BRPI0613004A2 - mutant expandase, polynucleotide, expression vector or cassette, host cell, method of producing a mutant expandase, and method of producing an α-lactam compound of interest - Google Patents

Abstract

MUTANT EXPANDASE, POLYNUCLEOTIDE, VECTOR OR EXPRESSION CASSETTE, HOST CELL, METHOD OF PRODUCING A MUTANT EXPANDASE AND METHOD OF PRODUCTING A <225> -LACTAMIC COMPOUND OF A INTEREST A variant of a polymorphic variant with expandase activity where the mutant expandase has at least 2.5-fold increased in vitro expansion for adipil-6-APA compared to a model polypeptide with expandase activity.

Description

MUTANT EXPANDASE, POLYNUCLEOTIDE, VECTOR OR EXCESSION CASSETTE, HOST CELL, METHOD OF PRODUCING A MUTANT EXPANDASE AND METHOD OF PRODUCING A β-LACMIC COMPOUND OF INTEREST

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

Beta-lactam antibiotics are the most important group of antibiotic compounds with a long history of clinical use. Within this group, prominences are penicillins and cephalosporins. Penicillins are naturally produced by various filamentous fungi such as Penicillium (eg P. chrysogenum). Cephalosporins are naturally produced by various microorganisms such as Acremonium (eg A.chrysogenum) and Streptomyces (eg Streptomycesclavuligerus).

As a result of classical lineage enhancement techniques, antibiotic production levels in P. chrysogenum and A. chrysogenum have improved markedly over the past decades. With the growing knowledge of biosynthetic pathways leading to cephalosporin penicillinase and the advent of recombinant DNA technology, new tools for improving production lines have become available. Most of the enzymes involved in β-lactam biosynthesis have been identified and their corresponding genes cloned, such as can be found in Ingolia eQueener, Med Res See (1989) 9: 245-264 (biosynthesis route and enzymes) and Aharonowitz, Cohen and Martin, Ann See Microbiol (1992) 46: 461-495 (gene cloning).

The first two steps in penicillin biosynthesis in P. chrysogenum are the condensation of three amino acids L-5-amino-5-carboxypentanoic acid (La-aminoadipic acid) (A), L-cysteine (C) and L-valine (V) notripeptide LLD-ACV, followed by cycling of this tripeptide to form isopenicillin N. This compound contains the typical β-lactam structure.

The third step involves the replacement of the hydrophilic L-5-amino-5-carboxypentanoic acid chain-side chain by the side chain by the action of the enzyme acyltransferase (AT).

In EP-A-044 818 O it has been described that the AT-mediated enzymatic disubstitution reaction is performed within a cell organelle, the micro-body. Observation that substantial quantities of deacetoxycephalosporin C (DAOC) may be formed by non-precursor P. chrysogenum transformants expressing deacetoxycephalosporin C synthase (EC 1.14.20.1 - hereinafter referred to as "expand") implies the presence of significant amounts of penicillin Ν, the substrate. natural for expandase in P.chrysogenum (Alvi et al., J. Antibiot (1995) 48: 338-340). However, the DAOC-Da-adipyl side chains cannot be easily removed.

Cephalosporins are much more expensive than penicillins. One reason is that some cephalosporins (eg cephalexin) are made from penicillins by a number of chemical conversions. Another reason is that until then only cephalosporins with a D-α-amino-adipyl side chain could be fermented. Cephalosporins C, certainly the most important starting material in this respect, is very soluble in water at any pH, thus implying costly long isolation processes using expensive and difficult-to-handle column technology. Cephalosporin C obtained in this manner was to be converted to therapeutically used cephalosporins by a number of chemical and enzymatic conversions.

Currently preferred methods in the industry for preparing the 7-amino-deacetoxyicephoporoporic acid intermediate (7-ADCA) involve complex chemical steps leading to the expansion and derivation of penicillin G. One of the chemical steps required to produce 7-ADCA involves the expansion of penicillin ring structure 5-membered to a 6-membered cephalosporin ring structure (see, for example, US 4,003,894). This complex chemical processing is both costly and harmful to the environment.

Consequently, there is a strong desire to replace chemical processes with enzymatic reactions such as enzymatic catalysis, preferably during fermentation. A key to replacing the chemical expansion process with a biological process is the central enzyme in the cephalosporin viabiosynthetic expandase.

The enzyme expandase of the bacterium Streptomycesclavuligerus (S. clavuligerus) has been found to perform, in some cases, expansions of the depenicillin ring. When introduced into P. chrysogenum, it can convert the structure of the penicillin ring to the cephalosporin ring structure, as described in Cantwell et al. Proc Proc Soc Lond B (1992) 248: 283-289. The well-characterized expandasefying enzyme (EP-A-0366354) was both biochemically and functionally, as was its corresponding gene. Both physical maps of the cefE gene (the gene encoding S. clavuligerus aenzyme expandase - EP-A-0341892), sequence of DNA and transformation studies on P.chrysogenum with cefE have been described. The amino acid DNA sequence of the S. clavuligerus expandase enzyme is shown in SEQ ID NO 1.

Another source of an expandase enzyme is the bacterium Nocardia lactamdurans (N. Iactamdurans, formally S.Iactamdurans). Both the biochemical properties of the DNA sequence enzyme and the gene encoding the enzyme have been described - see Cortes et al. , J Gen Microbiol (1987) 133: 3165-3174 and Coque et al., Mol Gen Genet (1993) 236: 453-458, respectively.

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

Table 1: Amino acid sequence identities of various expandases (data taken from Hsu et al.). <table> table see original document page 6 </column> </row> <table>

In the biosynthesis of cephalosporins in prokaryotic cells, deacetoxycephalosporin-C is subsequently converted to deacetyl cephalosporin-C by the enzyme deacetylcephalosporin C synthase also called dedeacetoxycephalosporin-C hydroxylase or hydrolase (EC1.14.11.26 - D) Genes encoding such hydroxylases are called cefE genes (for example, see Hsu et al.). Asexpandases found in eukaryotic cells, for example, Acremonium chrysogenum may catalyze the direct conversion of penicillin N to deacetoxycephalosporin-C due to the presence of both expandase and hydrolase activity, henceforth called cefEF (see Hsu et al.).

