MUTANT EXPANDASES

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).

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 are the condensation of the three amino acids L-5-amino-5-carboxypentanoic acid (L-α- aminoadipic acid) (A), L-cysteine (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

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 in prokaryotics 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 1.14.11.26 - 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 1.14.11.26 encoded by cefEF genes). 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. 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 2.5-fold improved in vitro expandase activity towards adipyl-6-APA in comparison with a 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. More preferably the in vitro expandase activity towards adipyl-6-APA of the mutant expandase is improved 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 11 -fold more preferably at least 12-fold, more preferably at least 13-fold, more preferably at least 14-fold, more preferably at least 15-fold more preferably at least 16- fold more preferably at least 17-fold more preferably at least 18-fold more preferably at least 19-fold, more preferably at least 20-fold. The invention also provides a mutant expandase which is preferably being modified at least at an amino acid position selected from the group consisting of positions 2, 59, 73, 89, 90, 99, 101, 105, 113, 155, 170, 177, 209, 213, 217, 244, 249, 251 , 277, 278, 280, 281 , 293, 300, 306, 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 the group consisting of positions 2, 59, 89, 90, 99, 105, 113, 170, 177, 209, 213, 217, 249, 251 , 278, 280 and 293. Most preferably, the mutant expandase is modified at least at an amino acid position selected from the group consisting of positions 2, 89, 90, 99, 105, 177, 251 , 278, 280 and 293.

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 sequence of the natural expandase enzyme. Most preferably, the invention provides a mutant expandase whereby the mutant expandase has an at least 2.5-fold improved in vitro expandase activity towards adipyl-6-APA in comparison with a model polypeptide with expandase activity, 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 11 -fold more preferably at least 12-fold, more preferably at least 13- fold, more preferably at least 14-fold, more preferably at least 15-fold more preferably at least 16-fold more preferably at least 17-fold more preferably at least 18-fold more preferably at least 19-fold, more preferably at least 20-fold and whereby the mutant expandase is preferably being modified at at least an amino acid position selected from the group consisting of positions 2, 59, 73, 89, 90, 99, 101 , 105, 113, 155, 170, 177, 209, 213, 217, 244, 249, 251, 277, 278, 280, 281, 293, 300, 306, 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. More preferably, the mutant expandase is modified at least at an amino acid position selected from the group consisting of positions 2, 59, 89, 90, 99, 105, 113, 170, 177, 209, 213, 217, 249, 251 , 278, 280 and 293. Most preferably, the mutant expandase is modified at least at an amino acid position selected from the group consisting of positions 2, 89, 90, 99, 105, 177, 251 , 278, 280 and 293.

The activity of expandase can be measured under various conditions. The concentration of the substrate (e.g. adipyl-6-APA) in the activity assay determines whether or not the expandase activity is measured under conditions wherein the V _max determines the activity (in case the substrate concentration is much higher, e.g. 5-20 fold than the K _M of the expandase for its substrate) or under conditions wherein in addition also the K _M is determining the activity of the expandase (in case the substrate concentration is roughly equal to or (much) lower - e.g. <0,05 - 0.2-fold - than the K _M of the expandase for its substrate). The improvement factor of the mutant expandases may be measured under these various conditions.

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 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 ! The present invention preferably provides mutant expandases that have modifications at least at o 1 or more amino acid positions selected from the group consisting of 2, 59, 73,

89, 90, 99, 101, 105, 113, 155, 170, 177, 209, 213, 217, 244, 249, 251 , 277, 278, 280, 281 , 293, 300, 307 and 311 o 1 or more amino acid positions selected from the group consisting of 2, 59, 89,

90, 99, 105, 113, 170, 177, 209, 213, 217, 249, 251 , 278, 280 and 293 o 1 or more amino acid positions selected from the group consisting 2, 89, 90, 99,

105, 177, 251 , 278, 280 and 293. 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+311 or 73+89+281+311 or 89+217+281+311 or 89+244+281+311 or 89+281+307+311 or 277+281+306+311 or 89+281+306+311 or 90+281+306+311. 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 311, more preferably at positions 2+89+281+306+311 or 2+155+281+306+311 or 73+89+213+281+311 or 89+78+218+307+311, 89+281+293+300+311 or 89+249+281+307+311. o 6 amino acid positions selected from the group consisting of

90+105+113+281+306+311. o 7 or more amino acid positions selected from the group consisting of 2, 73, 89,

105, 113, 155, 170, 177, 251, 277, 280, 281, 293, 300, 306, 307 and 311 , more preferably 73+89+281+293+300+307+311 or

105+113+155+177+281+306+311 or 155+177+277+280+281+306+ 311. 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+113+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+311. o 10 amino acid positions selected from the group consisting of 2, 59, 90, 101, 105, 113, 155, 177, 209, 277, 281 , 307 and 311 more preferably at

2+90+105+113+155+177+277+281+306+311. o 11 amino acid positions selected from the group consisting of 2, 90, 105, 113,

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

The invention furthermore provides mutant expandases as defined hereinbefore that are variants of the expandase of Streptomyces clavuligerus, the mutant expandase being modified at an amino acid position selected from the group consisting of positions D2, S59, M73, T89, N90, M99, Y101, T105, G113, C155, H170, P177, G209,

T213, Y217, H244, R249, L277, E280, C281 , T293, G300, R306, R307 and A311 whereby the letter preceding the number denotes the respective amino acid (one letter code) and the subsequent number the amino acid position in the amino acid sequence of the expandase enzyme of Streptomyces clavuligerus which is depicted in SEQ ID

NO: 1.

