Field of the invention

The present invention relates to mutant expandase enzymes, polynucleotides encoding such enzymes, to microorganisms transformed with said polynucleotides encoding said mutant expandase enzymes and with the use of such mutant expandase enzymes or such microorganisms in the ring expansion of 5-carboxypentanoyl-6- aminopenicillanic acid (adipyl-6-APA = ad-6-APA).

Background of the invention

Beta-lactam antibiotics constitute the most important group of antibiotic compounds with a long history of clinical use. Among this group, the prominent ones are the penicillins and cephalosporins. Penicillins are naturally produced by various filamentous fungi such as Penicillium (e.g. P. chrysogenum). Cephalosporins are naturally produced by various microorganisms such as Acremonium (e.g. A. chrysogenum) and Streptomyces (e.g. Streptomyces clavuligerus)

As a result of classical strain improvement techniques, the production levels of the antibiotics in P. chrysogenum and A. chrysogenum have increased remarkably over the past decades. With the increasing knowledge of the biosynthetic pathways leading to penicillins and cephalosporins, and the advent of recombinant DNA technology, new tools for the improvement of production strains have become available.

Most enzymes involved in β-lactam biosynthesis have been identified and their corresponding genes have been cloned, as can be found in lngolia and Queener, Med Res Rev (1989) 9:245-264 (biosynthesis route and enzymes), and Aharonowitz, Cohen, and Martin, Ann Rev Microbiol (1992) 46:461 -495 (gene cloning). The first two steps in the biosynthesis of penicillin in P. chrysogenum axe the condensation of the three amino acids L-5-amino-5-carboxypentanoic acid (L-α- aminoadipic acid) (A), L-cystein (C) and L-valine (V) into the tripeptide LLD-ACV, followed by cyclization of this tripeptide to form isopenicillin N. This compound contains the typical β-lactam structure.

The third step involves the replacement of the hydrophilic side chain of L-5-

amino-5-carboxypentanoic acid by a hydrophobic side chain by the action of the enzyme acyltransferase (AT).

In EP-A-0448180 has been described that the enzymatic exchange reaction mediated by AT takes place inside a cellular organelle, the microbody. The observation that substantial quantities of deacetoxycephalosporin C (DAOC) can be formed by non- precursed P. chrysogenum transformants expressing deacetoxycephalosporin C synthase (EC 1 .14.20.1 - DAOCS, further indicated herein as expandase) implies the presence of significant amounts of penicillin N, the natural substrate for expandase, in P. chrysogenum (Alvi et al., J Antibiot (1995) 48:338-340). However, the D-α-amino- adipyl side chains of DAOC cannot be easily removed.

Cephalosporins are much more expensive than penicillins. One reason is that some cephalosporins (e.g., cephalexin) are made from penicillins by a number of chemical conversions. Another reason is that, so far, only cephalosporins with a D-α- amino-adipyl side chain could be fermented. Cephalosporin C, by far the most important starting material in this respect, is very soluble in water at any pH, thus implying lengthy and costly isolation processes using cumbersome and expensive column technology. Cephalosporin C obtained in this way has to be converted into therapeutically used cephalosporins by a number of chemical and enzymatic conversions. The methods currently favored in industry to prepare the intermediate 7- amino-deacetoxycephaloporanic acid (7-ADCA) involve complex chemical steps leading to the expansion and derivatization of penicillin G. One of the necessary chemical steps to produce 7-ADCA involves the expansion of the 5-membered penicillin ring structure to a 6-membered cephalosporin ring structure (see for instance US 4,003,894). This complex chemical processing is both expensive and noxious to the environment.

Consequently, there is a great desire to replace such chemical processes with enzymatic reactions such as enzymatic catalysis, preferably during fermentation. A key to the replacement of the chemical expansion process by a biological process is the central enzyme in the cephalosporin biosynthetic pathway, expandase.

The expandase enzyme from the bacterium Streptomyces clavuligerus (S. clavuligerus) was found to carry out, in some cases, penicillin ring expansions. When introduced into P. chrysogenum, it can convert the penicillin ring structure into the cephalosporin ring structure, as described in Cantwell et al., Proc R Soc Lond B (1992) 248:283-289. The expandase enzyme has been well characterized (EP-A-0366354)

both biochemical and functional, as has its corresponding gene. Both physical maps of the cefE gene (the gene encoding the expandase enzyme of S. clavuligerus - EP-A- 0341892), DNA sequence and transformation studies in P. chrysogenum with cefE have been described. The DNA and amino acid sequence of the S. clavuligerus expandase enzyme are represented in SEQ ID NO 1 .

Another source for an expandase enzyme is the bacterium Nocardia lactamdurans (N. lactamdurans, formerly S. lactamdurans). Both the biochemical properties of the enzyme and the DNA sequence of the gene encoding the enzyme have been described - see Cortes et al., J Gen Microbiol (1987) 133:3165-3174 and Coque et al., MoI Gen Genet (1993) 236:453-458, respectively. The DNA and amino acid sequence of the N. lactamdurans expandase enzyme are represented in SEQ ID NO 2.

Recently, novel expandase (cefE) genes and enzymes have been found in Streptomyces jumonjinensis, Streptomyces ambofaciens and Streptomyces chartreuses - see Hsu et al. (2004), Appl. and Environm. Microbiol. 70, 6257-6263. Table 1 summarizes the amino acid sequence identities from several expandases and shows that the sequence identities range from 67 to 85%, depending on the source of the enzyme.

Table 1 : Amino acid sequence identities of several expandases (data taken from Hsu et al).

In the biosynthesis of cephalosporins, which takes mainly place in prokaryotic cells, the deacetoxycephalosporin-C is subsequently converted to deacetyl- cephalosporin-C by the enzyme deacetylcephalosporin C synthase also named deacetoxycephalosporin-C hydroxylase or hydroxylase (EC 1.14.1 1.26 - DACS).

Genes encoding such hydroxylases are named cefF-genes (e.g. see Hsu et al.) The expandase found in eukaryotic cells, e.g. Acremonium chrysogenum can catalyze the direct conversion of penicillin N to deacetoxycephalosporin-C due to possession of both expandase and hydrolyase activity, hence the encoded gene is termed cefEF(see Hsu et al.).