As defined herein, the term expandase relates to expandase enzymes (EC 1.14.20.1, encoded by genescefE) as well as expandases also processed in addition to hydrolase activity (EC 1.14.20.1 + EC 1.14.11.26coded by cefEF genes).

Since the expandase enzyme catalyzes the expansion of the 5-membered thiazolidine ring from penicillin N to the 6-membered DAOC aneldihydrothiazine, this enzyme may, of course, be a logical local candidate to replace the expansion ring of chemical processes. Unfortunately, the enzyme works in the penicillin N intermediate in the cephalosporin viabiosynthetic pathway, but not or very poorly in the readily available penicillins not expensive as those produced by P. chrysogenum, comopenicillin V or penicillin G. Penicillin N is not commercially available and even when expanded, its lateral chain. Da-amino adipyl cannot easily be removed by penicillin acylases.

The expandase enzyme has been reported to be able to expand penicillins with corresponding non-derivative 7-ADCA side chains. This feature of the expansion has been exploited in technology as disclosed in EP-A-0532341, W095 / 04148 and W095 / 04149. In these disclosures the in vitro chemical conversion of penicillin G to 7-ADCA has been replaced by the in vivo conversion of certain 6-aminopenicillanic acid derivatives (6-ΑΡΑ) into Penicilliumchrysogenum 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 an adipil side chain (hereinafter referred to as adipil) as a food stock, which is a substrate for the P acyltransferase enzyme. .chrysogenum. This leads to the formation of adipil-6-ΑΡΑ, which is converted by an expandase enzyme introduced in P. chrysogenum alignment to produce adipil-7-ADCA. Finally, removal of the adipil side chain is described, producing 7-ADCA Omo an end product. . In addition, EP-A-540210 teaches the similar production of adipil-7-ADAC and adipil-7-ACA.

As an alternative approach, expansion of penicillin G using cefE transformed P. chrysogenum was proposed in EP0828850. In addition, in order to improve penicillin G expansion, the use of S. clavuligerus mutant genecefE has been described. US5,919,680, WO 98/02551, WO 99/33994, WO 01/85951 and EP134 8759 all disclose mutant cefE genes allegedly encoding enzymes having a higher expandase activity on penicillin G.

However, these mutant expandases still do not provide a commercially attractive process for cephalosporin production.

Therefore, there is a need for further process improvement where cephalosporins are prepared from adipil-APA expansion. This process is characterized by efficient removal of the side chain material from the dacephalosporin core material. One step to further improve this process is to improve sobreadipil-6-de expansion activity.

In a first aspect, the invention provides a mutant expansion which is a variant of a polypeptide model with expandase activity. Preferably, the invention provides a mutant expandase, wherein the mutant expandase has at least 2.5-fold increased in vitro expandase activity for adipyl-6-ΑΡΑ as compared to a model polypeptide with expandase activity. Determination of in vitro expandase activity for adipil-6-APA is described in detail in the Materials and Methods. More preferably, the in vitro expandase activity for mutant expandase paraadipyl-6-expand is improved by at least 3-fold, more preferably at least 4-fold, more preferably at least 5-fold, more preferably at least 6-fold. times, more preferably at least 7 times, more preferably at least 8 times, more preferably at least 9 times, more preferably at least 10 times, more preferably at least 11 times, more preferably at, at least 12 times, more preferably at least 13 times, more preferably at least 14 times, more preferably at least 15 times, more preferably at least 16 times, more preferably at least 17 times more preferably at least 18 times, more preferably at least 19 times, more preferably at least 20 times. This invention also provides a mutant expandase which is preferably being modified at least in 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 sequence numbering of the expanded enzyme encoded by the Strptomyces clavuligerus cefE gene. Consequence of the Strptomyces clavuligerus cefE gene as well as the amino acid sequence encoded by said cefE gene are described 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, 277, 278, 280 and 293.

More preferably, the mutant expandase is modified at least one amino acid position selected from the group consisting of positions 2, 89, 90, 99.105, 177, 251, 278, 280 and 293.

By "altered or mutated expandase" in the context of the present invention is meant that any enzyme having expandase activity, which has not been obtained from a natural source and from which the amino acid sequence differs from the full amino acid sequence of the natural expandase enzyme.

More preferably, the invention provides a mutant expandase wherein the mutant expandase has at least 2.5-fold increased in vitro activity for adipyl-6-APA compared to a template polypeptide with expandase activity, more preferably at least 3-fold higher. preferably at least 4 times, more preferably at least 5 times, more preferably at least 6 times, more preferably at least 7 times, more preferably at least 8 times, more preferably at least 9 times times, more preferably at least 10 times, more preferably at least 11 times, more preferably at least 12 times, more preferably at least 13 times, more preferably at least 14 times, more preferably at least 15 times, more preferably at least 16 times, more preferably at least 17 times, more preferably at least 18 times, more preferably, p at least 19 times, more preferably at least 20 times and where the mutant expander is preferably being modified by at least one 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 amino acid sequence position enumeration of the enzyme cpE encoded by the Strptomycesclavuligerus cefE gene . 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. More preferably, the mutant expander is modified at least at an amino acid position selected from the group consisting of depositions 2, 89, 90, 99, 105, 177, 251, 278, 280 and 293.

Expansion activity can be measured under various conditions. The substrate concentration (eg adipil-6-ΑΡΑ) in the activity assay determines the activity of the expandase is measured under conditions where the maxmax determines the activity (if the substrate concentration is much higher, eg 5-20 times, than the Km of the expansion to its substrate) or under conditions where in addition the Km is determinant of. expandase activity (the case of substrate concentration will be approximately equal to or (much) below - for example, <0.05 - 0.02 times - than the Km of expandase for its substrate). The enhancement factor of mutant expandases can be measured under these various conditions.

