Field of the invention The present invention relates generally to novel acyltransferase polypeptides that are useful in the production of beta-lactam intermediates and antibiotic compounds.

Background of the invention

The most important classes of the beta-lactam antibiotics are the penicillins (penams) and the cephalosporins (ceph-3-ems). Penicillins are produced by filamentous fungi only (Penicillium chrysogenum or Aspergillus πidulans), whereas cephalosporins are produced by filamentous fungi (Acremonium chrysogenum) as well as bacteria (e.g. Streptomyces clavuligerus). All beta-lactam antibiotics share the common structural feature of a four-member beta-lactam ring. The naturally occurring penams and ceph-3- ems are synthesized from the same three amino acid precursors: L-alpha-aminoadipic acid (L-alpha-AAA), L-cystein and L-valine. The first two steps in the biosynthesis pathways of penicillins and cepalosporins are the same. In the first step, the amino acid precursors are condensed to the tripeptide delta-L-alpha-aminoadipyl-L-cysteinyl-D- valine (ACV). The requisite reaction cycle (e.g. recognition, activation and formation of peptide bonds) is catalysed by the multifunctional enzyme ACV synthetase (ACVS). In the second step the resulting linear tripeptide is cyclized by oxidative ring closure leading to the formation of a bicyclic ring structure, i.e. the four-member beta-lactam ring fused to the five-membered thiazolidine ring, which is characteristic of all penams. Cyclization is catalysed by the enzyme isopenicillin N synthase (IPNS). The resulting compound represents the first bioactive intermediate and is referred to as isopenicillin N (IPN). See generally, e.g., EP 0 422 790 (Miller et al.).

After IPN synthesis, the biosynthetic pathways leading to the production of the penicillins and cephalosporins diverge. In some fungi, for example in P. chrysogenum and in A. nidulans, the hydrophilic L-alpha-AAA side chain of IPN can be replaced by a hydrophobic acyl group in a third and final exchange step. The side chain may be of intracellular origin (e.g., hexonic acid or octenoic acid) or supplied exogenously. The only directly fermented penicillins of industrial importance are penicillin V and penicillin G, produced by adding the exogenous side chain precursors phenoxyacetic or phenylacetic acid, respectively. The exchange is catalyzed by acyl coenzyme A (CoA):isopenicillin N acyltransferase (AT) which is encoded by the penDE gene.

In contrast, the ceph-3-ems are produced by the isomerization of the L-alpha- AAA side chain of IPN to the D enantiomer by IPN epimerase to produce penicillin N which is the precursor of antibiotics comprising the ceph-3-em nucleus. For example, in the fungus A. crhrysogenum, the alpha-aminoadipic acid side chain of IPN is isomerized to produce penicillin N, after which the five-membered thiazolidine ring of the penicillin is "expanded" by deacetoxycephalosporin synthetase (DAOCS) expandase activity to produce deacetoxycephalosporin C (DAOC) which comprises the six-membered dihydrothiazine ring that is characteristic of the cephalosporins.

Many of the so-called natural beta-lactams (e.g., penicillin F, isopenicillin N, cephalosporin C, etc.) are of limited utility as antibiotics because they are unstable, difficult to purify from fermentation broth, have only limited antibiotic effect, and/or are produced in low yield. Replacing the side chains of these beta-lactams with other side chains leads to the formation of semisynthetic penicillins and cephalosporins, such as amoxycillin, ampicillin and cephalexin, which are more stable, easier to isolate, and have a higher antibiotic activity.

The large variety of side chains found in commercially significant beta-lactam compounds has placed increased importance on achieving more economic and efficient methods of preparing key intermediates for synthesis of various beta-lactam compounds. The cephalosporin intermediate 7-ADCA is an important intermediate for the synthesis of many semisynthetic cephalosporins. It is currently produced by either chemical derivatization of penicillin G or by a bioprocess as described in EP 0532341.

In EP 0532341 it is shown that adipyl-7-ADCA is formed by a Penicillium chrysogenum strain modified to express expandase and fed with the side chain precursor adipic acid. Subsequent removal of the adipyl side chain with a suitable enzyme leads to formation of 7-ADCA. Although the AT enzyme thus is capable of accepting other side chains than phenyl- or phenoxyacetic acid, it is unpredictable whether or not the capacity of the AT enzyme to accept other side chains than phenyl- or phenoxyacetic acid can be improved or its substrate specificity modified. Thus, an object of the present invention is to provide modified (variant) AT polypeptides with altered catalytic properties. Preferably, the variant AT polypeptides show improved properties in the production of beta-lactam antibiotics. AT variants with altered catalytic properties may show improved activity in desired reactions and/or decreased activity in undesirable reactions and which overall may lead to less by- products compared to desired products. AT variants with altered catalytic properties may

further show altered specificity, for instance a lower sensitivity for inhibition and/or an improved activity on substrates that are not converted or only converted to a low extent by the natural AT.

Figure Legends Figure 1. Atom coordinates of the mature AT model with PenG in its active site.

Figure 2: Alignment of the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3

Detailed description of the invention Throughout the description of the invention, polypeptides with acyl

CoA:isopenicillin N acyltransf erase (AT) activity are also called AT polypeptides or AT.

The model AT polypeptide as used in the present invention is selected from the group consisting of

• An AT polypeptide obtainable from Penicillium chrysogenum, preferably having an amino acid sequence according to SEQ ID NO: 1 and an AT polypeptide 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%.