As defined herein, the term expandase relates to expandase enzymes (EC 1.14.20.1 , encoded by cefE genes) as well as expandases also possessing in addition the hydroxylase activity (EC 1.14.20.1 + EC 1 .14.1 1.26 encoded by ce/EFgenes)

Since the expandase enzyme catalyses the expansion of the 5-membered thiazolidine ring of penicillin N to the 6-membered dihydrothiazine ring of DAOC, this enzyme would be of course a logical candidate to replace the ring expansion steps of the chemical process. Unfortunately, the enzyme works on the penicillin N intermediate of the cephalosporin biosynthetic pathway, but not or very inefficiently on the readily available inexpensive penicillins as produced by P. chrysogenum, like penicillin V or penicillin G. Penicillin N is commercially not available and even when expanded, its D- α-amino-adipyl side chain cannot be easily removed by penicillin acylases.

It has been reported that the expandase enzyme is capable of expanding penicillins with particular side chains to the corresponding 7-ADCA derivative. This feature of the expandase has been exploited in the technology as disclosed in EP-A- 0532341 , WO95/04148 and WO95/04149. In these disclosures the conventional chemical in vitro conversion of penicillin G to 7-ADCA has been replaced by the in vivo conversion of certain 6-aminopenicillanic acid (6-APA) derivatives in recombinant Penicillium chrysogenum strains transformed with an expandase gene.

More particularly, EP-A-0532341 teaches the in vivo use of the expandase enzyme in P. chrysogenum, in combination with a adipyl side chain (further referred to as adipyl) as a feedstock, which is a substrate for the acyltransferase enzyme in P. chrysogenum. This leads to the formation of adipyl-6-APA, which is converted by an expandase enzyme introduced into the P. chrysogenum strain to yield adipyl-7-ADCA. Finally, the removal of the adipyl side chain is described, yielding 7-ADCA as a final product. Furthermore, EP-A-540210 teaches the similar production of adipyl-7-ADAC and adipyl-7-ACA.

As an alternative approach, expansion of penicillin G using P. chrysogenum transformed with cefE has been proposed in EP0828850. Furthermore, in order to increase the expansion of penicillin G the use of mutant cefE gene of S. clavuligerus has been described. US 5,919,680, WO 98/02551 , WO 99/33994, WO 01/85951 and

EP 1348759 all disclose mutant cefE genes allegedly coding for enzymes having a higher expandase activity on penicillin G.

Nevertheless, these mutant expandases still do not provide for a commercially attractive process for the production of cephalosporins. Therefore, there is a need for further improving the process wherein cephalosporins are prepared from expansion of adipyl-6-APA. This process is characterized by an efficient removal of the side chain material from the cephalosporin core material. One step to further improve this process is improvement of the expandase activity on adipyl-6-APA.

Description of the Figures

1/3. A & B Amino acid alignment of improved expandase mutants; SCLAVEIW represents amino acid sequence of expandase of S. clavuligerus; NOCAEIW represents amino acid sequence of expandase of N. lactamdurans; For the other sequences the notation is as follows: 309EIWIT represents amino acid sequence of expandase of mutant expandase H309, and so son. 2/3. Schematic representation of vector plATWAn 3/3. HPLC analysis of expandase assay mixture according to Example 3 A. at t = O minutes

B. at t = 30 minutes

Detailed description of the invention

In a first aspect, the invention provides a mutant expandase that is a variant of a model polypeptide with expandase activity. Preferably, the invention provides a mutant expandase, whereby the mutant expandase has an at least 2-fold improved in vitro expandase activity towards adipyl-6-APA in comparison with the model polypeptide with expandase activity. The determination of the in vitro expandase activity towards adipyl-6-APA is described in detail in the Materials and Methods (Assay (1 ) - (3)). More preferably the in vitro expandase activity towards adipyl-6-APA of the mutant expandase is improved at least 2.5-fold, more preferably at least 3-fold, more preferably at least 4-fold, more preferably at least 5-fold, more preferably at least 6-fold, more preferably at least 7-fold, more preferably at least 8-fold, more preferably at least 9-fold, more preferably at least 10-fold, more preferably at least 1 1 -fold.

In some of the mutant expandase enzymes according to the invention parts of the amino acid sequence of the expandase of S. clavuligerus have been replaced by corresponding parts of the amino acid sequence of expandase of N. lactamdurans. The amino acid sequence of the CefE gene of N. lactamdurans strain ATCC27382 is represented in SEQ ID NO: 2.

In the description of these mutant expandases the amino acids are numbered according to the numbering of the homologous parts of the amino acid sequence of the expandase of S. clavuligerus. The homology of the expandase amino acid sequences of S. clavuligerus and N. lactamdurans is to be derived from Figure 1 and Table 1. With "altered or mutant expandase" in the context of the present invention is meant any enzyme having expandase activity, which has not been obtained from a natural source and for which the amino acid sequence differs from the complete amino acid sequences of the natural expandase enzymes of S. clavuligerus and N. lactamdurans.

The invention also provides a mutant expandase which is preferably being modified at least at an amino acid position selected from group 1 consisting of positions 2, 18, 59, 73, 74, 89, 90, 99, 101 , 105, 112, 113, 155, 170, 177, 209, 213, 217, 244, 249, 251 , 277, 278, 280, 281 , 284, 293, 300, 307 and 311 using the amino acid position numbering of the amino acid sequence of the expandase enzyme encoded by the cefE gene of Streptomyces clavuligerus. The nucleotide sequence of the cefE gene of Streptomyces clavuligerus as well as the amino acid sequence encoded by said cefE gene are depicted in SEQ ID NO: 1 . More preferably, the mutant expandase is modified at least at an amino acid position selected from group 2 consisting of positions 2, 18, 59, 89, 90, 99, 101 , 105, 112, 1 13, 170, 177, 209, 213, 217, 249, 251 , 278, 280, 284 and 293. The mutant expandase may also have been modified at least at an amino acid position selected from the group 1 which does not form part of group 2, together with at least an amino acid position selected from the group 2.