Modification at an amino acid position may comprise a substitution for another amino acid selected from the group consisting of 20 L-amino acids that occur in nature - see Table 2. Alternatively, modification of the amino acid position may comprise a deletion of the amino acid in said position. Further, modification to an amino acid position may comprise a substitution of one or more amino acids on the C-terminal or N-terminal side of said amino acid.

Table 2

<table> table see original document page 12 </column> </row> <table> <table> table see original document page 13 </column> </row> <table>

The model peptide with expandase activity as used in the present invention is selected from the group consisting of a polypeptide with an expanding activity, preferably having an amino acid sequence according to SEQ ID NO: 1 and polypeptides with an expanding activity having an amino acid sequence with a percent identity with an identity percentage of at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, such as the expansion enzymes that are summarized in Table 1.

The present invention preferably provides mutant pandases having modifications at least 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 and 311.

1 or more selected amino acid positions from the group consisting of 2, 59, 89, 90, 99, 105, 113, 170, 177,209, 213, 217, 249, 251, 278, 280 and 293. • 1 or more selected amino acid positions of the group consisting of 2.89, 90, 99, 105, 177, 251, 278, 280, and 293.

• 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.

• 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 or89 + 281 + 293.

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 or89 + 217 + 281 + 311 or 89 + 244 + 281 + 311 or 89 + 281 + 307 + 311 OU277 + 281 + 306 + 311 OR 89 + 281 + 306 + 311 OR 90+ 281 + 306 + 311.

• 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 2 + 89 + 281 + 306 + 311 or 2 + 155 + 281 + 306 + 311 or73 + 89 + 213 + 281 + 311 OR 89 + 78 + 218 + 307 + 311, 89 + 281 + 293 + 300 + 311 or 89 + 249 + 281 + 307 + 311.

• 6 amino acid positions selected from the group consisting of 90 + 105 + 113 + 281 + 306 + 311.

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.

• 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 nas2 + 90 + 99 + 105 + 113 + 281 + 306 + 311 or2 + 90 + 105 + 113 + 155 + 177 + 277 + 281.

• 9 amino acid positions selected from the group consisting of 2 + 90 + 105 + 113 + 155 + 177 + 281 + 306 + 311.

• 10 amino acid positions selected from the group consisting of 2, 59, 90, 101, 105, 113, 155, 177, 209,277, 281, 307 and 311, most preferably nas2 + 90 + 105 + 113 + 155 + 177 + 277 + 281 + 306 + 311.

• 11 amino acid positions selected from the group consisting of 2, 90, 105, 113, 155, 177, 277, 281, 293,307, 311.

The invention further provides mutant expandases as defined hereinbefore which are variants of the Streptomyces clavuligerus expander, the mutant expandase being randomized to the amino acid positions 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 where the preceding letter of the number denotes the respective amino acid (one letter code) and the subsequent number deposition of the amino acid sequence of the Streptomyces clavuligerus expandase enzyme which is described in SEQ IDNO: 1.

Preferred mutant expandases which are variants of Streptomyces clavuligerus expansion have modifications at least

• 1 or more selected amino acid positions from the group consisting of D2, S59, M73, T89, N90, M99, T105, G113, C155, Η170, Ρ177, G209, Τ213, Η224, R249, D251, L277, Α27 8, Ε280, C281, Τ293, G300, R306, R307 and Α311.

• 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.

• 1 or more amino acid positions selected from the group consisting of D2, T89, N90, M99, T105, P177, D251, A278, E280 and T293.

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

• 3 or more amino acid positions selected from the group consisting of D2, T89, C281, T293 and Ali.

• 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.

• 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.

6 or more amino acid positions selected from the group consisting of N90 + T105 + G113 + C281 + R306 + A311.

• 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.

• 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.

• 9 or more amino acid positions selected from the group consisting of D2 + N90 + T105 + G113 + C155 + P177 + C281 + R306 + A311.

• 10 or more amino acid positions selected from the group consisting of D2, S59, N90, T105, G113, T105, G113, C155, P177, G2 09, L277, C281, R306, R307, A311.

11 or more amino acid positions selected from the group consisting of D2, N90, T105, G113, C155, P177, L277, C2 81, R3 06, R307 and A311.

Preferred modifications to respective positions in Streptomyces clavuligerus naexpandase are as follows (using the one-letter amino acid code)

• D at position 2 according to SEQ ID NO 1 substituted by hydrophilic amino acids Υ, Ν, H, D, Q, E, K, R, S, or T, preferably Y7 N, Q, or H. Most preferred are substitutions D2Y, D2N and D2H.

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

• T at position 89 according to SEQ ID NO 1 substituted by K, R, A, C, Ρ, V, I, L or M, preferably by A, V, I, L, R or K. Most preferably AV or K. Most preferred is the T89K substitution.

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

M at position 99 according to SEQ ID NO 1 substituted by A, R, K, Q, Ε, N, D, S or T, preferably K, Q, E or T. Most preferred is M99T substitution.

T at position 105 according to SEQ ID NO 1 substituted by A, V, I, L, R, Κ, H, Q or N, preferably V, I, L, R or K. More preferred are substitutions 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 G113D substitution.

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

• P at position 177 according to SEQ ID NO 1 replaced by Κ, E, L, A, V, Τ, I, more preferably by L, A, V, Τ, I. More preferred are substitutions P177L and P177I.

T at position 213 according to SEQ ID NO 1 replaced by Q, G, A, V, I, R, S, more preferably by G, A, V, I, S. Most preferred is substitution T213A.

Y at position 217 according to SEQ ID NO 1 substituted by A, V, Η, Ρ, Τ, N or Q, more preferably by A, Ρ, N, Q or H. More preferred is Y217H substitution.