• An AT polypeptide having an amino acid sequence with a percentage identity of at least 75% with SEQ ID NO: 1 is obtainable from Emericella nidulans (formerly known as Aspergillus nidulans), preferably having an amino acid sequence according to SEQ ID NO: 2.

• An AT polypeptide having an amino acid sequence with a percentage identity of at least 95% with SEQ ID NO: 1 is obtainable from Penicillium nalgiovense, preferably having an amino acid sequence according to SEQ ID NO: 3.

Preferred model AT polypeptides are polypeptides having an amino acid sequence according to SEQ ID NO: 1 , SEQ ID NO: 2 and/or SEQ ID NO: 3. Most preferred is a model AT polypeptide having an amino acid sequence according to SEQ ID NO: 1. It is to be understood that the model AT polypeptides having an amino acid sequence according to SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 are not within the scope of the feature "variant AT polypeptides". Figure 2 shows an alignment of the amino acid sequences of SEQ ID NO: 1 , SEQ ID NO: 2 and SEQ ID NO: 3.

In a first aspect, the present invention provides polypeptides with AT activity that are variants of a model polypeptide with AT activity, the variant AT polypeptides being modified at one or more positions selected from the group I consisting of 28, 31 , 32, 33,

34, 35, 36, 37, 38, 39, 40, 41, 42, 47, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 118, 119, 120, 122, 123, 124, 125, 126, 129, 130, 145, 146, 147, 148, 149, 150, 151, 153, 154, 155, 165, 166, 167, 168, 169, 170, 171, 172, 180, 181, 182, 183, 184, 185, 189, 209, 210, 211, 212, 213, 245, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 269, 270, 271, 272, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 308, 309, 310, 311, 312, 313, 314, 315 and 332 using the position numbering according to SEQ ID NO: 1.

In one embodiment of the invention, a variant AT polypeptide is provided that is modified as compared to the model AT polypeptide at one or more positions selected from group Il consisting of 35, 36, 39, 41 , 89, 90, 93, 96, 97,101, 120, 122, 123, 148, 154, 166, 168, 182, 259, 260, 262, 302, 310 and 311. Amino acids at these positions belong to the primary interaction shell and have at least one atom within a distance of less than 4 Angstrom to one or more atoms of the substrate.

In a preferred embodiment, an AT polypeptide is provided that is modified at one or more positions selected from the group M-A consisting of positions 120, 122, 166 and 168, preferably having a modified activity and/or specificity with respect to an aliphatic and/or aromatic side chain. In the alternative or in addition, the conformation and/or spatial position of one or more of the amino acid at the positions 120, 122, 166 and 168 may be modified by modifying one or more of the amino acids that are in direct contact with the amino acid at the positions 120, 122, 166 and 168 and which are listed in Table 1 of Example 2.

In another preferred embodiment, an AT polypeptide is provided that is modified at one or more positions selected from the group M-B consisting of positions 89, 148, 154 and 182, preferably having a modified activity and/or specificity with respect to the nature of the side chain polar head group of the side chain, such as of the L-alpha- amino-adipyl side chain. In the alternative or in addition, the conformation and/or spatial position of one or more of the amino acid at the positions 89, 148, 154 and 182 may be modified by modifying one or more of the amino acids that are in direct contact with the amino acid at the positions 89, 148, 154 and 182 and which are listed in Table 1 of Example 2.

In another preferred embodiment, an AT polypeptide is provided that is modified at one or more positions selected from the group M-C consisting of positions 122, 123, 262, 302, 310, 311 , preferably having a modified activity and/or specificity with respect to the beta-lactam moiety of the substrate, for instance an improved capacity to accept the six-membered dihydrothiazine beta-lactam nucleus as a substrate. In the alternative

or in addition, the conformation and/or spatial position of one or more of the amino acid at the positions 122, 123, 262, 302, 310, 311 may be modified by modifying one or more of the amino acids that are in direct contact with the amino acid at the positions 122, 123, 262, 302, 310, 311 and which are listed in Table 1 of Example 2. In another preferred embodiment, an AT polypeptide is provided that is modified at one or more positions selected from the group M-C consisting of positions 35, 36, 39, 41 , 89, 90, 93, 96, 97, 101 , 259, 260, preferably having a modified activity and/or specificity with respect to the bind the coenzyme-A moiety. In the alternative or in addition, the conformation and/or spatial position of one or more of the amino acid at the positions 35, 36, 39, 41 , 89, 90, 93, 96, 97, 101, 259, 260may be modified by modifying one or more of the amino acids that are in direct contact with the amino acid at the positions 35, 36, 39, 41 , 89, 90, 93, 96, 97, 101, 259, 260 and which are listed in Table 1 of Example 2.

In another embodiment of the invention, a variant AT polypeptide is provided that is modified as compared to the model AT polypeptide at one or more positions selected from the group III consisting of 28, 31, 32, 33, 34, 37, 38, 40, 42, 47, 85, 86, 87, 88, 91, 92, 94, 95, 98, 99, 100, 102, 118, 119, 124, 125, 126, 129, 130, 145, 146, 147, 149, 150, 153, 155, 165, 169, 170, 171, 172, 180, 181, 183, 184, 185, 189, 209, 210, 211, 212, 213, 245, 257, 258, 261, 263, 264, 265, 266, 267, 269, 270, 271, 272, 297, 298, 299, 300, 301, 303, 304, 305, 306, 308, 309, 312, 313, 314, 315, and 332. Amino acids at these positions belong to the secondary interaction shell and have at least one atom within a distance of less than 4 Angstroms to one or more atoms of an amino acid belonging to the primary interaction shell, as identified above.

