EP4185703A1

EP4185703A1 - Improved process for production of clavulanic acid

Info

Publication number: EP4185703A1
Application number: EP21749793.2A
Authority: EP
Inventors: Peter Mrak; Matej Oslaj; Gregor Kopitar
Original assignee: Sandoz AG
Current assignee: Sandoz AG
Priority date: 2020-07-24
Filing date: 2021-07-23
Publication date: 2023-05-31
Also published as: WO2022018258A1

Abstract

The present invention relates to a process for the preparation of the β-lactamase inhibitor clavulanic acid by using a microorganism, comprising a step of targeted modifying said microorganism. The present invention is further directed to a process for preparing clavulanic acid by using a microorganism being capable of producing clavulanic acid, but at the same time exhibits reduced or no production of teleocidin A and/or congeners originating from the teleocidin biosynthetic pathway; to a process for preparing clavulanic acid by using a microorganism being capable of producing clavulanic acid, with this process comprising a step of testing said microorganism as to whether it is capable of producing clavulanic acid, but at the same time exhibits reduced or no production of teleocidin A, and/or other congeners originating from the teleocidin biosynthetic pathway; and to a method of testing the suitability of a strain of S. clavuligerus for being used in the production of clavulanic acid, this method comprising a step of assessing whether said strain produces teleocidin A and/or other congeners originating from the teleocidin biosynthetic pathway. The present invention further relates to a method of testing whether a culture broth that is used for culturing S. clavuligerus and producing clavulanic acid has low toxicity, comprising a step of detecting the presence or absence of teleocidin A in said culture broth. Finally, the present invention relates to S. clavuligerus that produces clavulanic acid but reduced or no teleocidin A and/or other congeners originating from the teleocidin biosynthetic pathway.

Description

IMPROVED PROCESS FOR PRODUCTION OF CLAVULANIC ACID

FIELD OF THE INVENTION

The present invention relates to a process for the preparation of the b-lactamase inhibitor clavulanic acid by using a microorganism, comprising a step of targeted modifying said microorganism. The present invention is further directed to a process for preparing clavulanic acid by using a microorganism being capable of producing clavulanic acid, but at the same time exhibits reduced or no production of the carcionogenic toxin teleocidin A and/or congeners originating from the teleocidin biosynthetic pathway; to a process for preparing clavulanic acid by using a microorganism being capable of producing clavulanic acid, with this process comprising a step of testing said microorganism as to whether it is capable of producing clavulanic acid, but at the same time exhibits reduced or no production of teleocidin A, and/or other congeners originating from the teleocidin biosynthetic pathway; and to a method of testing the suitability of a strain of S. clavuligerus for being used in the production of clavulanic acid, this method comprising a step of assessing whether said strain produces teleocidin A and/or other congeners originating from the teleocidin biosynthetic pathway.

The present invention further relates to a method of testing whether a culture broth that was used for culturing S. clavuligerus and producing clavulanic acid has low toxicity, comprising a step of detecting the presence or absence of teleocidin A in said culture broth.

Finally, the present invention relates to S. clavuligerus that produces clavulanic acid but reduced or no teleocidin A and/or other congeners originating from the teleocidin biosynthetic pathway.

BACKGROUND OF THE INVENTION

Historically b-lactam antibiotics such as penicillin and cephalosporin were among the first useful antibiotics discovered and remain at the forefront of clinical use to combat bacterial infections b-lactam resistant bacterial pathogens, however, started to emerge after application to general medical use mostly due to the wide-spread use of these antibiotics for more than 50 years. This has, in turn, drastically reduced their efficacy in combating bacterial infections. As a consequence, strategies aimed at overcoming acquired resistance have become of increasing interest. One of the best examples in broad clinical application is the development of b- lactamase inhibitors. The discovery of clavulanic acid (CA) was reported in 1976 (Reading and Cole, 1977), and CA has been shown to be a potent inhibitor of b-lactamases produced by staphylococci and plasmid-mediated b-lactamases of A. coli , as well as species from Klebsiella, Proteus, and Hemophilus (Brown et al ., 1976). Later CA was found to be active against broad spectrum of Gram-positive and Gram-negative bacteria, however with a low activity. Clavulanic acid, sharing a similar chemical structure with the b-lactam antibiotics, binds irreversibly with the enzyme b-lactamases to give a stable complex (Liras and Rodriguez - Garcia, 2000) thereby making it inactive. Co-formulation of CA with other broad-spectrum antibiotics which are themselves succeptible to b-lactamases became the prefered use of this important secondary metabolite (Brown, 1986).

Today, the use of well-established commercial products such as Augmentin^(R) and Timentin^(R), the combinationa of clavulanic acid and b-lactam antibiotics (Amoxyciline and Ticarcillin, respectively) is globally spread and often prescribed as a broad spectrum antibiotic of choice. In fact AUGMENTIN^(R) (GlaxoSmithKline) and its generic equivalents are the most widely prescribed anti-infective agents. The global market in antibiotics combined with clavulanic acid has attained sales close to 2 billion dollars yearly (Saudagar et al., 2008). The molecular structure of clavulanic acid is as follows:

Clavulanic acid or (2R,5R,Z)-3(2-hydroxyethylidene)-7-oxo-4-oxa-l-azabicyclo-heptane-2- carboxylic acid, is produced by several microorganisms, namely Streptomyces clavuligerus ATCC 27064 (S. clavuligerus , US4110165, BE827926), Streptomyces jumonjinensis (GB 1563103), Streptomyces katsurahamanus IFO 13716 FERM 3944 (JP83009679B) and Streptomyces sp. P6621 FERM 2804 (JP55162993A), although S. clavuligerus ATCC 27064 (equivalent to S. clavuligerus NRRL 3585) and it’s improved relatives are by far the most widely used and most studied in connection to clavulanic acid production. Hundreds of studies on the biology and genetics of S. clavuligerus ATCC 27064 (NRRL 3585), the original CA-producing strain, have been reported in recent decades. Among other, it has been found that S. clavuligerus as a member of Streptomycetes , produces other natural products apart from clavulanic acid. Holomycin, Clavams other than CA, Tunicamycin, Cephamycins etc., have been identified in S. clavuligerus culture broths (Medema et ah, 2010). Several independent genome sequencing projects from the first wave of next generation sequencing have been published (accession: PRJNA19249, PRJNA28551 and PRJNA42475). The availability of the genome sequence provided an insight into peculiarities of the genome, the incredible wealth of secondary metabolite clusters encoded within the main 6.7 Mb chromosome, the large 1.8 Mb linear plasmid and several other smaller genomic elements. Using bioinformatic tools, potential genetic clusters for 48 different secondary metabolites have been thus found in the genome of S. clavuligerus (Medema et al. 2010). Many additional genome-supported studies in transcriptomics, proteomics, metabolomics and regulatory networks, as well as genome mining approaches were done using these genome sequences, which consequently hold a “golden standard” status in the community.

As can be seen from the above, a lot of research has been done with regard to clavulanic acid and S. clavuligerus , the original CA-producing strain. However, despite all this knowledge, there is a huge - not only economic - interest, and thus there is still a need for, an improved process for preparing clavulanic acid, with this improvement being for example with regard to the amount of clavulanic acid produced, the general suitability of the process for preparing clavulanic acid, and/or safety aspects relating to the production process. Further, there is a need for an improved microorganism producing clavulanic acid, simply because CA is manufactured in such huge amounts annually of more than 1000 metric tons.

SUMMARY OF THE INVENTION

The present invention provides the following aspects, subject-matters and preferred embodiments, which respectively taken alone or in combination, contribute to solving the object of the present invention: 1. A process for preparing clavulanic acid by using a microorganism, comprising the steps of: a) providing a microorganism being capable of producing clavulanic acid and teleocidin A (also referred to herein as Lyngbyatoxin A) and/or other congeners originating from the teleocidin biosynthetic pathway; b) modifying said microorganism of step a) in that

• the production of teleocidin A and/or other congeners originating from the teleocidin biosynthetic pathway, is reduced when applying the same conditions as for producing clavulanic acid compared to its unmodified form, but the production of clavulanic acid at least essentially remains the same; c) Cultivating said microorganism of step b) or its descendants to produce clavulanic acid; and d) optionally isolating clavulanic acid.

In a preferred embodiment, the production of teleocidin A, and optionally additionally other congeners originating from the teleocidin biosynthetic pathway, is reduced.

2. The process of the preceding item, wherein the extent of reduction of teleocidin A and/or other congeners originating from the teleocidin biosynthetic pathway, is at least 50%; preferably, the extent of reduction is at least 60%, at least 70%, more preferably at least 80% or even more preferably at least 90%, or at least 95%.

3. The process according to any of the preceding items, wherein the modified microorganism resulting from step b) is not able to produce teleocidin A, and/or other congeners originating from the teleocidin biosynthetic pathway any more, compared to its unmodified form.

In a preferred embodiment, the modified organism resulting from step b) is not able to produce teleocidin A, and optionally additionally other congeners originating from the teleocidin biosynthetic pathway.

4. The process according to any of the preceding items, wherein the microorganism of step a) belongs to the genus Streptomyces , preferably the microorganism is selected from the group consisting of Streptomyces clavuligerus, preferably S. clavuligerus ATCC 27064, S. clavuligerus NRRL 3585, or S. clavuligerus K4567; Streptomyces katsurahamanus , preferably S. katsurahamanus IFO 13716 FERM 3994; Streptomyces jumonjinensis; and Streptomyces sp. P6621 FERM 2804; or the descendants thereof; more preferably, the microorganism is selected from the group consisting of S. clavuligerus ATCC 27064, S. clavuligerus K4567, and S. clavuligerus NRRL 3585, or the descendants thereof, and most preferably, the microorganism is S. clavuligerus K4567, or the descendants thereof.

S. clavuligerus K4567, which is a monoisloate from ATCC 27064, is deposited at the DSMZ with the deposition number DSM 33546.

5. The process according to any of the preceding items, wherein the modified microorganism of step b) is a result of any one of

(1) modifying protein(s) of the teleocidin biosynthetic pathway, preferably one or more proteins selected from the group consisting of TleA (a non-ribosomal peptide synthetase (NRPS)), TleB, and TleC, further preferred TleA, contained in the microorganism;

(2) modifying or deleting the nucleic acid of said microorganism encoding protein(s) of the teleocidin biosynthetic pathway, preferably encoding one or more proteins selected from the group consisting of TleA (a non-ribosomal peptide synthetase (NRPS)),

TleB, and TleC, further preferred TleA, respectively to the effect that the production of teleocidin A, and/or other congeners originating from the teleocidin biosynthetic pathway, is reduced, but the production of clavulanic acid at least essentially remains the same.

6. The process according to the previous item, wherein

(1) modifying protein(s) of the teleocidin biosynthetic pathway, preferably one or more proteins selected from the group consisting of TleA, TleB, and TleC, further preferred TleA, comprises applying a component that partially or completely inhibits the protein activity (function); preferably said component is a chemical or a compound that specifically inhibits the function of the protein(s) e.g. by chemically affecting the structure of the protein(s); e.g. said compound are aminoacyl-AMS ( aminoacyl- sulfoamoyladenosine) inhibitors or antibodies; preferably said compounds are aminoacyl-AMS ( aminoacyl- sulfoamoyladenosine) inhibitors; and (2) modifying or deleting the nucleic acid of one or more genes encoding protein(s) of the teleocidin biosynthetic pathway comprises introducing a mutation into said nucleic acid, such as deleting or inserting or replacing parts of sequence in said genes, modifying genetic regulatory elements such as promoters, translation factors, terminators, etc. which are involved in synthesis of the proteins, or destabilizing nucleic acid such as DNA or mRNA encoding said protein(s), e.g. by using antisense compounds such as antisense oligonucleotides.

7. The process according to item 6, wherein the component in (1) is a small molecule, a protein or peptide that interacts with said protein(s); or the component reacts with the protein(s) by modifying covalent bonds in said protein(s).

8. The process according to item 6 or 7, wherein (2) comprises reducing the production level of said protein(s), or completely preventing expression of a nucleic acid encoding said protein(s) in said microorganism, comprising manipulating cis- or trans-located transcriptional and/or translational factors, such as transcriptional promoters, transcriptional terminators, ribosome binding site, initiation codons, termination codons, or translational attenuators.

9. The process according to any of items 6 to 8, wherein in (2) modifying or deleting the nucleic acid results in reducing the production level of said protein(s), or completely preventing expression of said protein(s) in said microorganism, comprises the use of antisense compounds, preferably of antisense oligonucleotides.

10. The process according to any of items 6 to 9, wherein in (2) modifying or deleting the nucleic acid encoding said protein(s) comprises introducing mutation into the nucleic acid encoding said protein(s) to that it cannot express, or that it can only express reduced levels, or inactive or less active variants of the protein(s), preferably wherein introducing a mutation comprises deletion, insertion, substitution, and/or point mutation. 11. The process according to item 10, wherein modifying or deleting the nucleic acid encoding said protein(s) comprises introduction of one or more point mutations, e.g. substitution, insertion or deletion of a single or more nucleotides in said polynucleotide sequence, partial or complete deletion of said polynucleotide sequence, and/or partial or complete replacement of said polynucleotide sequence by a different, nucleotide sequence, which normally is not located in that position of that genome..

In a further embodiment, modifying the nucleic acid comprises completely deleting the teleocidin genetic cluster by applying the editing templates as depicted in SEQ ID NO: 4,

SEQ ID NO: 5, and SEQ ID NO: 6.

12. The process according to any of the preceding items, wherein

• TleA is encoded by the tleA gene;

• TleB is encoded by the tleB gene; and

• TleC is encoded by the tleC gene; preferably the TleA, TleB, and TleC respectively is encoded by a nucleic acid sequence that has a sequence identity of at least 65%, of at least 70%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90% and most preferably at least 95%, 97% or 98.5% to the nucleic acid as listed under SEQ. ID. NO: 1 (nucleic acid sequence encoding TleA), SEQ ID NO: 2 (nucleic acid sequence encoding TleB), and SEQ ID NO: 3 (nucleic acid sequence encoding TleC).

13. The process according to any of the preceding items, wherein prior to step c) spores of said modified microorganisms are prepared, preferably said spores are prepared on a sporulation medium.

14. The process according to any of the preceding items, wherein the spores, the microorganism, or its descendants, are used to inoculate a liquid medium.

15. The process according to any of the preceding items, wherein step c) comprises the use of said modified microorganism as seed microorganism and further comprises a fermentation process of said seed microorganism. 16. The process according to any of the preceding items, wherein the modified microorganism of step b) is stored in a frozen stock prior to cultivation in step c) and, optionally, step c) comprises the preparation of seed medium culture frozen stock.

17. The process according to item 15 or 16, wherein the fermentation process comprises the inoculation of a culture broth with the modified microorganism of step b), preferably the inoculation is carried out by aseptical transfer of the microorganism into a bioreactor comprising the culture broth.

The process step that takes place in a production medium is herein also referred to as “main fermentation process”.

18. The process according to item 17, wherein the culture broth is inoculated with the vegetative culture of the modified or treated microorganism.

19. The process according to any of items 15 to 18, wherein the fermentation process is carried out in a bioreactor, preferably under agitation and/or aeration, preferably under submerged aerobic conditions in an aqueous nutrient medium containing sources of carbon, nitrogen, phosphate and minerals.