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

• R in position 249 according to SEQ ID NO 1 replaced by G, S, Τ, N, D, Q, Ε, K, RH or C, more preferably G, S, D, N or C. Most preferred is substitution. • D at position 251 according to SEQ ID NO 1 replaced by G.

• L at position 277 according to SEQ ID NO 1 substituted by E, A, Q, K, R, H or T, more preferably Q, Ε, K, R, Η, T. Most preferred are L277Q, L277T and L277K substitutions. .

E is 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 the substitution E280G.

C in position 281 according to SEQ ID NO 1 substituted by Y, S, L, W, A, S, T or H, more preferably A, S, Tf W, H or Y. More preferred is C281Y substitution.

• T at position 293 according to SEQ ID NO 1 substituted by S, D, Ν, K or R, more preferably S, K or R. Most preferred is the substitution T293K.

• G in position 300 according to SEQ ID NO 1 substituted by A, S, Τ, N, D, Q, Ε, K, R, H or L, more preferably A, S, Τ, K, R, H or L More preferred are G300S, G300K and G300L replacements.

• R in position 306 according to SEQ ID NO 1 replaced by H or a deletion.

R in position 3 07 according to SEQ ID NO 1 substituted by Τ, K, A, S, V, G, D, N, Q, E, R 1 Η, I or a deletion, more preferably S, Τ, K, R, Η, I or a deletion. Most preferred are substitutions R307T, R307I and R307.

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

Highly preferred expandases are the mutant expandases which are summarized in Tables 3 and 4. Preferably, the mutant expandases provided by the present invention have an at least 2.5-fold improvement in the above-defined adipyl-6-ΑΡΑ activity. Alternatively, the mutant expandases provided by the present invention have an improvement in Pen-G expandase activity of at least 1.5-fold compared to a template polypeptide with expanding activity, more preferably at least 3-fold, more preferably at least , 4 times, more preferably at least 5 times, more preferably at least 6 times, more preferably at least 7 times, more preferably at least 8 times. Mutant expandases with improved Pen-G activity can be advantageously used in a process for the production of defenylacetyl-7-ADCA as described below. Preferably, mutant expandases provided by the present invention have improved expandase on both adipyl-6-ΑΡΑ as well as Pen-G. G is as previously defined herein.

The present invention also provides mutant expandases with decreased or even absent comiso-penicillin N (iPN) expandase activity. Preferably the activity expanding with either adipil-6-ΑΡΑ or Pen-G is unaffected, but more preferably the expansionase activity with tantoadipil-6-ΑΡΑ or Pen-G or both is improved as hereinbefore defined. The advantage of these more preferred mutant expandases is that the decrease or even absence of isase-penicillin N (iPN) expandase activity results in fewer byproducts in a fermentation process producing adipyl-7-ADCA or phenylacetyl-7-ADCA.

Preferred mutant expandases have decreased or even absent iso-penicillin N (iPN) activity as a substrate optionally combined with enhanced ad-6-expand expandase activity as a substrate has been modified at position 89 according to the amino acid numbering of SEQ ID No. 1 where the naturally occurring amino acid has been substituted, preferably by a positively charged amino acid such as lysine, arginine or histidine; more preferably being lysine.

A highly preferred mutant expandase is selected from a 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, H657, H658, H658, H660, H661, H662, G601, G602, G604, G605, G606, G607, G608, G606, G609 G611, G613, G614, H701, H702, H703, H704, H705, H706 (see Tables 3 and 4).

In a second aspect, the invention provides a polynucleotide encoding the mutant expandase of the present invention. The polypeptide encoding the mutant expandase according to the present invention may be any polynucleotide encoding the amino acid sequence itself according to the invention. Alternatively, the opolinucleotide of the invention may comprise a coding sequence in which the use of codon for various amino acids deviates from the use of codon in S. clavuligerus. For example, codon usage may be adapted for codon usage of a particular host cell, which will be or has been transformed as a DNA fragment encoding the altered expandase.

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

In a fourth aspect, the invention provides a transformed host cell transformed with the invention polynucleotide or the 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 mutant expanders of the invention are preferably host cells which are known in the art for efficient production of their protein or enzyme, either extracellular or intracellularly, for example microorganisms such as fungus, yeast and bacteria. Examples of preferred host cells include, but are not limited to, the following genera: Aspergillus (eg, A. niger, A. oryzea), Penicillium (eg, P. emersonii, P. ehrysogenum), Saccaromyces (eg, S. cerevisiae), Kluyveromyces (eg K. laetis), Baeillus (eg B. subtilis, B. ihenheniformis, B. amyloliquefaeiens). Seheriehia (E. eoli), Streptomyees (eg S.elavuligerus).

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

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

In a sixth aspect, the invention provides a process for the production of a beta-lactam compound of interest comprising culturing the transformed host cell according to the invention comprising culturing the transformed host cell according to the invention under conditions which contribute 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 producing phenylacetyl-7-ADCA or adipyl-7-ADCA by cultivating a selected Penicillium chrysogenum strain, which has been transformed with an inventive polynucleotide sequence encoding 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, H658, H658, H659, H660, H661, H662, G601, G603, G604, G605, G606, G607, G608, G609, G610, G610 G613, G614, H701, H702, H703, H704, H705, H706 (see Tables 3 and 4). For the production of phenylacetyl-7-ADCA, a mutantexpandase is selected that has a high improvement factor in Pen-G as a substrate. For deadipil-7-ADCA production, a mutant expandase is selected that has an improvement factor in ad-6-ΑΡΑ as a substrate.