Preferred mutant expandases that are variants of the expandase of Streptomyces clavuligerus have modifications at least at o 1 or more amino acid positions selected from the group consisting of D2, S59,

M73, T89, N90, M99, T105, G113, C155, H170, P177, G209, T213, Y217, H244, R249, D251, L277, A278, E280, C281, T293, G300, R306, R307 and A311 o 1 or more amino acid positions selected from the group consisting of D2, S59, T89, N90, M99, T105, G113, H170, P177, G209, T213, Y217, R249, D251 ,

A278, E280 and T293 o 1 or more amino acid positions selected from the group consisting D2, T89,

N90, M99, T105, P177, D251 , A278, E280 and T293. 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 A311 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 A311 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 A311 o 6 amino acid positions selected from the group consisting of

N90+T105+G113+C281 +R306+A311. o 7 or more amino acid positions selected from the group consisting of D2, M73, T89, T105, G113, 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, G113, 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,

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

G113, C155, P177, L277, C281 , R306, R307, A311.

Preferred modifications at the respective positions in the expandase of Streptomyces clavuligerus are the following (using the one-letter code for amino acids) • D at position 2 according to SEQ ID NO 1 replaced by hydrophilic amino acids Y, N, H, D, Q, E, K, R, S or T, preferably by Y, N, Q or H. Most preferred are replacements D2Y, D2N and D2H.

• M at position 73 according to SEQ ID NO 1 replaced by A, V, I, L, N, Q, H, K or R, preferably N, Q, H, L or I. Most preferred are replacements M73H and M73I.

• T at position 89 according to SEQ ID NO 1 replaced by K, R, A, C, P, V, I, L, or M, preferably A, V, I, L, R or K, more preferably A, V or K. Most preferred is replacement T89K.

• N at position 90 according to SEQ ID NO 1 replaced by H, R, K, N, Q, W, S, C, T or Y, preferably K, R, N, S, W. Most preferred are replacements N90W and N90S.

• M at position 99 according to SEQ ID NO 1 replaced by A, R, K, Q, E, N, D, S or T. more preferably K, Q, E or T. Most preferred is replacement M99T. • T at position 105 according to SEQ ID NO 1 replaced by A, V, I, L, R, K, H, Q or N, preferably by V, I, L, R or K. Most preferred are replacements T105I and T105K.

• G at position 113 according to SEQ ID NO 1 replaced by A, P, Q, K, R, E, D or N, more preferably A, D, E, Q, K or R. Most preferred is replacement G113D.

• C at position 155 according to SEQ ID NO 1 replaced by A, N, S, T, V, W, more preferably A, S, T, V or W. Most preferred is replacement C155W.

• P at position 177 according to SEQ ID NO 1 replaced by K, E, L, A, V, T, I, more preferably L, A, V, T, I. Most preferred are replacements P177L and P177I.

• T at position 213 according to SEQ ID NO 1 replaced by Q, G, A, V, I, R, S, more preferably G, A, V, I, S. Most preferred is replacement T213A. • Y at position 217 according to SEQ ID NO 1 replaced by A, V, H, P, T, N or Q, more preferably A, P, N, Q or H. Most preferred is replacement Y217H.

• H at position 244 according to SEQ ID NO 1 replaced by N, Q, K or R, more preferably K or R. Most preferred is replacement H244R.

• R at position 249 according to SEQ ID NO 1 replaced by G, S, T, N, D, Q, E, K, R, H or C, more preferably G, S, D, N or C. Most preferred is replacement R249C.

• D at position 251 according to SEQ ID NO 1 replaced by G.

• L at position 277 according to SEQ ID NO 1 replaced by E, A, Q, K, R, H or T more preferably Q, E, K, R, H, T. Most preferred are replacements L277Q, L277T and L277K • E at position 280 according to SEQ ID NO 1 replaced by G, A, Q, K, R or H, more preferably G, Q or R. Most preferred is replacement E280G.

• C at position 281 according to SEQ ID NO 1 replaced by Y, S, L, W, A, S, T or H, more preferably A, S, T, W, H or Y. Most preferred is replacement C281 Y.

• T at position 293 according to SEQ ID NO 1 replaced by S, D, N, K or R, more preferably S, K or R. Most preferred is replacement T293K.