It is also possible that a solid state fermentation process or a continuous fermentation process is carried out.

20. The process according to any of items 15 to 19, wherein the fermentation process is carried out at a pH in the range of about 5.8 to 7.3, preferably 6.1 to 7.1 and at a temperature in the range of from 19°C to 31°C, preferably of from 21°C to 30°C; and optionally additionally, the production cultures of the fermentation process are incubated for 80 to about 300 hours, more preferably for about 130 to 280 hours.

21. Process for the preparation of an intermediate product or final pharmaceutical dosage form comprising clavulanic acid or salts or derivatives thereof, comprising the following steps: a) preparing clavulanic acid or salts or derivatives thereof by applying the process of any of the preceding items; b) combining said clavulanic acid or salts or derivatives with excipients.

22. A process for preparing clavulanic acid by using a microorganism, comprising the steps of: a) providing a modified S. clavuligerus strain being capable of producing clavulanic acid,

- wherein said modified S. clavuligerus strain is producing clavulanic acid at least essentially in the same quantity, if compared to the production of clavulanic acid of a respective non- modified S. clavuligerus reference strain, and

- wherein said modified S. clavuligerus strain exhibits reduced or no production of teleocidin A and/or other congeners originating from the teleocidin biosynthetic pathway, if compared to the production of teleocidin A and/or other congeners from the teleocidin biosynthetic pathway of said respective non-modified S. clavuligerus reference strain wherein the step of providing includes a step of selecting said modified S. clavuligerus strain out of a mixture of S. clavuligerus strains, wherein a non-selected S. clavuligerus strain represents said reference strain; b) cultivating said microorganism of step a) or its descendant to produce clavulanic acid; and c) optionally isolating clavulanic acid.

23. A process for preparing clavulanic acid by using a microorganism, comprising the steps of: a) providing a microorganism being capable of producing clavulanic acid; b) testing said microorganism of step a) as to whether it is capable of producing clavulanic acid, but at the same time exhibits reduced or no production of teleocidin A, and/or other congeners originating from the teleocidin biosynthetic pathway, when compared to strain S. clavuligerus K4567; c) cultivating said microorganism of step b), or its descendant to produce clavulanic acid, if the conditions in b) are fulfilled; and d) optionally isolating clavulanic acid.

In a preferred embodiment, in step b) the microorganism is additionally tested as to whether the production of clavulanic acid is at least essentially remaining the same; preferably the production of clavulanic acid is at least essentially the same when compared to the production of clavulanic acid of the respective reference strain.

24. Method of testing the suitability of a strain of S. clavuligerus for being used in the production of clavulanic acid, comprising the steps of:

A) providing a strain of S. clavuligerus that is capable of producing clavulanic acid; and

B)

Bi) cultivating the strain of A);

Bii) detecting by using suitable means if the strain of S. clavuligerus produces teleocidin A, and/or other congeners originating from the teleocidin biosynthetic pathway; and Biii) determining that, if in step ii) it is assessed that said strain does not produce teleocidin A or at least reduced levels of teleocidin A, and/or other congeners originating from the teleocidin biosynthetic pathway, when compared to strain S. clavuligerus K4567, the strain of S. clavuligerus is suitable for being used in the production of clavulanic acid; or

C)

Ci) testing the strain of A) for the presence or absence of genes encoding Tie A, TleB, and/or TleC, preferably Tie A, by detecting nucleic acids encoding Tie A, TleB, and/or TleC, preferably wherein the step of detecting comprises or is a step of carrying out a polymerase chain reaction (PCR) that specifically amplifies nuclei acid sequences that are specific for genes encoding TleA, TleB, and/or TleC, preferably for genes encoding TleA;

Cii) determining that, if in step Ci) it is assessed that said strain does not comprise genes encoding TleA, TleB, and/or TleC, preferably TleA, the strain of A) is suitable for being used in the production of clavulanic acid;

D)

Di) cultivating the strain of A);

Dii) assessing by using antibodies or antibody fragments or molecules comprising antibodies or antibody fragments specific against TleA, TleB, and/or TleC, preferably against TleA, whether the respective proteins are present or not; Diii) determining that, if in step Dii) it is assessed that said strain does not produce TleA, TleB, and/or TleC, preferably does not produce TleA, or at least reduced levels of TleA,

TleB, and/or TleC, when compared to strain S. clavuligerus K4567, the strain of A) is suitable for being used in the production of clavulanic acid; or

E)

Ei) cultivating the strain of A);

Eii) detecting the biological acticity of TleA, TleB, and/or TleC by using respective suitable bioassays;

Eiii) determining that, if in step Eii) a biological acitivity of TleA, TleB, and/or TleC is detected, the strain of A) is not suitable for being used in the production of clavulanic acid.

25. The method of item 24, wherein in Ci), genomic DNA isolated from the S. clavuligerus strain to be tested (strain of A) can be used as template, or wherein direct PCR methods such as colony PCR methods can be used.

26. The method according to item 24 or 25, wherein the PCR is carried out by using the primer sets 7 (amplified region is the tleA gene; SEQ ID NOs: 19 and 20); 12 (amplified region is the tleC gene; SEQ ID NOs: 29 and 30); and 13 (amplified region is the tleB gene; SEQ ID NOs: 31 and 32).

27. Method of testing whether a culture broth that is used for culturing S. clavuligerus and producing clavulanic acid, contains low amounts of toxins originating from products of the teleocidin biosynthetic pathway and is thus suitable for being further processed, comprising the steps of: aa) detecting the presence or absence of teleocidin A in said culture broth; bb) determining that, if in step aa) no teleocidin A is present, said culture broth is suitable for being further processed.

28. S. clavuligerus or its descendant, obtained by the steps of a) providing a microorganism being capable of producing clavulanic acid and teleocidin A (also referred to herein as Lyngbyatoxin A) and/or other congeners originating from the teleocidin biosynthetic pathway; b) modifying said microorganism in that • the production of teleocidin A, and/or other congeners originating from the teleocidin biosynthetic pathway, is reduced or abolished compared to the unmodified form of the microorganism, but the production of clavulanic acid at least essentially remains the same; c) obtaining said modified microorganism.

The modifying step in step b) can be carried out as disclosed elsewhere herein. In particular, the modification is carried out as disclosed in any of items 5 to 12 herein.

29. A S. clavuligerus strain or its descendants, characterized in that it is capable of producing clavulanic acid, but not teleocidin A, and/or other congeners originating from the teleocidin biosynthetic pathway.

30. The S. clavuligerus strain or its descendants of item 29, characterized in that said strain produces essentially at least the same amount of clavulanic acid, preferably at least 3%, more preferably at least 5%, 10% or 15% more of clavulanic, when compared to strain ATCC 27064 when applying respectively the same suitable conditions for producing clavulanic acid.

31. The process according to any of the preceding items, wherein the presence or absence or reduction of teleocidin A and/or other congeners originating from the teleocidin biosynthetic pathway; or the presence or absence of TleA, TleB, and/or TleC, can be determined by any suitable means that are known to a person skilled in the art, such as by applying PCR, or suitable chromatographic methods, e.g. by applying HPLC methods; UV detectors; or MS detectors; preferably by applying HPLC methods; or by applying antibodies that are specific for TleA, TleB, or TleC.

32. A process for the preparation of a pharmaceutical dosage form comprising clavulanic acid and a b-lactam antibiotic, wherein the process comprises preparing the clavulanic acid by using a method as defined in any one of items 1 to 27 or 31, and a step of combining clavulanic acid and b-lactam antibiotic to obtain said pharmaceutical dosage form.

33. Use of the strain as defined in item 29 or 30 for producing clavulanic acid. 34. S. clavuligerus strain deposited at the DSMZ with the deposition number DSM 33546.

35. Process for the preparation of a pharmaceutical dosage form comprising clavulanic acid and a b-lactam antibiotic, comprising a step of using the S. clavuligerus strain as defined in item 29 or 30, or 34, and a step of combining the clavulanic acid and the b -lactam antibiotic to obtain said pharmaceutical dosage form.

Other objects, features, advantages and aspects of the hereby invention will be immediately understood by those skilled in the art from the following description, as will various changes and modifications within the spirit and scope of the disclosed invention. The following description and specific examples, however, while indicating preferred embodiments of the invention, are given by way of illustration only.

DEFINITIONS OF TERMS USED WITHIN THE MEANING OF THE PRESENT INVENTION

Within the meaning of the present invention, the expression "unmodified form" e.g. of a microorganism denotes a microorganism that has not been modified, for example that has not been modified with regard to the expression and/or function of protein(s) of the teleocidin biosynthetic pathway, preferably one or more proteins selected from the group consisting of TleA, a non-ribosomal peptide synthetase (NRPS), TleB, and TleC, further preferred TleA. An unmodified microorganism is e.g. the wild type form of said microorganism.

Within the meaning of the present invention, the expression "essentially the same" defines a deviation of up to 20%, preferably of up to 10%, more preferably of up to 7% and even more preferably of up to 3% of a given value.

Accordingly, the expression "essentially no" defines that there is an amount/activity of the respective matter (such as protein) present (or left) that corresponds to at most 20%, preferably at most 10%, more preferably at most 7%, and even more preferably at most 3% of the respective unmodified (e.g. wild type) amount/activity. The teleocidins in S. clavuligerus cultures can be analysed as e.g. disclosed in the example part (example 3) herein. It is preferred to carry out HPLC, more preferably as disclosed in example 3, method a.

The activity of the teleocidin A can be determined for example by any of the methods known in the art which measure activity of protein kinase C in presence of the analysed sample. In case of presence of teleocidin, activity of protein kinase C is proportionally increased (Fujiki et al., 1984).

The isolation of teleocidin A e.g. of the fermentation broth can be carried out as described in example 4 herein.

The clavulanic acid can be analysed e.g. by modified HPLC methods (Agilent technologies, 2007), and quantified by comparison to authentic pure clavulanic lithium, as disclosed in example 3 herein.

“Essentially no” also includes the absence of the respective matter/amount/activity, preferably meaning below the detection limit of the methods described in this application for that matter/ amount/ activity.

Further, the expression "essentially the same" defines that two values differ by a maximum of at most 20%; preferably at most 10%, more preferably at most 7%, and even more preferably at most 3%.

Within the meaning of the present invention, the term “microorganism” denotes any entity of microscopic or submicroscopic size that is capable of carrying on living processes, for instance bacteria.

Within the meaning of the present invention, the term “microorganisms being capable of producing clavulanic acid” denotes microorganisms that have the capability of clavulanic acid production. In general, microorganisms that have the capability of producing clavulanic acid are in possession of the respective necessary cellular machinery, such as proteins, in particular enzymes, and genes for producing clavulanic acid. With respect to clavulanic acid, enzymes typically involved in the biosynthesis thereof are for instance N²-(2-carboxyethyl)arginine synthase, b-lactam synthetase, clavaminate synthase, proclavaminate amidino hydrolase, and clavulanate dehydrogenase.

Teleocidin (Lyngbyatoxin) is biologically very potent compound. Human poisoning with Lyngbyatoxin was reported several times. Mostly, the poisoning came from ingesting marine turtle meat. Most recently, the poisoning between 1993 and 1996 in Madagascar affected 414 cases with 29 fatalities (Champetier et al, 1998). The turtle acquire the toxin from the blue- green algae Lyngbya majuscula (Yasumoto, 1998). Actinobacterial teleocidin (teleocidin A-l is identical to lyngbyatoxin A, Caerdellina et al. 1979) was discovered independently and is now known to be produced by several Streptomyces species (Takashima and Sakai, 1960).

The most potent activity of Lyngbyatoxin is activation of protein kinase C, acting as one of the most potent tumor promoting agents (Fujuki et al. 1984). Initially the compound was reported to be extremely toxic to wide range of organisms. In mice, LD50 intravenously was 400ug/kg, LD50 orally was 2mg/kg. In rabbit, lOOug/kg intravenously caused death within 5 minutes (Takashima and Sakai, 1960; Ito et al. 2002). Later, the compound was also reported as strong skin irritant and blistering agent. Finally, the tumor promoting activity was observed in several testing environments (mice, human cell lines) in the dosage between 2.5 and 10 pg/kg (Fujiki et al. 1981) There are numerous reports on biological activity, all emphasizing potency of the compound in various tumor promoting mechanisms .

In conclusion, the Lyngbyatoxin and its analogs are extremely potent toxins and tumor promoting agents which can have severe adverse effects already in very small dosage.

A microorganism that is capable of producing teleocidin A (and optionally additionally teleocidin B), and optionally additionally further congeners originating from the teleocidin biosynthetic pathway, is in possession of a functional teleocidin biosynthetic pathway. In other words, the microorganism is in possession of the respective necessary cellular machinery, such as proteins, in particular enzymes, and genes for producing the teleocidin(s). Examples of genes that are necessary for producing teleocidins are genes tie A, tleB and tleC encoding the proteins TleA, TleB, and TleC. Some details of the teleocidin synthesis pathway including some proteins and enzymes involved in this pathway are depicted in Fig. 4. All essential parts of the teleocidin synthesis pathway are encoded by the genes of tie A (SEQ. ID #1), tleB (SEQ. ID #2) and tleC (SEQ. ID #3). Further within the meaning of the present invention, the term “modifying” shall denote any kind of manipulation in or on the microorganism, in particular a manipulation of its genome and/or structure, wherein, after the manipulation has stopped, the microorganism remains modified. In particular, said modification can be provided, or passed over, to the descendants of the microorganism. A preferred type of modification is a manipulation of the microorganism by genetic engineering of its genome.

Generally, and unless otherwise indicated, a "reference microorganism" is a microorganism that is the same type of microorganism that is modified, however with the difference that the reference microorganism is not modified in this specific aspect. A modified microorganism can for instance be a modified S. clavuligerus. In this case, a corresponding reference microorganism is an unmodified (e.g. wild type, parent strain) microorganism S. clavuligerus.

Thus, within the meaning of present invention, and unless otherwise indicated, the term "reference strain” is the same strain that is being subjected to modification and/or selection from within its population, however with the difference that the reference strain is not modified or selected from within its population in this specific aspect. A modified or selected microorganism can for instance be a modified or selected S. clavuligerus. In this case, a corresponding reference strain is an unmodified or non-selected (e.g. wild type, parent strain) microorganism S. clavuligerus. Another example is when one subjects to modification or selection from within its population an improved strain for production of clavulanic acid such as F613-1 (Cao et al., 2016). When evaluating the resulting strain of such modification or selection the term “respective reference strain” relates to the unmodified or non-selected S. clavuligerus F613 - 1.

Strain ATCC 27064 as available today (in the year 2020) is a strain that does not express teleocidin A; and a mono isolate obtained from old stock strain ATCC 27064 (old stock labeled “Dec. 15.

1986” from the ATTC), the mono isolate being referred to herein as S. clavuligerus K4567 is a strain that exhibits teleocidin A expression. The strain is deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) under the deposition number DSM 33546. Within the meaning of the present invention, the term “descendant” denotes the progenitors of the originally modified or treated microorganism. Further, a descendant can for instance also be a spore of the microorganism, “decendants” of a microorganisms are the offsprings of that microorganism which are the result of the proliferation of said microorganism, regardless if this proliferations occurs via cell devision or via formation of spores which subsequently can “grow” to said microorganism.