In a further embodiment, a variant AT polypeptide is provided that is modified at one or more positions selected from the group IV consisting of 31 , 32, 40, 42, 47, 86, 92, 94, 97, 98, 100, 118, 123, 124, 126, 130, 147, 155, 166, 170, 172, 181, 192, 185, 211, 212, 213, 245, 257, 264, 265, 298, 300, 304, 306, 309, 313, 314 and 315, being a position wherein an amino acid has at least one side chain atom within a distance less than 4 Angstroms to one or more side chain atoms of an amino acid of the primary shell. In another embodiment of the invention, a variant AT polypeptide is provided that is modified as compared to the model AT polypeptide at a position selected from the positions of groups M-A, M-B, IV-A and IV-B group consisting of 31 , 32, 35, 36, 86, 89, 90, 93, 94, 118, 120, 122, 123, 126, 130, 147, 148, 151, 154, 155, 166, 168, 181, 182, 185, 211 , 213, 314 and 315, preferably having a modified activity and/or specificity with

respect to the nature of the side chain of the substrate as compared to that of the model AT polypeptide.

Preferred embodiments of the present invention are variant AT polypeptides, which are modified at one or more positions selected from the group consisting of 35, 90, 94, 97, 98, 120, 123, 124, 166 and 170, more preferably at positions 35, 97, 124 and 166, most preferably at position 97.

Most preferred embodiments are variant AT polypeptides having an amino acid sequence as depicted in SEQ ID NO: 1 and which are modified at one or more positions selected from the group consisting of L35, T90, Y94, K97, A98, W120, F123, S124, Y166 and H 170, more preferably at positions L35, K97, S124 and Y166, most preferably at position K97. Highly preferred embodiments are variant AT polypeptides having an amino acid sequence as depicted in SEQ ID NO:1 and which have one or more of the following modifications: L35M, T90L, Y94F, K97R, K97V, A98E, W120M, F123M, S124T, Y166F and/or H 170N. The variant AT polypeptide according to the invention is modified as compared to a model AT in such a way that the polypeptide according to the invention, when aligned to the model AT, contains a modification selected from the group consisting of: a substitution of an amino acid as present in the model AT for a different amino acid, a deletion of an amino acid as present in the model AT, or an insertion of an amino acid. The alignment of the polypeptide according to the invention to a model AT is done in such a way as to obtain a maximal amount of homologous (identical) residues between the polypeptide according to the invention and the model AT. In a preferred embodiment of the invention, the modification is a substitution.

The number of modifications may be at least one, preferably at least 2, more preferably at least 3, more preferably at least 4, more preferably at least 5, more preferably at least 10, more preferably at least 20.

In the present invention, a denotation like e.g. "97RV" means that the amino acid in position 97 of the model AT in question is substituted with either R or V. The nature of the original amino acid residue may depend on the model AT that is used. A denotation like e.g. "K97R" means that a specific amino acid residue present in the model AT, e.g. K, is substituted with a different amino acid, e.g. R.

A polypeptide of the invention may comprise all of the modifications set out above. In addition, the polypeptide of the invention may comprise additional modifications that concern positions in the polypeptide wherein a modification does not affect the folding or activity of the polypeptide. Typically, such modifications may be

conservative substitutions, i.e. substitutions wherein a non-polar, polar uncharged, polar charged or aromatic amino acid is substituted for a different amino acid from the same category.

Thus, in one embodiment, the polypeptide of the invention may encompass a polypeptide with at least 90, preferably at least 91, more preferably at least 92, more preferably at least 93, more preferably at least 94, more preferably at least 95, more preferably at least 96, more preferably at least 97, more preferably at least 98 or most preferably at least 99% sequence homology (identity) to a variant AT polypeptide comprising one or more of the modifications set out above. Polypeptides of the invention may be produced by synthetic means although usually they will be made recombinantly by expression of a polynucleotide sequence encoding the polypeptide in a suitable host organism.

Polypeptides of the invention may be provided in a form such that they are outside their natural cellular environment. Thus, they may be substantially isolated or purified or produced in a cell or cell compartment in which they do not occur in nature, e.g. a cell of other fungal species, animals, yeast or bacteria.

Polypeptides of the invention may be analysed by any suitable assay known to the skilled person to measure an improvement as compared to a model AT known in the art. A suitable assay is an HPLC assay. Accordingly, the activity of a variant AT polypeptide can be measured using the following functional groups as alternative for the phenylacetyl side chain:

• phenylacetyl side chains with substitution of one or more hydrogen atoms of the ring by different atoms or groups of atoms,

• acyl side chains in which the benzene ring has been replaced by other cyclic moieties such as 2- or 3-thiopheneacetyl acid, 2-,3-furoylacetyl, cyclopentyl acetyl, cyclopentenoic acetyl, cyclohexylacetyl, pyridylacetyl, etc.,

• acyl side chains in which the benzene ring is replaced by non-cyclic aliphatic moieties such as propionic acid, butyric acid, pentanoic acid, pentadienoic acid (glutaric acid), hexanedienoic acid (adipic acid), heptanoic acid, etc., • acyl side chains in which the benzene ring is maintained but with longer or shorter acyl chain such as benzoyl, phenylpropionyl, phenyl butyryl, phenoxyacetyl, thiophenoxyacetyl, etc

• acyl side chains in which the benzene ring is maintained but with substitutions on the α-carbon of the phenylacetyl group e.g. the α proton is replaced by a hydroxy, methyl, ethyl, amino, halogen or nitro group, optionally combined with

substitution of one or more hydrogen atoms of the benzene ring by different atoms or groups of atoms.

