KR20220031043A - Streptomyces clabuligerus - Google Patents

Abstract

The present invention relates to a Streptomyces clavuligerus strain comprising two point mutations in the ccaR promoter region and a substitution mutation in the ccaR gene, wherein the mutation in the ccaR promoter region is of SEQ ID NO: 1 a C to T point mutation at the site corresponding to position 48 and a G to A point mutation at the site corresponding to position 143 of SEQ ID NO: 1; The mutation in the ccaR gene is an arginine to tryptophan substitution at the site corresponding to position 32 of SEQ ID NO:2. The invention also relates to a method of using said Streptomyces clavuligerus strain to produce clavulanic acid.

Description

Streptomyces clabuligerus

The present invention generally relates to Streptomyces clavuligerus [S. Clavuligerus ( S. clavuligerus )] relates to the improvement of clavulanic acid production. In particular, the present invention relates to the ccaR promoter region and S. Clavuligerus, supra S. A vector comprising a ccaR promoter region and a gene comprising the mutation to make clavigerus and the S. A method for producing clavulanic acid using clavuligerus, and the S. A pharmaceutical formulation prepared using clavulanic acid produced using clavuligerus is provided.

β-lactam antibiotics, such as penicillins, are the most widely used class of antibiotics (Bush & Bradford (2016) Cold Spring Harb Perspect Med ). However, certain pathogens have developed resistance to β-lactam antibiotics by producing β-lactamase, thereby reducing the effectiveness of β-lactam antibiotics.

Clavulanic acid is S. It is a β-lactam compound structurally related to penicillin produced as a fermentation product by Clavuligerus. Clavulanic acid is S. It was isolated from Clavuligerus and characterized as a novel β-lactamase inhibitor by Reading and Cole in 1976 (Reading & Cole, (1977) Antimicrob. Agents Chemother. ). The addition of clavulanic acid has been shown to enhance the antibacterial activity of β-lactamase-labile antibiotics.

Since then, clavulanic acid has been widely used in the pharmaceutical industry in combination with β-lactam antibiotics to treat infections caused by β-lactamase producing pathogens that may become resistant to β-lactam antibiotics. It is understood that the β-lactam structure of clavulanic acid binds to the active site of β-lactamase produced by the pathogen instead of β-lactam antibiotics, resulting in irreversible inhibition of the enzyme (Labia and Peduzzi, (1985)) Drugs Exp. Clin. Res ; Georgopapadkou (2004) Exp. Opin. Investig. Drugs ).

s. Improvement of the clavuligerus strain plays an important role in reducing the production cost in the industrial fermentation process of clavulanic acid. Strains development strategies to obtain higher yields of clavulanic acid in industrial fermentation have traditionally relied heavily on random mutagenesis and selection techniques. However, S. With a growing understanding of the key biosynthetic pathways and regulatory mechanisms involved in clavulanic acid production by clavuligerus, strain development strategies also included a knowledge-based rational approach (Paradkar (2013) Journal of Antibiotics ).

Thus, there continues to be a need to develop additional Streptomyces clavulanic acid producing higher yields of clavulanic acid and improved processes using these strains to produce higher yields of clavulanic acid at lower cost. do.

Surprisingly , the present inventors found that S. The clavuligerus strain is wild-type (WT) S. It was found to produce higher titers of clavulanic acid at a faster rate compared to clavuligerus. The present disclosure will allow for an improved process for clavulanic acid production, particularly on an industrial scale, wherein higher titers of clavulanic acid are used in wild-type S. It is produced at a lower cost compared to Clavuligerus.

According to a first aspect of the present invention, S. comprising two point mutations in the ccaR promoter region and a substitution mutation in the ccaR gene. provided, wherein the point mutation in the ccaR promoter region comprises a C (cytosine) to T (thymine) mutation at the site corresponding to position 48 of SEQ ID NO: 1 and SEQ ID NO: : comprises a G (guanine) to A (adenine) point mutation at the site corresponding to position 143 of 1; The mutation in the ccaR gene is an arginine to tryptophan substitution at the site corresponding to position 32 of SEQ ID NO:2.

According to a further aspect of the invention, the S. of the invention for use in the production of clavulanic acid. Clavuligerus is served.

According to another aspect of the present invention, there is provided a vector comprising a nucleic acid sequence comprising a ccaR promoter region comprising two point mutations and a ccaR gene comprising a substitution mutation, wherein the point mutation in the ccaR promoter region comprises the sequence a C to T mutation at the site corresponding to position 48 of SEQ ID NO: 1 and a G to A mutation at the site corresponding to position 143 of SEQ ID NO: 1; The substitution mutation in the ccaR gene is an arginine to tryptophan substitution at the site corresponding to position 32 of SEQ ID NO:2.

According to another aspect of the present invention, S. Growing clavuligerus and the S. A method for producing clavulanic acid is provided, comprising the step of recovering clavulanic acid produced by clavuligerus or its progeny.

According to a further aspect of the present invention, there is provided a method for producing a pharmaceutical formulation comprising a β-lactam antibiotic and clavulanic acid, the method comprising: (a) the S. Growing Clavuligerus; (b) said S. recovering clavulanic acid produced by clavuligerus or its progeny; and (c) preparing a pharmaceutical formulation by combining the clavulanic acid recovered in step (b) with a β-lactam antibiotic.

Figure 1: Effect on fermentation of combined ccaR mutants M1, M2 and M3: titer profile of clavulanic acid.
Figure 2: Effect of ccaR mutation: fermentation titer at 65 hours.
Figure 3: Effect of ccaR mutation on fermentation: titer profile of clavulanic acid.
Figure 4: Effect of combined ccaR mutations M1, M2 and M3 on fermentation viscosity profile.
Figure 5: Effect of ccaR mutation on fermentation viscosity profile.

The present invention provides S. by introducing mutations in the ccaR promoter region and gene. It provides a significant improvement to the clavulanic acid production process using clavuligerus. S. of the present invention. The clavulanic acid produced by the clavuligerus strain can be combined with a β-lactam antibiotic, such as amoxicillin, to produce AUGMENTIN.

The biosynthesis of clavulanic acid can be divided into early and late synthesis, and therefore, according to its function, genes involved in clavulanic acid synthesis can also be classified into early and late genes.

s. The clavigerus ccaR gene is located within the cepamycin C gene cluster and upstream of the blp gene (Perez-Llarena et al. (1997) J. Bacteriol. ). The nucleotide sequence of the ccaR promoter region, the ccaR gene and the linked blp gene can be obtained from GenBank (Accession No. Z81324).

The ccaR gene encodes CcaR, a positively acting transcriptional regulator that controls the production of both clavulanic acid and cepamycin C (Alexander & Jensen (1998) J. Bacteriol .; Santamarta et al. (2011) Mol. Microbiol. ) . Clavulanic acid and cefamycin C production were found in S. It was abolished in the clavigerus ccaR knockout mutant and restored upon 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. By directly regulating the expression of the ceaS2-bls-pah-cas2 polycistronic transcript, the product is involved in the 'early' reaction pathway, resulting in the formation of clavamic acid. By indirectly regulating the expression of claR , the expression of genes involved in the 'late' reaction pathway that eventually converts clavamic acid to clavulanic acid is regulated. Moreover, CcaR has been shown to bind upstream of ccaR and self-regulate its expression.

The present inventors described wild-type S. An improved S. producing higher titer of clavulanic acid over a shorter fermentation period compared to the clavuligerus strain. To develop the clavuligerus strain, three mutations in the ccaR promoter region and gene described below were identified.

Mutation 1 (M1) is a C to T point mutation at the site corresponding to position 48 of SEQ ID NO: 1 (48C>T).

Mutation 2 (M2) is a G to A point mutation at the site corresponding to position 143 of SEQ ID NO: 1 (143G>A).

Mutation 3 (M3) is an arginine to tryptophan amino acid substitution at the site corresponding to position 32 of SEQ ID NO: 2 (R32W).