Methods of modifying, such as inactivating, genes in microorganisms, e.g. as referred to in step (b) above, are well known in the art, for example, a method is described in the handbook "Practical Streptomyces genetics" (Kieser et al ., 2000). A similar but more preferred method which includes use of both positive and negative selection markers was reported recently (Dubeau, et al ., 2009). Both methods are based on homologous recombination mechanisms which, in the outcome of double recombination, results in exchange of the target sequence in the genome for the sequence introduced externally and positioned between the homologous regions. Therefore, preferably homologous sequences flanking the region to be modified in the genome are used. These regions are selected according to the desired effect. For instance for deletion, the sequences are positioned distant to each other for the fragment length which is targeted for deletion. The above mentioned homologous sequences can be suitably obtained by conventional cloning methods (such as PCR, polymerase chain reaction) based on the published sequence.

A further method of modifying the genome of a specific microorganism is applying the CRISPR/Cas-system of genome editing.

Within the meaning of the present invention, the term "function" or "activity" of a protein denotes the function or activity a protein in its unmodified (e.g. wild type) form, in regard to this specific function or activity. This function or activity can be modified by the measures disclosed herein, e.g. by applying a component that partially or completely inhibits the (e.g. enzymatic) activity (function) of protein(s) of the teleocidin biosynthetic pathway, preferably one or more proteins selected from the group consisting of TleA, TleB, and TleC, further preferred TleA, contained in the microorganism. Said component can for example be a chemical or a compound that specifically inhibits the function of the protein(s) e.g. by chemically affecting the structure of the protein(s). The effect of modifying the function or activity of said protein(s) is that the production or effect of teleocidin A is reduced, but the production of clavulanic acid at least essentially remains the same.

The function or activity can be determined by suitable methods that are known to a person skilled in the art. An example of such method would be in vitro activity tests. These may consist of putting in contact cell free extracts of the analysed cultures with suitable amounts of L- tryptophan, S-adenosylmethionine, L-valine and ATP under suitable conditions and measuring formation of the N-methyl-valyl-tryptophane. The conditions and concentrations in such tests are preferably similar to physiological conditions at which the analysed culture normally produces teleocidins. The measurement of N-methyl-valyl-tryptophane is most preferably made by HPLC or LC-MS chromatography.

Within the meaning of the present invention the term "reduced", e.g. "reduced" activity, "reduced" function, or "reduced" amount, denotes that the total activity, function, or amount of a matter (e.g. TleA, TleB, or TleC protein; teleocidin A and/or other congeners originating from the teleocidin biosynthetic pathway) is at most 40%, preferably at most 30%, more preferably at most 20%, even more preferably at most 10% or at most 5% of the total activity, function and/or amount of the unmodified (e.g. wild type) form of this matter (e.g. TleA, TleB, or TleC protein; teleocidin A and/or other congeners originating from the teleocidin biosynthetic pathway).

Within the meaning of the present invention, the expression "reduced function or activity" also comprises an essentially complete abolishing of the function, activity, or amount.

An essentially complete abolished function, activity, or amount cannot be determined, as it is below the detection limit.

Within the meaning of the present invention, the expression "congeners originating from the teleocidin biosynthetic pathway" denotes a compound that is synthesized by the teleocidin biosynthetic pathway, and that is related to teleocidin A with regard to origin, structure, or function. An example of a congener originating from the teleocidin biosynthetic pathway is teleocidin B.

"Sequence identity" or “% identity” or percentage identity refers to the percentage of residue matches between at least two polypeptide or polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences. For purposes of the present invention, the sequence identity between two amino acid sequences or nucleotide is determined using the NCBI BLAST program version 2.2.29 (Jan-06-2014) (Altschul et ah, 1997)). Sequence identity of two amino acid sequences can be determined with blastp set at the following parameters: Matrix: BLOSUM62, Word Size: 3; Expect value: 10; Gap cost: Existence = 11, Extension = 1; Filter = low complexity activated; Filter String: L; Compositional adjustments: Conditional compositional score matrix adjustment. For purposes of the present invention, the sequence identity between two nucleotide sequences is determined using the NCBI BLAST program version 2.2.29 (Jan-06-2014) with blastn set at the following exemplary parameters: Word Size: 11; Expect value: 10; Gap costs: Existence = 5 , Extension = 2; Filter = low complexity activated; Match/Mismatch Scores: 2,-3; Filter String: L; m. If the context of the text does not make it clear if polypeptide- or polynucleotide-sequence identity is meant, it is always meant polypeptide-sequence identity.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is now described in more detail by preferred embodiments and examples, which are however presented for illustrative purpose only and shall not be understood as limiting the scope of the present invention in any way.

All patent applications, patents and literature references cited herein are hereby incorporated by reference in their entirety. In practicing the present invention, many conventional techniques in molecular biology, microbiology, and recombinant DNA are used. These techniques are well known and are explained in, for example, Ausubel, 1995; Sambrook and Russell, 2001; Kieser et ah, 2000 and Dyson, 2011.

In the inventors' laboratory, the inventors have independently sequenced and compared genomes of S. clavuligerus ATCC 27064 cultures, which were obtained from ATCC (American Type Culture Collection) on 3 occasions: in 1991, 1996 and 2003. Although the bulk of the sequence between the genomes was identical, they have found additional -138 kb of DNA sequence within the main chromosome of the S. clavuligerus ATCC 27064 culture acquired in 1991 (labeled “Dec. 15. 1986” from the ATTC). Similarity searches for this fragment in the published genomes of S. clavuligerus ATCC 27064 (PRJNA 19249, PRJNA28551 and PRJNA42475) and even in the proprietary and public nucleotide databases gave no clue about its origin until a recent publication of the genome sequence of an industrial S. clavuligerus strain F613-l(Cao et ah, 2016). This genome provided the first similarity hit for the mysterious 138 kb sequence (Fig. 2). A subsequent study by the same group has provided some insight into the plasticity of the genome of S. clavuligerus , however gave no explanation for the origin of the 138 kb region (Li etal., 2018).

The analysis of the 138 kb region for secondary metabolite gene clusters with AntiSMASH 3.0 predicted presence of several secondary metabolite gene clusters, including a putative teleocidin cluster. The genetic organization of this cluster (Fig. 3) is identical to the recently reported situation in S. blastmyceticus (Abe, 2018), however, the gene context into which the teleocidin cluster is placed is completely different. The identity scores compared to S. blastmyceticus TleA, TleB and TleC are 68%, 84% and 66%, respectively. Interestingly, in analogy with the S. blastmyceticus , a methyltransferase gene tleD, which is needed for conversion of teleocidin A to teleocidin B (Abe, 2018), is not associated with the cluster, nor can be found within the 138 kb DNA region. Instead, a putative TleD homologue with 66% identity is encoded by a SAM-dependent methyltransferase (RefSeq: WP 003953057) and conserved in the giant linear plasmid pSCL4 of all published genomes of S. clavuligerus strains.

The analysis of the cultures with LC-MS has shown presence of teleocidin A as the major, and teleocidin B as the minor indolactam compound (Fig. 4) in the 1986 vial of S. clavuligerus ATCC 27064, but not in cultures acquired from ATCC in later years. The indolactams were found mostly associated with the mycelium of the S. clavuligerus cultures and only traces could be detected in culture supernatants. HRMS data [M+H⁺] for Teleocidin A (C₂₇H₄₀N₃O₂; calc.: 438.3115; found: 438.3114, D=0.2 ppm) and Teleocidin B (C₂₈H₄₂N₃O₂; calc.: 452.3272, found: 452.3269, D=0.7 ppm) as well as UV-absorption spectra and chromatographic behavior of these compounds are in agreement with authentic samples and the published data (Hitotsuyanagi etal. 1984; Takashima etal. 1962). In addition, the inventors have observed the presence of compounds with m/z [M+H⁺] = 454 and characteristic UV absorption spectra that could indicate teleocidin-related compound. HRMS data [M+H⁺] suggest molecular formula C₂₇H₃₉N₃O₃ (calculated: 454.3064, measured: 454.3062, D=0.4 ppm). This formula corresponds to 2-oxo-teleocidin A (JBIR-31), a teleocidin analog found in Streptomyces sp. NBRC 105896 (Izumikawa et al, 2009).

Whole-broth extracts were used to isolate and confirm the identity the major compound. Subsequent HPLC -prep purification gave ~15 mg of teleocidin (lyngbyatoxin) A in form of off- white to brownish amorphous powder. The 1H-NMR, 13C-NMR and HRMS spectra were found in agreement with the published data (Table 1; Cardelina et al, 1979; Takashima et al. 1962)

Shake flask testing of randomly picked single colonies from the plated culture of S. clavuligerus ATCC 27064 (the 1986 vial) has shown that only a third of the population produces teleocidins. The inventors have therefore performed two subsequent sub-plating cycles and only then obtained monoisolates of entirely teleocidin-positive populations (e.g. K4567). Homogenous populations of teleocidin-negative phenotype were obtained already at the first sub-plating round. In parallel, the inventors have performed colony PCR (primer pair 6) for the presence of the teleocidin genes in each of the tested colonies and found that production of teleocidins is without exception linked to this genotype. The monoisolates showed stable genotypes and phenotypes for several successive sub-plating rounds.

A monoisolate obtained from this culture after two additional sub-plating rounds, the strain K4567, was sequenced again. A detailed investigation of the raw reads on the insertion joints has excluded possibility of stand-alone genetic element or contamination with heterogeneous DNA. A whole-genome alignment was made between the genome of S. clavuligerus K4567 and S. clavuligerus F613-1 (Cao et. al., 2016). 99.9 % identity was found over the region of 138kb from position 441669 to 579837 relative to latter genome.

The obtained monoisolate of S. clavuligerus ATCC 27064, that is the S. clavuligerus K4567, is deposited at the DSMZ under the deposition number DSM 33546.

The 138 kb region is inserted at position 6298508 of the chromosome relative to the PRTN1A19249, which is at present the most complete public genome sequence of S. clavuligerus ATCC 27064. Upon investigation of the insertion joint, no inverted repeats, palindromes or other elements that could indicate involvement of an integrase or transposase could be found. Instead, a moderately conserved direct repeat of 19 bp is positioned at the borders of the 138 kb region (Fig. 2). This could facilitate excision of the 138 kb region by homologous recombination (perhaps a repair after a double-strand break or recombinase-assisted event), to resolve into the situation found in the published ATCC genomes, having one of the direct repeats conserved at the excision site.

The teleocidin-positive monoisolates were then cultivated in shake flasks in comparison to teleocidin-negative population and S. clavuligerus ATCC 27064 strains from the more recent ATCC stocks. Cultures were analyzed with Liquid Chromatography-Mass Spectroscopy (LC- MS), at the day 4 and 7 of cultivation. The maximal amounts of accumulated teleocidin A was exceeding 100 mg L^'1 and the relative amount of teleocidin B increased to ~10 % area under the curve with ageing of the culture.

Fig. 5 shows LC-UV and LC-MS analysis of cultures of S. clavuligerus ATCC 27064 and S. clavuligerus K4567. In comparison, an authentic sample of teleocidin A is also shown. We have thus isolated a second, stable genotype strain of S. clavuligerus ATCC 27604, designated S. clavuligerus K4567, from the mixed genotype culture found in the vials provided by ATCC in 1986. The K4567 strain is deposited at the DSMZ under the deposition number DSM 33546. Most importantly, this genotype harbors teleocidin biosynthetic genes, which are located on a distinct 138 kb chromosomal region and supports accumulation of significant amounts of these highly toxic, tumor-promoting indolactam-terpenoid secondary metabolites, which act through activation of protein kinase C (Fujiki et ah, 1981; Fujiki et ak, 1984).

Teleocidins are water-insoluble and unstable in acidic environment (Takashima et al ., 1962) . This is fortunate because the established industrial processes for production of CA use three key steps (Saudagar et ak, 2008; EP06125246), which effectively prevent contamination of CA with teleocidins. i. The mycelium is most often separated from the aqueous CA solution by means of filtration or centrifugation, leading teleocidins which are associated with mycelium phase to the waste stream, ii. The aqueous CA solution is then acidified to pH ~2, to allow extraction of CA to water-immiscible organic solvent. This acidification would degrade any teleocidin carry-over from the filtration step. iii. CA is precipitated from organic solvent by formation of salt with addition of organic or inorganic base, rendering CA insoluble in organic solvents. Again, teleocidins cannot be carried over through this purification stage. The risk of teleocidin carry-over to the final CA product is therefore minor. Indeed, the inventors could not detect teleocidins in the tested commercial products containing CA salts (data not shown).

On the other hand, there is a strong concern about the environmental impact coming from the waste mycelium streams, potentially contaminated with teleocidin(s). In fact, there has already been a report where workers in industrial fermentation process with another Streptomyces complained about skin irritation, which was subsequently attributed to teleocidins (Sugimura, 1986). These toxins, which are also produced by a filamentous blue-green alga Moorea producens (formerly Lyngbya majuscula ) are known to cause acute dermatitis called ‘swimmer's itch’ (Cardelina et ak, 1979; Osborne et ak, 2001). Occasional acute poisonings by ingestion of marine turtle meat {Chelonia mydas ) have been reported in Indo-Pacific, several with fatal outcomes (Champetier, 1998). At least in one case, the intoxication was linked to the accumulation of algal teleocidin A (lyngbyatoxin A) in the food chain (Yasumoto, 1998). Little is known about the effects of long-term exposure of humans to sub-toxic doses of teleocidins, however given their tumor-promoting activity (Fujiki et al ., 1981; Osborne et al ., 2001) and toxicity (Ito et al. , 2002), caution is appropriate. With improper waste mycelium management strategy and more critically with potential reuse of the waste mycelium biomass (e.g. as fertilizer, animal feedstock etc.), teleocidins may enter human or animal diet. Therefore, a risk assessment should be made for each specific situation where large amounts of S. clavuligerus biomass are being generated.

Due to its capability to produce CA, this organism has been disseminated from a single initial source to laboratories and industrial facilities all over the world. Despite the fact that teleoci din- producing variant of S. clavuligerus ATCC 27064 apparently lost from the subsequent collection stocks, and has remained obscure to the scientific community, it appears that this genotype was not entirely lost. At least one of the industrial S. clavuligerus strains for commercial production of CA, originating from ATCC 27064, holds the genetic potential for production of teleocidins (Cao et al., 2016). This and the sheer scale of CA production globally, raises questions about safety and environmental impact, which need to be addressed appropriately.

Clearly, one of the more elegant solutions is to use an industrial microorganism for CA production which does not have the capability to produce teleocidins. Such organism may be obtained from the teleocidin-producing parent strains by several methods.

Traditional strain improvement is frequently used to obtain industrial fermentation strains with desired properties. This has depended largely on random mutagenesis and selection techniques. Unfortunately, development of new generations of high producing strains, with this approach, often takes 5 years or more (Nielsen, 1997). To apply this approach, a relatively high throughput method of distinguishing between teleocidin-producing and non-producing strain is needed, in order to achieve the goal in a reasonable timeframe. Such methods are provided with this invention.