In another preferred embodiment, the invention provides a process for the production of 7-ADCA comprising the process for producing phenylacetyl-7-ADCA or adipyl-7-ADCA as described hereinbefore, followed by, after further purification of said 7-ADCA derivatives, a process step in which the phenylacetyl or adipyl defenylacetyl-7-ADCA and adipyl-7-ADCA side chain respectively are cleaved off then generating 7-ADCA and the liberated acid of the side chains. Said cleavage may be obtained by chemical means or more preferably enzymatically using an acylase enzyme. Acyls suitable for adipyl side chain cleavage are obtainable from several Pseudomonastal species such as Pseudomonas SY-77 or Pseudomonas SE-83. Suitable acylases for cleavage of the phenylacetyl side chains are penicillin acylases from Escherichia coli or Alcaligenes feacalis.

In a seventh aspect, the invention provides for the production of a cephalosporin from the corresponding penicillin where expansion of the penicillin 5-membered thiazolidine ring to the cephalosporin 6-membered dihydrothiazine ring occurs in an in vitro process catalyzed by a mutant expandase of the invention. In a preferred process, adipyl-6-ΑΡΑ is expanded to corresponding adipil-7-ADCA by a mutant expandase of the invention, preferably a mutant expandase that has a high improvement factor adipil-6-ΑΡΑ as a substrate. In another preferred process, Pen-G is expanded to phenylacetyl-7-ADCA corresponding to a mutant expandase of the invention, preferably a mutant expandase having a high improvement factor adipyl-6-ΑΡΑ as a substrate. The process may be followed by, after optional purification of said 7-ADCA derivatives, a process step in which adipyl-7-ADCA and phenylacetyl-7-ADCA side chains are cleaved off generating 7-ADCA and released side chain acids - see supra. The desired 7-ADCA end product may be recovered according to methods known in the art involving chemical or enzymatic cleavage of the adipyl-7-ADCA or phenylacetyl-7-ADCA side chain and optionally the resulting 7-ADCA may be further purified and / or crystallized.

In an eighth aspect, the invention provides a method for obtaining the mutant expandases of the invention wherein the process comprises the following steps: 1. Mutagenesis of a cloned gene encoding a model polypeptide with expandase activity then obtained a collection of mutagenized genes encoding mutant asexpandases;

2. Expression of the mutagenized gene collection encoding the mutant expandases in an appropriate host and scanning the mutant expandase collection for enhanced activity with an appropriate substrate;

3. Optionally by repeating steps 1 and 2 times or several times using either the model encoding expandase activity model opolipeptide or one or more mutagenized genes encoding mutant expandases with expandase activity on the appropriate substrate.

Cloning of genes encoding a model polypeptide with expandase activity can be performed according to methods known in the art.

Preferred model polypeptides with activity expansion are selected from the group consisting of a polypeptide with activity expandase obtainable from Streptomyces clavuligerus, preferably have an amino acid sequence according to SEQ ID NO: Iepolipeptides with activity expandase having a percent amino acid sequence 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%, more preferably at least 95%, such as the expandase enzymes which are summarized in Table 1.

Any mutagenesis techniques that can be applied which result in mutations over the complete gene. Appropriate techniques are error-susceptible PCR (EP) and / or by using saturated mutation primer PCR (sMPP) exactly according to WO3 / 010183.

Materials and methods

1. General

• Oligonucleotides were synthesized by Invitrogen (Carlsbad CA, USA).

· DNA sequencing was performed by SEQLAB (Gottingen, Germany) or Baseclear (Leiden, the Netherlands).

• Restriction enzymes were purchased from Invitrogen.

• Protein assays were performed according to the method described by Bradford, Μ. M. (1976) Anal. Biochem.72: 284-54.

• Electromax Escherichia coli DHlOB competent cells were obtained from Invitrogen. The protocol was delivered by the manufacturer.

• SDS-PAGE electrophoresis was performed on the system supplied by Invitrogen. NuPAGE Novex Bis-Tris Gels (Invitrogen).

SimplyBlue SafeStain Microwave Protocol.

• To create a CefE expression vector, the ampicillin selectable marker in pBAD / Myc-His (commercially available from Invitrogen ™) is replaced by the zeozine selective marker. Additionally, the maltose protein ligand is fused to the S. clavuligerus wild type expandase gene and ligated behind the pBAD promoter resulting in the pBAD-MH-Zeo-MBP-ScEwt vector. Wild type CefE is exchanged for the library of error-prone mutants.2. Primary Scan on Microtiter Plates (MTP) Growth and Induction

The E. coli expansionase library is plated on Luria-Bertani (LB) medium plates with low salt concentration Agar + 25 pg / ml Zeocine and incubated overnight at 37 ° C. 150 µl titration plates (MTP) of low salt LB medium and 25 pg / ml Zeocin are inoculated from the plates and incubated for 36 hrs at 250 ° C and 550 rpm.

From the MTP, 5 pL is inoculated into deep well plate with 1 ml 2 * TY medium (tryptone and yeast extract) + 50 pg / ml Zeocin + arabinose. To the culture remaining in the MTP, 100 µl of 50% glycerol is added and frozen at -800 ° C as glycerol stock.

The deep well plate is incubated for 30 hr at 250 ° C and 550 rpm. Deep well plate cultures are centrifuged at 2750 rpm (Heraeus multifuge 4 kr.) For 10 min. The supernatant is discarded and the precipitate is frozen at -20 ° C.

Cell Free Extract (CFE)

The frozen precipitates obtained in the previous section are resuspended in 200 µL extraction buffer (50 mMTris / HCl pH = 7.5; 5 mM DTT; 0.1 mg / mL DNAse; 5 mM MgSO4H2) and 0.5 mg / mL lysozyme). To resuspend precipitates, the plates are shaken. The plates are incubated for 30 minutes at room temperature and centrifuged for 10 minutes at 2750 rpm.

3. Secondary sweep in shake flasksGrowth and Induction

Selected colonies are inoculated from the plate in 10 mL low salt LB + 25 pg / mL Zeocin and incubated overnight at 37 ° C, 280 rpm. The grown culture is inoculated in 100 ml low salt LB with 25 pg / ml Zeocina at an optical density of (600 nm) between 0.010 and 0.050 (Biochrom Ultraspec 2000). After growth at 37 ° C, 280rpm, the cells are recovered at an optical density at 600nm between 0.4-0.6. Thereafter, arabinose is added (0.2% final concentration) and induced overnight at 27 ° C at 220 rpm. The cultures are centrifuged, the supernatant is discarded and the precipitate is frozen at -20 ° C.

