strains, method for producing a pharmaceutical formulation using the S. clavuligerus strains.
  • x means, for example, x ⁇ 10%, 5%, 2% or 1%.
  • promoter region refers to nucleic acid sequence comprising any regulatory region required for gene function or expression.
  • Streptomyces clavuligerus refers to Streptomyces clavuligerus and strains thereof.
  • wild-type or “WT” as used herein in the context of "Streptomyces clavuligerus ", “S. clavuligerus, “S. clavuligerus strain” refers to a S. clavuligerus strain found in its natural, non- mutated form (/.e. S. clavuligerus - culture collection ATCC 27064).
  • WT as used herein in the context of S. clavuligerus in particular refers to a strain that does not comprise mutations in the ccaR promoter region and ccaR gene, namely, Ml, M2 or M3.
  • ccaR and "ccaR gene” are used interchangeably and as used herein refers to a ccaR gene.
  • CcaR refers to the protein encoded by the ccaR gene.
  • point mutation refers to alteration of a single base pair in a nucleotide sequence. For example, one base may be substituted for another. Such a point mutation may have one of three effects.
  • the base substitution can be a silent mutation where the altered codon corresponds to the same amino acid.
  • the base substitution can be a missense mutation where the altered codon corresponds to a different amino acid.
  • the base substitution can be a nonsense mutation where the altered codon corresponds to a stop signal.
  • substitution mutation refers to the replacement of one amino acid protein with a different amino acid. It may also refer to the replacement of one base pair in a nucleotide sequence for another base pair.
  • hours of fermentation and “log hour” in the context of fermentation as used herein, refer to the number of hours of fermentation since point of inoculation of the production media with a seed culture.
  • fermentation broth refers to a mixture of fermentation media and cells fermented therein, in particular S. clavuligerus.
  • vector or "nucleic acid vector” refers to a vehicle which is able to artificially carry genetic material into another cell, where it can be replicated and/or expressed.
  • Mutation 1 (Ml) - a point mutation from C to T at position 48 of SEQ ID NO: 1;
  • Mutation 2 (M2) - a point mutation from G to A at position 143 of SEQ ID NO: 1; and Mutation 3 (M3) - an arginine to tryptophan amino acid substitution at position 32 of SEQ ID NO:
  • the position of Ml and M2 are in the ccaR promoter region.
  • the position of M3 is within the ccaR gene.
  • Primers NC_18_22 and NC_18_23 were used to amplify the ccaR gene and surrounding 4Kb DNA (2Kb each side) by PCR using Q5TM polymerase. These primers contain a Hindlll and Xbal site, respectively.
  • Three PCRs were carried out using different genomic DNA as a template - either from Streptomyces clavuligerus WT strain, strain 3 (SC3) or strain 4 (SC4). Using these different genomic preps will yield products containing no mutations, M1+M2 and all three mutations (M1+M2+M3), respectively.
  • PCR products were cloned into pKC1132 containing a codA gene, which may be used as a selection tool, at its Hindlll and Xbal site and transformed into E. coli NEB10. Colonies were screened by colony PCR using primers NC_18_22 and NC_18_23. Plasmids were extracted from colonies yielding a correct size PCR product and these named pCLV23 (WT sequence), pCLV24 (SC3 sequence/Ml+M2) and pCLV25 (SC4 sequence/Ml+M2+M3). Plasmids were sequenced to check that the cloned DNA was correct.
  • pCLV25 contained the correct sequence.
  • pCLV23 was found to have a point mutation upstream of ccaR in the orflO gene.
  • pCLV26, pCLV27 and pCLV28 also contained this mutation.
  • pCLV24 was found to have an unwanted mutation in the ccaR gene.
  • a mutagenesis PCR was carried out on pCLV24 using primers QC_correct_ccaRmut_F and QC_correct_ccaRmut_R. Resulting plasmids were sent for sequencing and of these a plasmid selected which contained the correct sequence in the ccaR gene. The final plasmid was designated pCLV30 (M1+M2).
  • ccaR mutant Streptomyces clavuHgerus ccaR mutant plasmids prepared as outlined in Example 1 were used to prepare ccaR mutant strains of S. clavuligerus.
  • Each plasmid was transformed into electro-competent £ coli ET12567 [pUZ8002].
  • This strain of £ coli is dam- dcm- thus yielding unmethylated plasmid DNA and harbours the pUZ8002 plasmid containing oriT (an origin of transfer gene) enabling conjugation to take place.
  • ET12567 is resistant to chloramphenicol;
  • pUZ8002 is resistant to kanamycin.
  • All constructs used contained the mutated ccaR region flanked by 2Kb of DNA sequences identical to that found upstream and downstream of ccaR as found on the S. clavuligerus genome, known as the homologous arms, which were required to facilitate homologous recombination between the plasmid and S. clavuligerus genome.
  • After conjugation of plasmid DNA into S. clavuligerus mutants had undergone a single-crossover event between the plasmid and S. clavuligerus genome - yielding strains which contained both the original copy of ccaR and the mutated ccaR and plasmid DNA.