• G at position 300 according to SEQ ID NO 1 replaced by A, S, T, N, D, Q, E, K, R, H or L, more preferably A, S, T, K, R, H or L. Most preferred are replacements G300S, G300K and G300L.

• R at position 306 according to SEQ ID NO 1 replaced by H or a deletion. • R at position 307 according to SEQ ID NO 1 replaced by T, K, A, S, V, G, D, N, Q, E, R, H, I or a deletion, more preferably S, T, K, R, H, I or a deletion. Most preferred are replacements R307T, R307I and R307deletion.

• A at position 311 according to SEQ ID NO 1 replaced by D.

Highly preferred mutant expandases are the mutant expandases that are summarized in Table 3 and 4. Preferably, the mutant expandases provided by the present invention have an at least 2.5-fold improved expandase activity on adipyl-6- APA as defined before. Alternatively, the mutant expandases provided by the present invention have an improved expandase activity on Pen-G of at least 1.5-fold in comparison with a model polypeptide with expandase activity, 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. 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. Preferably, the mutant expandases provided by the present invention have an improved expandase on both adipyl-6-APA as well as Pen-G as defined hereinbefore.

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 producing adipyl-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 positively charged amino acid such as lysine, arginine or histidine; most preferred being lysine. A highly preferred mutant expandase is selected from the group consisting of

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, G611 , G613, G614, H701, H702, H703, H704, H705, H706 (see Table 3 and 4).

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. 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. 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. 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. A highly preferred mutant expandase is selected from the group consisting of 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, G611 , G613, G614, H701 , H702, H703, H704, H705, H706 (see Table 3 and 4). 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 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 cephalorin from a corresponding penicillin whereby the expansion of the 5-membered thiazolidine ring of the penicillin to the 6-membered dihydrothiazine ring of the cephalorin 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 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 (Gδttingen, Germany) or by Baseclear (Leiden, The Netherlands).

• Restriction enzymes were purchased from Invitrogen.

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

• Escherichia coli DH 1OB 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

• To create the CefE expression vector, the ampicillin selection marker in pBAD/Myc- His (commercially available from Invitrogen™) is replaced by the zeozin selection marker. Additionally, the maltose binding protein is fused to the wild type S. clavuligerus expandase gene and ligated behind the pBAD promoter resulting in the vector pBAD-MH-Zeo-MBP-ScEwt. The wild type CefE is exchanged by the library of error prone mutants.

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°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°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 -80 ⁰ C as glycerol stock. The deep well plate is incubated for 30 hrs at 25°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 -20 ⁰ C.

Cell free extract (CFE) The frozen pellets obtained in the previous section are thawed in 200μl extraction buffer (50 rtiM Tris/HCI pH=7.5; 5 rtiM DTT; 0.1 mg/ml DNAse; 5 rtiM 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.

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°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°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°C at 220 rpm. The cultures are centrifuged, the supernatant is discarded and the pellet is frozen at -20 ⁰ C.

Cloning of the E. coli expression vectors

The fusion product is ligated in the pBAD/MH MBP-ScEwt Dest zeo-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\.

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

4 Expandase assay

A total of 300 μl of a reaction mixture consisting of 50 rtiM Tris/HCI pH 7.5; 1 rtiM DTT; 2.7 rtiM Ascorbate; 0.05 rtiM ATP; 24 rtiM α-ketoglutarate; 0.06 rtiM FeSO ₄ .7H ₂ O; 4 rtiM adipyl-6-amino-penicillanic acid (ad-6-APA) was added to 75 μl CFE and incubated at 29°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. Under these conditions, the expandase activity is measured close to V _max conditions since the K _M of the S. clavuligeris wild type enzyme for ad-6-APA is ca 0.4 mM. Alternatively, the assay may be carried out using 0.4 mM ad-6-APA (~ K _M of the S. clavuligeris wild type enzyme for ad-6-APA).

The adipyl-6-APA was obtained from 6-APA and adipic acid catalyzed by glutaryl 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

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).

Example 2

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.

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).

Example 3

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 using Pen-G at 7 rtiM as a substrate yielded 13 additional mutant expandases (G601-G611 and G613-G614; see Table 3)

Example 4

4 ^rd generation of improved expandases (H700 series) Ten mutants obtained in the 3 ^rd generation Example 3 (G606, G612, H602, H606, H650, H651, H653, H655, H658, H661) were used as templates for the construction of the 4 ^th generation expandase library. This library was constructed in the same way as the 3 ^rd generation expandase library.

The screening of this library yielded 6 different expandase mutant genes that were significantly improved (up to 20-fold) when tested for their expandase activity on ad-6-APA at 0.4 rtiM concentration (Table 4, H701-H706).

Example 5

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 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.

Table 3 shows that 46 mutant expandases have been obtained which have improvement factors in the range of 1.5 to 10.6-fold.

Table 4