A highly preferred mutant expandase is modified at least at amino acid position 89, in combination with any of the amino acid positions of the expandase, preferably with an amino acid position selected from group 1 or an amino acid position selected from group 2 or an amino acid position selected from group 1 which does not form part of group 2, together with at least an amino acid position selected from the group 2. Most preferably, the invention provides a mutant expandase whereby the

mutant expandase has an at least 2-fold improved in vitro expandase activity towards adipyl-6-APA in comparison with the model polypeptide with expandase activity, more preferably at least 2.5-fold, more preferably at least 3-fold, more preferably at least 4- fold, more preferably at least 5-fold, more preferably at least 6-fold, more preferably at least 7-fold, more preferably at least 8-fold, more preferably at least 9-fold, more preferably at least 10-fold, more preferably at least 1 1 -fold and whereby the mutant expandase is preferably being modified at at least an amino acid position selected from the group 1 defined above using the amino acid position numbering of the amino acid sequence of the expandase enzyme encoded by the cefE gene of Streptomyces clavuligerus. More preferably, the mutant expandase is modified at least at an amino acid position selected from the group 2. The mutant expandase with the improved expandase activity may also have been modified at least at an amino acid position selected from the group 1 together with at least at an amino acid position selected from the group 2. A highly preferred mutant expandase with improved expandase activity is modified at least at amino acid position 89, in combination with any of the amino acid positions of the expandase, preferably with an amino acid position selected from group 1 or an amino acid position selected from group 2 or an amino acid position selected from group 1 together with at least at an amino acid position selected from group 2. The modification at an amino acid position may comprise a substitution by another amino acid, selected from the group of 20 L-amino acids that occur in Nature - see Table 2. Alternatively, the modification at an amino acid position may comprise a deletion of the amino acid at said position. Furthermore, the modification at an amino acid position may comprise a substitution of one or more amino acids at the C-terminal or N-terminal side of said amino acid.

Table 2

The model polypeptide with expandase activity as used in the present invention is selected from the group consisting of a polypeptide with expandase activity, preferably having an amino acid sequence according to SEQ ID NO: 1 or having an amino acid sequence according to SEQ ID NO: 2 and polypeptides with expandase activity having an amino acid sequence with a percentage identity with SEQ

ID NO: 1 of at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, most preferably at least 95%, or with SEQ ID NO: 2 of at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, most preferably at least 95%, such as the expandase enzymes that are summarized in Table 1 . Most preferred as model polypeptide with expandase activity as used in the present invention is a polypeptide with expandase activity, having the amino acid sequence according to SEQ ID NO: 1 or having the amino acid sequence according to SEQ ID NO: 2.

The present invention preferably provides mutant expandases that have modifications at least at o 2 or more amino acid positions selected from the group consisting of 2, 89, 277,

281 and 300, more preferably at positions 2+281 or 89+281 or 277+300. o 3 or more amino acid positions selected from the group consisting of 2, 89, 281 ,

293 and 311 , more preferably at positions 2+89+281 or 89+281 +311 or 89+281 +293. o 4 or more amino acid positions selected from the group consisting of 2, 73, 89,

90, 217, 244, 277, 280, 281 , 306, 307 and 311 , more preferably at positions 2+277+280+281 or 2+281 +307+31 1 or 73+89+281 +31 1 or 89+217+281 +311 or

89+244+281 +311 or 89+281 +307+311 or 277+281 +306+31 1 or 89+281 +306+311 or 90+281 +306+31 1. o 5 or more amino acid positions selected from the group consisting of 2, 73, 89, 90, 155, 213, 249, 278, 281 , 293, 300, 306, 307 and 31 1 , more preferably at

positions 2+89+281 +306+311 or 2+155+281 +306+31 1 or 73+89+213+281 +31 1 or 89+78+218+307+31 1 , 89+281 +293+300+311 or 89+249+281 +307+311 . o 6 amino acid positions selected from the group consisting of

90+105+1 13+281 +306+31 1. o 7 or more amino acid positions selected from the group consisting of 2, 73, 89,

105, 1 13, 155, 170, 177, 251 , 277, 280, 281 , 293, 300, 306, 307 and 311 , more preferably 73+89+281 +293+300+307+31 1 or

105+113+155+177+281 +306+31 1 or 155+177+277+280+281 +306+ 31 1. o 8 or more amino acid positions selected from the group consisting of 2, 89, 90, 99, 105, 113, 155, 177, 277, 281 , 307 and 311 more preferably at

2+90+99+105+1 13+281 +306+311 or 2+90+105+113+155+177+277+281 o 9 amino acid positions selected from the group consisting of

2+90+105+113+155+177+281 +306+31 1. o 10 amino acid positions selected from the group consisting of 2, 59, 90, 101 , 105, 1 13, 155, 177, 209, 277, 281 , 307 and 31 1 more preferably at

2+90+105+113+155+177+277+281 +306+311 . o 1 1 amino acid positions selected from the group consisting of 2, 90, 105, 1 13,

155, 177, 277, 281 , 293, 307, 311 .

The present invention relates to substitutions of certain of the amino acids of expandase. Preferably such substitutions involve replacement of:

• histidine at position 18 according to SEQ ID NO 1 by cystein, serine, threonine, asparagine, glutamine, tyrosine, lysine and arginine; more preferably serine, threonine, asparagine, glutamine, lysine and arginine; most preferably arginine; • methionine at position 74 according to SEQ ID NO 4 by threonine or by a hydrophobic amino acid such as valine, leucine, isoleucine and phenylalanine or by an amphiphilic amino acid such as tyrosine, tryptophan, histidine, glutamine and asparagine, more preferably threonine or isoleucin;

• Threonine at position 89 according to SEQ ID NO 1 preferably by a charged amino acid, preferably a negatively charged amino acid such as aspartic acid or glutamic acid, most preferably a positively charged amino acid such as lysine, arginine or hystidine; most preferred being lysine.

• serine at position 112 according to SEQ ID NO 1 preferably by threonine;

• histidine at position 244 according to SEQ ID NO 1 by glutamine or asparagine, more preferably glutamine;

• aspartic acid at position 284 according to SEQ ID NO 1 and at position 285 in SEQ ID NO 4 by an amino acid with a polar side chain such as serine, threonine, asparagine, glutamine, glutamic acid, histidine, lysine, arginine and tyrosine, more preferably asparagine, glutamine, glutamic acid, lysine and arginine; most preferred being asparagine

• glycine at position 300 according to SEQ ID NO 1 by an amino acid with a small residue, such as alanine, serine, threonine, cystein, valine, isoleucine, leucine, asparagine and aspartic acid, more preferably valine.