Accordingly, the activity of a variant AT polypeptide can be measured using the following functional groups as alternative for the beta-lactam moiety: 7-ADCA (7-amino-3- deacetoxycephalosporanic acid), 7-ACA (7-aminocephalosporanic acid), 7-amino-3- deactecylcephalosporanic acid, 7-amino-3-carbamoylcephalosporanic acid (7-ADCCA). Accordingly the activity of a variant AT polypeptide can be measured using the following functional groups as alternative for the α-a mi no-ad ipoyl moiety: α-D-aminoadipoyl, adipoyl, glutaryl, etc. Accordingly the activity of a variant AT polypeptide can be measured using the following functional groups as alternative for the CoA moiety in the acyl-CoA thioester acyl side chain donor : Acyl side chain precursors in which the CoA is replaced by other SH-containing molecules such as for example glutathion, ACV, cystein. In addition acyl esters e.g. phenylacetylmethyl ester, phenyl acetyl ethyl ester etc, might be explored as acyl donors. In a second aspect, the invention provides the use of the 3-dimensional structure of an AT polypeptide to identify positions in the AT polypeptide that are relevant for the catalytic properties of the AT polypeptide and to identify those positions in AT polypeptides that are at least 70% homologous thereto. The 3-dimensional structure may be determined of an enzymatically active AT or from an inactive precursor, obtained by selective mutagenesis, such as the C103A mutant of the AT from Penicillium chrysogenum. The 3-dimensional-structure of an enzymatically active AT may be derived from an inactive precursor by computer modelling, such as from the C103A mutant of the AT from Penicillium chrysogenum.

1. Superpositioning of the 3-dimensional structure of the AT C103A precursor onto a given beta-lactam acylase such that alanine at position 103 of the AT C103A precursor overlaps with the matching atoms of the N-terminal amino acid of the beta-lactam acylase beta-subunit.

2. Replacing A103 in silico by cystein in such way that the position of the corresponding atoms is maintained. 3. In silico cleaving of the peptide bond preceding AT C103 so that a heterodimer is formed having a α-subunit that ends at residue 102 and a beta-subunit that starts at C103.

4. Forcing residues 88-102 to adopt a α-helical conformation.

5. Selecting a substrate and a bond of interest, which may be an amide bond, an ester bond or a thioester bond.

6. Obtaining the 3-dimensional structure of the selected substrate - when the coordinates of a substrate are not available from a database one could obtain the 3-dimensional model for the substrate using modelling software such as Insight™ and Discover™ 7. Docking the substrate into the active site of the AT model generated in step 4 in such a way that it mimics the transition state tetrahedral intermediate where the AT C103 sulphur atom forms a covalent bond with the carbonyl carbon atom of the scissile bond and the carbonyl oxygen is positioned in the oxyanion binding pocket forming hydrogen bonds with main chain amide of A168 and the side chain amide of N246. It should be clear that the docking is not limited to a particular energy minimization method. Alternative methods known in the art to optimise docking of substrates into proteins may also be used. The final results might show small differences with respect to atom coordinates, but this will not effect the identification of modification targets very drastically. 8. Selecting amino acids that have at least one atom within a distance of less than 4 Angstroms to one or more atom of the substrate (belonging to the primary interaction shell), exploring the corresponding positions by mutagenesis and selecting enzymes with altered catalytic properties. Preferred are those amino acids, which have at least one side chain atom within a distance less than 4 Angstroms to the substrate.

9. Preferably selecting amino acids that have at least one atom within a distance of less than 4 Angstroms to one or more atoms of the amino acids selected in step 7 (belonging to the secondary interaction shell), exploring the corresponding positions by mutagenesis and selecting enzymes with an altered catalytic properties. More preferably, at least one side chain atom of the selected amino acids is within a distance less than 4 Angstroms to one or more side chain atoms of the amino acids selected in step 7. The amino acids belonging to the secondary interaction shell consist of residues that can modulate the local 3- dimensional structure and/or conformational mobility of amino acids in the primary shell.

In a third aspect, the present invention provides a polynucleotide (e.g. isolated and/or purified) comprising a polynucleotide sequence encoding the polypeptide of the invention. The polynucleotides of the present invention further include any degenerate versions of a polynucleotide sequence encoding the polypeptide of the invention. For example, the skilled person may, using routine techniques, make nucleotide

substitutions that do not affect the protein sequence encoded in the polynucleotides of the invention to reflect the codon usage of any particular host organism in which the polypeptides of the invention are to be expressed.

The polynucleotide sequence of the invention may be RNA or DNA and includes genomic DNA, synthetic DNA or cDNA. Preferably, the polynucleotide is a DNA sequence.

Polynucleotides of the invention can be synthesized according to methods well known in the art. They may be produced by combining oligonucleotides synthesized according to and along the nucleotide sequence of the polynucleotide of the invention. Alternatively, they may be synthesized by mutagenising a parental polynucleotide at any desired position.