Cloning of E. coli expression vectors

The fusion product is ligated into the pBAD / MH MBP-ScEwtzeo-zeo vector by an EcoRI / SalI digestion. This replaces the expandase wild gene with the library genes. To predict significant numbers of wild constructs, the linked constructs were digested with SmaI.

E. coli transformation with the ligation mixture yielded libraries of approximately 12,000-13,000 colonies. To test library quality, a random set of colonies were selected and constructs were digested with restriction enzymes. This showed that approximately 28% of the library revealed an aberrant pattern. Sequence analyzes were then performed on 10 regular clones to determine mutation frequencies. Sequences showed that so many saturation and error susceptibility mutations were present at a satisfactory level.

4 Expandase AssayA total of 300 µl of a reaction mixture consisting of 50 mM Tris / HCl pH 7.5; 1 mM DTT; 2.7mM Ascorbate; 0.05 mM ATP; 24 mM α-ketoglutarate, 0.06 mM FeSO 4 · 7H 2 O; 4 mM adipyl-6-amino-penitionic acid (ad-6-ADCA) was added to 75 µL of CFE and incubated at 290 ° C for the desired time. Adding 60 µl maleic acid with 10 g / l EDTA stopped the reaction. Adipyl-7-amino-deacetoxy-cephalosporinic acid formation (ad-7-ADCA) was detected using 1 H-NMR. Under these conditions, the expandase activity is measured close to the Vmax conditions provided that the Km of S. clavuligerus para ad-6-ΑΡΑ enzyme is ca 0.4 mM. Alternatively, the assay can be performed using 0.4 mM ad-6-ΑΡΑ (~ KM of S. clavuligerus enzyme for ad-6-ΑΡΑ).

Adipi-6-APA was obtained from 6-APA and glutaryl acylase catalyzed adipic acid (EC 3.5.1.11).

Alternatively, adipyl-6-ΑΡΑ may be obtained by cultivating, for example, an appropriate Penicilliumchrysogenum strain in the presence of adipic acid as a precursor.

EXAMPLES

Example 1

1st generation of improved expandases (H400 series).

The first generation of the expandase library was constructed by performing error susceptibility (EP) PCR on the CefEde Streptomyces clavuligerus gene (SEQ ID No. 1). After scanning this library by enhanced conversion of ad-6-ΑΡΑ to ad-7-APA, 3 different mutant genes were selected. The mutants exhibited an ad-6-expansão expansion activity greater than 2.5 times (H4 01, H4 02, H4 03: see Table 3).

Example 2

2nd generation of improved expandases (H500 series).

The second generation of the expansionase library was constructed by the use of Saturated Mutation Primer (sMPP) PCR exactly according to WO 03/010183. Mutant genes selected from the first generation (H401, H402 and H403) were used as templates for the construction of the 2nd generation library.

SMPP was performed by using Taqpolimerase, then introducing additional (random) mutations and increasing variation in the library. Drawn primers ringed at the mutation positions and were saturated at the mutations found at the expandase sites.

1st generation. In addition, an upstream and downstream universal oligonucleotide was designed into which a NdeI and NsiI site were introduced respectively. This facilitates cloning into the Penicillium paratest expression vector of mutant expansases in vivo.

The library was grown and expansionase expression was induced as described in the material and methods section. Ad-7-ADCA formation was determined using NMR as described in the material and methods section.

Approximately 150 improved mutant expandases were selected and retested for their ad-6-ex expansion activity. Based on the results of this retest, 17 mutants were selected for final testing in the shake flasks.

In total, 15 of the 17 selected points showed significantly improved expansion activity with ad-6-APA as a substrate. To exclude false selection of expression mutants, protein levels were quantified on SDS-PAGE. This confirmed that the improved activity was not due to the stronger expression expandase in E. coli, but that the improvement can be attributed to an improved specific activity of the mutant expander.

In total, 8 mutant expandases were identified which exhibited a 4.5-fold improvement factor in their ad-6-expand above expandase (compared to the wild S. clavuligerus expandase - SEQ ID NO 1.). These mutants were designated H501 - H508 (see Table 3).

Example 3

3rd generation of enhanced expandases (H600 series).

The 8 mutants obtained from the 2nd generation Example 2 (H5 01 -H508) were used as templates for the construction of the 3rd generation expandase library. This library was built in the same way as the 2nd generation expand library. Additionally, the SKA signal sequence at the C-terminal of the expandases has been changed to SKD, causing expansionase expression only in the cytoplasm when expressed in Penicillium.

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

Scanning the same library for a second time using 7 mM Pen-G as a substrate yielded 13 additional mutant expanders (G601-G611 and G613-G614; see Table 3).

Example 4

4th generation of improved expandases (H700 series).

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

Scanning of this library yielded 6 different mutant deexpandase genes that were significantly improved (over 20-fold) when tested for their 0.4 mM ad-6-expand expansion activity (Table 4, H701-H706).

Example 5

Activity of mutant expandases enhanced 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 iPN assay as a substrate was performed as described in the materials and methods section except that ad-6-ΑΡΑ was replaced by the same iPN concentration (4 mM). Pen-G assay as a substrate was performed as described in the material section and methods except that ad-6-ΑΡΑ was replaced by 7 mM Pen-G. Table 3 summarizes the activities of the various iPN and Pen-G expandants.

While most of the mutant expandases tested exhibited a 5-fold improvement in their expandasecom iPN activity, mutants carrying the T89K mutation lose virtually all activity against iPN. The activity against Pen-G of several mutant expandases tested was both the same as wild-type expandase factor. upgrade is 1) or upgrade above 8 times. The data also show that there is no correlation between improvement factors for a single mutant with ad-6-ΑΡΑ ePen-G as a substrate. The rate between the bestlooking factors for ad-6-ΑΡΑ and Pen-G ranges from 0.1 (eg H654) to 1.2 (eg H503).