According to one aspect of the present invention, there is provided Streptomyces clavuligerus comprising two point mutations in the ccaR promoter region and a substitution mutation in the ccaR gene, wherein the point mutation in the ccaR promoter region is SEQ ID NO: 1 a C to T mutation at a site corresponding to 48 of and a G to A mutation at a site corresponding to 143 of SEQ ID NO: 1; The mutation in the ccaR gene is an arginine to tryptophan substitution at the site corresponding to 32 of SEQ ID NO:2.

In one embodiment, the arginine to tryptophan amino acid substitution is the result of a C to T point mutation (344C>T) at the site corresponding to position 344 of SEQ ID NO:1.

In one embodiment, S. Clavuligerus comprises the nucleic acid sequence of SEQ ID NO:3. S. comprising a nucleic acid sequence having at least 80%, 85%, 90%, 95% or greater identity to SEQ ID NO:3. Clavuligerus is disclosed, wherein the nucleic acid sequence comprises M1; M2; M3; M1 and M3; M1 and M2; M2 and M3; or M1, M2 and M3.

In one embodiment, the nucleic acid sequence of the ccaR promoter region and the nucleic acid sequence encoding the ccaR gene comprising at least one of M1, M2 and M3 is S. integrated into the chromosomal DNA of the clavuligerus strain. In another embodiment, the nucleic acid sequence encoding the ccaR gene comprising the nucleic acid sequence of the ccaR promoter region and one or more of M1, M2 and M3 is S. It is located extrachromosomally in Clavuligerus strains.

S. comprising the ccaR promoter region and gene sequence. Multiple sequences of different strains of Clavuligerus are available. The skilled person will readily recognize the respective positions corresponding to M1, M2 and M3 in the context of these different sequences. For example, S. Clavuligerus F613-1 is an industrial strain. The genome of F613-1 was sequenced in 2016 and S. The complete genome sequence of Clavuligerus was provided (GenBank Accession No. CP016559.1 ). The skilled person will readily ascertain that within this sequence the positions of M1, M2 and M3 correspond to positions 2,011,673, 2,011,768 and 2,011,969, respectively. As a further example, S. Within the Clavuligerus F1D-5 strain chromosome (GenBank Accession No. CP032052.1 ), the positions of M1, M2 and M3 correspond to positions 2,013,392, 2,013,487 and 2,013,688, respectively. Within Genbank Accession No. AH006362 (S. clavuligerus ATCC 27064), the positions of M1, M2 and M3 correspond to positions 14,042, 14,137 and 14,338, respectively.

A significant advantage seen in the strain after introduction of ccaR promoter region and gene mutations was the rate of adhesion of clavulanic acid. In other words, the S. Clavuligerus strains are wild-type S. It produces a higher titer of clavulanic acid when compared to the clavuligerus strain, and this higher titer is reached within a reduced fermentation time. The mutant S. of the present invention. This increased productivity of clavulanic acid by the clavuligerus strain is key to enabling a more efficient downstream process in the extraction and purification of clavulanic acid as the product to impurity ratio is improved. Additionally, the capacity and speed of industrial plants for producing pharmaceutical formulations comprising clavulanic acid are increased, thereby reducing production costs.

S. of the present invention. Clavuligerus strains are wild-type S. It produces a higher titer of clavulanic acid when compared to the clavuligerus strain. In one embodiment, S. Clavuligerus is at least about 0.5 g/L, at least about 0.75 g/L, at least about 1 g/L, at least about 1.25 g/L, at least about 1.5 g/L, at least about 1.75 g/L, at least about 2 g/L Titers of clavulanic acid greater than L or greater than about 2.5 g/L can be produced. In a further embodiment, S. Clavuligerus is capable of producing a titer of clavulanic acid of about 2.1 g/L or greater.

In one embodiment, at least about 0.5 g/L, at least about 0.75 g/L, at least about 1 g/L, at least about 1.25 g/L, at least about 1.5 g/L, at least about 1.75 g/L, at least about 2 g S. capable of producing a titer of clavulanic acid of at least /L, at least about 2.1 g/L, or at least about 2.5 g/L. Clavuligerus is produced after 65 hours of fermentation. In a further embodiment, an S. that is capable of producing a titer of clavulanic acid of at least about 2.1 mg/L. Clavuligerus is produced after 65 hours of fermentation. In one embodiment, the titer is 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 of fermentation. Produced after hours, 105 hours, 110 hours, 115 hours, 120 hours, 125 hours, 130 hours, 135 hours or 140 hours.

In one embodiment, S. Clavuligerus is wild-type (WT) S. 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 when compared to clavuligerus , 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 It is possible to produce a titer of more clavulanic acid. In some embodiments, wild-type S. This percentage increase in titer of clavulanic acid as compared to clavuligerus is produced after 65 hours of fermentation. In some embodiments, wild-type S. This percentage increase in the titer of clavulanic acid as compared to clavuligerus is about 40, 45, 50, 55, 60, 65, 70, 72, 75, 80, 85, 90, 95, 100, 105, Produced after 110, 115, 120, 125, 130, 135 or 140 hours. Preferably, in one embodiment, the S. Clavuligerus is a wild-type S. Compared to clavuligerus, it is possible to produce a titer of clavulanic acid of about 1000% or more after 65 hours of fermentation.

A further significant advantage conferred by strains comprising the ccaR promoter region and gene mutations was the reduction in viscosity of the fermentation culture. s. Clavuligerus is a filamentous organism and the morphology of the fermentation culture is an important parameter affecting gas and heat transfer efficiency in fermentation. Viscosity measurements of cultured samples are used to represent the difficulties and energies associated with controlling dissolved oxygen and maintaining temperature in large-scale fermentations. A desirable feature of the culture is high titer without very high viscosity.

Accordingly, in some embodiments, Streptomyces clavuligerus of the present invention has a viscosity of a fermentation broth comprised therefrom by at least about 10%, at least about 15%, at least about 20%, at least about 25% compared to wild-type Streptomyces clavuligerus. or more, about 30% or more, about 35% or more, or about 40% or more.

In one embodiment, a Streptomyces clavigerus of the present invention is capable of reducing the viscosity of a fermentation broth by at least about 40% after 65 hours of fermentation as compared to wild-type Streptomyces clavigerus. In these embodiments, the fermentation broth comprises Streptomyces clavuligerus of the present invention.

In one embodiment, the Streptomyces clavuligerus of the present invention is capable of a viscosity of a fermentation broth consisting of about 35 centipoise or less, about 30 centipoise or less, 25 centipoise or less, or 20 centipoise or less. . In one embodiment, Streptomyces clavigerus of the present invention has a viscosity of a fermentation broth comprising of at least about 5 centipoise, at least about 7 centipoise, at least about 10 centipoise relative to the viscosity of wild-type Streptomyces clavuligerus. or more, 13 centipoise or more, 15 centipoise or more, 17 centipoise or more, or 20 centipoise or more. In one embodiment, Streptomyces clavigerus of the present invention has a viscosity of a fermentation broth comprising of at least about 6 centipoise, at least 13 centipoise, or at least 17 centipoise relative to the viscosity of wild-type Streptomyces clavuligerus. can be reduced In one embodiment, said viscosity, or decrease 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 comprising it of about 28 centipoise or less after 65 hours of fermentation. In one embodiment, the Streptomyces clavuligerus of the present invention is capable of making the viscosity of a fermentation broth comprising it about 25 centipoise or less after 65 hours of fermentation.

Thus, mutant S. An improved process for producing clavulanic acid comprising a clavuligerus strain is provided.

s. Clavuligerus may comprise one or more mutations selected from M1, M2 and M3. s. Clavigerus may comprise two mutations selected from M1, M2 and M3. s. Clavigerus may comprise two mutations selected from M1, M2 and M3, wherein one of the two mutations is M2. For example, S, including M2 and M1. Clavuligerus or S including M2 and M3. Clavuligerus. s. Clavuligerus may comprise a single mutation selected from M1, M2 and M3.

According to a further aspect of the invention, the S. of the invention for use in the production of clavulanic acid. Clavuligerus is served.

In a further aspect of the invention there is provided a vector comprising a nucleic acid sequence comprising a ccaR promoter region comprising two point mutations and a ccaR gene comprising a substitution mutation, wherein the mutation in the ccaR promoter region is SEQ ID NO: a C to T point mutation at the site corresponding to position 48 of 1 and a G to A point mutation at the site corresponding to position 143 of SEQ ID NO: 1; The mutation in the ccaR gene is an arginine to tryptophan substitution at the site corresponding to position 32 of SEQ ID NO:2.