Polynucleotides of the invention may be used to obtain polynucleotides encoding a further modified polypeptide, e.g. by subjecting polynucleotides of the invention to additional mutagenesis techniques. Site-directed mutagenesis may be used to alter the polynucleotides of the invention at one or more specific positions. Gene shuffling technology (for instance as disclosed in WO95/22625, WO98/27230, WO98/01581, WO00/46344 and/or WO03/010183) may be used to obtain polynucleotide variants with a random combination of any variant position present in any member of a starting population of polynucleotides, said starting population including one or more polynucleotides according to the invention.

The invention also provides vectors comprising a polynucleotide of the invention, including cloning and expression vectors or cassettes.

In an expression vector or cassette, the polynucleotide of the invention is operably linked to a regulatory sequence that is capable of providing for the expression of a polypeptide from its coding sequence by the host cell. The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A regulatory sequence such as a promoter, an enhancer or another expression regulatory signal "operably linked" to a coding sequence is positioned in such a way that expression of a polypeptide from its coding sequence is achieved under conditions compatible with the regulatory sequences.

Promoters/enhancers and other expression regulatory signals may be selected to be compatible with the host cell for which the expression cassette or vector is designed.

If the polypeptide is produced as a secreted protein, the polynucleotide sequence encoding a mature form of the polypeptide in the expression cassette is operably linked to a polynucleotide sequence encoding a signal peptide.

The DNA sequence encoding the polypeptide of the invention is preferably introduced into a suitable host as part of an expression cassette. For transformation of the suitable host with the expression cassette, transformation procedures are available which are well known to the skilled person. The expression cassette can be used for transformation of the host as part of a vector carrying a selectable marker, or the expression cassette may be co-transformed as a separate molecule together with the vector carrying a selectable marker. The vector may comprise one or more selectable marker genes.

For most filamentous fungi and yeasts, the expression construct is preferably integrated in the genome of the host cell in order to obtain stable transformants. In that case, the constructs are either integrated at random loci in the genome, or at predetermined target loci using homologous recombination.

Thus, a further aspect of the invention provides host cells transformed with or comprising a polynucleotide or vector of the invention.

Suitable host cells are host cells that allow for a high expression level of a polypeptide of interest. Such host cells are usable in case the AT polypeptides needs to be produced and further to be used, e.g. in in vitro reactions.

Alternatively, suitable host cells are cells capable of production of beta-lactam compounds, i.e. host cells possessing the biosynthetic pathway(s) leading to penam and/or cephem compounds.

The host cells may for example be prokaryotic (for example bacterial), fungal or yeast cells.

A heterologous host may be chosen wherein the polypeptide of the invention is produced in a form that is substantially free from other polypeptides with a similar activity as the polypeptide of the invention. This may be achieved by choosing a host that does not normally produce such polypeptides with similar activity. In a further aspect the invention provides a process for preparing a polypeptide according to the invention by cultivating a host cell (e.g. transformed with an expression vector as described above) under conditions to provide for expression (by the vector) of the polypeptide according to the invention, and optionally recovering the expressed polypeptide. Preferably the polypeptide is produced as a secreted protein in which case the polynucleotide sequence encoding a mature form of the polypeptide in the

expression construct is operably linked to a polynucleotide sequence encoding a signal peptide.

In a further aspect, the invention provides a process for preparing a beta-lactam compound by cultivating a host cell capable of production of beta-lactam compounds (e.g. transformed with an expression vector as described above) under conditions to provide for expression of the polypeptide of the invention and conducive to the production of a beta-lactam, and optionally recovering the beta-lactam compound. The beta-lactam compound that is produced may a deacylated beta-lactam compound, i.e. a beta-lactam nucleus as 6-APA or 7-ADCA, or may be a 6-acylated penam or 7-acylated cephem compound.

The recombinant host cells according to the invention may be cultured using procedures known in the art. For each combination of a promoter and a host cell, culture conditions are available which are conducive to expression of the polypeptide of the invention. After reaching the desired cell density or titre of the polypeptide the culture is stopped and the polypeptide is recovered using known procedures. Additionally, fermentation conditions may be established conducive to the production of a beta- lactam.

The fermentation medium may comprise a known culture medium containing a carbon source (e.g. glucose, maltose, molasses), a nitrogen source (e.g. ammonium sulphate, ammonium nitrate, ammonium chloride, organic nitrogen sources e.g. yeast extract, malt extract, peptone), and other inorganic nutrient sources (e.g. phosphate, magnesium, potassium, zinc, iron, etc.). Optionally, an inducer may be included.

The selection of the appropriate medium may be based on the choice of expression host and/or based on the regulatory requirements of the expression construct. Such media are known to those skilled in the art. The medium may, if desired, contain additional components favouring the transformed expression hosts over other potentially contaminating microorganisms.

The fermentation can be performed over a period of 0.5-30 days. It may be a batch, continuous or fed-batch process, suitably at a temperature in the range of between 0 and 45 ⁰ C and, for example, at a pH between 2 and 10. Preferred fermentation conditions are a temperature in the range of between 20 and 37 ⁰ C and/or a pH between 3 and 9. The appropriate conditions are usually selected based on the choice of the expression host and the protein and/or beta-lactam compound to be expressed.