Since molecular genetics techniques have become increasingly sophisticated, the ability to modify existing pathways or to create non-native pathways has advanced rapidly. Progress in genetics, transcriptional analysis, proteomics, metabolic reconstructions and metabolic flux analysis offer genetic engineering as an alternative approach for strain improvement in a targeted manner (Baltz, 2001). Duplication, replacement or deletion of specific genes can be achieved by inserting molecular tools, targeting the desired gene(s), in the chromosome by homologous recombination or by site-specific integration. Targets for genetic engineering are usually the genes thought to be involved in rate limiting steps, the genes involved in undesired pathways and global or specific pathway regulators.

Specifically, disruption or deletion of one or several biosynthetic genes for teleocidin is a preferred method of interfering with the functionality (that is, the ability of the teleocidin pathway to result in functional teleocidin such as teleocidin A, and/or teleocidin B, and/or other congeners that originate from the teleocidin biosynthetic pathway) of the teleocidin pathway. Several methods, described in more detail below are known in the art to achieve targeted disruption of gene function in Streptomyces, and specifically in S. clavuligerus.

On the other hand it is obvious to persons skilled in the art that productivity of strains with long history in strain development (also known as high-producer strains) is often influenced by genetic manipulation in unanticipated manner. In other words, the higher the titers and the more finely tuned the processes, the higher the risk of unexpected negative effects of a given genetic manipulation can be expected. In addition, it has been postulated that the presence of the additional 138 kb genomic region in the industrial strain F613-1, is one of the factors determining fitness of the strain for industrial use, which includes high productivity of clavulanic acid (Li, 2018).

Therefore it was surprising to find that e.g. no negative effects in terms of clavulanic acid productivity were found by introducing significant deletions, centering around and within the teleocidin gene cluster in the 138 kb genomic region. This even includes the most drastic modification presented within this invention, deletion of the whole 138kb genomic region.

Hence, the present invention is based on the surprising find that interfering with the function of teleocidin genes tie A , tleB and/or tleC or the deletion of the distinct 138 kb genomic region on which they are positioned not only abolishes the production of teleocidin but also maintains high productivity of clavulanic acid of the microorganism. In particular, the invention is based on the surprising find that interfering with the function of teleocidin non-ribosomal peptide synthase TleA encoded by the tleA gene in S. clavuligerus results in a microorganism that is still capable of producing at least essentially the same amount of clavulanic acid when compared to its unmodified (non-interfered) form, but at the same time exhibits reduced or even abolished production of teleocidin A and/or other congeners from the teleocidin biosynthetic pathway.

The present invention therefore provides an improved process for the production of clavulanic acid, with the proviso that the microorganism that is used in this process and that is capable of producing clavulanic acid, is modified in order to selectively (targetedly) reduce or abolish the microorganism's production of teleocidin A and/or other congeners originating from the teleocidin biosynthetic pathway, while, at the same time, the production of clavulanic acid at least essentially remains the same when compared to its unmodified form.

Additionally, by applying the process of the present invention, the produced product itself can have an improved quality (e.g. there is no risk of contaminations caused by teleocidin A and/or other congeners from the teleocidin biosynthetic pathway). This is all the more advantageous if the clavulanic acid is intended to be used in the field of pharmaceutics, e.g. in human medicine: Testing and potential purifications are not necessary. Furthermore, the safety of the production environment is improved, for example health risks of employees dealing with clavulanic acid production and/or risks for the environment that are in connection with teleocidin A and/or other congeners from the teleocidin biosynthetic pathway are reduced or even completely abolished.

Furthermore, it has been found that by applying the process according to the present invention, the yield of clavulanic acid at least essentially remains the same, or can even be improved. It is all the more surprising that even if strains are used that have a history in strain development (also known as high-producer strains, that for instance have many years of random mutagenesis and selection cycles), the yield in clavulanic acid essentially remains the same or can even be improved, despite the manipulation on the genetic cluster being involved in teleocidin biosynthesis.

Thus, the present invention relates to a process for preparing clavulanic acid by using a microorganism, comprising the steps of: a) providing a microorganism being capable of producing clavulanic acid and teleocidin A (also referred to herein as Lyngbyatoxin A) and/or other congeners originating from the teleocidin biosynthetic pathway; b) modifying said microorganism of step a) in that

• the production of teleocidin A and/or other congeners originating from the teleocidin biosynthetic pathway, is reduced when applying the same conditions as for producing clavulanic acid compared to using unmodified microorganisms, but the production of clavulanic acid at least essentially remains the same; c) Cultivating said microorganism of step b) or its descendants to produce clavulanic acid; and d) optionally isolating clavulanic acid.

In step (a), a microorganism is chosen, wherein the microorganism can be a commercially available microorganism or a microorganism that has been genetically engineered to have desired properties such as for example a high rate of reproduction.

A preferred microorganism for carrying out the present invention is a microorganism belonging to the group of clavulanic acid producing organisms. A suitable microorganism of step a) preferably belongs to the genus Streptomyces , preferably the microorganism is selected from the group consisting of Streptomyces clavuligerus , preferably S. clavuligerus ATCC 27064, S. clavuligerus NRRL 3585, or S. clavuligerus K4567; Streptomyces katsurahamanus , preferably S. katsurahamanus IFO 13716 FERM 3994; Streptomyces jumonjinensis ; and Streptomyces sp. P6621 FERM 2804; or the descendants of said microorganisms; more preferably, the microorganism is selected from the group consisting of S. clavuligerus ATCC 27064, S. clavuligerus K4567, and S. clavuligerus NRRL 3585, or the descendants thereof, and most preferred, the microorganism is S. clavuligerus K4567, or the descendants thereof.

In order to identify microorganisms other than the above that are suitable within the present invention, or whether a microorganism is capable of producing clavulanic acid (CA) and teleocidin A and/or other congeners originating from the teleocidin biosynthetic pathway, potential microorganisms can be tested to determine their capability of producing clavulanic acid and teleocidin A and/or other congeners originating from the teleocidin biosynthetic pathway. Suitable testing methods are known to a person skilled in the art and include e.g. analyzing of cultures of the respective microorganisms with HPLC or LC-MS (see methods described elsewhere herein). In step b), the microorganism is modified to the effect that the production of teleocidin A and/or other congeners originating from the teleocidin biosynthetic pathway is reduced when applying the same conditions as for producing CA compared to its unmodified form. At the same time, the production of CA at least essentially remains the same.

It is preferred that the extent of reduction of teleocidin A and/or other congeners originating from the teleocidin biosynthetic pathway, is at least 50%; preferably, the extent of reduction is at least 60%, at least 70%, more preferably at least 80% or even more preferably at least 90%, or at least 95%.

In a particular preferred embodiment, the modified microorganism resulting from step b) is not able to produce teleocidin A, and/or other congeners originating from the teleocidin biosynthetic pathway any more, compared to the unmodified microorganism.

In general, the production of teleocidin A and/or other congeners originating from the teleocidin biosynthetic pathway can be reduced or abolished by interfering with the function of factors of said pathway that are necessary for the production of teleocidin A and/or other congeners originating from the teleocidin biosynthetic pathway. Such factors are for example the proteins TleA, TleB, or TleC. TleA, TleB, and TleC are enzymes (TleA is a non-ribosomal peptide synthetase, TleB is a P-450 monooxygenase, and TleC is a prenyltransferase) that play an essential role in teleocidin biosynthesis, such as teleocidin A, and teleocidin B.

Such an interference may be accomplished by interfering with the function of said protein(s)/enzyme(s). Interfering with the function of the enzyme may be on the protein or nucleic acid level. It is to be understood that in the context of the present invention a complete inactivation of the enzyme or gene function is preferred but not necessary. Also a partial inactivation of the function will result in a reduced amount of produced teleocidin, albeit not to the extent as in case of a complete inactivation.

Hence, in an embodiment of the present invention, the modified microorganism of step b) is a result of any one of (1) modifying protein(s) of the teleocidin biosynthetic pathway, preferably one or more proteins selected from the group consisting of TleA, TleB, and TleC, further preferred TleA, contained in the microorganism; (2) modifying or deleting the nucleic acid of said microorganism encoding protein(s) of the teleocidin biosynthetic pathway, preferably one or more proteins selected from the group consisting of TleA, TleB, and TleC; particularly preferred TleA. It is the effect of this modification (interference with the function of the protein(s)) that the production of teleocidin A, and/or other congeners originating from the teleocidin biosynthetic pathway, is reduced, but at the same time, the production of clavulanic acid at least essentially remains the same.

Teleocidin A, and/or other congeners originating from the teleocidin biosynthetic pathway (such as teleocidin B), if present, can typically be found in the culture broth (which includes the biomass, e.g. the mycelium) that is used for cultivating the microorganism of step a) and primarily are located in the mycelium of the microrganism. The presence and/or amount of said teleocidins and/or other congeners can be determined by any suitable means that is known to a person skilled in the art, for example by means (methods) indicated elsewhere herein. Such means (methods) are within the routine skill of a person skilled in the art.

There are several different methodical approaches known in the art describing interference with function of proteins. For instance, compounds or chemicals may be used that specifically inhibit the protein function such as e. g. specific antibodies or other proteins or peptides that bind to or chemically modify the protein to be inactivated. Also chemical modification of the protein structure affecting its function may be performed. As mentioned above, said inhibition may be only partial thus leaving some residual protein activity.

In one embodiment a method of interfering with the function of the protein(s) (enzyme(s)) is to interfere with or destabilize nucleic acids such as DNA or the messenger RNA encoding said enzyme, thereby reducing expression levels. For instance, antisense compounds, particularly antisense oligonucleotides, may be used to inhibit the expression of nucleic acid molecules. These antisense compounds specifically hybridize with the nucleic acids and prevent them from fulfilling their function, i.e. being transcribed into mRNA in case of DNA or translated into a protein in case of mRNA.

Another approach used to interfere with the function of proteins can be undertaken by manipulating expression levels of said protein by interfering with transcriptional and/or translational factors regardless the factors being cis or trans. Non limiting examples of such factors are, transcriptional promoters or parts thereof, transcriptional terminators and other cis located transcriptional signals. An equally successful approach is to manipulate translational cis factors such as ribosome binding sight, initiation and termination codons, translational attenuators and other translational cis elements. Another method is to manipulate the function of in trans located transcription and translation factors, such as specific transcriptional regulators and translation initiation factors.

Alternatively, the function of the nucleic acids may be modified by way of mutation of the nucleic acid sequence thereby resulting in a protein with no or reduced activity. By introducing mutations to the sequence either the translation of the protein may be completely blocked or a mutated protein with corresponding mutations in its amino acid sequence will result. Such a protein will either be completely inactive or will display a reduced functional activity. It is well within the general knowledge of a person skilled in the art to select for those mutations that will have the desired effect. Merely established standard technology is required to generate such mutated nucleic acids and to select for mutations suitable in the context of the present invention. As used herein, the term "mutation" encompasses any change in the nucleic acid sequence such as deletions, insertions, substitutions and point mutations. Single nucleotides may be changed but also larger portions of the sequence or the complete sequence may be affected.

In a preferred embodiment of the invention, such mutation is introduced into the gene encoding TleA, TleB, and/or TleC. TleA is encoded by the tleA gene; TleB is encoded by the tleB gene; and TleC is encoded by the tleC gene. Preferably, the TleA, TleB, and TleC respectively is encoded by a nucleic acid sequence that has a sequence identity of at least 65%, of at least 70%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90% and most preferably at least 95%, 97% or 98.5% to the nucleic acid as listed under SEQ. ID. NO: 1 (nucleic acid sequence encoding TleA), SEQ ID NO: 2 (nucleic acid sequence encoding TleB), and SEQ ID NO: 3 (nucleic acid sequence encoding TleC).

The method of inactivating a gene in microorganism is well known in the art, for example as described in the handbook "Practical Streptomyces genetics" (Kieser et ah, 2000, Practical Streptomyces genetics, A laboratory Manual. ISBN0-7084-0623-8). A similar but more preferred method which includes use of both positive and negative selection markers was reported recently (Dubeau, etal. , 2009). Both methods are based on homologous recombination mechanisms which, in the outcome of double recombination, results in exchange of the target sequence in the genome for the sequence introduced externally and positioned between the homologous regions. Therefore, preferably homologous sequences flanking the region to be modified in the genome are used. These regions are selected according to the desired effect e.g. for deletion, the sequenced are positioned distant to each other for the fragment length which is targeted for deletion. The above mentioned homologous sequences can be suitably obtained by conventional cloning methods (such as PCR) based on the published sequence.

Accordingly following from the above, in a preferred embodiment of the present invention, the modified microorganism of step b) is a result of e.g. (1) modifying the protein(s) of the teleocidin biosynthetic pathway, wherein this modification comprises applying a component that partially or completely inhibits the protein activity (function); preferably said component is a chemical or a compound that specifically inhibits the function of the protein(s) e.g. by chemically affecting the structure of the protein(s); e.g. said compound is selected from aminoacyl-AMS ( aminoacyl- sulfoamoyladenosine) inhibitors or antibodies; preferably said compound is selected from aminoacyl-AMS ( aminoacyl- sulfoamoyladenosine) inhibitors. The component of (1) can be any component that interacts or reacts with the protein(s), e.g. by modifying covalent bonds in said protein. Such a component can e.g. be a small molecule. A small molecule is a low molecular weight (< 900 Daltons) organic compound that is able to regulate a biological process, for example it is able to inhibit a specific function of a protein or disrupt protein-protein interactions.

It is also preferred that the modified microorganism of step b) is a result of (2) modifying or deleting the nucleic acid of one or more genes encoding protein(s) of the teleocidin biosynthetic pathway, comprising a step of introducing a mutation into said nucleic acid, such as deleting or inserting or replacing parts of sequence in said genes, modifying genetic regulatory elements such as promoters, translation factors, terminators, etc. which are involved in synthesis of the proteins, or destabilizing nucleic acid such as DNA or mRNA encoding said protein(s), e.g. by using antisense compounds such as antisense oligonucleotides.

Modifying or deleting the nucleic acid of one or more genes encoding protein(s) of the teleocidin biosynthetic pathway of option (2) above can also comprise reducing the production level of said protein(s), or completely preventing expression of a nucleic acid encoding said protein(s) in said microorganism, comprising manipulating cis- or trans-located transcriptional and/or translational factors, such as transcriptional promoters, transcriptional terminators, ribosome binding site, initiation codons, termination codons, or translational attenuators.

In a further preferred embodiment, modifying or deleting the nucleic acid of one or more genes encoding protein(s) of the teleocidin biosynthetic pathway of option (2) above can also result in reducing the protein level of said protein(s), or even in complete preventing the expression of said protein(s) in said microorganism, and can comprise the use of antisense compounds, preferably antisense oligonucleotides.

In a particular preferred embodiment, modifying or deleting the nucleic acid encoding said protein(s) in option (2) above comprises introducing mutation into the nucleic acid encoding said protein(s) to that it cannot express, or that it can only express reduced levels, or inactive or less active variants of the protein(s), preferably wherein introducing a mutation comprises deletion, insertion, substitution, and/or point mutation. Hence, modifying or deleting the nucleic acid encoding said protein(s) comprises introduction of one or more point mutations, e.g. substitution, insertion or deletion of a single or more nucleotides in said polynucleotide sequence, partial or complete deletion of said polynucleotide sequence, and/or partial or complete replacement of said polynucleotide sequence by a different, nucleotide sequence, which normally is not located in that position of that genome.