Table 3. Mutant expandases and their expanded activities against ad-6-ΑΡΑ, Pen-G, and iPN. Each mutant is identified by its code; The mutations were introduced into the S. clavuligerus model expandase (SEQ IDNO 1). The expandase activity is expressed as a better factor compared to the S. clavuligerus model expandase (expandase activity = 1 by definition) according to in vitro expandase assays described in Materials and Methods and Example 4.

<table> table see original document page 34 </column> </row> <table> <table> table see original document page 35 </column> </row> <table> <table> table see original document page 36 < / column> </row> <table> <table> table see original document page 37 </column> </row> <table>

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

Table 4

<table> table see original document page 37 </column> </row> <table> SEQUENCE LIST

<110> DSM IP Assets B.V.

<120> MUTANT EXPANDASES

<130> 24867WO

<160> 1

<170> PatentIn in version 3.1

<210> 1

<211> 936

15 <212> DNA

<213> Streptomyces clavuligerus

<220>

20 <221> exon

<222> (1). . (933) <223>

<4 0 0> 1

25 atg gac acg acg gtg ccc acc ttc age ctg gcc gaa ctc cag cag ggc 48

Met Asp Thr Thr Val Pro Thr Phe Be Read Wing Glu Read Gln Gln Gly15 10 15

ctg cac cag gag gag ttc cgc agg tgt ctg agg gac aag ggc ctc ttc 96

3 0 Read His Gln Asp Glu Phe Arg Arg Cys Read His Arg Asp Lys Gly Read Phe20 25 30

tat ctg acg gac tgc ggt ctg acc gac acc

Tyr Leu Thr Asp Cys Gly Leu Thr Asp Thr Glu Leu Lys Ser Ala Lys35 35 40 45

gac till gtc till gac ttc ttc gag cac ggc age gag gcg gag aag cgc 192

Asp Ile Val Ile Asp Phe Phe Glu His Gly Ser Glu Wing Glu Lys Arg50 55 60

40

gcc gtc acc tcg ccc gtc ccc acc atg cgc cgc

Val Thr Wing Be Pro Val Pro Thr Met Arg Arg Gly Phe Thr Gly Leu65 70 75 80

45 gag tcg gag age acc gcc cag till acc aat acc ggc age tac tcc gac 288

Glu Be Glu Be Thr Wing Gln Ile Thr Asn Thr Gly Be Tyr Ser Asp85 90 95

tac tcg atg tgc tac tcg atg ggc acc gcg gac aac ctc ttc ccg tcc 336

50 Tyr Be Met Cys Tyr Be Met Gly Thr Wing Asp Asn Leu Phe Pro Ser100 105 110

ggt gac ttc gag cgg until tgg acc cag tac ttc gac cgc cag tac acc 3 84

Gly Asp Phe Glu Arg Ile Trp Thr Gn Tyr Phe Asp Arg Gln Tyr Thr55 115 120 125

gcc tcc cgc gcg gtc gcc cgg gag gtc ctg cgg

Wing Ser Arg Wing Val Wing Arg Wing Glu Val Leu Arg Wing Thr Gly Thr Glu130 135 140

60

ccp gac ggg ggg gtc gag gcc ttc ctc gac tgc gag ccg ctg ctg cgg 480Pro Asp Gly Gly Val Glu Ala Phe Leu Asp Cys Glu Pro Leu Arg145 150 155 160

ttc cgc tac ttc ccg cag gtc ccc gag cac cgc age gcc gag gag cag 528

Phe Arg Tyr Phe Pro Gln Val Pro Glu His Arg Be Wing Glu Glu Gln

165 170 175

ccc ctg cgg atg gcg ccg cac tac gac ctg tcg atg gtc acc ctc until 576

Pro Leu Arg Met Wing Pro His Tyr Asp Leu Being Met Val Thr Leu Ile180 185 190

cag cag aca ccc tgc gcc aac ggc ttc gtc age ctc cag gcc gag gtc 624

Gln Gln Thr Pro Cys Wing Asn Gly Phe Val Ser Leu Gln Wing Glu Val

195 200 205

ggc ggc gcg ttc acg gac ctg ccc tac cgt ccg gac gcc gtc ctc gtc 672

Gly Gly Phe Thr Wing Asp Leu Pro Tyr Arg Pro Asp Wing Val Leu Val210 215 220

ttc tgc ggc gcc up to gcg acc ctg gtg acc ggc ggc cag gtc aag gcc 720

Phe Cys Gly Wing Ile Wing Thr Read Val Gly Gly Gln Val Lys Wing Ala225 230 235 240

ccc cgg cac cat gtc gcg gcc ccc cgc agg gac cag ata gcg ggc age 768

Pro Arg His His Val Wing Ala Pro Arg Arg Asp Gln Ile Ala Gly Ser

245 250 255

age cgc acc tcc agt tg ttc ttc ctc cgt ccc aac gcg gac ttc acc 816

Be Arg Thr Be Ser Val Phe Phe Leu Arg Pro Asn Wing Asp Phe Thr260 265 270

ttc tcc gtc ccg ctg gcg cgc gag tgc ggc ttc gat gtc age ctg gac 864

Phe Ser Val Pro Leu Arg Glu Cys Gly Wing Phe Asp Val Ser Leu Asp275 280 285

ggc gag acc gcc acg ttc cag gat tgg until ggg ggc aac tac gtg aac 912

Gly Glu Thr Wing Thr Phe Gln Asp Trp Ile Gly Gly Asn Tyr Val Asn290 295 300

until cgc cgc aca tcc aag gea tga 936

Ile Arg Arg Thr Ser Lys Ala305 310

Claims (23)