After fermentation, if necessary, the cells can be removed from the fermentation broth by means of centrifugation or filtration. After fermentation has stopped and/or after removal of the cells, the polypeptide of the invention or the produced beta-lactam compound may be recovered using conventional means. Recovery may include purification and/or extraction and/or crystallization steps.

Conveniently, the polypeptide of the invention or the beta-lactam compound is combined with suitable (solid or liquid) carriers or diluents including buffers to produce a polypeptide or beta-lactam compound composition. The polypeptide or the beta-lactam compound may be attached to or mixed with a carrier, e.g. immobilized on a solid carrier. Thus the present invention provides in a further aspect a composition comprising a polypeptide of the invention or a beta-lactam compound. This may be in a form suitable for packaging, transport and/or storage, preferably where the activity of the polypeptide is retained. Compositions may be of paste, liquid, emulsion, powder, flake, granulate, pellet or other extrudate form.

EXAMPLE 1

Determination of the three-dimensional structure model for the mature AT in complex with penicillin-G.

Crystallization, X-ray diffraction and structure determination

Crystallization and preliminary X-ray diffraction of a C103A mutant of AT, i.e. an inactive precursor of AT, was carried out as described by Hensgens et al. (2002), Acta Cryst. D58, 716-718. Since mature wild type AT could not be crystallized because it aggregates easily, the C103A mutant was used instead. This mutation prohibits the autocatalytic cleavage of the peptide bond between G 102 and C103. As a consequence maturation towards the active AT enzyme is prevented.

The 3-dimensional structure of the AT C103A precursor was determined using the dataset recorded from crystals of the C103A mutant obtained in hanging drop vapour diffusion experiments employing 1.6M (NH ₄ J ₂ SO ₄ , 0.1 M NaCI and 0.1 M HEPES- NaOH buffer pH=7.5 as precipitant. Data collection was done on a MacScience DIP2030 image-plate detector using a Cu Ka omitting rotating anode generator. Data processing was done using DENZO and SCALEPACK (Methods. Enzymol. 276, 307-326). Ultimately the phase problem could be solved after crystallization of the L-Selenium- Methionine derivative of the AT C103A mutant by performing a Multiple Wavelength

Anomalous Dispersion experiment using synchrotron radiation. Positions of the selenium atoms were determined with SHELXC (Methods Enzymol. 1997, 277, 319-343). Initial phases were calculated using SHARP (Methods Enzymol 1997, 276, 472-494). Phase extension and model building was dine using ARP/WARP (Acta Crystallogr. D60, 2004, 2222-2229).

Homology search based on 3-dimensional-structure

Since the amino acid sequence of AT does not show any significant homology with another amino acid sequence of a protein with known 3-drimensioanl structure, a homology search was carried out on the basis of the 3-dimensional-structure. Hereto, the 3-dimensional-structure of the AT C103A precursor was superpositioned onto the 3- dimensional-structures of all proteins from the Brookhaven Protein Data. This was carried out by based on a direct comparison of 3D structures in order to detect remote homology often undetectable by sequence comparison. This has been done by using the VAST algorithm (Gibrat, J-F., Madej, T., Bryant, S. H. (1996) Surprising similarities in structure comparison. Current Opinion in Structural Biology. 6, 377-385; ^^U^^ϊJ]^MM ₁ BM _i ^^I^M^M^^Ik3M3§M^^M) but could also have been on the DaIi server (htte^/wwWjebLac.uk/^i/indexJTtal) and at the CE server The 3-dimensional structure coordinate files were viewed using various software packages, e.g. RasMol Swiss PDB viewer and Insight™

It was surprisingly found that the AT C103A precursor of AT was folded in a very similar way as Penicillin G Acylases (PAC), Glutaryl-Cef acylases (GAC) and Penicillin V acylases (PVA). The 3-dimensional structure coordinates of PAC (e.g. entry codes 1PNK, 1PNL, 1 PNM, 1FXH, 1 FXV, 1 E3A etc), GAC (e.g. entry codes 1FM2, UVZ, UWO, 1KEH, etc) and PVA (e.g. entry codes 2PVA and 3PVA) are available from the Protein Data Bank (H. M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T.N. Bhat, H. Weissig, I.N. Shindyalov, P.E. Bourne: Nucleic Acids Research, 28 pp. 235-242 (2000)).

Upon the correct superpositioning of the 3-dimensional structure of the AT C103A precursor onto that of one of the beta-lactam acylase listed above, it was observed that the alanine at position 103 of the AT C103A precursor coincides with the N-terminal serine of the beta-subunits of PAC and GAC and the N-terminal cystein of PVA. The serine and cystein residues located at the N-terminus of the beta-subunit are