It is possible to modify or delete a nucleic acid encoding one or more protein(s) of the teleocidin biosynthetic pathway, or the whole teleocidin genetic cluster forming the teleocidin biosynthetic pathway that finally results in teleocidin A and/or other congeners originating from the teleocidin biosynthetic pathway. An example of nucleic acid sequence encoding one or more protein(s) of said pathway is the nucleic acid sequence encoding TleA, TleB, and/or TleC, wherein said sequence has a sequence identity of at least 65%, of at least 70%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90% and most preferably at least 95%, 97% or 98.5% to the nucleic acid as listed under SEQ. ID. NO: 1 (nucleic acid sequence encoding TleA), SEQ ID NO: 2 (nucleic acid sequence encoding TleB), and SEQ ID NO: 3 (nucleic acid sequence encoding TleC). In a further embodiment, modifying the nucleic acid comprises completely deleting the teleocidin genetic cluster, e.g. by applying the editing templates as depicted in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6. Applying the editing templates results in deleting parts thereof or deleting the complete teleocidin genetic cluster which comprises a deletion of up to 138 kb.

In step c), the modified microorganism from step a), or its descendants, is cultivated to - finally - produce clavulanic acid. Cultivation of the microorganism can be carried out by methods known to a person skilled in art. Cultivation processes of Streptomyces clavuligerus are for example described in W00005397, US6100052, WO 9739137, and others.

Preferably, the cultivation step c) comprises a main fermentation process and optionally a step of producing a culture of seed microorganisms prior to the main fermentation process. The main fermentation process is carried out in a production medium. The seed microorganisms are present in a seed medium.

Preferably the main fermentation process in step c) is carried out in a bioreactor, in particular under agitation and/or aeration. In a preferred embodiment, the process for the production of clavulanic acid is carried out under submerged aerobic conditions in an aqueous nutrient medium (production medium), containing sources of assimilable carbon, nitrogen, phosphate and minerals.

The main fermentation process can be carried out according to any suitable method that is known to a person skilled in the art. In general, the main fermentation process comprises the inoculation of production medium with a culture of the microorganism of step a), in particular a culture of seed microorganism. Inoculation can for example be carried by aseptical transfer into the bioreactor (also referred to herein as “reactor”). The seed culture can for example be prepared from a frozen stock, in particular a seed medium culture frozen stock. A seed medium culture frozen stock refers to a microorganism grown in a seed medium and stored as a frozen vial to be used later as an inoculum for further cultivation. It is preferred to employ the vegetative culture of the microorganism for inoculation.

Further, the seed culture can also be prepared from spores of the modified or treated microorganism. The spores are prepared according to any suitable method that is known to a person skilled in the art, for instance the modified or treated microorganism is cultivated on a sporulation medium. The obtained spores are used to inoculate a liquid medium, for example a seed medium. After cultivation, the culture broth can be stored as a frozen stock by freezing below -20°C in an appropriate vial.

The addition of nutrient medium (production medium) in the main fermentation process into the bioreactor can be carried out once or more, batch-wise or in a continuous way. Addition of nutrient medium (production medium) can be carried out before and/or during the fermentation process. The preferred sources of carbon in the nutrient media can be selected from dextrin, starch, glycerol, maltose, sucrose or oil as exemplified below. The preferred sources of nitrogen in the nutrient media are yeast extract, soymeal protein isolates or concentrates, soybean meal, bacterial peptone, casein hydrolysate, ammonium sulphate or any of the proteinogenic amino acids individually or in a mixture. Inorganic/mineral salts such as calcium carbonate, sodium chloride, sodium or potassium phosphate, magnesium, manganese, zinc, iron and other salts may also be added to the medium.

The main fermentation process in step c) can be carried out at a pH in the range of about 5.8 to 7.3 and temperature in the range of 19 to 30 °C. Preferably, the pH is in the range of about 6.1 to 7.1 and the temperature is preferably in the range of about 21 to 29 °C. Preferably, the production cultures are incubated for 80 to about 300 hours, more preferably for about 130 to 280 hours.

The production of clavulanic acid may be performed in aerobic conditions with agitation and aeration of production medium. Agitation and aeration of the culture mixture may be accomplished in a variety of ways, which are known to a person skilled in the art. The agitation of production medium may be provided by a propeller or similar mechanical device and varied to various extents according to fermentation conditions and scale. The aeration rate can be varied in the range of 1.0 to 2.5 VVM (gas volume flow per unit of liquid volume per minute (volume per volume per minute)) with respect to the working volume of the bioreactor.

Further, known additives for the fermentative process may be added, in particular in the main fermentation process. To prevent excessively foaming of the culture medium, anti-foaming agents may be added, such as silicone oil, fatty oil, plant oil and the like. Particularly, a silicone- based anti-foaming agent may be added during the fermentation process to prevent excessively foaming of the culture medium.

Further preferred, prior to step c), spores of said modified or treated microorganism, preferably a strain of S. clavuligerus as defined elsewhere herein, are prepared. The spores can be prepared according to any suitable method that is known to a person skilled in the art, preferably said spores are prepared on a sporulation medium, e.g. on a sporulation medium as described herein. Said spores, the microorganism or its descendant can then be used to inoculate a liquid medium. In a further embodiment, step c) comprises the use of said modified, preferably genetically modified, microorganism, as seed microorganism and further comprises a fermentation process of said seed microorganism.

It is also possible that the microorganism of step a) is stored in a frozen stock, preferably by freezing below -20°C in an appropriate vial, prior to cultivation in step c). Optionally, step c) comprises the preparation of seed medium culture frozen stock.

In principle, the cultivation of seed microorganism can be carried out under the conditions (e.g. pH and temperature) similar to the main fermentation process (described under step c)).

Preferably, the production of seed microorganism (which can for instance be used in the main fermentation process) for the production of clavulanic acid starts from a frozen stock of said modified microorganism. In this respect, the process according to the present invention comprises the preparation of frozen stock of modified microorganism, preferably of Streptomyces clavuligerus, and optionally seed medium culture frozen stock of modified microorganism. This preparation of inoculum may be carried out using methods known in the state of art. Preferably this frozen stock of modified strain of microorganism is used to produce a vegetative or seed culture by inoculation to a seed medium. The production of vegetative culture of described microorganism should start with inoculation of a relatively small quantity of seed medium with the frozen stock.

Additionally preferred, the fermentation process comprises the inoculation of a culture broth with the modified microorganism, preferably with the vegetaticve culture of said microorganism. The inoculation can be carried out by any suitable method that is known to a person skilled in the art; in a preferred embodiment, the inoculation is carried out by aseptical transfer of the microorganism into a bioreactor comprising the culture broth.

Additionally preferred, the fermentation process is carried out in a bioreactor, preferably under agitation and/or aeration, preferably under submerged aerobic conditions in an aqueous nutrient medium containing sources of carbon, nitrogen, phosphate and minerals. The preferred sources of carbon in the nutrient media can be selected from dextrin, starch, glycerol, maltose, sucrose or oil as exemplified below. The preferred sources of nitrogen in the nutrient media are yeast extract, soymeal protein isolates or concentrates, soybean meal, bacterial peptone, casein hydrolysate, ammonium sulphate or any of the proteinogenic amino acids individually or in a mixture. Inorganic/mineral salts such as calcium carbonate, sodium chloride, sodium or potassium phosphate, magnesium, manganese, zinc, iron and other salts may also be added to the medium.

It is also possible that a solid state fermentation process is carried out.

The fermentation process can be carried out at a pH in the range of about 5.8 to 7.3, preferably 6.1 to 7.1, and at a temperature in the range of from 19°C to 30°C, preferably of from 21°C to 29°C.

The production culture of the fermentation process can be incubated for 80 to about 300 hours, preferably for about 130 to 280 hours.

In an optional step d), the clavulanic acid is isolated.

The isolation of clavulanic acid in step d) can be carried out by using suitable techniques that are known to a person skilled in the art. Clavulanic acid from the fermentation broth can be separated and purified by conventional methods commonly used for recovery of biologically active substances. A number of methods used for recovery of clavulanic acid from fermentation broth and subsequent conversion to pharmaceutically acceptable salts of clavulanic acid are for example described in Saudagar etal. , 2008.

Clavulanic acid is a drug that functions as a beta-lactam-inhibitor. It is often combined with antibiotics, e.g. from the penicillin-group, in order to overcome possible antibiotic resistances in bacteria that secrete beta-lactamases. Beta-lactamases have the potential to inactivate beta- lactam antibiotics, e.g. most of the antibiotics from the penicillin group.

Hence, the present invention further refers to a process for the preparation of an intermediate product or a final pharmaceutical dosage form comprising clavulanic acid, comprising the following steps: a) preparing clavulanic acid or salts or derivatives thereof by applying the process of preparing clavulanic acid as disclosed elsewhere herein; b) combining said clavulanic acid or salts or derivatives thereof with pharmaceutically acceptable excipients and, optionally, a beta-lactam antibiotic.

In a preferred embodiment, the clavulanic acid or salts or derivatives thereof is combined with pharmaceutically acceptable excipients and one or more beta-lactam antibiotic(s).

All pharmaceutically acceptable salts of clavulanic acid can be used. In a preferred embodiment, clavulanic acid as a salt of potassium (potassium clavulanate) is used.

Any suitable pharmaceutically acceptable excipients that are known to a skilled person can be used.

By making use of the surprising finding of the present invention, it is further possible to specifically and targetedly select a microorganism that is capable of producing clavulanic acid, but at the same time exhibits reduced or no production of teleocidin A, and/or other congeners originating from the teleocidin biosynthetic pathway, when compared to the production of teleocidin A, and/or other congeners originating from the teleocidin biosynthetic pathway of strain S. clavuligerus ATCC 27064 (K4567), in the production of clavulanic acid.

Hence, the present invention also relates to such a microorganism as such, and to the use of such a microorganism for producing clavulanic acid.

Further, the present invention also relates to a process for preparing clavulanic acid by using a microorganism, comprising the steps of: a) providing a microorganism being capable of producing clavulanic acid, but at the same time exhibiting reduced or no production of teleocidin A, and/or other congeners originating from the teleocidin biosynthetic pathway, when compared to the production of teleocidin A, and/or other congeners originating from the teleocidin biosynthetic pathway of strain S. clavuligerus K4567, with the production of clavulanic acid at least essentially remaining the same when compared to the production of clavulanic acid of the respective reference strain; wherein the step of providing includes a step of selecting said microorganism out of a mixture of microorganisms of said strain; b) cultivating said microorganism of step a) or its descendant to produce clavulanic acid; and c) optionally isolating clavulanic acid. The mixture of microorganisms of step a) comprises microorganisms that are capable of producing CA, but at the same time exhibit reduced or no production of teleocidin A, and/or other congeners originating from the teleocidin biosynthetic pathway, when compared to the production of teleocidin A, and/or other congeners originating from the teleocidin biosynthetic pathway of strain S. clavuligerus K4567, with the production of clavulanic acid at least essentially remaining the same when compared to the production of clavulanic acid of the respective reference strain, and microorganisms that still produce teleocidin A, and/or other congeners originating from the teleocidin biosynthetic pathway essentially at the same level, when compared to the respective reference strain. In an embodiment of the present invention, the mixture of microorganisms of step a) comprises modified and unmodified microorganisms as disclosed elsewhere herein.

The step of selecting said microorganism out of a mixture of microorganisms comprises determining the production of teleocidin A, and/or other congeners originating from the teleocidin biosynthetic pathway. Determining the production (e.g. the amount and/or activity of teleocidin A, and/or other congeners originating from the teleocidin biosynthetic pathway) can be carried out as disclosed elsewhere herein.

Steps a), b) and c) can be carried out as disclosed elsewhere herein.

The present invention further refers to a process for preparing clavulanic acid by using a microorganism, comprising the steps of: a) providing a microorganism being capable of producing clavulanic acid; b) testing said microorganism of step a) as to whether it is capable of producing clavulanic acid, but at the same time exhibits reduced or no production of teleocidin A, and/or other congeners originating from the teleocidin biosynthetic pathway, when compared to strain S. clavuligerus K4567, preferably with the production of clavulanic acid at least essentially remaining the same; c) cultivating said microorganism of step b), or its descendant to produce clavulanic acid, if the conditions in b) are fulfilled; and d) optionally isolating clavulanic acid.

Strain S. clavuligerus ATCC 27064, as available at present is an example of a strain that does not express teleocidin A; and

Strain S. clavuligerus K4567 is an example of a strain that exhibits teleocidin A expression. In a preferred embodiment, the testing in step b) comprises determining whether said microorganism produces teleocidin A, and/or other congeners originating from the teleocidin biosynthetic pathway. Determining whether said microorganism produces teleocidin A, and/or other congeners originating from the teleocidin biosynthetic pathway can be carried out by applying suitable methods that are known to a person skilled in the art, e.g. the methods as disclosed elsewhere herein.

Steps c) and d) can be carried out as disclosed elsewhere herein.

The present invention further refers to a method of testing the suitability of a strain of S. clavuligerus for being used in the production of clavulanic acid, comprising the steps of:

B)

Bi) cultivating the strain of A);

C)

Ci) testing the strain of A) for the presence or absence of genes encoding TleA, TleB, and/or TleC, preferably TleA, by detecting nucleic acids encoding TleA, TleB, and/or TleC, preferably wherein the step of detecting comprises or is a step of carrying out a polymerase chain reaction (PCR) that specifically amplifies nuclei acid sequences that are specific for genes encoding TleA, TleB; and/or TleC, preferably for genes encoding TleA;

Cii) determining that, if in step Ci) it is assessed that said strain does not comprise genes encoding TleA, TleB, and/or TleC, the strain of S. clavuligerus is suitable for being used in the production of clavulanic acid;

D)

Di) cultivating the strain of A);

Dii) assessing by using antibodies specific against TleA, TleB, and/or TleC whether the respective proteins are present or not; Diii) determining that, if in step Dii) it is assessed that said strain does not produce TleA, TleB, and/or TleC, preferably does not produce TleA, or at least reduced levels of TleA, TleB, and/or TleC, when compared to strain S. clavuligerus K4567, the strain of A) is suitable for being used in the production of clavulanic acid; or

E)

Ei) cultivating the strain of A);

The nuclei acid sequences that are specific for the gene encoding TleA, TleB, and TleC are depicted in SEQ. ID. NO: 1 (sequence encoding TleA), SEQ ID NO: 2 (sequence encoding TleB), and SEQ ID NO: 3 (sequence encoding TleC).

Detecting (determining) whether the strain of S. clavuligerus produces teleocidin A, and/or other congeners originating from the teleocidin biosynthetic pathway, can be carried out by any suitable method that is known to a person skilled in the art. Particularly, reference is made to the respective methods disclosed elsewhere herein.

Assessing by using antibodies, antibody-fragments or molecules comprising such antibodies or antibody-fragments, which are specifically binding to TleA, TleB, and/or TleC whether the respective proteins are present or not can be carried out bay any suitable method that is known to a person skilled in the art. Western blot, ELISA and other well-known immunological techniques may be used.

In step Ci), genomic DNA can be isolated from the S. clavuligerus strain to be tested and can be used as template for the PCR. It is also possible that direct PCR methods such as colony PCR methods are applied.