In one embodiment, the amino acid substitution from arginine to tryptophan is the result of a C to T point mutation at the site corresponding to position 344 of SEQ ID NO:1.

In one embodiment, the vector is a plasmid. In another embodiment, the vector is linear DNA. In another embodiment, the vector is a bacteriophage. Other vectors capable of artificially transporting genetic material into another cell in which it can be replicated and/or expressed are well known in the art and can 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 a ccaR gene.

In one embodiment, the vector comprises the nucleic acid sequence of SEQ ID NO:3.

S. comprising a nucleic acid sequence having at least 80, 85, 90, 95% or greater identity to SEQ ID NO:3. Clavuligerus is disclosed, wherein the nucleic acid sequence comprises M1 and M2, M2 and M3, or M1, M2 and M3.

In a further embodiment, the vector of the invention comprises the S. Used to produce Clavuligerus.

Suitably, the mutated ccaR promoter region and gene of the invention can be obtained by conventional cloning methods (such as PCR) based on the sequences provided herein.

Further suitably, the vectors of the present invention are S. using methods known in the art. The S. Used to make Clavuligerus. For example, techniques and basic vectors used in Streptomyces biology are described in Practical Streptomyces Genetics (T. Keizer, MJ Bibb, MJ Buttner, KF Chater, DA Hopwood), a manual published by the John Innes Center. (2000))].

To date, the knowledge base of S. Strategies for improving Clavuligerus strains can be broadly classified into three approaches: (1) increasing precursor flow into pathways, (2) increasing gene dose of key biosynthetic and/or regulatory genes, and (3) pathways. Elimination of competing reactions that convert the stream of

For example, glyceraldehyde-3-phosphate (G3P) and arginine are two essential precursors in the clavulanic acid pathway. In addition to the clavulanic acid production pathway, G3P can alternatively enter the glycolytic pathway and then into the Krebs cycle, where it is transported by glyceraldehyde-3-phosphate dehydrogenase ( gap ). gap-1 mutant S. The clavuligerus strain showed an 80 to 110% increase in clavulanic acid production compared to the strain with wild-type gap-1 (Li & Townsend (2006) Metab. Eng. ).

Moreover, increasing the gene dose of cas2 and ccaR increased the production level of clavulanic acid (Hung et al. (2007) J. Micrbiol. Biotechnol. ).

cvm1 encodes an enzyme involved in the conversion of clavamic acid to 3S, 5S clavam, which, unlike the 3R and 5R stereochemistry of clavulanic acid, does not have β-lactamase inhibitory properties. Inactivation of cvm1 increased clavulanic acid production levels (Paradkar et al. (2001) Appl. Environ. Microbiol. ).

In some embodiments, the S. Clavuligerus strains further include genetic modifications known in the art. In one embodiment, the vector of the invention comprises an additional genetic mutation (as compared to wild-type S. clavigerus). It is used to introduce ccaR promoter region and gene mutations into clavuligerus strains.

Genetic modifications in the art may be targeted mutations based on knowledge-based approaches or mutations resulting from random mutagenesis and selection approaches. These genetic modifications known in the art are combined with the ccaR promoter region and gene mutations of the present invention, when compared with each genetic modification known in the art alone or with the ccaR promoter and gene mutations of the present invention alone, S. It is expected to provide further improvements to the clavuligerus strain, such as a further increased titer of clavulanic acid.

Mutations in the ccaR promoter region and gene disclosed herein include S. can be introduced into a clavuligerus strain. This can be done using vectors carrying the ccaR promoter region and gene mutations disclosed herein.

Alternatively, mutations known in the art can be used in the S. can be introduced into a clavuligerus strain. Such strains can be produced using routine genetic engineering methods known in the art.

S. suitable for introducing ccaR promoter region and gene mutations of the present invention. Mutant strains of Clavuligerus are described in WO98/33896 (SmithKline Beecham Plc; Governors of the University of Alberta).

In some embodiments, the S. The clavigerus strain contains a wild-type ccaR promoter region and a copy of the gene. In other words, the ccaR promoter region and gene comprising the mutations described herein are in addition to, but not replace, the wild-type ccaR promoter region and gene. In some embodiments, an S. of the invention comprising a mutation in the ccaR promoter region and gene. The clavuligerus strain contains a wild-type ccaR promoter region and one or more copies of the gene.

In yet a further aspect of the present invention, clavulanic acid comprising the steps of growing a Streptomyces clavuligerus of the present invention and recovering the clavulanic acid produced by the modified Streptomyces clavuligerus or its progeny. A method for producing an acid is provided.

s. Methods for growing clavuligerus and recovering clavulanic acid are known in the art. For example, S. Suitable conditions for fermentation of clavuligerus and extraction of clavulanic acid are described in WO01/87891 (SmithKline Beecham PLC) and Ser et al. (2016) Front. Microbio. ) was described.

In one embodiment, the method produces a titer of clavulanic acid of at least about 0.5 g/L, at least about 1 g/L, at least about 1.5 g/L, at least about 2 g/L, or at least about 2.5 g/L .

Preferably, in one embodiment, the titer of clavulanic acid is produced by the method after 65 hours of fermentation. In some embodiments, the titer of said clavulanic acid is about 50, 55, 60, 65, 70, 72, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, Produced by the method after 135 or 140 hours.

In one embodiment, the method comprises wild-type (WT) S. 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 when compared to clavuligerus , 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 Produces more clavulanic acid. In some embodiments, wild-type S. This percentage increase in titer of clavulanic acid as compared to clavuligerus is produced after 65 hours of fermentation. In some embodiments, wild-type S. This percentage increase in the titer of clavulanic acid as compared to clavuligerus is about 40, 45, 50, 55, 60, 65, 70, 72, 75, 80, 85, 90, 95, 100, 105, Produced after 110, 115, 120, 125, 130, 135 or 140 hours. Preferably, in one embodiment, the S. Clavuligerus is a wild-type S. Compared to clavuligerus, it is possible to produce a titer of clavulanic acid of about 1000% or more after 65 hours of fermentation.

In some embodiments, the viscosity of a fermentation broth comprising a mutant Streptomyces clavigerus strain of the invention is at least about 10%, at least about 15%, at least about 20%, at least about 25 compared to wild-type Streptomyces clavuligerus. % or more, about 30% or more, about 35% or more, or about 40% or more. In one embodiment, said decrease in viscosity is obtained after 65 hours of fermentation. In one embodiment, said decrease in viscosity is about 40, 45, 50, 55, 60, 65, 70, 72, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, obtained after 125, 130, 135, or 140 hours.

In one embodiment, the viscosity of a fermentation broth comprising a mutant Streptomyces clavigerus strain of the invention is reduced by at least about 40% after 65 hours of fermentation as compared to wild-type Streptomyces clavuligerus.

In one embodiment, the viscosity of a fermentation broth comprising a mutant Streptomyces clavigerus strain of the 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. In one embodiment, the viscosity of a fermentation broth comprising a mutant Streptomyces clavuligerus strain of the invention is about 28 centipoise or less after 65 hours of fermentation. In one embodiment, the viscosity of a fermentation broth comprising a mutant Streptomyces clavuligerus strain of the invention is about 25 centipoise or less after 65 hours of fermentation.

In another aspect of the invention, the steps of:

(a) growing a modified Streptomyces clavuligerus of the present invention;

(b) recovering clavulanic acid produced by the modified Streptomyces clavuligerus or its progeny; and

(c) preparing a pharmaceutical formulation by combining the clavulanic acid recovered in step (b) with a β-lactam antibiotic;

A method for producing a pharmaceutical formulation comprising a β-lactam antibiotic and clavulanic acid is provided.

In a preferred embodiment, the β-lactam antibiotic is amoxicillin.