usually referred to as serine/cystein B1 where B refers to the B-subunit and 1 to amino acid position 1 in the B-subunit. In PAC and GAC this serine B1 is crucial to catalysis. The Ser B1 amino group is believed to act as the general acid/base and its side chain gamma-hydroxyl group acts as a nucleophile in the reaction. In PVA the N-terminal cystein fulfils this role. Via computational mutagenesis, the alanine at position 103 of AT C103A was replaced by the cystein residue found in wild type AT and the bond peptide between residues 102 and 103 was cleaved, using the software package Insight™ (ΪMS^lMM3S£§lϊϊ§MMBl)- It was confirmed that the AT cystein now almost perfectly coincides with the PAC and GAC serine B1 and the PVA cystein B1. For PAC as well as GAC structural data is available on the precursor of the mature enzymes. The comparison between the GAC precursor (PDB entry code 1 KEH) and the AT mutant precursor shows even a higher degree of similarity than observed for the mature GAC (e.g. PDB entry 1FM2). The connecting loop in GAC between the alpha- and the beta-subunit is only nine amino acids longer than the corresponding loop in AT. After the cleavage of the peptide bond preceding the B1 serine in GAC eleven amino acids are removed from the C-terminus of the newly formed C-terminus of the N- terminal subunit (usually referred to as the alpha subunit). The removal of these nine amino acids opens up the active site so that the newly formed N-terminus of the C- terminal subunit (the beta-subunit serine B1) becomes accessible to the solvent. Contrary to GAC in AT no further digestion of the peptide chain takes place after cleavage of the bond preceding C103. G102 becomes the new C-terminal residue of the D-subunit after maturation. So, for AT the proteolysis of the C-terminal residues of the D-subunit alone does not explain how in the mature enzyme the catalytic cystein B1 becomes accessible to reactants from the outside solvent. However, a closer look at the AT precursor reveals that the amino acids 94-102 preceding the cystein B1 are surrounded by a substantial amount of solvent and that there are relativity few interactions with the rest of the protein. It was noticed that fragment 88-102 exhibits a high helical propensity that makes the helical turn 88-92 a good nucleation site for turning the whole fragment 88-102 into a helix. Therefore the fragment 92-102 was smoothly forced towards extending the helical turn 88-92 into an α-helix comprising residues 88-102 using energy minimization methods using the software package Discover™ (httfiΛwwj∞JasOTi/). The resulting helix did match quite well with the remaining part of the AT precursor structure which had been held fixed during these energy minimizations. Surprisingly, as a result of the C-terminal part of the α-subunit adopting a helical conformation, an active site had formed allowing reactants in the

solvent to enter the active site and approach the catalytic cystein B1.

In conclusion, upon the computational cleavage of the peptide bond preceding cystein 103 and the subsequent reorganization of the residues forming the new C- terminus of the α-subunit, a substrate binding site was formed which allows substrates to approach the catalytic N-terminal nucleophile C103. The obtained active site model allows for the identification of amino acids that are crucial for the catalytic properties and substrate specificity of the AT enzyme.

Apart from the conservation of the N-terminal nucleophile, the superposition of the AT precursor mutant C103A with beta-lactam acylases showed features, which were believed to be crucial for catalytic activity:

• It was observed that the main chain amide of AT Ala168 and the side chain amide group of AT Asn246 coincide with the peptide amide of penicillin G acylase Ala B69 and the side chain amide of penicillin G acylase Asn B241 , respectively. In penicillin G acylase these two amide groups form the so-called oxy-anion binding site that stabilizes the tetrahedral intermediate in the transition state. Likely, the AT oxyanion binding site facilitates the enzymatic cleavage of peptide and ester bonds via stabilization of the tetrahedral intermediate in a similar manner as is observed for the acylases. • Furthermore, the AT model shows two amino acid residues, Asp121 and Arg268, forming hydrogen bonds with the N-terminal Cys103 amino group and the side chain carbonyl oxygen of Asn246, while Thr104 next to the N-terminal nucleophile makes an additional hydrogen with the Arg268 side chain. These interactions are very conserved in beta-lactam acylases, e.g. in pen V acylase the corresponding residues are Asp20, Arg228 and Ser2 showing similar interactions as Asp121 , Arg268 and

Thr104.

A preliminary estimate of the binding pocket was obtained from calculating the accessible surface around the catalytic cystein, but a high number of options remained for accurate positioning the substrate. However, given the identified position of the catalytic cystein and the identification of the oxyanion hole in the AT model, the degrees of freedom for modelling the substrate were drastically reduced when considering the transition state. Because in the transition state the carbonyl carbon adopts an sp3 hybridisation, the transition state intermediate is usually referred to as the tetrahedral intermediate. In the tetrahedral intermediate the carbonyl carbon forms a covalent bond with the cystein sulphur and the carbonyl oxygen forms hydrogen bonds with the amides

of the oxyanion-binding site. This fixes the orientation of the substrate to a large extent and leaves mainly rotational freedom for the side chain and the beta-lactam moiety.

Modelling of penicillin G into the active site of AT. The coordinates of the PenG (penicillin G) molecule were taken from the Protein

Data Bank entry 1 FXV. After positioning PenG roughly in AT active site, in silico a covalent bond between the Cys103 sulfhydryl and the carbonyl carbon of the scissile bond was formed. In this process the carbonyl carbon adopts an sp3-hybridisation. The position of the phenylacetyl side chain and the beta-lactam 6-APA (6-amino-penicllinic acid) moiety were optimised using energy minimization while hydrogen bonding of the carbonyl oxygen with the amide groups was strictly maintained. The coordinates of the mature AT model with PenG in its active site are given in Figure 1.

Modelling of isopenicillin N into the active site of AT. The coordinates of the IPN (isopenicillin N) molecule were taken from the Protein Data Bank entry 1QJE. After positioning IPN roughly in AT active site, in silico a covalent bond between the Cys103 sulfhydral and the carbonyl carbon of the scissile bond was formed. In this process the carbonyl carbon adopts an sp3-hybridisation. The position of the alpha-L-aminoadipoyl side chain and the beta-lactam 6-APA moiety were optimized using energy minimization while hydrogen bonding of the carbonyl oxygen with the amide groups was strictly maintained.