It is a routine task for a person skilled in the art to design suitable primers for amplifying the respective nucleic acid sequences. In a preferred embodiment, the PCR is carried out by using the primer sets disclosed in table 6. More preferred, the primer sets 7, 12, and 13 are used. However, in a preferred embodiment, the PCR is carried out by using the primer sets 7 (amplified region is the tleA gene; SEQ ID NOs: 19 and 20); 12 (amplified region is the tleC gene; SEQ ID NOs: 29 and 30); and 13 (amplified region is the tleB gene; SEQ ID NOs: 31 and 32).

Determining and applying suitable PCR conditions is a routine task for a person skilled in the art.

With regard to cultivating conditions and isolating methods, reference is made to the respective disclosure elsewhere herein.

The present invention further relates to a method of testing whether a culture broth that is used for culturing S. clavuligerus and producing clavulanic acid, has low toxicity, meaning contains no or only low amounts of teleocidin, and is thus suitable for being further processed, comprising the steps of: aa) detecting the presence or absence of teleocidin A in said culture broth; bb) determining that, if in step aa) no teleocidin A is present, said culture broth is suitable for being further processed.

Steps aa) and bb) can be carried out as disclosed elsewhere herein.

Further, the present invention refers to S. clavuligerus or its descendant, obtained by the steps of a) providing a microorganism being capable of producing clavulanic acid and teleocidin A (also referred to herein as Lyngbyatoxin A) and/or other congeners originating from the teleocidin biosynthetic pathway; b) modifying said microorganism in that

• the production of teleocidin A, and/or other congeners originating from the teleocidin biosynthetic pathway, is reduced or abolished compared to its unmodified form, but the production of clavulanic acid at least essentially remains the same; c) obtaining said modified microorganism.

The modifying step in step b) can be carried out as disclosed elsewhere herein. In particular, the modification is carried out as disclosed in any of items 5 to 12 herein. Finally, the present invention refers to a S. clavuligerus strain or its descendants, characterized in that it is capable of producing clavulanic acid, but not teleocidin A, and/or other congeners originating from the teleocidin biosynthetic pathway.

In a preferred embodiment, the S. clavuligerus strain or its descendants produces essentially at least the same amount of clavulanic acid, preferably at least 3%, more preferably at least 5%, 10% or 15% more of clavulanic, when compared to strain S. clavuligerus ATCC 27064 when applying respectively the same suitable conditions for producing clavulanic acid.

According to the present invention such a S. clavuligerus strain or its descendants can be beneficially used for producing clavulanic acid. Further useful according to the present invention is the use of defined S. clavuligerus strain or its descendants in preparing clavulanic acid, and then combine the thus obtained clavulanic acid with a b-lactam antibiotic to prepare a pharmaceutical dosage form comprising clavulanic acid and the b-lactam antibiotic.

The invention is further described by reference to the following examples. These examples are provided for illustration purposes and are not intended to be limiting.

EXAMPLES

EXAMPLE 1. Maintenance and general manipulation with S. clavuligerus

Media and growth conditions used for general manipulation and maintenance of S. clavuligerus are listed. Specific procedures are given at appropriate examples below. a) SNK-t medium

SNK-t medium is a liquid medium used for general cultivation of S. clavuligerus where CA production is not necessary. Said medium is used for example for obtaining S. clavuligerus biomass for purpose of DNA isolation, re-cultivation in selection procedures as well as a seed phase for testing purposes.

Table 2. : The composition of the SNK-t medium.

The pH was adjusted to 6.8 with using 5M HC1. Medium is sterilized at 121±2°C, 120±10 kPa for 15 minutes. After sterilization, 500ml sterilized and cooled solution of Maltose (DIFCO BD) - lOg/L was added to a cooled medium.

SNK-t medium was used with culture conditions as follows:

- Deep-well microtiter plate (square well): 500 pL of medium, inoculation with 2 % suspension of S. clavuligerus spores or, alternatively, another liquid culture or frozen culture. Incubation was carried out on a rotary shaker at 28 °C and 260 rpm (2.5 cm excenter) for 24-48 hours under aerobic conditions.

- 100 ml Erlenmeyer shake flask: 15 ml of medium, inoculation with 2 % suspension of S. clavuligerus spores or, alternatively, another liquid culture or frozen culture. Incubation was carried out on a rotary shaker at 28 °C and 260 rpm (2,5 cm excenter) for 24-48 hours under aerobic conditions.

- 250 ml shake flask: 50 ml of medium, inoculation with 2 % suspension of S. clavuligerus spores or, alternatively, another liquid culture or frozen culture. Incubation was carried out on a rotary shaker at 28 °C and 260 rpm (2.5 cm excenter) for 24-48 hours under aerobic conditions. b) KKA-2L medium

KKA-2L medium is a solid medium used as general medium for spore preparation, subcultivation of the strains, imposing and /or maintaining selection pressures, isolation of single colony derived strains etc.

Table 3. : The composition of the KKA-2L medium.

The pH is naturally -1.0, there is no pH adjustment needed prior to sterilization. Sterilization was performed at 121±2 °C, 220±10 kPa for 20 minutes b) ISP-2 medium

ISP-2 medium is a solid medium used for plating of liquid culture dilutions. Characteristically it allows faster growth of the S. clavuligerus colonies. The pH was adjusted to 7.0 with 1M NaOH prior to sterilization. Sterilization was performed at 121±2°C, 220±10 kPa for 20 minutes.

EXAMPLE 2. Identification of teleocidin genes by PCR

Presence of teleocidin genes was detected in the genome of the host microorganism by PCR. Isolated genomic DNA was used as PCR template (such as in example 5, 2. a) or was alternatively used for direct PCR methods (also known as colony PCR). For routine confirmation, we developed a quick freeze-thaw protocol for preparing “colony PCR” DNA template with S. clavuligerus (liquid culture or homogenized colony from agar plate can be used). Small amount of cultures in TE buffer (Tris-EDTA buffer) were frozen for at least 5 min, at -80 °C, followed by 5 min incubation time at 80 °C. Mixtures were centrifuged and 1-2 pL of the supernatant was used in PCR as the DNA template. Below is shown the list of primer pairs (Table 6), which were successfully used for detection of various parts of teleocidin gene cluster and its surrounding. The tests were positive both on isolated DNA and with colony PCR protocols.

A typical PCR protocol was as follows:

Reaction was performed using an Eppendorf Mastercycler Gradient thermocycler. The PCR reaction was carried out with PCR Extender System (5PREME) and the buffer provided by the manufacturer (lOx Tuning buffer with Magnesium) in the presence of 200 pM dNTP, 4 % DMSO, 2 pM of each primer, approximately 50 ng of template genomic DNA and 1 unit of enzyme in a final volume of 25 pL for 30 cycles.

The thermal profile started with denaturation step at 98 °C for 120 sec followed by 95 °C for 120 sec. The thermal cycle for the following 30 cycles was 95°C for 20 sec (denaturation step), 62 °C for 15 sec (annealing step), and 72 °C for 150 sec (extension step). The final elongation step was carried out at 72 °C for 7 min.

Table 6: PCR primer sets, useful for detection of various parts of teleocidin gene cluster and its flanking regions

Alternatively, Colony PCR was performed using the Mastercycler Vapo protect (Eppendorf). The PCR reaction was carried out with Q5® High-Fidelity DNA Polymerase (New England Biolabs) followed by the manufacturer protocol. Reaction mixture contained lx Q5 Reaction Buffer, 200 mM dNTP, 0.5 pM of each primer, IX Q5 High GC Enhancer, 0,5 unit of Q5 High- Fidelity DNA Polymerase, 2 pL of template genomic DNA and demineralized water to a final volume of 25 pL. The thermal profile started with denaturation step at 98 °C for 180 sec.

The thermal cycle for the following 35 cycles was 95°C for 20 sec (denaturation step), 62 °C for 20 sec (annealing step), and 72 °C for 150 sec (extension step). The final elongation step was carried out at 72 °C for 7 min. EXAMPLE 3. Analytical methods.

Analysis of teleocidins in S. clavuligerus cultures can be performed using several methods.

Although various chromatographic methods can be used, HPLC was used as preferred method.

Non-limiting examples of HPLC methods which were used and found suitable are: a. The fermentation broth was extracted with methanol 1:4 and centrifuged. Clear supernatant (5 pL) was injected onto a Phenomenex Kinetex C18 column (100 x 2.1 mm, 1.7 pm) with a flow of 0.4 mL min^'1, column temperature 40 °C and UV diode array multi -wavelength detection. Mobile phase A (MPA) was 0.1% (v/v) (v/v = volume per volume) formic acid in 1% acetonitrile, mobile phase B (MPB) was 0.1% (v/v) formic acid in 80% acetonitrile. The gradient profile for the method was started at 70 % MPB and after 1.2 min progressed linearly to 100 % MPB in 5.3 min, with 3 min hold time and final re-equilibration for 1.4 min. b. The fermentation broth was extracted with acetonitrile 1:4 and centrifuged. Clear supernatant (5 pL) was injected onto a Merck Purospher STAR RP-18e, (100 x 2.1mm, 2.0 pm) with a flow of 0.4 mL min^'1, column temperature 60 °C and UV diode array multiwavelength detection. Mobile phase A (MPA) was 0.1% (v/v) formic acid in 1% acetonitrile, mobile phase B (MPB) was 0.1% (v/v) formic acid in 80% acetonitrile. The gradient profile for the method progressed from 0 % to 7.6 % MPB in 1.2 min, then to 36,7 % MPB at 3.5 min and holding until 5.8 min after which the gradient progressed to 100 % MPB at 7 min with 5 min hold time and final re-equilibration for 2 min.

Detection can be achieved either by UV or MS detectors, in the latter case, direct injection may be used instead of chromatography. a. TSQ Quantum MS (Thermo Scientific) equipped with ESI source was used for detection. Positive ionization (source voltage 4 kV, vaporizer temperature 125 °C capillary temperature 300 °C, sheat gas 42 AU, auxiliary gas 5 AU (AU = arbitrary units) and full-scan monitoring with m/z range 70-500 was used. The compounds were detected primarily in protonated form [M+H]+; teleocidin A (lyngbyatoxin A), m/z = 438; teleocidin B, m/z = 452. b. HRMS was performed either after chromatography as described above, or by direct injection into Q Exactive MS instrument (Thermo Scientific) equipped with HESI ion source operating in positive mode with the same ESI settings as above. c. PDA UC absorption detector can be used either in full-range mode (e.g. 190 - 500 nM) or at specific wavelengths suitable for detection of teleocidin (e.g. 300 - 320 nM). d. UV detectors with monochromators can be used as well, preferably at wavelengths suitable for detection of teleocidin (e.g. 300 - 320 nM).

Clavulanic acid was analyzed in culture supernatants with a modified HPLC method (Agilent technologies, 2007), and quantified by comparison to authentic pure clavulanate lithium. Phosphoric aci d/water/ acetonitrile (0.05/75/25) was used as mobile phase on Zorbax SB-Aq column (Agilent, USA) with detection at 220 nm. Detailed methods for detection and quantification of clavulanic acid are describe in Agilent Technologies, 2007, "Compendium of HPLC Applications for Traditional Chinese Medicine and Chemical Drugs in China Pharmacopoeia." Agilent Technologies, March 1. 316. www. agil ent.com/chem . or in the U S. Pharmacopeia, USP29, USP monographs: Clavulanate Potassium (http://www.pharmacopeia.cn/v29240/usp29nf24s0 m 18000.html)

EXAMPLE 4. Isolation of teleocidin A

The fermentation broth was extracted with a mixture of n-butanol:acetone:diethyl ether (1:1.5:1). After evaporation of diethyl ether and acetone, the remaining extract formed 3 separate phases. The top n-butanol phase was separated and mixed with water. The azeotrope was evaporated to concentrate the material to ~20 g L^'1. This was subjected to preparative chromatography: 4 mL was injected onto a Thermo Syncronis Cl 8 column (250 x 21.2 mm, 5 pm, flow: 26 mL min-1, column temperature: 60 °C). Mobile phase A (MPA) was 1% acetonitrile and mobile phase B (MPB) was 80% acetonitrile. The method was isocratic for 12 min, followed with a gradient from 70% MPB to 100 % MPB in 13 min. A 10 min holding time (100% MPB) was completed with a final re-equilibration for 6 min. lOmL fractions were collected and analyzed. Fractions containing teleocidin A, were pooled and solvents evaporated. This material (~60 mg), was dissolved in acetonitrile and injected again for a polishing run. The purest fractions were pooled and solvents evaporated. 15 mg of this material was dissolved in 0.7 mL of CDC13 and characterized by 1H and 13C NMR (Bruker Advance III 500MHz). COSY, HSQC and HMBC was used to support assignation.

EXAMPLE 5. Construction of teleocidin deletion plasmids E Construction of pKONC plasmid

The plasmid pKONC was designed as a suicide plasmid for Streptomyces. Only E. colt replicative element is included in the plasmid (originating from pSET151) but no replicative element conferring replication in Streptomyces. Therefore upon transformation and exposure to selection pressure only those cells of S. clavuligerus form colonies, that have the plasmid integrated into the chromosome. Two selection markers are included into the plasmid: aac(3)IV gene conferring apramycin resistance, originating from pSET152, a well-known plasmid for use in Streptomyces (Kieser et al, 2000) and codA counterselection marker gene originating from pMG302M, producing a very toxic 5-fluoro-uracyl in presence of 5-fluoro-cytosine (Dubeau, et al ., 2009). codA gene was amplified using PCR amplification of pMG392M plasmid DNA using a Eppendorf Mastercycler Gradient thermocycler. The PCR reaction was carried out with PCR Extender System (5PRIME) and the buffer provided by the manufacturer (lOx tuning buffer with Magnesium) in the presence of 200 mM dNTP, 4% DMSO, 2 pM of each primer, approximately 50 ng of template DNA and 1 unit of enzyme in a final volume of 25 pL for 30 cycles.

The thermal profile started with denaturation step at 94 °C for 10 min. The thermal cycle for the following 30 cycles was 94 °C for 45 sec (denaturation step), 58 °C for 45 sec (annealing step), and 72 °C for 150 sec (extension step). The final elongation step was carried out at 72 °C for 7 min. Oligonucleotide primer pair used was: The PCR-amplified product was analyzed by agarose gel electrophoresis and its length agreed with the predicted length (1373 bp). The fragment was cloned into the pGEM-T easy PCR cloning vector (Promega) yielding plasmids pGEM/codA. The sequence analysis of the cloned PCR fragment confirmed its respective sequence as expected from the primer design procedure. aac(3)IV gene was amplified using PCR amplification of pSET152 plasmid DNA (Kieser et al., 2000) using an Eppendorf Mastercycler Gradient thermocycler. The PCR reaction was carried out with PCR Extender System (5PRIME) and the buffer provided by the manufacturer (lOx tuning buffer with Magnesium) in the presence of 200 mM dNTP, 4% DMSO, 2 pM of each primer, approximately 50 ng of template DNA and 1 unit of enzyme in a final volume of 25 pL for 30 cycles.

The thermal profile started with denaturation step at 94 °C for 10 min. The thermal cycle for the following 30 cycles was 94 °C for 45 sec (denaturation step), 58 °C for 45 sec (annealing step), and 72 °C for 150 sec (extension step). The final elongation step was carried out at 72 °C for 7 min. Oligonucleotide primer pair used was:

The PCR-amplified product was analyzed by agarose gel electrophoresis and it’s length agreed with the predicted length (1011 bp). The fragment was cloned into the pGEM-T easy PCR cloning vector (Promega) yielding plasmids pGEM/Apr2. The sequence analysis of the cloned PCR fragment confirmed its respective sequence as expected from the primer design procedure.