The invention furthermore provides mutant expandases as defined hereinbefore that are variants of the group consisting of the wild type expandases of S. clavuligerus, S. jumonjinensis, S. ambofaciens, S. chartreuses and N. lactamdurans. Most preferred are mutant expandases as defined hereinbefore that are variants of the wild type expandase of S. clavuligerus or N. lactamdurans. Most preferred are mutant expandases as defined hereinbefore that are variants of the wild type expandase of S. clavuligerus. Preferred mutant expandases that are mutants of the expandase of Streptomyces clavuligerus have modifications at least at o 2 or more amino acid positions selected from the group consisting of D2, T89,

L277, C281 and G300. o 3 or more amino acid positions selected from the group consisting of D2, T89,

C281 , T293 and A31 1 o 4 or more amino acid positions selected from the group consisting of D2, M73,

T89, N90, Y217, H244, L277, E280, C281 , R306, R307 and A31 1 o 5 or more amino acid positions selected from the group consisting of D2, M73, T89, N90, C155, T213, R249, A278, C281 , T293, R306, R307 and A31 1 o 6 amino acid positions selected from the group consisting of

N90+T105+G1 13+C281 +R306+A31 1. o 7 or more amino acid positions selected from the group consisting of D2, M73, T89, T105, G1 13, C155, H170, P177, D251 , L277, E280, C281 , T293, G300, R306, R307 and A311 o 8 or more amino acid positions selected from the group consisting of D2, T89,

N90, M99, T105, G1 13, C155, P177, L277, C281 , R306, R307 and A311 . o 9 amino acid positions selected from the group consisting of

D2+N90+T105+G113+C155+P177+C281 +R306+A311 . o 10 amino acid positions selected from the group consisting of D2, S59, N90,

10

T105, G1 13, T105, G113, C155, P177, G209, L277, C281 , R306, R307, A311 . o 11 amino acid positions selected from the group consisting of D2, N90, T105, G1 13, C155, P177, L277, C281 , R306, R307, A311 .

Preferred modifications at the respective positions are D2N, D2H, D2Y, S59G,

M73H, M73I, T89A, T89K, T89V, N90W, N90S, M99T, T105I, Y101 F, G1 13D, C155W, H170Y, P177L, G209A, T213A, Y217H, H244R, R249C, D251 G, L277Q, L277T, A278V, E280G, C281Y, T293K, G300S, R306deletion, R307deletion, R370T and A311 D. In this notation, the letter following the number represents the amino acid (one letter code) present in the mutant expandase. The deletion at position R306 or R307 mean that the original arginine at position 306 or 307 is no longer present in the mutant expandase. Highly preferred mutant expandases are the mutant expandases that are summarized in Table 3.

Preferably, the mutant expandases provided by the present invention have an improved expandase activity on adipyl-6-APA as defined herein before, the mutant expandases provided by the present invention have likewise, an improved expandase activity on Pen-G. Preferably, the mutant expandases provided by the present invention have an improved expandase on both adipyl-6-APA as well as Pen-G. Mutant expandases with an improved activity on Pen-G can be used advantageously in a process for the production of phenylacetyl-7-ADCA as described further below.

The present invention also provides mutant expandases with a decreased or even absent expandase activity with iso-penicillin N (iPN). Preferably the expandase activity with either adipyl-6-APA or Pen-G is not affected, but more preferably the expandase activity with either adipyl-6-APA or Pen-G or both is improved as defined hereinbefore. The advantage of these more preferred mutant expandases is that the decreased or even absent expandase activity with iso-penicillin N (iPN) results in less byproduct in a fermentation process to produce ad-7-ADCA or phenylacetyl-7-ADCA.

Preferred mutant expandases have a decreased or even absent expandase activity with iso-penicillin N (iPN) as a substrate optionally combined with an improved expandase activity on ad-6-APA as a substrate and have been modified at position 89 according to the amino acid numbering of SEQ ID No 1 . whereby the naturally occurring amino acid has been replaced, preferably by a charged amino acid, preferably a negatively charged amino acid such as aspartic acid or glutamic acid, most preferably a positively charged amino acid such as lysine, arginine or hystidine; most preferred being lysine.

1 1

A highly preferred mutant expandase is selected from the group consisting of H101 , H106, H1 1 1 , H122, H127, H262, H301 , H305, H308, H309, H401 , H402, H403, H501 , H502, H503, H504, H505, H506, H507, H508, H601 , H602, H603, H604, H605, H606, H607, H608, H609, H650, H651 , H652, H653, H654, H655, H656, H657, H658, H659, H660, H661 , H662, G601 , G602, G603, G604, G605, G606, G607, G608, G609, G610, G61 1 , G613 and G614 (see Table 3).

In a second aspect, the invention provides a polynucleotide encoding the mutant expandase of the present invention. The polynucleotide encoding the mutant expandase according to the present invention can be any polynucleotide that encodes the proper amino acid sequence according to the invention. This implies that it may correspond to the cefE gene of S. clavuligerus except for the nucleotides encoding the modifications at the respective amino acid positions. Alternatively, it may correspond to the cefE gene of S. clavuligerus except for the polynucleotide fragments encoding the parts of the amino acid sequence corresponding with sequences of N. lactamdurans expandase. Alternatively, the polynucleotide of the invention may comprise a coding sequence in which the codon usage for the various amino acids deviates from the codon usage in S. clavuligerus and/or in N. lactamdurans. For example, the codon usage may be adapted to the codon usage of a particular host cell, which will or has been transformed with the DNA fragment encoding the altered expandase.

In a third aspect, the invention provides an expression vector or expression cassette comprising the polynucleotide of the invention as defined hereinbefore.

In a fourth aspect, the invention provides a transformed host cell, transformed with the polynucleotide of the invention or the expression vector or expression cassette of the invention. The transformed host cell may be used for the production of the mutant expandase of the invention or the host cell may be used for the production of a beta-lactam compound of interest. Host cells for the production of the mutant expandase of the invention are preferably host cells which are known in the art for their efficient protein or enzyme production, either extracellular or intracellular^, for example microorganisms such as fungi, yeast and bacteria. Examples of preferred host cells comprise, but are not limited to, the following genera: Aspergillus (e.g. A. niger, A. oryzea), Penicillium (e.g. P. emersonii, P. chrysogenum), Saccharomyces (e.g. S. cerevisiae), Kluyveromyces (e.g.

12

K. lactis), Bacillus (e.g. B. subtilis, B. licheniformis, B. amyloliquefaciens). Escherichia, (E. coli), Streptomyces (e.g. S. clavuligerus).

Host cells for the production of a beta-lactam compound of interest are preferably host cells that are known in the art for their efficient beta-lactam compound production. Examples of preferred host cells comprise, but are not limited to, to the following genera: Penicillium (e.g. P. chrysogenum), Acremonium (e.g. A. chrysogenum), Streptomyces (e.g. S. clavuligerus), Nocardia (e.g. N. lactamdurans), Lysobacter (e.g. L lactamgenus) and Flavobacterium species.