The product co-amoxiclav is marketed as augmentin by GlaxoSmithKline to treat bacterial infections. It contains the combination of the β-lactam antibacterial agent amoxicillin and clavulanic acid. The product is provided in a variety of pharmaceutical formulations, such as tablets, capsule powders and sachets containing free-flowing granules. The pharmaceutical formulation may comprise different ratios of β-lactam antibiotics, such as amoxicillin or ticarcillin and clavulanic acid. In one embodiment, clavulanic acid is combined with the β-lactam antibiotic in a ratio ranging from 3:1 to 30:1. The specific ratio may depend on the type of agent, dosing regimen, route of administration, and/or target indication.

In some embodiments, a pharmaceutical formulation of the invention comprises amoxicillin trihydrate and potassium clavulanate.

Suitable pharmaceutical formulations comprising amoxicillin and clavulanic acid for use in the present invention have been described in WO94/27557 (SmithKline Beecham Corporation) and WO98/35672 (SmithKline Beecham Laboratoires Pharmaceutiques).

Also disclosed herein are pharmaceutical formulations comprising clavulanic acid produced by the methods described herein, further comprising a β-lactam antibiotic. Preferably, the β-lactam antibiotic is amoxicillin. In one embodiment, the pharmaceutical agent is augmentin.

In yet a further aspect, there is provided a genetically engineered microorganism producing clavulanic acid comprising two point mutations in the ccaR promoter region and a substitution mutation in the ccaR gene, wherein the point mutation in the ccaR promoter region is SEQ ID NO: : a C to T point mutation at the site corresponding to position 48 of 1 and a G to A point mutation at the site corresponding to position 143 of SEQ ID NO: 1; The substitution mutation in the ccaR gene is an arginine to tryptophan substitution at the site corresponding to position 32 of SEQ ID NO:2.

In one embodiment, the genetically engineered microorganism is Streptomyces clavuligerus. In a further embodiment, the ccaR gene is integrated into the chromosomal DNA of said microorganism.

In one embodiment, the arginine to tryptophan substitution is the result of a C to T point mutation at the site corresponding to position 344 of SEQ ID NO:1. In another embodiment, the genetically engineered microorganism comprises the nucleic acid sequence of SEQ ID NO:3. In one embodiment, the genetically engineered microorganism has a titer of clavulanic acid of at least about 0.5 g/L, at least about 1 g/L, at least about 1.5 g/L, at least about 2 g/L, or at least about 2.5 g/L. can produce In one embodiment, the genetically engineered microorganism is capable of producing a titer of clavulanic acid of at least about 2.1 g/L. In one embodiment, the titer of clavulanic acid is produced after 65 hours of fermentation. In one embodiment, the genetically engineered microorganism is at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600% after 65 hours of fermentation as compared to a wild-type (WT) microorganism. It may produce a titer of clavulanic acid of greater than, about 700% or greater, about 800% or greater, about 900% or greater, or about 1000% or greater. Reference to a wild-type microorganism will be understood to refer to a microorganism of the same species as the genetically engineered microorganism, ie a non-genetically engineered microorganism of the same species. In one embodiment, the genetically engineered microorganism is capable of producing a titer of clavulanic acid of at least about 1000% after 65 hours of fermentation as compared to a wild-type microorganism. In one embodiment, the genetically engineered microorganism has a viscosity of the fermentation broth of at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30% after 65 hours of fermentation when compared to a wild-type (WT) microorganism. or more, about 35% or more, or about 40% or more. In another embodiment, the microorganism is capable of reducing the viscosity of the fermentation broth by at least about 40% after 65 hours of fermentation as compared to a wild-type microorganism.

In one aspect, a genetically engineered microorganism described herein for use in the production of clavulanic acid is provided.

In a further aspect, a method comprising: growing a genetically engineered microorganism described herein; and recovering clavulanic acid produced by the genetically engineered microorganism or progeny thereof.

In one embodiment of the method, a titer of clavulanic acid of at least about 0.5 g/L, at least about 1 g/L, at least about 1.5 g/L, at least about 2 g/L, or at least about 2.5 g/L is produced . In a further embodiment, a titer of clavulanic acid of at least about 2.1 g/L is produced. In yet a further embodiment, the titer of clavulanic acid is produced after 65 hours of fermentation. In one embodiment of the method, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, after 65 hours of fermentation as compared to a wild-type (WT) microorganism; A titer of clavulanic acid of at least about 700%, at least about 800%, at least about 900%, or at least about 1000% is produced. In one embodiment, a titer of clavulanic acid of at least 1000% is produced after 65 hours of fermentation as compared to the wild-type microorganism. In one embodiment, the viscosity of the fermentation broth comprising the genetically engineered microorganism is at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30% after 65 hours of fermentation as compared to a wild-type microorganism. , by at least about 35%, or by at least about 40%. In one embodiment, the viscosity of the fermentation broth comprising the genetically engineered microorganism is reduced by at least about 40% after 65 hours of fermentation as compared to the wild-type microorganism.

In another aspect, the steps of:

(a) growing the genetically engineered microorganism described herein;

(b) recovering clavulanic acid produced by the genetically engineered microorganism or its progeny; and

(c) preparing a pharmaceutical formulation by combining the clavulanic acid recovered in step (b) with a β-lactam antibiotic;

A method for producing a pharmaceutical formulation comprising a β-lactam antibiotic and clavulanic acid is provided.

In one embodiment, the β-lactam antibiotic is amoxicillin. In one embodiment, the pharmaceutical agent is augmentin.

The aforementioned S. All aspects and embodiments of the clavuligerus strain also apply to the genetically engineered microorganism that produces clavulanic acid. For the avoidance of doubt, these aspects and embodiments are also described in S. A method of producing clavulanic acid using a clavuligerus strain, S. methods of producing a pharmaceutical formulation using the clavuligerus strain.

Justice

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents and publications mentioned herein are incorporated by reference in their entirety.

The term “comprising” encompasses “comprising” or “consisting of, for example, a composition “comprising” X may consist solely of X or include something additional, such as X + Y.

The term “consisting essentially of” limits the scope of the present feature to those that do not materially affect the specified material or step and the basic characteristic(s) of the claimed feature.

The term “consisting of” excludes the presence of any additional component(s).

The term “about” in reference to a numerical value x means, for example, x ± 10%, 5%, 2% or 1%.

The term “promoter region” as used herein refers to a nucleic acid sequence comprising any regulatory region necessary for gene function or expression.

The terms "Streptomyces clavuligerus", "S. clavuligerus", "S. clavuligerus strain" are used interchangeably and refer to Streptomyces clavuligerus and strains thereof.

The term “wild-type” or “WT” as used herein in the context of “Streptomyces clavuligerus”, “S. found S. Clavuligerus strain (ie, S. clavuligerus - Culture Collection ATCC 27064). s. Wild-type as used herein in the context of clavuligerus refers, inter alia, to a strain that does not contain mutations in the ccaR promoter region and the ccaR gene, ie M1, M2 or M3.

The terms " ccaR " and " ccaR gene " are used interchangeably and as used herein refer to the ccaR gene.

The term “CcaR” as used herein refers to the protein encoded by the ccaR gene.

As used herein, the term “point mutation” refers to an alteration of a single base pair within a nucleotide sequence. For example, one base may be replaced by another. These point mutations can have one of three effects. First, the base substitution may be a silent mutation in which the altered codon corresponds to the same amino acid. Second, the base substitution may be a missense mutation in which the altered codon corresponds to a different amino acid. Third, the base substitution may be a nonsense mutation in which the altered codon corresponds to a stop signal.

As used herein, the term “substitution mutation” refers to the replacement of one amino acid protein with a different amino acid. It can also refer to replacing one base pair with another in a nucleotide sequence.

As used herein, the terms “fermentation time” and “log time” in the context of fermentation refer to the fermentation time after the time the production medium is inoculated with the seed culture.

The term "fermentation broth" as used herein refers to a fermentation medium and cells fermented therein, particularly S. refers to a mixture of clavuligerus.

The term “vector” or “nucleic acid vector” refers to a vehicle capable of artificially transporting genetic material into another cell in which it can be replicated and/or expressed.

The invention will now be described in more detail with reference to the following non-limiting examples.