  • Mutant strains subsequently undertook a second homologous recombination event during DNA replication in which the plasmid DNA is lost, and the mutated copy of ccaR is exchanged for the original copy of ccaR. These mutants are referred to as double-crossovers. This second recombination event may also result in the excision of the plasmid and mutated copy of ccaR, giving rise to strains which had reverted to the wild-type strain.
  • S. clavuligerus mutants were patched onto three L3M9 plates and incubated at 26°C for 2 weeks giving a lawn of growth on each plate. Spores were subsequently harvested by placing 3 mL 10% sucrose onto each plate and, using a sterile 10 pL loop, scraping off spores and placing them into a sterile 25 mL tube. The total volume of each spore suspension was made to 15 mL with 10% sucrose, then using a sterile sonication probe was sonicated at an amplitude of 14 microns for 30 seconds. Sonicated spore suspensions were subsequently aliquoted into cryovials and stored at -70°C until required for fermentation.
  • ccaR mutant strains of S. clavuligerus generated as outlined in Example 2 were tested at 2 L micro-fermenter scale for clavulanic acid accretion and growth profiles against WT (SC2) S. clavuligerus working stock controls. (Testing was carried out across five fermenter runs comprising ten vessels per run. Each run included WT (SC2) controls to account for any run to run variation).
  • SC2 WT
  • Pre-inoculation media pH was adjusted to 7.0 using 17% v/v ammonium hydroxide solution and controlled at pH 6.8 throughout with this solution. Temperature was maintained at 26°C, agitation at 1400 rpm and airflow at 0.8 wm (volume of air/volume of liquid/ minute). A 32% w/v maltodextrin feed was introduced from 24 hours at a rate of 1.4 mls/hr and maintained throughout. A 5.67% w/v potassium phosphate monobasic feed solution was introduced from 0 hours at a rate of 0.3 mls/hr until 24 hours, then increased to 1.1 mls/hr until 48 hours at which time it was terminated. Fermentations were sampled throughout for clavulanic acid titre and terminated at approximately 140 hours as described below.
  • the assay involves a chemical reaction between clavulanic acid and imidazole detecting the change in absorbance when the two are mixed is measured at 313 nm.
  • Reagent Blank REAG / DIL
  • Viscosity was measured using a Brookfield DV-II+ viscometer set to 20 rpm. 1 mi- fermentation broth was placed into the centre of the viscometer sample cup and the motor switched on - the torque required to turn the spindle submerged in the fermentation broth giving a measurement of viscosity. Measurements were taken after a few seconds once the readings had stabilised.
  • Ml and M3 either alone or in combination gave an increase in titre but not to the same extent as with combining these with M2, giving titre increases over the WT control at 65 log hours for Ml, M3 and M1+M3 of 163%, 145% and 133% respectively.
  • the ccaR mutations have reduced both the viscosity at time of peak production and also the peak viscosity observed (see Table 2, FIG. 4 and FIG. 5). At 65 log hours, the greatest reduction in viscosity was produced during fermentation of strains carrying all three mutations (M1+M2+M3), followed by strains carrying M2 only, with 41% and 32% reduction in viscosity as compared to the WT control.
  • error bars indicate ⁇ one standard deviation for data acquired from multiple fermentation runs.
  • SEQ ID NO: 1 Nucleic acid sequence of a WT ccaR promoter region and gene.
  • SEQ ID NO: 2 Amino acid sequence of a WT ccaR gene.
  • SEQ ID NO: 3 Nucleic acid sequence of a ccaR promoter region and ccaR gene comprising mutations described herein.
  • SEQ ID NO: 4 Amino acid sequence of a ccaR gene comprising a substitution mutation described herein.
  • SEP ID NO: 1 Amino acid sequence of a ccaR gene comprising a substitution mutation described herein.

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Abstract

L'invention concerne une souche de Streptomyces clavuligerus comprenant deux mutations ponctuelles dans une région de promoteur ccaR et une mutation de substitution dans le gène ccaR, les mutations dans la région de promoteur ccaR étant une mutation ponctuelle C à T au niveau d'un site correspondant à la position 48 de SEQ ID NO : 1 et une mutation ponctuelle G à A au niveau d'un site correspondant à la position 143 de SEQ ID NO : 1 ; et la mutation dans le gène ccaR étant une substitution arginine en tryptophane au niveau d'un site correspondant à la position 32 de SEQ ID NO : 2. L'invention concerne également un procédé d'utilisation de ladite souche de Streptomyces clavuligerus pour produire de l'acide clavulanique.
EP20737125.3A 2019-07-05 2020-07-03 Streptomyces clavuligerus Pending EP3994154A1 (fr)

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GB1909698.1A GB2585246B (en) 2019-07-05 2019-07-05 Streptomyces clavuligerus
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PCT/EP2020/068761 WO2021004912A1 (fr) 2019-07-05 2020-07-03 Streptomyces clavuligerus

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