In a fifth aspect, the invention provides a process for the production of the mutant expandase of the invention comprising cultivating the transformed host cell according to the invention under conditions conducive to the production of the mutant expandase and, optionally, recovering the mutant expandase. The recovered mutant expandase may be used advantageously in an in vitro process to produce a desired cephalosporin from a corresponding penicillin, for instance the recovered mutant expandase may be used in a process to produce phenylacetyl-7-ADCA from Pen-G or adipyl-7-ADCA from adipyl -6-APA.

In a sixth aspect, the invention provides a process for the production of a beta- lactam compound of interest comprising cultivating the transformed host cell according to the invention under conditions conducive to the production of the beta-lactam compound of interest and, optionally, recovering the beta-lactam compound. Preferred beta-lactam compounds belong to the group of cephalosporins such as phenylacetyl-7- ADCA, adipyl-7-ADCA, adipyl-7-ADAC and adipyl-7-ACA. In a preferred embodiment, the invention provides a process for the production of phenylacetyl-7-ADCA or adipyl-7- ADCA by cultivating a selected strain of Penicillium chrysogenum, that has been transformed with a selected polynucleotide of the invention that encodes a mutant expandase of the invention. For the production of phenylacetyl-7-ADCA, a mutant expandase is selected that has a high improvement factor on Pen-G as a substrate. For the production of adipyl-7-ADCA, a mutant expandase is selected that has a high improvement factor on ad-6-APA as a substrate.

In another preferred embodiment, the invention provides a process for the production of 7-ADCA comprising the process for the production of phenylacetyl-7- ADCA or adipyl-7-ADCA as described herein before, followed by, after optional purification of said 7-ADCA-derivatives, a process step in which the phenylacetyl or

13

adipyl side chain of phenylacetyl-7-ADCA and adipyl-7-ADCA respectively is cleaved off thereby generating 7-ADCA and the liberated side chains acid. Said cleavage can be obtained by chemical means or, more preferably, enzymatically using an acylase enzyme. Suitable acylases for the cleavage of the adipyl side chain are obtainable from various Pseudomonas species such as Pseudomonas SY-77 or Pseudomonas SE-83. Suitable acylases fro the cleavage of the phenylacetyl side chains are the penicillin acylases from Escherichia coli or Alcaligenes feacalis.

In a seventh aspect, the invention provides a process for the production of a cephalosporin from a corresponding penicillin whereby the expansion of the 5- membered thiazolidine ring of the penicillin to the 6-membered dihydrothiazine ring of the cephalosporin occurs in an in vitro process, catalyzed by a mutant expandase of the invention. In a preferred process, adipyl-6-APA is expanded to the corresponding adipyl-7-ADCA by a mutant expandase of the invention, preferably a mutant expandase that has a high improvement factor adipyl-6-APA as a substrate. In another preferred process, Pen-G is expanded to the corresponding phenylacetyl-7-ADCA by a mutant expandase of the invention, preferably a mutant expandase that has a high improvement factor on Pen-G as a substrate. The process may be followed by, after optional purification of said 7-ADCA- derivatives, a process step in which the side chains of adipyl-7-ADCA and phenylacetyl-7-ADCA are cleaved off thereby generating 7-ADCA and the liberated side chains acid - supra vide.

The desired end product 7-ADCA can be recovered according to methods known in the art involving chemical or enzymatic cleavage of the side chain of adipyl-7- ADCA or phenylacetyl-7-ADCA and optionally the resulting 7-ADCA can be further purified and/or crystallized.

In an eight aspect, the invention provides a method to obtain the mutant expandases of the invention whereby the process comprises the following steps: 1 . Mutagenesis of a cloned gene encoding a model polypeptide with expandase activity thus obtaining a collection of mutagenised genes encoding the mutant expandases; 2. Expression of the collection of mutagenised genes encoding the mutant

14

expandases in a suitable host and screening the collection of mutant expandases for an improved activity with a suitable substrate;

3. Optionally repeating steps 1 and 2 one or several times using either the gene encoding the model polypeptide with expandase activity or one or more of the mutagenised genes encoding mutant expandases with an improved activity on the suitable substrate.

Cloning of the gene encoding a model polypeptide with expandase activity can be carried out according to methods known in the art.

Preferred model polypeptides with expandase activity are selected from the group consisting of a polypeptide with expandase activity obtainable from Streptomyces clavuligerus, preferably having an amino acid sequence according to SEQ ID NO: 1 and polypeptides with expandase activity having an amino acid sequence with a percentage identity with SEQ ID NO: 1 of at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, most preferably at least 95%, such as the expandase enzymes that are summarized in Table 1.

Any mutagenesis technique can be employed which results in mutations over the entire gene. Suitable techniques are error prone (EP) PCR (Polymerase Chain Reaction) and/or by using saturated Mutation Primer PCR (sMPP) exactly according to WO 03/010183.

Materials and Methods

1. General

Oligonucleotides were synthesized by Invitrogen (Carlsbad CA, US).

DNA sequencing was carried out by SEQLAB (Gόttingen, Germany) or by Baseclear (Leiden, The Netherlands).

Restriction enzymes were purchased from Invitrogen.

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The protein assays were carried out according to the method described by Bradford, M. M. (1976) Anal Biochem. 72:248-54

Escherichia coli DH1 OB electromax competent cells were obtained from Invitrogen. The protocol was delivered by the manufacturer.

SDS-PAGE electrophoresis was carried out in the system supplied by Invitrogen. NuPAGE Novex Bis-Tris Gels (Invitrogen). SimplyBlue SafeStain Microwave protocol

Overview of used constructs

1. Cloning of the E. coli expression vectors

The fusion product is ligated in the pBAD/MH MBP-ScEwt Dest zeo vector by an EcoRI/Sa/l digestion. This replaces the wild type expandase gene by the library genes. To prevent significant numbers of wild type constructs, the ligation constructs were digested with Sma\.

2. Construction of the libraries

Transformation of E. coli with the ligation mix yielded libraries of roughly 12,000-13,000 colonies. To test the quality of the libraries, a random set of colonies was selected and the constructs were digested with restriction enzymes. This showed that approximately 26% of the library revealed an aberrant pattern. Next, sequence analysis was performed on 10 regular clones to determine mutation frequencies.

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Sequences showed that both saturation and error prone mutations were present at a satisfactory level.