Example

Example 1

ccaR Mutant Plasmid Construction

The three mutations in the ccaR promoter region and the ccaR gene, individually or in combination, were evaluated for their effect on clavulanic acid production. Mutations are as follows:

Mutation 1 (M1)—C to T point mutation at position 48 of SEQ ID NO:1;

Mutation 2 (M2)—G to A point mutation at position 143 of SEQ ID NO:1; and

Mutation 3 (M3)—Arginine to tryptophan amino acid substitution at position 32 of SEQ ID NO:2.

The positions of M1 and M2 are within the ccaR promoter region. The location of M3 is within the ccaR gene.

Creating the initial construct

Amplify the ccaR gene and surrounding 4 Kb DNA (2 Kb on each side) by PCR with Q5™ polymerase using primers NC_18_22 and NC_18_23. These primers contain HindIII and XbaI sites, respectively. Three PCRs were performed using different genomic DNAs from the Streptomyces clavuligerus wild-type strain, strain 3 (SC3) or strain 4 (SC4) as templates. Using these different genomic preps will yield products containing no mutations at all, products containing M1+M2 and products containing all three mutations (M1+M2+M3), respectively.

The PCR product was cloned into pKC1132 containing the codA gene, which can be used as a selection tool, at its HindIII and XbaI sites, and E. It was 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 to yield PCR products of the correct size, which were named pCLV23 (WT sequence), pCLV24 (SC3 sequence/M1+M2) and pCLV25 (SC4 sequence/M1+M2+M3). Plasmids were sequenced to check that the cloned DNA was correct.

Creating a plasmid containing one mutation

· primer sets ccaR_QCM1_F and ccaR_QCM1_R; ccaR_QCM2_F and ccaR_QCM2_R; Three 'mutagenic' PCRs were set up using ccaR_QCM3_F and ccaR_QCM3_R to create M1, M2 and M3 mutations, respectively, using pCLV23 as a template. These include the fact that a high concentration of template DNA is used (1 μl standard plasmid prep in 25 μl PCR reaction), that a low concentration of primer is used (1 μl of 1 mM primer stock in 25 μl PCR reaction), the annealing temperature is always It differs from standard PCR in that it is 60° C. and PCR is performed only 12 times.

· After PCR, each mixture was digested with DpnI to remove the template DNA and E. It was transformed into E. coli NEB10. Plasmids were prepared and sequenced from 6 colonies of each mutagenesis. From each, plasmids containing the correct mutation were picked and these plasmids were named pCLV26 (M1), pCLV27 (M2) and pCLV28 (M3).

Sequencing Results and Subsequent Mutagenesis

Among the original plasmids constructed, pCLV25 contained the correct sequence. pCLV23 was found to have a point mutation upstream of ccaR in the orf10 gene. As a result, pCLV26, pCLV27 and pCLV28 also contained these mutations. pCLV24 was found to have an unwanted mutation in the ccaR gene.

Mutagenesis PCR was performed on pCLV23, pCLV26, pCLV27 and pCLV28 using primers QC_correct_orf10_F and QC_correct_orf10_R. The resulting plasmid was sent for sequencing, among which plasmids containing the correct sequence in the orf10 gene were selected, and the final plasmids were pCLV29 (WT seq), pCLV31 (M1), pCLV32 (M2) and pCLV33 ( M3) was designated.

Mutagenesis PCR was performed on pCLV24 using primers QC_correct_ccaRmut_F and QC_correct_ccaRmut_R. The resulting plasmid was sent for sequencing, among which the plasmid containing the correct sequence in the ccaR gene was selected. The final plasmid was designated pCLV30 (M1+M2).

To obtain a mutant containing all repeats of the mutation, two additional plasmids were needed to cover the mutations M1+M3 and M2+M3. To add either M1 or M2 to a plasmid that already contains M3, the primer sets ccaR_QCM1_F and ccaR_QCM1_R; Mutagenesis PCR was performed using pCLV33 (M3) as a template using ccaR_QCM2_F and ccaR_QCM2_R, respectively. The resulting plasmids were sequenced, and a plasmid containing the desired mutation was selected from among them. The final plasmids were designated pCLV34 (M1+M3) and pCLV35 (M2+M3).

Primer Table:

Final construct:

Example 2

ccaR Mutant Streptomyces clavuligerus

Using the ccaR mutant plasmid prepared as described in Example 1, S. A ccaR mutant strain of Clavuligerus was prepared.

1. Preparation of plasmid DNA

· Each plasmid is electrically competent. It was transformed into E. coli ET12567 [pUZ8002]. These are. As the E. coli strain is dam- dcm- , it yields unmethylated plasmid DNA and carries the pUZ8002 plasmid containing the oriT (origin of the transgene) to which splicing can occur. ET12567 is resistant to chloramphenicol; pUZ8002 is resistant to kanamycin.

2. Conjugation of plasmid DNA

Day 1

· this. Each plasmid [pUZ8002] in E. coli ET12567 was inoculated into 5 mL lysogeni broth (LB) medium containing 50 μg/mL apramycin, 50 μg/mL kanamycin and 10 μg/mL chloramphenicol. This was incubated at 35° C. at 250 rpm overnight.

Day 2

· 2 mL each overnight. E. coli cultures were inoculated into 250 mL straight edge flasks containing 30 mL lysogeni broth (LB) + apramycin, kanamycin and chloramphenicol. This was incubated at 35° C. at 250 rpm until OD ₆₀₀ = about 0.6.

· 10 mL of each tooth. The E. coli culture was poured into 50 mL centrifuge tubes and centrifuged at 4000 rpm for 5-10 minutes. Then, the resulting pellet was washed 3 times in 10 mL LB. final this. The E. coli pellet was resuspended in 500 μL LB.

S per plasmid to be conjugated. Two plates of Clavigerus spores were harvested by pipetting 3 mL LB onto these plates, using a loop to remove the spores and then pipetting the LB from the plate into a 2 mL microcentrifuge tube. The spores were centrifuged at 7000 rpm for 10 minutes, and the resulting pellet was washed once in LB. The final pellet was resuspended in 250 μL LB. The spores were incubated at 50° C. for 10 minutes and then immediately placed on ice.

· 250 µL of E. The E. coli suspension was mixed with each aliquot of spores. This mixture was then spread onto L3M9 agar plates and incubated overnight at 26°C.

L3M9:

Day 3

Each plate was covered with apramycin and fosfomycin - 15 μL of 100 mg/mL apramycin stock and 45 μL of 50 mg/mL fosfomycin stock in 700 μL spread evenly onto each plate and incubate the plates at 26°C for 7-10 days.

3. Double Cross Acquisition

Apramycin-resistant colonies achieved by conjugation are single crossover - these colonies are S. It will have a copy of the mutated ccaR in the entire vector integrated into the chromosome of clavigerus and will still contain the original copy of ccaR (WT). A second homologous recombination event is required to remove the inserted plasmid DNA along with the original copy of ccaR .

Colonies were picked using sterile wooden cocktail sticks and patched onto L3M9 plates covered with apramycin and fosfomycin. They were incubated at 26° C. for 7-10 days.

· Once the patch has grown and spore formation has begun, the strain is re-patched onto L3M9 plates containing apramycin alone and incubated at 26°C for 7-10 days.

Patches were patched twice in succession onto L3M9 plates containing no antibiotics.

Spores were harvested from the final L3M9 plate by pipetting 3 mL 10% sucrose onto the final L3M9 plate, using a loop to remove the spores and then pipetting this spore suspension into a sterile syringe containing cotton wool. The spores were pushed through a syringe and the resulting spore suspension was diluted to 10 ^-8 . Dilutions were plated onto L3M9 plates covered with 300 μL 10 mg/mL 5-fluorocytosine (the plasmid used for this work expresses CodA, which converts 5-fluorocytosine to the toxic 5-fluorouracil) Thus, a colony capable of growing on 5-fluorocytosine will lose this plasmid from its chromosome). Plates were incubated at 26° C. for 7-10 days.

Colonies were picked and replicates plated (as patches) onto L3M9 plates with and without apramycin and L3M9 plates with no apramycin. After 5 days, patches that did not grow on plates containing apramycin were S. It was considered to have lost the plasmid integrated into the Clavuligerus chromosome (which underwent a secondary recombination event).