The plasmid pGEM/CodA (as described above) was cleaved using restriction endonucleases Ndel and Xbal (both Promega) according to instruction of the manufacturer. The mixture was resolved on the agarose gel electrophoresis gel. The resulting 1368 bp fragment was purified from the agarose gel. In parallel the plasmid pGEM/Apr2 was cleaved with the restriction endonucleases Ndel and Xbal (Both Promega) so that 3987 bp was isolated from the band cut out of an agarose electrophoresis band. The fragments were purified of the remaining agarose with help of Wizard® Gel and PCR Clean-up System (Promega).

The fragments were assembled in a DNA ligation reaction using T4 DNA Ligase (Promega) according to manufacturer’s instructions. The ligation mixture was used to transform competent E. coli JM109 (Promega) and transformed clones were selected based on their resistance to 50 pg/mL Ampicilin. The resulting clones were verified using restriction enzyme analysis using several distinct restriction endonucleases. The final construct was designated pGEM/codA_Apr2 with overall length of 5357 bp.

Plasmid pSET151 (Kieser et al., 2000) was than cleaved using restriction endonuclease EcoRI and Ndel (both Promega) according to instruction of the manufacturer but under conditions of partial clevage. Fragments were separated on agarose gel electrophoresis. 5897 bp fragment containing the larger part of the plasmid was purified of the remaining agarose with help of Wizard® Gel and PCR Clean-up System (Promega). In parallel the plasmid pGEM/coda_Apr2 was cleaved with the restriction endonucleases Ndel and EcoRI (both Promega) so that 2373bp fragment containing aac(3)IV and codA genes was isolated from the band cut out of an agarose electrophoresis band. The fragment was purified of the remaining agarose with help of Wizard® Gel and PCR Clean-up System (Promega).

The fragments were assembled in a DNA ligation reaction using T4 DNA Ligase (Promega) according to manufacturer’s instructions. The ligation mixture was used to transform competent E. coli JM109 (Promega) and transformed clones were selected based on their resistance to 50 pg/mL ampicillin. The resulting clones were verified using restriction analysis with several distinct restriction endonucleases. The final construct was designated pSET151/codA_Apr2 with overall length of 8270 bp.

Finally the plasmid pSET151/codA_Apr2 was cleaved using restriction endonuclease Dral (Promega) according to instruction of the manufacturer. Fragments were separated by agarose gel electrophoresis. 5428 bp fragment containing the larger part of the plasmid was purified of the remaining agarose with help of Wizard® Gel and PCR Clean-up System (Promega) and selfligated in a DNA ligation reaction using T4 DNA Ligase (Promega) according to manufacturer’s instructions. The ligation mixture was used to transform competent E. coli JM109 (Promega) and transformed clones were selected based on their resistance to 25 pg/mL Apramycin. The resulting clones were verified using restriction enzyme analysis using several distinct restriction endonucleases. The final construct was designated pKONC with overall length of 5428 bp which differs from pSET151/codA_Apr2 in loss of a fragment containing genes xylE, tsr and bla.

2. Construction of pKONC LynC, pKONC LynO and pKONC LynS deletion plasmids Three deletion strategies were used for inactivation of the teleocidin gene functions:

- LynS strategy deleting the whole tleA gene (NRPS) and small parts of tleB and tleC genes.

LynC strategy deleting the complete teleocidin gene cluster, including tleA, tleB and tleC

- LynO strategy deleting the distinct 138 kb genomic region which contains the teleocidin biosynthetic cluster.

These strategies were done by selection of the homologous fragments flanking the deletion sites. The 5’ and 3’ homologous fragments together form an editing template. Graphic representations of the locations of homologous flanking fragments are depicted in figure 6. a) Preparation of Streptomyces clavuligerus ATCC 27064 (K4567) genomic DNA

Spores of Streptomyces clavuligerus K4567 were used to inoculate 50 ml of SNK-t medium in a 250 ml Erlenmeyer flask. Cultures were grown for 24 hours at 28 °C with shaking (260 rpm). Mycelium was recovered by centrifugation and genomic DNA was prepared using Wizard® Genomic DNA Purification Kit (Promega) according to the instructions of the kit manufacturer. DNA was resuspended in 100 pL TE buffer (Sambrook, and Russell, 2000). b) PCR amplification of 5’ and 3’ flanking fragments (editing templates) for pKONC LynC and pKONC LynO 5’ and 3’ flanking fragments for pKONC LynC and pKONC LynO deletion plasmids were amplified using S. clavuligerus K4567 genomic DNA as a template. Reaction was performed using an Eppendorf Mastercycler Gradient termocycler. The PCR reaction was carried out with PCR Extender System (5PRIME) and the buffer provided by the manufacturer (lOx Tuning buffer with Magnesium) in the presence of 200 mM dNTP, 4 % DMSO, 2 pM of each primer, approximately 50 ng of template genomic DNA and 1 unit of enzyme in a final volume of 25 pL for 30 cycles.

The thermal profile was started with a denaturation step at 98 °C for 120 sec followed by 95 °C for 120 sec. The thermal cycle for the following 30 cycles was 95°C for 20 sec (denaturation step), 62 °C for 15 sec (annealing step), and 72 °C for 150 sec (extension step). The final elongation step was carried out at 72 °C for 7 min.

For the amplification of the 5’ flanking fragment of pKONC LynC deletion plasmid, the following oligonucleotide primer pair was used:

SEQ ID NO: 37 lynO L F EcoRI SEQ ID NO: 38 lynC L R BamHI

For the amplification of the 3’ flanking fragment of pKONC LynC deletion plasmid, the following oligonucleotide primer pair was used:

SEQ ID NO: 39 lynC_R_F_BamHI SEQ ID NO: 40 lynC_R_R_HindIII

For the amplification of the 5’ flanking fragment of pKONC LynO deletion plasmid, the following oligonucleotide primer pair was used:

SEQ ID NO: 41 lynO L F EcoRI SEQ ID NO: 42 lynO_L_R_Bamffl For the amplification of the 3’ flanking fragment of pKONC LynO deletion plasmid, the following oligonucleotide primer pair was used:

SEQ ID NO: 43 lynO R F B amHI

SEQ ID NO: 44 lynO R R Hindlll

The PCR-amplified products obtained were analyzed by agarose gel electrophoresis. They were found to agree with their corresponding predicted lengths.

The PCR fragments were cloned into the pGEM-T easy cloning vector (Promega) yielding plasmids containing 3’ and 5’ flanking fragments (editing template). The sequence analysis of the cloned PCR fragments confirmed its respective sequence as expected from the primer design procedure. c) DNA synthesis of 5’ and 3’ flanking fragments (editing templates) for pKONC_LynS

5’ and 3’ flanking fragments (editing template) for pKONC LynS were synthetized by Genewiz, Inc. The fragments are derived from sequence between genomic positions 560501 and 561936 for the 5’ and 549866 and 551315 for the 3’ flanking fragments (relative to the F613-1 genome, PRJNA329150; NZ_CP016559), respectively. For cloning purposes EcoRI and Hindlll restriction sites were added on 5’ and 3’ end of the synthetic fragment. d) Final assembly of pKONC LynO, pKONC LynC, pKONC LynS deletion plasmids

The plasmid pKONC (as described above) was cleaved using restriction endonucleases EcoRI and Hindlll (both Promega) according to instruction of the manufacturer. The mixture was separated on the agarose gel electrophoresis gel. The resulting 5377 bp fragment was purified from the agarose gel. In parallel 5’ and 3’ flanking fragments (editing template) for pKONC LynC and pKONC LynO were excised from the pGEM plasmids using restriction endonucleases (Promega). EcoRI and BamHI for 5’ flanking fragments, BamHI and Hindlll for 3’ flanking fragments.

For pKONC LynS fragment 5’ and 3’ flanking fragments were excised together from synthetic fragment using EcoRI and Hindlll restriction endonucleases. Excised fragments were isolated from the band cut out of an agarose electrophoresis and purified using Wizard® Gel and PCR Clean-up System (Promega).

The fragments were assembled in a single step in a DNA ligation reaction using T4 DNA Ligase (Promega) according to manufacturer’s instructions. The ligation mixture was used to transform competent E. coli JM109 (Promega) and transformed clones were selected based on their resistance to 25 pg/mL Apramycin. The resulting clones were verified using restriction enzyme analysis using several distinct restriction endonucleases. The final constructs were designated pKONC LynC with overall length of 8780 bp and containing editing template according to SEQ. ID. # 4, pKONC LynO with overall length of 9038 bp and containing editing template according to SEQ. ID. # 5, and pKONC LynS with overall length of 8288 bp and containing editing template according to SEQ. ID. # 6. The plasmid map of the design for pKONC is illustrated in Fig. 7.

EXAMPLE 5. Introduction of LynO. LynC and LynS deletion strategies into S. clavuliserus genome

Streptomyces clavuligerus K2731 and K549 are descendants of the teleoci din-positive genotype of S. clavuligerus ATCC 27064 (e.g. S. clavuligerus ATCC 27064 (K4567), having an improved productivity of clavulanic acid and preserved teleocidin A and teleocidin B production. In the following examples these strains served as reference or parent (wild-type) strains for genetic modifications.

1. Conjugal transfer of deletion plasmids to S. clavuliserus K2731 and K549

Plasmid constructs pKONC LynO, pKONC LynC, pKONC LynS were introduced by transformation into electro-competent A. coli strain ET12567 containing the conjugation helper plasmid pUZ8002. The plasmid pUZ8002 contains all the necessary genes for construction of conjugative pili, however it lacks the origin of transfer and, thus, remains in the host cell. Conjugation procedure was done as described in Kieser et al., 2000, using S. clavuligerus K2731 spores. KKA-2L agar plates were used for selection of exoconjugants and apramycin (Sigma) was used for selection at 60 μg/mL. Transformation with pKONC deletion plasmids typically yielded 10-50 exconjugants on apramycin selection agar plates. As the pKONC deletion plasmids are unable to replicate in S. clavuligerus , the exoconjugants must have had the plasmid integrated into the genome. Only the top part of exoconjugant colonies were transferred in a dense spread to KKA-2L medium supplemented with 60 pg/rnL apramycin (KKA-2L Apr) and grown at 25°C for 10 days. Exoconjugants were tested by a PCR method, described later in detail, and were shown that they all contain a single crossover genotype as expected.

2. Selection of deletion mutants

Selection for stable secondary recombinant strains of S. clavuligerus with deletions in LynO, LynC and LynS genomic regions was achieved by counterselection to CodA (Cytosine- deaminase) marker gene with fluorocytosine (Sigma) and therefore loss of all genetic markers together with the pKONC plasmid backbone. The approach is known by skilled persons as scarless gene deletion. (Dubeau et al., 2009)

Spores of S. clavuligerus exconjugants with integrated pKONC LynO, pKONC LynC, and pKONC_LynS (grown at 25°C on KKA-2L medium supplemented with 60 pg/mL Apramycin; KKA-2L Apr) were inoculated into liquid SNK-t medium with Apramycin (60 pg/ml) and grown in deep-well microtiter plates at 28°C and 260 rpm to produce a dense mycelium culture. After 24h, this culture was sub-cultivated into parallel wells of fresh deep-well microtiter plates, one containing SNK-t medium supplemented with 60 pg/L apramycin and the other containing SNK-t medium supplemented with 100 pg/L 5-fluoro-cytosine (FC) medium and grown at 25°C and 260 rpm until growth appeared in the wells containing 5-fluoro-cytosine. The cultures were sub -cultivated repeatedly, as soon as growth appeared in the SNK-t FC containing wells into parallel wells in a manner described above (the 1% inoculum used, was always taken from the preceding generation in SNK-t FC wells).

The procedure was repeated for at least five and for up to as many as fifteen cycles of selection, until growth was no longer appearing in the wells with SNK-t supplemented with apramycin. The cultures were then plated onto ISP-2 medium containing 100 pg/mL 5-fluoro-cytosine in appropriate dilutions yielding approximately 100 colonies per plate. After 5-10 days, the colonies were spread in parallel onto KKA-2L and KKA-2L Apr agar plates. A large majority of the transferred strains showed growth only on KKA-2L but not on KKA-2L Apr agar plates, indicating loss of genetic markers from the genome and thereby successful secondary recombination. Approximately 30 independently obtained secondary recombinants were isolated for each parent strain and each deletion strategy.

3. Confirmation of LynO, LynC, and LynS deletion by genetic characterization PCR was used as a method for confirmation of correct integration and homologous recombination events during the selection pressure cycles. A set of confirmation oligonucleotide primers were designed which distinguish between wild type, single crossover situation and the final double recombination situation in the LynO, LynC, and LynS locus in the S. clavuligerus genome. The expected lengths of the confirmation PCR products are listed in Tables 8, 9 and 10.

DNA templates were prepared in accordance to the “colony PCR” approach. Specifically, the spores of the tested strains were cultivated in SNK-t medium at 25°C and 260 rpm for 48h, followed by separation of the mycelium by filtration, and partial permeabilization of the mycelium using resuspension in TE buffer (Sambrook, and Russell, 2000), supplemented with 2.5 pg/mL RNase (Roche, 500 pg/mL stock solution). The mycelium was filtered off and the liquid phase was used as a DNA template for the PCR reactions. The PCR reaction was carried out with PCR Extender System (5PRIME) on an Eppendorf Mastercycler Gradient thermocycler with the buffer provided by the manufacturer (lOx Tuning buffer with Magnesium) in the presence of 200 pM dNTP, 4% DMSO, 2 pM of each primer, approximately 1 pL of DNA template and 0.5 unit of polymerase enzyme in a final volume of 12.5 pL for 30 cycles.

The thermal profile started with denaturation step at 98 °C for 3 min followed by 95 °C for 120 sec. The thermal cycle for the following 30 cycles was 95 °C for 20 sec (denaturation step), 61 °C for 15 sec (annealing step), and 72 °C for 3 min (extension step). The final elongation step was carried out at 72°C for 7 min. PCR products were analyses on agarose gel electrophoresis determining presence and length of PCR products. The primer sequences used for confirmation of LynO deletion in S. clavuligerus and the expected lengths of PCR products are listed below.

Table 8: The expected lengths of the confirmation PCR products for LynO deletion

The primer sequences used for confirmation of LynO deletion in S. clavuligerus and the expected lengths of PCR products are listed below.

Table 9: The expected lenghts of the confirmation PCR products for LynC deletion

The primer sequences used for confirmation of LynC deletion in S. clavuligerus and the expected lengths of PCR products are listed below.

Table 10: The expected lengths of the confirmation PCR products for LynC deletion

5 strains with LynO deletion, 4 strains with LynC deletion and 6 strains with LynS deletion were confirmed for S. clavuligerus K2731 as a parent strain. When using S. clavuligerus K549 as a parent strain, 10 strains with LynO deletion, 39 strains with LynC deletion and 28 strains with LynS deletion were confirmed.

These strains were additionally tested for absence of genetic marker cytosine deaminase and marker for apramycin resistance using PCR and all were found negative. Strains which reverted to the wild type genotype were discarded. All deletion strains have shown absence of teleocidin production.