3. Expression and screening of mutant expandases

3.1. Screening of the cefE library for improved activity

E. coli TG1 cells containing the library A were plated and grown on agar plates. Colonies are picked robotically into 96-wells microtiter plates containing liquid medium (2YT, 30 μg/ml chloramphenicol, 05% glucose), and grown to saturation by shaking at 37 ⁰ C. Cells are subcultured in 96 well megatiter plates; 1 :40 inoculum into 2YT, 30 μg/ml chloramphenicol and induced by 0.2 mM IPTG shaking at 28 ⁰ C for 24 h. Cells are pelleted and washed with an equal volume of buffer (5 mM morpholine pH7.5). After spinning, the cells are resuspended in lysis mix for primary screening Expandase assay (1 )).

3.2. Primary screening in microtitre plates (MTP)

Growth and induction

The expandase E. coli library is plated on Luria-Bertani (LB) medium plates low salt agar + 25 μg/ml Zeocine and incubated overnight at 37 ⁰ C. Microtitre plates (MTP) with 150 μl LB low salt medium and 25 μg/ml Zeocine are inoculated from the plates and incubated 36 hrs at 25 ⁰ C and 550 rpm.

From the MTP, 5 μl is inoculated in deep well plate with 1 ml 2 ^* TY (tryptone and yeast extract) medium + 50 μg/ml Zeocine + arabinose. To the remaining culture in the MTP, 100 μl 50% glycerol is added and frozen at -8O ⁰ C as glycerol stock.

The deep well plate is incubated for 30 hrs at 25 ⁰ C and 550 rpm. The deep well plate cultures are centrifuged at 2750 rpm (Heraeus multifuge 4 kr.) for 10 min. The supernatant is discarded and the pellet is frozen at -2O ⁰ C.

Cell free extract (CFE)

The frozen pellets obtained in the previous section are thawed in 200μl extraction buffer (50 mM Tris/HCI pH=7.5; 5 mM DTT; 0.1 mg/ml DNAse; 5 mM

MgSO ₄ H ₂ O and 0.5 mg/ml lysozyme). To resuspend pellets, the plates are shaken.

The plates are incubated for 30 minutes at room temperature and centrifuged for 10 minutes at 2750 rpm.

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3.3 Secondary screening in shake flasks

Growth and induction

Selected colonies are inoculated from plate in 10 ml LB low salt + Zeocin 25 μg/ml and incubated overnight at 37 ⁰ C, 280 rpm. The grown culture is inoculated in 100 ml LB low salt with 25 μg/ml Zeocine at an optical density (600 nm) between 0.010 and 0.050 (Biochrom Ultraspec 2000). After growth at 37 ⁰ C, 280 rpm, the cells are harvested at an optical density at 600 nm between 0.4-0.6. Then, arabinose is added (final concentration 0.2%) and induced overnight at 27 ⁰ C at 220 rpm.

4. Expandase assays

4.1 Expandase assay (1) - Screening assay (primary assay):

Expandase assay buffer: 5 mM morpholine buffer pH 7,5 and cofactor mix (final concentrations 50 μM ATP, 1 mM DTT, 60 μM FeSO4, 2,7 mM ascorbate), 500 μM ad-6-APA, 1/10 ^th volume of E. coli cell lysate and 2.4 mM α-KG. Each cofactor was made in buffer pH 7.5, which was critical for activity. The expandase reaction was found to be linear for up to 3 hours with respect to adipyl-7ADCA formation/detection. The optimal temperature for the in vitro expandase reaction was determined to be approximately 1 1 ⁰ C, probably due to instability of the enzyme or production at higher temperatures. Nevertheless, actual screening was performed at a temperature between room temperature and 25 ⁰ C, which is more reflective of the desired reaction condition temperature.

Screening of the family shuffled libraries for improved 7-ADCA formation was determined by Flow Injection Analysis-MS (FIA-MS, negative mode, Mass Transition [M-H] ^' 341 >295). Final analysis: LC column: using solvent conditions between 0.1% formic acid in 40% MeOH to 100% MeOH. Two channels (two mass transitions [M-H ]) are used to monitor for each reaction product. Samples are analyzed after 30 minutes of reaction.

4.2 Expandase assay (2) - Secondary expandase assay

Expandase assay buffer: 5 mM morpholine buffer pH 7,5 and cofactor mix (final concentrations: 50 μM ATP, 1 mM DTT, 60 μM FeSO4, 2,7 mM ascorbate), 2 mM

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adipyl-6-APA, 1/10 ^th volume of E. co// cell lysate and 2.4 mM α-ketoglutarate. Each cofactor was made in buffer pH 7.5, which was critical for activity. The conversion was performed at 29 ⁰ C and samples were taken between 0 and 30 minutes. The reaction was stopped with 60% (v/v) acetonitrile final concentration. The formed adipyl-7-ADCA was analysed on HPLC. (Mobile phase: 10% acetonitrile in 10 mM potassium phosphate buffer pH 3.0) Column: X-Terra™ RP18 3.5μ. 3.9x100 mm detected at 214 and 254 nm (254 nm to analyse expanded antibiotics). HPLC data analysis: software: Waters Millennium ^{32 ®} 3.20.

4.3 Expandase assay (3) - Expandase assay

A total of 300 μl of a reaction mixture consisting of 50 mM Tris/HCI pH 7.5; 1 mM DTT; 2.7 mM Ascorbate; 0.05 mM ATP; 24 mM a-ketoglutarate; 0.06 mM FeSO ₄ .7H ₂ O; 4 mM adipyl-6-amino-penicillanic acid (ad-6-APA) was added to 75 μl CFE and incubated at 29 ⁰ C for the desired time. Adding 60 μl maleic acid with 10 g/l EDTA stopped the reaction. The formation of adipyl-7-amino-desacetoxy- cephalosporinic acid (ad-7-ADCA) was detected using ¹ H-NMR.

The adipyl-6-APA was obtained from 6-APA and adipic acid catalyzed by penicillin acylase (EC 3.5.1.11 ). Alternatively, adipyl-6-APA can be obtained by culturing for instance a suitable Penicillium chrysogenum strain in the presence of adipic acid as precursor.