All constructs used were plasmids and S. Known as the homology arms required to promote homologous recombination between the clavuligerus genomes, S. It contained a mutated ccaR region flanked by a DNA sequence of 2 Kb identical to that found upstream and downstream of ccaR as found on the clavigerus genome. Plasmid DNA is S. After conjugation into Clavuligerus, the mutant was combined with a plasmid and S. It underwent a single crossover event between the clavigerus genomes - resulting in strains containing both the original copy of ccaR and the mutated ccaR and plasmid DNA. The mutant strain subsequently underwent a second homologous recombination event during DNA replication in which plasmid DNA was lost and a mutated copy of ccaR was exchanged for the original copy of ccaR . Such mutants are referred to as double crossovers. This second recombination event can also result in cleavage of the plasmid and the mutated copy of ccaR , resulting in a strain reverted to the wild-type strain.

One of the genes located in the homology arms in the plasmid used to mutate each strain was orf10 . Although the function of these genes involved in clavulanic acid synthesis is not yet known, S. Knockout of these genes in clavuligerus results in mutants incapable of producing clavulanic acid (Jensen et al., (2000) Antimicrob. Agents Chemother. ). Point mutations were made to these genes during the cloning step to create the ccaR mutant constructs - the ccaR promoter region and the region flanking the gene in S. It had to be restored to have 100% homology with the region found at the same location on the clavuligerus genome (see Example 1).

4. Double Cross Sequencing

Sequencing was required to confirm whether the strain carried a mutated copy of ccaR or the original wild-type copy of ccaR .

· Use a sterile loop to scrape the apramycin-sensitive patch and place it in 50 μL DMSO. It was frozen at -70°C, thawed, vortexed, and then frozen twice more.

The resulting mixture was used as a template in a PCR reaction using PHUSION polymerase and primer sets NC_20 and NC_21, yielding an approximately 1.5 Kb PCR product covering the mutation introduced into the strain. PCR products were cleaned up and Sanger sequenced using sequencing primers ccaR_seq or ccaR_seq_rev that anneal within the PCR products.

· The obtained sequence is S. Alignment to the Clavuligerus ccaR gene and promoter sequence was performed to evaluate the presence of the correct mutation.

5. Preparation of Working Spore Stocks

· s. Clavuligerus mutants were patched onto three L3M9 plates and incubated at 26° C. for 2 weeks to provide growing turf on each plate. Subsequently, spores were harvested by placing 3 mL of 10% sucrose on each plate, scraping the spores using a sterile 10 μL loop and placing them in a sterile 25 mL tube. The total volume of each spore suspension was made to 15 mL with 10% sucrose and then sonicated at an amplitude of 14 micrometers for 30 s using a sterile sonication probe. The sonicated spore suspension was then aliquoted into cold vials and stored at -70°C until needed for fermentation.

To calculate the number of colony forming units per mL (cfu/mL) in each spore suspension, thaw one cryovial and make a dilution series of 10 ^-8 in polysorbate 80 (0.1% v/v). . 100 μL from 10 ⁻⁷ and 10 ⁻⁸ dilutions were plated onto trypsinized soy agar plates and incubated at 26° C. for 5 days. Thereafter, colonies were counted and cfl/mL was calculated.

Example 3

Fermenter assay for clavulanic acid

Fermentation

S. produced as described in Example 2. The ccaR mutant strain of clavigerus was transformed into wild-type (SC2) S. Clavulanic acid adhesion and growth profiles were tested on a 2 L micro-fermenter scale against a clavuligerus working stock control. (Tests were run over 5 fermentor runs, containing 10 vessels per run. Each run included a wild-type (SC2) control to account for any inter-run variability).

100 mL S13A seed medium (20 g/L SOFARINE skim soy flour, 10 g/L dextrin, 0.6 g/L monobasic potassium phosphate, 5 g/L rapeseed oil, pH adjusted to 7.0 with 10 M sodium hydroxide) A ⁵⁰⁰ mL Erlenmeyer flask containing The flask was incubated at 26° C. and 220 rpm.

After 52 hours, 3% volume is 1.4 L S8A2 final stage medium (38.5 g/L SOFARINE skim soy flour, 10 g/L dextrin, 0.175 g/L potassium phosphate monobasic, 23 g/L rapeseed oil, 1 g /L Foamdoctor™ Antifoam, 0.1 g/L Magnesium Chloride Hexahydrate, 0.03 g/L Ferrous Sulfate Heptahydrate, 0.005 g/L Zinc Chloride, 0.005 g/L Cupric Chloride, 0.005 g/L Manga Sulfate Nizhny monohydrate) was inoculated into a 2 L micro fermenter. The medium pH prior to inoculation was adjusted to 7.0 using 17% v/v ammonium hydroxide solution and controlled to pH 6.8 throughout this solution. The temperature was maintained at 26° C., the agitation was maintained at 1400 rpm, and the airflow was maintained at 0.8 vvm (volume of air/volume of liquid/min). A 32% w/v maltodextrin feed was introduced at a rate of 1.4 mls/hr from 24 hours and maintained throughout. A 5.67% w/v monobasic potassium phosphate feed solution was introduced at a rate of 0.3 mls/hr from 0 to 24 hours, then increased to 1.1 mls/hr by 48 hours, at which point it was terminated. Fermentation was sampled throughout for titer of clavulanic acid and ended at approximately 140 hours as described below.

Determination of the concentration of clavulanic acid

· 1.5 mL of shaken broth was poured into a disposable Eppendorf tube and centrifuged at 13,000 rpm at 4°C for 10 minutes.

· The supernatant was diluted 1/20 in a COBAS cup using a Hamilton™ diluter. Duplicate dilutions were performed for each sample.

The diluted samples were run on a Mira S Plus automated analyzer using the parameters as described below. The assay involves a chemical reaction between clavulanic acid and imidazole, which detects a change in absorbance measured at 313 nm when the two are mixed.

summary

Measurement mode: ABSORB

Reaction Mode: R-S

Calibration Mode: SLOPE AVG

Reagent blank: REAG/DIL

Cleaner: None

Wavelength: 813 nm on Lab 101 Mira S Plus (serial number: 35-8663)

413 nm on MFU Mira S Plus (serial number: 36-3139)

Decimal position: 0

Unit: μg/mL

analyze

Post dilution factor: none

Concentration factor: none

Sample Cycle: 1

Volume: 8.0 μL

Diluent Name: H ₂ O

Volume: 40.0 μL

Reagent Cycle: 1

Volume: 280 μL

calculate

Sample Limit: None

Reaction direction: increase

Inspection: did

Conversion Factor: 1.00000

Offset: 0.00000

Low Test Coverage: No

High: 80000 μg/mL

Normal Range Low: No

High: 80000 μg/mL

Number of steps: 1

Calculation Step A: Endpoint

First reading: T1 Last: 3

correction

Calibration Interval: Each Run

blank

Reagent Range Low: -0.0500 A

High: 0.0500 A

Low blank range: -0.0500 ΔA

High: 0.0500 ΔA

Standard Force: 1

STD-1: 17460 μg/mL (MFU) 4300 μg/mL (Lab 101)

STD-2: 17460 μg/mL 4300 μg/mL

STD-3: 17460 μg/mL 4300 μg/mL

Duplicate: single

Deviation: 3.0%

control

CS1 Force: None

CS2 Force: None

CS3 Force: None

Note: This assay should be performed at 30°C.

Determination of the viscosity of fermentation broth

Viscosity was measured using a Brookfield DV-II+ viscometer set at 20 rpm. A 1 mL fermentation broth was placed in the center of the viscometer sample cup and the motor was started - a torque was required to rotate the spindle submerged in the fermentation broth to measure the viscosity. Measurements were taken after a few seconds once the readings were stable.

result

The biggest advantage seen in the mutant after introduction of the ccaR mutation was the rate of adhesion of clavulanic acid. For mutants containing all three mutations (M1+M2+M3), it was found that the titer of clavulanic acid peaked at 65 log hours of fermentation and then leveled off thereafter ( FIGS. 1 and 3 ). Reference). Therefore, the 65 log time time point will be used to compare the productivity of clavulanic acid across all strains.

