Process for clavulanic acid production

Abstract

Novel bacterial genes, microorganisms and processes for improving the manufacture of 5R clavams, eg. clavulanic acid.

1 . DNA comprising one or more genes specific for 5S clavam biosynthesis in  S. clavuligerus  and which is not essential for 5R clavam biosynthesis.  
     
     
         2 . DNA according to  claim 1  as identified in FIG. 1 (SEQ ID No. 1).  
     
     
         3 . DNA according to  claim 1  having the sequence or substantially the sequence shown in FIG. 1 as orfup3, orfup2, orfup1, orfdwn 1, orfdwn2 or orfdwn3 (SEQ ID Nos. 2-7).  
     
     
         4 . DNA according to  claim 1  having the sequence or substantially the sequence shown in FIG. 1 as orfup1 (SEQ ID No. 4).  
     
     
         5 . DNA which hybridises under conditions of high stringency with the DNA of any one of  claims 1  to  4 .  
     
     
         6 . A vector comprising the DNA of  claim 1  in which one or more of the genes specific for 5S clavam biosynthesis has been disrupted or otherwise made defective.  
     
     
         7 . A vector according to  claim 6  containing one or more defective genes which is pCEC060, pCEC061, pCEC056 or pCEC057.  
     
     
         8 . A vector according to  claim 7  which is pCEC061.  
     
     
         9 . A host containing the vector of  claim 6 .  
     
     
         10 . A host according to  claim 9  which is capable of producing raised levels of clavulanic acid.  
     
     
         11 . A host according to  claim 9  which is capable of producing low or no levels of 5S clavam.  
     
     
         12 . A host according to  claim 9  which is  S. clavuligerus.    
     
     
         13 .  S. clavuligerus  comprising DNA corresponding to an open reading frame flanking cas1 which DNA has been disrupted or otherwise made defective.  
     
     
         14 .  S. clavuligerus  according to  claim 13  wherein the open reading frame is selected from the group consisting of orfup3, orfup2, orfup1, orfdwn1, orfdwn2 and orfdwn3.  
     
     
         15 . A process for improving 5R clavam production in a suitable microorganism comprising manipulation of DNA as defined in any 1 and its inclusion in the said microorganism.  
     
     
         16 . A process according to  claim 15  wherein said suitable microorganism is  S. clavuligerus.    
     
     
         17 . A process for improving 5R clavam production in  S. clavuligerus  comprising disrupting or otherwise making defective DNA regions flanking cas1.  
     
     
         18 . A process according to  claim 15  wherein said DNA corresponds to open reading frames selected from the group consisting of orfup3, orfup2, orfup1, orfdwn1, orfdwn2 and orfdwn3.  
     
     
         19 . A process according to  claim 15  wherein said DNA corresponds to open reading frame orfup1.  
     
     
         20 . A process according to  claim 15  wherein said 5R clavam is clavulanic acid.  
     
     
         21 . A process for the identification of a microorganism suitable for high 5R clavam production comprising a preliminary screening for microorganisms with low or no 5S clavam production.  
     
     
         22 . A process according to  claim 21  wherein the microorganism is  S. clavuligerus.    
     
     
         23 . A process according to  claim 22  wherein the 5R clavam is clavulanic acid.  
     
     
         24 . A process according to  claim 21  wherein one or more genes specific for the production of 5S clavams is defective.  
     
     
         25 . A microorganism which is capable of 5R clavam production and low or no 5S clavam production obtainable by the process of  claim 15 .  
     
     
         26 . A microorganism obtainable by the process of  claim 25  which is capable of producing clavulanic acid but which does not produce clavam-2-carboxylate and/or 2-hydroxymethylclavam.  
     
     
         27 . A microorganism obtained by the process of  claim 15  which is strain 56-1A, 56-3A, 57-2B, 57-1C, 60-1A, 60-2A, 60-3A, 61-1A, 61-2A, 61-3A or 61-4A.  
     
     
         28 . Clavulanic acid obtainable by the fermentation of a microrganism as defined in  claim 25 .  
     
     
         29 . Clavulanic acid according to  claim 28  which is free of clavam-2-carboxylate.  
     
     
         30 . Clavulanic acid according to  claim 28  in the form of its potassium salt.  
     
     
         31 . Clavulanic acid which is free of any 5S clavam.  
     
     
         32 . Clavulanic acid which is free of any clavam-2-carboxylate.  
     
     
         33 . A composition comprising potassium clavulanate according to  claim 30  in combination with a beta-lactam antibiotic.  
     
     
         34 . A composition according to  claim 33  in which the beta-lactam antiobiotic is amoxycillin.  
     
     
         35 . A process for the preparation of a composition comprising potassium clavulanate and amoxycillin which process comprises producing clavulanic acid from a microorganism according to  claim 25  and thereafter converting it to the potassium salt and combining the potassium salt with amoxycillin.