EXAMPLES

Example 1

Cloning of wild type expandase

The cefE gene was PCR cloned from S. clavuligerus genomic DNA and ligated in GFP-fusion vectors, the so-called pCK series, and used to transform E. coli

TG1 (Amersham Pharmacia Biotech, Inc. NJ USA). Several colonies from the plated transformations were selected and checked for an active clone. Several active clones were identified. Clone '778' appeared to be a wild type gene but interestingly, one other clone, clone "779", revealed strongly improved activity on adipyl-6-APA expansion. The product of clone "779" was sequenced and revealed three amino acid mutations

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(H18R, D284N and G300V). The adipyl-7-ADCA production of the clones analysed by Flow Injection Analysis-Mass Spectrometry (FIA/MS) showed that the Sc-cefE mutant "779" was superior to the wild-type parents. Measurement of GFP fluorescence suggests that the improvement of the expandase enzyme product of clone "779" is not due to improved expression of cefE therein. Hence, it was concluded that the improvement of the expandase enzyme product of clone "779" is an improvement of the activity as a result of the amino acid substitutions.

pCK derived GFP fusion vectors

Fusion tag vector series (called pCK series) containing a His tag fused to the 3' end of expandase with green fluorescent protein (GFP) fusion at the 3' end of the HIS tag were constructed. The GFP containing vectors enable high throughput normalisation of expandase activity data in subsequent assays with respect to protein concentration. The vectors contain the p15A origin of replication (low copy number), and a lac promoter that is repressed in the presence of glucose.

Example 2

Family shuffling and Screening of wild-type N. lactamdurans and

S. clavuliαerus cefE genes - Library A

The expandase genes were shuffled according to the process described in EP752008. Expandase mutant libraries were made using the GFP-fusion tag vectors (pCK). Only the CefE ORF was shuffled, while other sequences where not changed. After moving the shuffled pool of expandases into the pCK vectors, one library set (Library A) was made using the two parental wild type expandase genes (S. clavuligerus and N. lactamdurans) plus clone "779".

Initial plating of a sample of the library showed that the libraries contained at least 70% green fluorescent colonies. Due to this, it was not considered necessary to pre-screen out non-fluorescent colonies before proceeding to the first FIA/MS screens. DNA sequencing of randomly picked colonies form library set A indicated that all parents were represented in the library, so this library was selected for initial screening. 720 clones from library A were screened and 14 clones (mutant expandases

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H101 , H102, H104, H106, H108, H11 1 , H1 12, H114, H1 15, H1 17, H120, H122, H125, H127) were retested and the preliminary results showed an expandase activity improvement compared to the S. clavuligerus expandase (clone "778") as judged by in vitro expandase assay (1 ). These clones were chosen for further testing. Normalization of the expandase activity with respect to expandase-GFP concentration (i.e. correcting for differences in the expandase protein concentration) confirmed that these clones were improved in specific activity.

Example 3

Family shuffling of wild-type N. lactamdurans and S. clavuligerus cefE genes.

Library B

Library B was constructed shuffling wild type N. lactamdurans and S. clavuligerus cefE as parent genes. The "779" hit sequence was not used in construction of this library. This expandase library showed to have a significant level of inactive or very poorly active clones in the library. In order to remove inactive clones from further testing, a FACS-based pre-screen was used for the library B screening to improve the percentage of active clones that enter the expandase assays. After FACS sorting based on their fluorescence by GFP, selected cells were recovered under repressing conditions. DNA prepared from these cultures was used to transform

E. coli TG1. These expandase constructs used are also fusion proteins with GFP as described for library A.

70-80 percent of the library was GFP-active. In total, 2200 clones from this library were then screened using the in vitro expandase assay and FIA-MS detection of adipyl-7-ADCA formation.

Fourteen possible hits were selected (H200 series). Four of these hits, amongst them H262, were confirmed through retesting procedure including retransformation and in vitro testing and analysis MS.

Example 4

Construction of chimeric expandase seguences using shuffled expandases

Library C

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Using traditional molecular biological techniques and bioinformatics tools, the hit sequences from the two above described libraries (libraries A and B) were recombined, resulting in library C (H300 series). Three resulting chimeras were tested for activity, amongst them expandase H301. Six remaining clones (amongst them: H305, H307, H308, H309) were not tested as GFP fusion product nor analysed by FIA-MS. These clones were directly recloned in the pSJ vector series fused with maltose binding protein (MBP) upstream of the Ce/E mutant genes and analysed, after the In vitro assay, on HPLC.

Example 5

Recloninq of genes encoding improved expandases using pSJ based vector series and enzymatic activity of the improved expandases

pSJOδ was used as cloning vector for the expandase libraries. This plasmid was constructed by replacing the expandase gene (as Ndel-Nsil fragment) by a small oligonucleotide introducing a Notl restriction site. S. clavuligerus cefE was cloned as Nde\-Nsi\ fragment in pSJOδ, resulting in pSJ05. pSJ05 contained an additional Nde\ site just upstream of the Λ/s/l site. This site was removed to prevent possible interference with further cloning of the shuffled genes in the Nde\-Nsi\ sites, resulting in pSJ11 . Next, maltose binding protein (MBP) was fused to the 5' end of ce/E resulting in pSJ10. A plasmid containing MBP-cefE fusion genes for Nocardia lactambdurans (pSJ01 ) was also constructed. DNA sequence analysis confirmed all sequences.

Improved clones from the A, B and C library were recloned from the pCK- vectors into the pSJ10 vector (MBP-cefE behind the tac promoter). This was done by amplifying the improved expandases by PCR. The forward (5'-end) primer introduces a Nde\ and the reverse (3'-end) primer introduces a Λ/s/l site. After digestion with Nde\ and Λ/s/l the hit was cloned in the E. coli vector (pSJ10) and in the Penicillium chrysogenum vector (p IATWAn- Figure 2/3) Table 2.

Finally, a few clones of each were sequenced and one of each, containing the verified correct sequence, was used for further analysis.

The DNA and amino acid sequences of the best expandase hits are included in the sequence listing - see Table 2 for an overview.

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Induction of expression

E. coli NM554 was grown overnight on 2xTY medium with chloramphenicol (30 μg/ml) at 37 ⁰ C. The cells were diluted to an optical density at 540 nm (OD ₅₄₀ ) of 0.010-0.020 in fresh 2xTY with chloramphenicol (30 μg/ml). When cells reached an optical density at 540 nm of 0.4-0.6, IPTG was added (final concentration 0.5 mM) and expression was induced overnight at 27 ⁰ C.

The cells were washed and resuspended in extraction buffer (5 mM DTT, 50 mM Tris/HCI pH 7.5 with lysozym) and sonicated (on ice water). After centrifugation the supernatant is used for the secondary expandase assay (2).