1. Mutant expandase characterized in that it is a variant of a model polypeptide with expandase activity where the mutant expandase has at least 2.5-fold improved invitro expandase activity compared to a model polypeptide with expandant activity. 2. Mutant expandase characterized by being a variant of a model polypeptide with expandase activity where the mutant expandase has been modified to at least one 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 amino acid sequence position numbering of the enzyme expanded by the Streptomyces clavuligerus cefE gene (SEQID NO: 1). Mutant expandase according to claim 2, characterized in that the mutant expandase has been modified in at least one 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, more preferably from the group consisting of positions 2, 89, 90, 99, 105, 177, -251, 278, 280 and 293 . Mutant expandase according to claim 3, characterized in that it has been modified by at least one amino acid position selected from the group as defined in claim 2 or which has been modified to a selected amino acid position from any one. groups as defined in claim 3. Mutant expandase according to any one of Claims 1, 2, 3 or 4, characterized in that it comprises at least 2 or more amino acid positions selected from the group consisting of positions - 2, 89, 277, 281 and 300. more preferably at positions 2 + 281 or 89 + 281 or 277 + 300. Mutant expandase according to any one of claims 1, 2, 3, 4 or 5, characterized in that it comprises at least 3 or more amino acid positions selected from the group consisting of positions 2, - 89, 281, 293 and 311, more preferably at positions 2 + 89 + 281 or 89 + 281 + 311 or 89 + 281 + 293. Mutant expander according to any one of Claims 1, 2, 3, 4, 5 or 6, characterized in that the fat comprises at least 4 or more amino acid positions selected from the group consisting of depositions 2, 73, 89, 90. , 217, 244, 277, 281, 306, 307 and 311, more preferably at positions 2 + 277 + 280 + 281 or -2 + 281 + 306 + 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. Mutant expander according to any one of Claims 1, 2, 3, 4, 5, 6 or 7, characterized in that it comprises at least 5 or more amino acid positions selected from the group consisting of depositions 2, 73, 89. , 155, 213, 249, 281, 293, 300, 306 and 311, more preferably at positions 2 + 89 + 281 + 306 + 311or 2 + 155 + 281 + 306 + 311 or 73 + 89 + 213 + 281 + 311 or -89 + 281 + 293 + 300 + 311 or 89 + 249 + 281 + 306 + 311. Mutant expandase according to any one of Claims 1, 2, 3, 4, 5, 6, 7 or 8, characterized in that it comprises at least 6 amino acid positions selected from the group consisting of the -90 + 105 positions. + 113 + 281 + 306 + 311. 10. Mutant expansion according to any one of Claims 1, 2, 3, 4, 5, 6, 7, 8 or 9, characterized in that it comprises at least 7 or more amino acid positions selected from the group consisting of the depositions. , 89, 105, 113, 155, 177, 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 -5 + 177 + 277 + 280 + 281 + 3 06 + 311. Mutant expandase according to any one of Claims 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, characterized in that it comprises at least 8 or more amino acid positions selected from the group consisting of of positions 2, 90, 99, 105, 113, 155, 177, -277, 281, 306 and 311, more preferably nas-2 + 90 + 99 + 105 + 113 + 281 + 306 + 311 or-2 + 90 + 105 + 113 + 155 + 177 + 277 + 281. Mutant expandase according to any one of Claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11, characterized in that it comprises at least 9 amino acid positions selected from the group consisting of of positions 2 + 90 + 105 + 113 + 155 + 177 + 281 + 306 + 311. Mutant expandase according to claim 1, characterized in that it comprises at least 10 amino acid positions selected from the group consisting of positions 2 + 90 + 105 + 113 + 155 + 177 + 281 + 306 + 311. Mutant expandase according to any one of claims 2, 3 and 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13, characterized in that it has an in vitro expandase activity against adipil-6-ΑΡΑ. at least 2.5 times better compared to a model polypeptide with expandase activity. Mutant expandase according to any one of Claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or -14, characterized in that the mutant expandase is a variant of the expandase. cefE-encoded S. clavuligerus as described in SEQ ID No. 1. Mutant expandase according to claim 15, characterized in that the mutant expandase has been modified in at least one 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, D251, L277, A278, E280, -C281, T293, G300, R306, R307 and A311, more preferably at least , 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, more preferably from the group consisting of positions 2, 89, 90, 99, 105, 177, -251, 278, 280 and 293. A polynucleotide encoding the mutant expandase of any one of claims 1, -2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16. An expression vector or cassette characterized by the polynucleotide comprising claim 17. A host cell characterized by being transformed with the polynucleotide of claim 17 or the vector or cassette of claim 18. A method of producing a mutant expandase of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, -14, 15 or 16 which comprises cultivating a host cell. claim 19 under conditions which contribute to the production of the mutant expandase and optionally recovering the polypeptide. Method of producing a β-LACTAMIC COMPOUND of interest characterized in that it comprises culturing a host cell of claim 20 under conditions that contribute to the production of the β-LACTAMIC Compound and optionally recovering the β-LACTAMIC COMPOUND. Method according to claim 21, characterized in that it further comprises N-deactivation of the β-LACTAMIC COMPOUND to produce a N-deactivated β-LACTAMIC COMPOUND. Method of obtaining the mutant expandase of any one of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, -13, 14, 15 or 16 characterized in that the method comprises the following steps: - mutagenesis of a cloned gene encoding an expanding polypeptide model polypeptide, preferably encoding the expandase from S. clavuligerus, then obtaining a collection of mutagenized genes encoding the mutantexpandases.- expression of the collection of mutagenized genes encoding the mutant expandases in a host mutant expansionase collection for improved activity with an appropriate substrate, preferably ad-6-ΑΡΑ, - optionally repeating steps 1 and 2 or several times, thereby using mutagenesis of the gene encoding model opolipeptide with expandase activity and / or one or more. mutagenized genes encoding mutant expandases with improved activity on the appropriate substrate.