EXAMPLE 6. Shake-flask testing of the deletion strains originating from S. clavuligerus K549

General characterization of the strains

The S. clavuligerus K549 A LynO, S. clavuligerus K549 A LynC and S. clavuligerus K549ALynS strains were first evaluated by morphological apearance on several different agar media. From the PCR confirmed deletion mutants, those with most similar appearance as compared to the respective parent strain (K549) were selected for further testing.

Growth conditions media and sampling methods Spores of the tested strains obtained from 1 square cm of the confluent KKA-2L agar plate culture were used as inoculum for the seed phase. The seed phase was cultivated on a rotary shaker in 250 mL shake flasks containing SNK-t medium at 28 °C and 260 rpm (2.5 cm excenter). After 40-48 h the culture was ready for transfer to the production medium CM (Ortiz, et al ., 2007). The production medium CM (Ortiz, et al ., 2007), 15 mL in 100 mL shake flask, was inoculated with 300 mΐ of the seed medium culture and incubated on a rotary shaker at 25 °C and 260 rpm (2.5 cm excenter) for 6 days.

Table 11.: The composition of the CM medium

VIOPS = 3-(A^f-morpholino)propanesulfonic acid

The pH was adjusted to 6.8 with 5M NaOH prior to sterilization. Sterilization was performed at 121±2°C, 220±10 kPa for 15 minutes.

A large number of parallel shake flasks were inoculated so that each one flask was discarded after sampling while others were left incubating until the next sampling and so on.

Results

Cultures were analyzed with HPLC and LC-MS, periodically during a period from 24 to 120 hours of cultivation. Comparison of the shake flask cultures of S. clavuligerus K549 A LynO, S. clavuligerus K549 A LynC and S. clavuligerus K549 ALynS strains with the respective reference strain K549 (teleocidin-producing strain) showed complete absence of teleocidin production in all deletion mutants, regardless of the length of the deleted region (Fig. 8: LC-MS chromatograms). The same result was obtained with the LynS, LynO and LynC deletion mutants originating from S. clavuligerus K2731. Fig. 8 shows LC-MS chromatograms of the cultures of (A) S. clavuligerus K549, S. clavuligerus K549 ALynS, S. clavuligerus K549 ALynC and S. clavuligerus K549 ALynO, and (B) S. clavuligerus K2731, S. clavuligerus K2731 ALynS, S. clavuligerus K2731 ALynC and S. clavuligerus K2731 ALynO, after 96h of cultivation. LC-MS ESI chromatograms, with full scan in the m/z range 200-500.

Production of clavulanic acid by cultures of S. clavuligerus parent strain K549 and deletion mutants K549 ALynS, K549 ALynO and K549 ALynC showed no significant difference in clavulanic acid productivity. If there was any difference, it was in favor of the deletion strains regarding a very slightly higher productivity of CA (Figure 9). The productivity of clavulanic acid is shown as relative amount of clavulanic acid given as % wherein the highest amounts produced by wild type strain (S. clavuligerus parent strain) are defined as 100% (Table 12).

Table 12. : Relative amounts of clavulanic acid given as % wherein the highest amounts produced by the parent strain (S. clavuligerus K549) is defined as 100%.

EXAMPLE 7. Shake-flask testing of the deletion strains originating from S. clavuligerus K549 and K2731 in industrial media

S. clavuligerus K2731 ALynO, S. clavuligerus K2731 ALynS and S. clavuligerus K549 ALynC deletion mutants were tested against their respective reference strains on an undisclosed high- productivity industrial medium. The production medium (15 mL in 100 mL shake flask), was inoculated with 300 mΐ of the seed medium culture (as in previous example) and incubated on a rotary shaker at 25 °C and 260 rpm (2.5 cm excenter) for 6 days. A large number of parallel shake flasks were inoculated so that each one flask was discarded after sampling while others were left incubating until the next sampling and so on. At least 9 independent production cultures were sampled and analyzed for each culture tested at each time point. Cultures were analyzed with HPLC and LC-MS, periodically from 74 to 144 hours of cultivation.

Table 13: Relative amounts of clavulanic acid given as % wherein the highest amounts produced by the parent strain (S. clavuligerus K2731) is defined as 100%. At least 9 independent experiments are included in the calculations.

Comparison of the deletion strains S. clavuligerus K2731 A LynO, S. clavuligerus K2731 A LynS with their respective parent strain K2731 (teleocidin-producing strain) showed no significant difference in productivity. Relative normalized values for the above experiment are shown in Table 13 and Figure 10.

Production of clavulanic acid by cultures of deletion mutant S. clavuligerus K549 A LynC showed no significant difference in clavulanic acid productivity, compared to its parent K549 (Figure 11). The productivity of clavulanic acid is shown as relative amount of clavulanic acid given as % wherein the highest amount produced by wild type strain (S. clavuligerus parent strain K549, the respective reference strain) is defined as 100% (Table 14).

Table 14: Relative amounts of clavulanic acid given as % wherein the highest amount produced by the parent strain (S. clavuligerus K549) is defined as 100%. At least 9 independent experiments are included in the calculations.

DESCRIPTION OF THE FIGURES

Fig. 1 shows the molecular structure of clavulanic acid.

Fig. 2 shows whole genome alignments indicating the presence of extra 138kb region.

Fig. 3 illustrates the genetic organization of the S. clavuligerus teleocidin biosynthetic gene cluster.

Fig. 4 shows the biosynthesis and molecular structure of teleocidin A and teleocidin B.

Fig. 5 shows an LC-UV and LC-MS analysis of cultures of S. clavuligerus ATCC 27064 and S. clavuligerus K4567. In comparison, authentic sample of teleocidin A is also shown.

Fig. 6 is a schematic representation of positions of pKONC LynC, pKONC LynO and pKONC LynS homologous flanking fragments (editing templates) in S. clavuligerus genome. Fig. 7 illustrates the genetic map of the pKONC LynC, pKONC LynO and pKONC LynS plasmids for disruption of teleocidin biosynthesis.

Fig. 8 shows LC-MS chromatograms of the cultures of (A) S. clavuligerus K549, S. clavuligerus K549 ALynS, S. clavuligerus K549 ALynC and S. clavuligerus K549 ALynO, and (B) S. clavuligerus K2731, S. clavuligerus K2731 ALynS, S. clavuligerus K2731 ALynC and S. clavuligerus K2731 ALynO, after 96h of cultivation. LC-MS ESI chromatograms, with full scan in the m/z range 200-500.

Fig. 9 shows the production of clavulanic acid by cultures of S. clavuligerus K549 (parent strain, empty squares) compared to: (A) S. clavuligerus K549 ALynS (solid squares), (B) S. clavuligerus K549 ALynO (solid diamonds) and (C) S. clavuligerus K549 ALynC (solid circles) as a function of cultivation time.

Fig. 10 shows the production of clavulanic acid by cultures of S. clavuligerus K2731 (parent strain, empty squares) compared to: (A) S. clavuligerus K2731 ALynS (solid squares), (B) S. clavuligerus K2731 ALynO (solid diamonds) as a function of cultivation time.

Fig. 11 shows the production of clavulanic acid by cultures of S. clavuligerus K549 (parent strain, empty squares) compared to S. clavuligerus K546 ALynC (solid circles). REFERENCES

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Patents cited:

EP06125246, W00005397, US6100052, WO 9739137

Claims

1. A process for preparing clavulanic acid by using a microorganism, comprising the steps of: a) providing a microorganism being capable of producing clavulanic acid and teleocidin A (also referred to herein as Lyngbyatoxin A) and/or other congeners originating from the teleocidin biosynthetic pathway; b) modifying said microorganism of step a) in that

2. The process of the preceding claim, wherein the extent of reduction of teleocidin A and/or other congeners originating from the teleocidin biosynthetic pathway, is at least 50%; preferably, the extent of reduction is at least 60%, at least 70%, more preferably at least 80% or even more preferably at least 90%, or at least 95%.

3. The process according to any of the preceding claims, wherein the microorganism of step a) belongs to the genus Streptomyces, preferably the microorganism is selected from the group consisting of Streptomyces clavuligerus, preferably S. clavuligerus ATCC 27064, S. clavuligerus NRRL 3585, or S. clavuligerus K4567; Streptomyces katsurahamanus, preferably S. katsurahamanus IFO 13716 FERM 3994; Streptomyces jumonjinensis; and Streptomyces sp. P6621 FERM 2804; or the descendants thereof; more preferably, the microorganism is selected from the group consisting of S. clavuligerus ATCC 27064, S. clavuligerus K4567, and S. clavuligerus NRRL 3585, or the descendants thereof, and most preferred, the microorganism is S. clavuligerus K4567, or the descendants thereof.

4. The process according to any of the preceding claims, wherein the modified microorganism of step b) is a result of any one of

(2) modifying or deleting the nucleic acid of said microorganism encoding protein(s) of the teleocidin biosynthetic pathway, preferably one or more proteins selected from the group consisting of TleA (a non-ribosomal peptide synthetase (NRPS)), TleB, and TleC, further preferred TleA, respectively to the effect that the production of teleocidin A, and/or other congeners originating from the teleocidin biosynthetic pathway, is reduced, but the production of clavulanic acid at least essentially remains the same.

5. The process according to the previous claim, wherein

(1) modifying protein(s) of the teleocidin biosynthetic pathway, preferably one or more proteins selected from the group consisting of TleA, TleB, and TleC, further preferred TleA, comprises applying a component that partially or completely inhibits the protein activity (function); and

(2) modifying or deleting the nucleic acid of one or more genes encoding protein(s) of the teleocidin biosynthetic pathway comprises introducing a mutation into said nucleic acid.

6. The process according to claim 5, wherein in (2) modifying or deleting the nucleic acid of the gene(s) encoding said protein(s) comprises introducing mutation into the nucleic acid of the gene(s) encoding said protein(s) to that it cannot express, or that it can only express reduced levels, or inactive or less active variants of the protein(s), preferably wherein introducing a mutation comprises deletion, insertion, substitution, and/or point mutation.

7. Process for the preparation of an intermediate product or final pharmaceutical dosage form comprising clavulanic acid or salts or derivatives thereof, comprising the following steps: a) preparing clavulanic acid or salts or derivatives thereof by applying the process of any of the preceding claims; b) combining said clavulanic acid or salts or derivatives with excipients.

8. A process for preparing clavulanic acid by using a microorganism, comprising the steps of: a) providing a modified S. clavuligerus strain being capable of producing clavulanic acid,

- wherein said modified S. clavuligerus strain is producing clavulanic acid at least essentially in the same quantity, if compared to the production of clavulanic acid of a respective non-modified S. clavuligerus reference strain, and

- wherein said modified S. clavuligerus strain exhibits reduced or no production of teleocidin A and/or other congeners originating from the teleocidin biosynthetic pathway, if compared to the production of teleocidin A and/or other congeners from the teleocidin biosynthetic pathway of said respective non-modified S. clavuligerus reference strain, wherein the step of providing includes a step of selecting said modified S. clavuligerus strain out of a mixture of S. clavuligerus strains, wherein a non- selected S. clavuligerus strain represents said reference strain_^ b) cultivating said microorganism of step a) or its descendant to produce clavulanic acid; and c) optionally isolating clavulanic acid.

9. A process for preparing clavulanic acid by using a microorganism, comprising the steps of: a) providing a microorganism being capable of producing clavulanic acid; b) testing said microorganism of step a) as to whether it is capable of producing clavulanic acid, but at the same time exhibits reduced or no production of teleocidin A, and/or other congeners originating from the teleocidin biosynthetic pathway, when compared to strain S. clavuligerus K4567; c) cultivating said microorganism of step b), or its descendant to produce clavulanic acid, if the conditions in b) are fulfilled; and d) optionally isolating clavulanic acid.

10. Method of testing the suitability of a strain of S. clavuligerus for being used in the production of clavulanic acid, comprising the steps of:

B)

Bi) cultivating the strain of A);

Bii) detecting by using suitable means if the strain of S. clavuligerus produces teleocidin A, and/or other congeners originating from the teleocidin biosynthetic pathway; and

Biii) determining that, if in step ii) it is assessed that said strain does not produce teleocidin A or at least reduced levels of teleocidin A, and/or other congeners originating from the teleocidin biosynthetic pathway, when compared to strain S. clavuligerus K4567, the strain of S. clavuligerus is suitable for being used in the production of clavulanic acid; or

C)

Ci) testing the strain of A) for the presence or absence of genes encoding TleA, TleB, and/or TleC by detecting nucleic acids encoding TleA, TleB; and/or TleC;

Cii) determining that, if in step Ci) it is assessed that said strain does not comprise genes encoding TleA, TleB, and/or TleC, the strain of A) is suitable for being used in the production of clavulanic acid; or

D)

Di) cultivating the strain of A);

Dii) assessing by using antibodies or antibody fragments or molecules comprising antibodies or antibody fragments specific against TleA, TleB, and/or TleC whether the respective proteins are present or not;

Diii) determining that, if in step Dii) it is assessed that said strain does not produce TleA, TleB, and/or TleC, or at least reduced levels of TleA, TleB, and/or TleC, when compared to strain S. clavuligerus K4567, the strain of S. clavuligerus is suitable for being used in the production of clavulanic acid.

11. The method of claim 10, wherein in Ci), genomic DNA isolated from the S. clavuligerus strain to be tested can be used as template, or wherein direct PCR methods such as colony PCR methods can be used.

12. Method of testing whether a culture broth that is used for culturing S. clavuligerus and producing clavulanic acid, has low toxicity and is thus suitable for being further processed, comprising the steps of: aa) detecting the presence or absence of teleocidin A in said culture broth; bb) determining that, if in step aa) no teleocidin A is detected, said culture broth is suitable for being further processed.

13. S. clavuligerus or its descendant, obtained by the steps of a) providing a microorganism being capable of producing clavulanic acid and teleocidin A (also referred to herein as Lyngbyatoxin A) and/or other congeners originating from the teleocidin biosynthetic pathway; b) modifying said microorganism in that

• the production of teleocidin A, and/or other congeners originating from the teleocidin biosynthetic pathway, is reduced or abolished compared to the unmodified form of the microorganism, but the production of clavulanic acid at least essentially remains the same; c) obtaining said modified microorganism.

14. A S. clavuligerus strain or its descendants, characterized in that it is capable of producing clavulanic acid, but not teleocidin A, and/or other congeners originating from the teleocidin biosynthetic pathway.

15. The S. clavuligerus strain or its descendants of claim 14, characterized in that said strain produces essentially at least the same amount of clavulanic acid, preferably at least 3%, more preferably at least 5%, 10% or 15% more of clavulanic, when compared to strain ATCC 27064 when applying respectively the same suitable conditions for producing clavulanic acid.

16. Use of the S. clavuligerus strain or its descendants as defined in claim 14 or 15 for producing clavulanic acid.

17. A process for the preparation of a pharmaceutical dosage form comprising clavulanic acid and a b-lactam antibiotic, wherein the process comprises preparing the clavulanic acid by using a method as defined in any of claims 1 to 6, 8, and 9, or by using the S. clavuligerus strain or its descendants as defined in claim 16, and a step of combining clavulanic acid and b-lactam antibiotic to obtain said pharmaceutical dosage form.