The presence of all three mutations (M1+M2+M3) in the ccaR promoter region and gene yielded the highest titer of clavulanic acid, at 65 log times of 1031% compared to the control (wild-type S. clavigerus). (see Table 1, Figure 1, Figure 2 and Figure 3). The next highest titer was achieved with the strain carrying the M2 mutation alone (808% of the control at 65 log times), which appears to be the most influential of the three mutations, but in order to fully maximize the effect on titer, It is emphasized that the other two mutations need to be combined with this. The presence of M1 and M3 alone or in combination increased titers, but not to the same extent as when combined with M2, and 163 titers compared to wild-type controls at 65 log times for M1, M3 and M1+M3, respectively. %, 145% and 133%.

<Table 1>

Data for viscosities at 65 log hours, average peak viscosities, percentage viscosities at 65 log hours, and percentage peak viscosities compared to wild-type controls as observed with the mutant strains are provided in Table 2.

<Table 2>

The ccaR mutation reduced both the viscosity at the time of peak production and the observed peak viscosity (see Table 2, Figures 4 and 5). At 65 log hours, the greatest decrease in viscosity was seen during fermentation of the strains carrying all three mutations (M1+M2+M3), followed by the strain carrying only M2 in terms of viscosity compared to the wild-type control. 41% and 32% reduction.

Low viscosity combined with high productivity is a highly desirable characteristic in Streptomyces clavuligerus fermentation.

1, 2, 3 and 4, error bars represent ±1 standard deviation for data obtained from multiple fermentation runs.

sequence list

SEQ ID NO: 1: Nucleic acid sequence of wild-type ccaR promoter region and gene.

SEQ ID NO: 2: Amino acid sequence of wild-type ccaR gene.

SEQ ID NO: 3: Nucleic acid sequence of the ccaR promoter region and ccaR gene comprising the mutations described herein.

SEQ ID NO: 4: amino acid sequence of the ccaR gene comprising the substitution mutations described herein.

(Location of the mutation (M1=48, M2=143, M3=344) is underlined in bold)

(The location of the M3 mutation (R32W) is underlined in bold)

SEQUENCE LISTING <110> GlaxoSmithKline Intellectual Property (No.2) Limited <120> Streptomyces clavuligerus <130> PB66783 <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 1039 <212> DNA <213> Streptomyces clavuligerus <400> 1 cccgtcgacg tcccttccca cagccttccc acccacccgt cccgactcgc cgtgaagccc 60 cgggttcttc cgggttcacc gaggctgtcc caaatcgtcc atgccttgag ggtcccgctg 120 cgtgatcgaa ccgtaaccct tggaatttct gtggattaag cgtaaacatg ggtgccgaca 180 ccaaggatta cgccgaagcc atgtccaccc ctctcggcga gggcgtggtt ccttcacaag 240 ggggaccgcc atgaacacct ggaatgatgt gacgatccgg ctcctggggc cggtgacact 300 cgtgaaaggt tccgtaccga tacccatccg cgggcagcga cagcggcgat tcctcgcctc 360 attagcgctg cgaccgggcc aggtcatctc caaggaagcg atcatcgaag actcctggga 420 cggggagcca ccactgaccg tttcgggcca gttgcagacg tcggcctgga tgatccggac 480 cgcgctggcg gaggcggggc tgccccgcga cgccctcggc tcccacgacc gcggctacga 540 actgcgcgtc ctgccggact ccatcgacct cttcgtcttc cgggaggccg tgcgcgccgt 600 gcgggacctg cacgcacgcg gtcagcacca ggaggcgtcc gaacggctcg acacggcgct 660 cgccctgtgg aaggggcccg ccttcgcgga tgtgacctcc agtcggctgc ggctgcgggg 720 cgagaccctg gaggaggagc ggaccgccgc ggtcgagctg cgcgccctga tcgatgtcgg 780 cctcggctac tacggggacg cgatcacccg gctgtcggag ctcgtcgatc acgacccgtt 840 ccgtgaggac ctgtatgtga gcctgatgaa ggcctactac gcggagggcc gccaggccga 900 cgcgatccag gtcttccacc gcgcgaagga catcctgcgg gagcagatcg gcatcagccc 960 cggcgagcgg atgacaaggg tcatgcaggc catcctgcgt caggacgagc aggtcctgcg 1020 ggtcggtacc ccggcctga 1039 <210> 2 <211> 262 <212> PRT <213> Streptomyces clavuligerus <400> 2 Met Asn Thr Trp Asn Asp Val Thr Ile Arg Leu Leu Gly Pro Val Thr 1 5 10 15 Leu Val Lys Gly Ser Val Pro Ile Pro Ile Arg Gly Gln Arg Gln Arg 20 25 30 Arg Phe Leu Ala Ser Leu Ala Leu Arg Pro Gly Gln Val Ile Ser Lys 35 40 45 Glu Ala Ile Ile Glu Asp Ser Trp Asp Gly Glu Pro Pro Leu Thr Val 50 55 60 Ser Gly Gln Leu Gln Thr Ser Ala Trp Met Ile Arg Thr Ala Leu Ala 65 70 75 80 Glu Ala Gly Leu Pro Arg Asp Ala Leu Gly Ser His Asp Arg Gly Tyr 85 90 95 Glu Leu Arg Val Leu Pro Asp Ser Ile Asp Leu Phe Val Phe Arg Glu 100 105 110 Ala Val Arg Ala Val Arg Asp Leu His Ala Arg Gly Gln His Gln Glu 115 120 125 Ala Ser Glu Arg Leu Asp Thr Ala Leu Ala Leu Trp Lys Gly Pro Ala 130 135 140 Phe Ala Asp Val Thr Ser Ser Arg Leu Arg Leu Arg Gly Glu Thr Leu 145 150 155 160 Glu Glu Glu Arg Thr Ala Ala Val Glu Leu Arg Ala Leu Ile Asp Val 165 170 175 Gly Leu Gly Tyr Tyr Gly Asp Ala Ile Thr Arg Leu Ser Glu Leu Val 180 185 190 Asp His Asp Pro Phe Arg Glu Asp Leu Tyr Val Ser Leu Met Lys Ala 195 200 205 Tyr Tyr Ala Glu Gly Arg Gln Ala Asp Ala Ile Gln Val Phe His Arg 210 215 220 Ala Lys Asp Ile Leu Arg Glu Gln Ile Gly Ile Ser Pro Gly Glu Arg 225 230 235 240 Met Thr Arg Val Met Gln Ala Ile Leu Arg Gln Asp Glu Gln Val Leu 245 250 255 Arg Val Gly Thr Pro Ala 260 <210> 3 <211> 1039 <212> DNA <213> <220> <223> ccaR promoter region and ccaR gene comprising mutations <400> 3 cccgtcgacg tcccttccca cagccttccc acccacccgt cccgacttgc cgtgaagccc 60 cgggttcttc cgggttcacc gaggctgtcc caaatcgtcc atgccttgag ggtcccgctg 120 cgtgatcgaa ccgtaaccct tgaaatttct gtggattaag cgtaaacatg ggtgccgaca 180 ccaaggatta cgccgaagcc atgtccaccc ctctcggcga gggcgtggtt ccttcacaag 240 ggggaccgcc atgaacacct ggaatgatgt gacgatccgg ctcctggggc cggtgacact 300 cgtgaaaggt tccgtaccga tacccatccg cgggcagcga cagtggcgat tcctcgcctc 360 attagcgctg cgaccgggcc aggtcatctc caaggaagcg atcatcgaag actcctggga 420 cggggagcca ccactgaccg tttcgggcca gttgcagacg tcggcctgga tgatccggac 480 cgcgctggcg gaggcggggc tgccccgcga cgccctcggc tcccacgacc gcggctacga 540 actgcgcgtc ctgccggact ccatcgacct cttcgtcttc cgggaggccg tgcgcgccgt 600 gcgggacctg cacgcacgcg gtcagcacca ggaggcgtcc gaacggctcg acacggcgct 660 cgccctgtgg aaggggcccg ccttcgcgga tgtgacctcc agtcggctgc ggctgcgggg 720 cgagaccctg gaggaggagc ggaccgccgc ggtcgagctg cgcgccctga tcgatgtcgg 780 cctcggctac tacggggacg cgatcacccg gctgtcggag ctcgtcgatc acgacccgtt 840 ccgtgaggac ctgtatgtga gcctgatgaa ggcctactac gcggagggcc gccaggccga 900 cgcgatccag gtcttccacc gcgcgaagga catcctgcgg gagcagatcg gcatcagccc 960 cggcgagcgg atgacaaggg tcatgcaggc catcctgcgt caggacgagc aggtcctgcg 1020 ggtcggtacc ccggcctga 1039 <210> 4 <211> 262 <212> PRT <213> <220> <223> ccaR gene comprising a substitution mutation <400> 4 Met Asn Thr Trp Asn Asp Val Thr Ile Arg Leu Leu Gly Pro Val Thr 1 5 10 15 Leu Val Lys Gly Ser Val Pro Ile Pro Ile Arg Gly Gln Arg Gln Trp 20 25 30 Arg Phe Leu Ala Ser Leu Ala Leu Arg Pro Gly Gln Val Ile Ser Lys 35 40 45 Glu Ala Ile Ile Glu Asp Ser Trp Asp Gly Glu Pro Pro Leu Thr Val 50 55 60 Ser Gly Gln Leu Gln Thr Ser Ala Trp Met Ile Arg Thr Ala Leu Ala 65 70 75 80 Glu Ala Gly Leu Pro Arg Asp Ala Leu Gly Ser His Asp Arg Gly Tyr 85 90 95 Glu Leu Arg Val Leu Pro Asp Ser Ile Asp Leu Phe Val Phe Arg Glu 100 105 110 Ala Val Arg Ala Val Arg Asp Leu His Ala Arg Gly Gln His Gln Glu 115 120 125 Ala Ser Glu Arg Leu Asp Thr Ala Leu Ala Leu Trp Lys Gly Pro Ala 130 135 140 Phe Ala Asp Val Thr Ser Ser Arg Leu Arg Leu Arg Gly Glu Thr Leu 145 150 155 160 Glu Glu Glu Arg Thr Ala Ala Val Glu Leu Arg Ala Leu Ile Asp Val 165 170 175 Gly Leu Gly Tyr Tyr Gly Asp Ala Ile Thr Arg Leu Ser Glu Leu Val 180 185 190 Asp His Asp Pro Phe Arg Glu Asp Leu Tyr Val Ser Leu Met Lys Ala 195 200 205 Tyr Tyr Ala Glu Gly Arg Gln Ala Asp Ala Ile Gln Val Phe His Arg 210 215 220 Ala Lys Asp Ile Leu Arg Glu Gln Ile Gly Ile Ser Pro Gly Glu Arg 225 230 235 240 Met Thr Arg Val Met Gln Ala Ile Leu Arg Gln Asp Glu Gln Val Leu 245 250 255 Arg Val Gly Thr Pro Ala 260