Expression of the expandase in the cell free extract was determined by SDS-PAGE.

Table 2. H 100, H200 and H300 series expandase hits, their mutation(s). their E. coli and Penicillium expression vectors and their expandase activity (assay 2).

^* Specific activity (nmol 7-ADCA.min ^' .mg ^' protein)

^** I. F. = Improvement factor; equal the ratio of the specific activity of the mutant expandase and the specific activity of the expandase from S. clavuligeris

Example 6

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Expression of expandase H 122 and H 127 in Penicillium chrvsoaenum

Expandase variants H 122 and H 127 were cloned into vectors for Penicillium chrysogenum expression (plAT series plasmids, Table 2). Shuffled expandase genes cloned into a plasmid vector with an E. coli replicon and selectable marker. A promoter compatible with expression in P. chrysogenum, i.e., the IPNS promoter, was used to express the expandase gene. Additionally a selectable marker for Penicillium chrysogenum is present (amdS).

The expression constructs were obtained by digesting the expression construct, plAT127 (expandase H127) and plAT122 (expandase H122), with Notl. A high producing Penicillium chrysogenum is co-transformed with the linear fragments together with the amdS expression cassette (Hind\\\ digest).

By precursing with adipate, adipyl -7-ADCA production can be tested in strains expressing active expandase genes. The cephalosporin production of the strain expression expandase H 127 was compared to the strain expressing wild type expandase in batch regime with lactose as carbon source. The adipyl-7-ADCA production is increased between 10-50% and the total β-lactam production was increased by 0-30%, depending on the lactose and the adipate concentration. Additionally, expandase H 122 was expressed in Penicillium chrysogenum.

Comparison of the cephalosporin production with the strain expressing wild type expandase showed that the adipyl -7-ADCA production is increased between 0 to 50% and the total beta-lactam production varied between -20 to 0% depending on the lactose and the adipate concentration in the media.

Example 7

1 ^st generation improved expandases (H400 series).

The first generation expandase library was constructed by performing error prone (EP) PCR (Polymerase Chain Reaction) on wild type Streptomyces clavuligerus CefE gene (SEQ ID No.1 ). After screening this library for improved conversion of ad-6- APA to ad-7-ADCA, 3 different mutant genes were selected. The mutants exhibited an improved ad-6-APA expansion activity up to 2.5-fold (H401 , H402, H403: see Table 3).

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Example 8

2 ^nd generation improved expandases (H500 series)

The second-generation expandase library was constructed by using saturated Mutation Primer PCR (sMPP) exactly according to WO 03/010183. The selected mutant genes from the first generation (H401 , H402 and H403) were used as templates for the construction of the 2 ^nd generation library. The sMPP was performed by using Taq polymerase, thereby introducing additional mutations (random) and increasing the variation of the library. Designed primers annealed at the mutation positions and were saturated at the mutations found in the 1 ^st generation expandase hits. Additionally, a universal forward and reverse primer was designed in which an Nde\ and Λ/s/l site were introduced respectively. This facilitated cloning into Penicillium expression vector for testing of the mutant expandases in vivo.

The library was grown and expression of the expandase was induced as described in the materials and methods section. The formation of ad-7-ADCA was determined using NMR as described in the materials and methods section. Approximately 150 improved mutant expandases were selected and retested for their ad-6-APA expandase activity. Based upon the results of this retest, 17 mutants were selected for final tests in shake flasks and analysis by HPLC as described in the Materials and Methods.

In total, 15 of the 17 selected hits showed a significant improved expandase activity with ad-6-APA as a substrate. To exclude the false positive selection of expression mutants, protein levels were quantified on SDS-PAGE. This confirmed that the improved activity was not due to stronger expression of expandase in E. coli, but that the improvement could be attributed to an improved specific activity of the expandase mutant. In total, 8 mutant expandases were identified which exhibited an improvement factor of their ad-6-APA expandase up to 4,5-fold (compared to the wild type expandase of S. clavuligerus - SEQ ID No 1 .). These mutants are designated H501 - H508 (see Table 3).

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Example 9

3 ^rd generation of improved expandases (H600 series)

The 8 mutants obtained in the 2 ^nd generation Example 2 (H501 - H508) were used as templates for the construction of the 3 ^rd generation expandase library. This library was constructed in the same way as the 2 ^nd generation expandase library. Additionally, the SKA signaling sequence at the C-terminus of the expandases was changed to SKD, causing expression of expandase solely in the cytosol when expressed in Penicillium.

The screening of this library yielded 22 different expandase mutant genes that were significantly improved between 5-fold and 11 -fold compared to S. clavuligerus expandase (Table 3, H600 series).

Screening of the same library for a second time yielded 13 additional mutant expandases (G601 - G611 and G613 - G614; see Table 3)

Example 10

Activity of improved mutant expandases with iso-penicillin N (iPN) and Penicillin-G (Pen-G) as a substrate.

The activity of several mutant expandases was measured using iPN and Pen- G as a substrate. The assay with iPN as a substrate was carried out as described in the materials and methods section except that ad-6-APA was replaced by the same concentration of iPN (4 mM). The assay with Pen-G as a substrate was carried out as described in the materials and methods section except that ad-6-APA was replaced by 7 mM Pen-G. Table 3 summarizes the activities of the various expandase mutants for iPN and Pen-G.

Whereas most of the expandase mutants tested exhibited a 5-fold improvement of their expandase activity with iPN, mutants that carry the T89K mutation lost virtually all activity towards iPN.

The activity towards Pen-G of the various expandase mutants tested was either the same as the wild type expandase (improvement factor is 1 ) or improved up to 8-fold. The data further show that there is no correlation whatsoever between the improvement factors obtained for a single mutant with ad-6-APA and Pen-G as a substrate. The ratio between the respective improvement factors for ad-6-APA and

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Pen-G vary in a range from 0.1 (e.g. H654) to 1 .2 (e.g. H503).

Table 3. Mutant expandases and their expandase activity toward ad-6-APA, Pen-G and iPN. Each mutant is identified by its code; the mutations are introduced in the model expandase from S. clavuligerus (SEQ ID NO 1 ). The expandase activity is expressed as improvement factor compared to the model expandase from S. clavuligerus (expandase activity = 1 by definition) according to the in vitro expandase assays described in the Materials and Methods and Example 4.

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Table 3 shows that mutant expandases have been obtained which have improvement factors in the range of 1.5 to 10.6-fold.

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