Claims (25)

Streptomyces clavuligerus comprising two point mutations in the ccaR promoter region and a substitution mutation in the ccaR gene, wherein the point mutation in the ccaR promoter region corresponds to position 48 of SEQ ID NO: 1 a C to T point mutation at the site and a G to A point mutation at the site corresponding to position 143 of SEQ ID NO: 1; The substitution mutation in the ccaR gene is a substitution of arginine to tryptophan at the site corresponding to position 32 of SEQ ID NO: 2 Streptomyces clavuligerus. The Streptomyces clavuligerus of claim 1 , wherein the arginine to tryptophan substitution is the result of a C to T point mutation at the site corresponding to position 344 of SEQ ID NO:1. 3. Streptomyces clavuligerus according to claim 1 or 2, comprising the nucleic acid sequence of SEQ ID NO:3. 4. The clavula according to any one of claims 1 to 3, wherein at least about 0.5 g/L, at least about 1 g/L, at least about 1.5 g/L, at least about 2 g/L, or at least about 2.5 g/L Streptomyces clabuligerus capable of producing acid titers. 5. Streptomyces clavuligerus according to any one of claims 1 to 4, which is capable of producing a titer of clavulanic acid of at least about 2.1 g/L. The Streptomyces clavuligerus according to claim 4 or 5, wherein the titer of clavulanic acid is produced after 65 hours of fermentation. 7. The method of any one of claims 1 to 6, wherein at least about 100%, at least about 200%, at least about 300%, at least about 400%, after 65 hours of fermentation when compared to wild-type (WT) Streptomyces clavuligerus; Streptomyces clavuligerus capable of producing a titer of clavulanic acid of at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or at least about 1000%. 7. A Streptomyces clavuligerus according to any one of claims 1 to 6, which is capable of producing a titer of clavulanic acid of at least about 1000% after 65 hours of fermentation when compared to wild-type Streptomyces clavuligerus. 9. The method of any one of claims 1 to 8, wherein the viscosity of the fermentation broth after 65 hours of fermentation when compared to wild-type (WT) Streptomyces clavuligerus is about 10% or more, about 15% or more, about 20% or more, Streptomyces clabuligerus, which is capable of reducing by at least about 25%, at least about 30%, at least about 35%, or at least about 40%. 10. The Streptomyces clavuligerus of claim 9, which is capable of reducing the viscosity of the fermentation broth by at least about 40% after 65 hours of fermentation as compared to wild-type Streptomyces clavuligerus. 11. Streptomyces clavuligerus according to any one of claims 1 to 10 for use in the production of clavulanic acid. A vector comprising a nucleic acid sequence comprising a ccaR promoter region comprising two point mutations and a ccaR gene comprising a substitution mutation, wherein the point mutation in the ccaR gene promoter region corresponds to position 48 of SEQ ID NO: 1 a C to T mutation at the site and a G to A mutation at the site corresponding to position 143 of SEQ ID NO: 1; The vector 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 vector of claim 12 , wherein the arginine to tryptophan substitution is the result of a C to T point mutation at the site corresponding to position 344 of SEQ ID NO:1. 14. The vector according to claim 12 or 13, comprising the nucleic acid sequence of SEQ ID NO:3. 15. A vector according to any one of claims 12 to 14 for use in producing the Streptomyces clavuligerus according to any one of claims 1 to 10. Growing the Streptomyces clavigerus of any one of claims 1 to 10; and
Recovering clavulanic acid produced by the Streptomyces clavuligerus or its progeny
A method for producing clavulanic acid, comprising a. 17. The method of claim 16, wherein a titer of clavulanic acid of at least about 0.5 g/L, at least about 1 g/L, at least about 1.5 g/L, at least about 2 g/L, or at least about 2.5 g/L is produced. method. The method of claim 16 , wherein a titer of clavulanic acid of at least about 2.1 g/L is produced. 19. The method according to claim 17 or 18, wherein the titer of clavulanic acid is produced after 65 hours of fermentation. 20. The method of any one of claims 16 to 19, wherein at least about 100%, at least about 200%, at least about 300%, at least about 400%, after 65 hours of fermentation, compared to wild-type (WT) Streptomyces clavuligerus, wherein a titer of clavulanic acid of at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or at least about 1000% is produced. 21. The method according to any one of claims 16 to 20, wherein a titer of clavulanic acid of at least 1000% is produced after 65 hours of fermentation when compared to wild-type Streptomyces clavuligerus. 22. The method according to any one of claims 16 to 21, wherein the viscosity of the fermentation broth comprising Streptomyces clavigerus is at least about 10%, at least about 15% after 65 hours of fermentation as compared to wild-type Streptomyces clavuligerus. , by at least about 20%, at least about 25%, at least about 30%, at least about 35%, or at least about 40%. 23. The Streptomyces clavuligerus of claim 22, wherein the viscosity of the fermentation broth comprising Streptomyces clavigerus is reduced by at least about 40% after 65 hours of fermentation as compared to wild-type Streptomyces clavuligerus. (a) growing the Streptomyces clavuligerus of any one of claims 1 to 10;
(b) recovering clavulanic acid produced by the Streptomyces clavuligerus or its progeny; and
(c) preparing a pharmaceutical formulation by combining the clavulanic acid recovered in step (b) with a β-lactam antibiotic;
A method for producing a pharmaceutical formulation comprising a β-lactam antibiotic and clavulanic acid, comprising: 25. The method of claim 24, wherein the β-lactam antibiotic is amoxicillin.

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