Modelling of phenylacetyl-CoA into the active site of AT.

The coordinates of the coenzyme A molecule (CoA) were taken from the Protein Data Bank entry 1 NDI. The CoA moiety was connected to the phenylacetyl moiety via a thioether bond resulting in phenylacetyl-CoA. In the previously determined models for tetrahedral intermediates formed between AT and PenG and between AT and IPN respectively, the side chains of PenG or IPN were removed computationally leaving AT complexed with 6APA. It was observed that the removal of the acyl side chain creates enough space in the active site for phenylacetyl-CoA to enter the active site and to bind with its phenylacetyl moiety in the acyl side chain binding groove while the scissile thioester bond is close to the catalytic cysteine. As was done for the PenG substrate it is also possible to create the transition state tetrahedral intermediate between AT and the phenylacetyl moiety while 6APA remains bound. The position of the phenylacetyl side chain and the CoA moiety were optimized using energy minimization while hydrogen

bonding of the carbonyl oxygen with the amide groups was strictly maintained. Upon cleavage of the thioester bond and dissociation of the CoA moiety, 6APA is ideally positioned to carry out a nucleophilic attack on phenylacetyl cysteinyl thioester. The model for the ternary complex of AT with phenylacetyl-CoA and 6APA is in agreement with the acylation of 6APA proceeding via an acyl-cysteine enzyme intermediate instead of direct nucleophilic attack of the phenylacetyl-CoA by 6APA.

EXAMPLE 2

Identification of amino acids positions in AT for mutagenesis

From the mature AT model with PenG in the active site (for the atomic coordinates see Figure 1) amino acid positions were identified to form the active site of the mature AT and having contact with PenG. These amino acid positions can be divided into the following groups:

Group I: All positions of groups II, III and IV: 28, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 47, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 118, 119, 120, 122, 123, 124, 125, 126, 129, 130, 145, 146, 147, 148, 149, 150, 151 , 153, 154, 155, 165, 166, 167, 168, 169, 170, 171, 172,

180, 181, 182, 183, 184, 185, 189, 209, 210, 211 , 212, 213, 245, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 269, 270, 271 , 272, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 308, 309, 310, 311 , 312, 313, 314, 315 and 332. Group II: primary interaction shell comprising all positions where at least one of the side chain atoms of the amino acids at those positions is within 4 Angstrom of at least on of the atoms of (see Table 1 ).

• the phenyl acetyl side chain of the substrate (Group M-A)

• alpha-amino-adipyl side chain of the subtrate (Group M-B) • the beta-lactam moiety of the substrate (Group M-C)

• the phenyl-acetyl-CoA substrate (Group M-D)

Group III: Secondary interaction shell comprising all positions where at least one of any atoms of the amino acids at those positions is within 4 Angstrom of at least one of any atoms of the amino acids of the primary interaction shell. This group contain the positions of Group I minus the positions of Group II:

28, 31, 32, 33, 34, 37, 38, 40, 42, 47, 85, 86, 87, 88, 91, 92, 94, 95, 98, 99, 100, 102, 118, 119, 124, 125, 126, 129, 130, 145, 146, 147, 149, 150, 153, 155, 165, 169, 170, 171, 172, 180, 181 , 183, 184, 185, 189, 209, 210, 211, 212, 213, 245, 257, 258, 261 , 263, 264, 265, 266, 267, 269, 270, 271, 272, 297, 298, 299, 300, 301, 303, 304, 305, 306, 308, 309, 312, 313, 314, 315, and 332

Group IV: secondary interaction shell comprising only those positions where at least one of the side chain atoms of the amino acids at those positions is within 4 Angstrom of at least one of the side chain atoms of the amino acids of the primary interaction shell (see Table 1).

Table 1 shows that

• the amino acid selected from the group consisting of the amino acids at position 120, 122, 166 and 168 forms part of the active site of AT and has contact with the phenylacetyl side chain of PenG.

• the amino acid selected from the group consisting of the amino acids at 89, 148, 154 and 182 forms part of the active site of AT and has contact with the polar head group of the amino-adipic acid side chain of the substrate.

• the amino acid selected from the group consisting of the amino acids at position 122, 123, 262, 302, 310 and 311 forms part of the active site of AT and has contact with the beta-lactam moiety of PenG.

• the amino acid selected from the group consisting of 35, 36, 39, 41, 89, 90, 93, 96, 97, 101 , 259, 260 forms part of the active site of AT and has contact with the acetyl CoA side chain of the substrate. The right hand column shows amino acids positions in AT that have contact with the respective amino acids at the positions listed in the middle column. For instance, the amino acid at position 120 has contact with the amino acid at positions 118, 130, 148, 154, 155, 314 and 315. All the amino acids of Group Il of Group IV are candidates to be mutated in order to generate AT mutants with altered catalytic properties. Furthermore, each mutation at one of the amino acid position from Group Il may be combined with one or more of the amino acid positions of Group IV in the corresponding row.

Table 1. Amino acid positions in AT which are in contact with various parts of the substrate

The catalytic key residues C103, T104, D121, N246 and R268 also belonging to the primary interaction shell are excluded from modification.

EXAMPLE 3

Modification AT acyl side chain specificity in favour of linear aliphatic chains

During synthesis of Pen-G, AT catalyses the exchange of alpha-L-aminoadipic acid for phenylacetic acid. In order to produce penicillins with different side chains the specificity of AT must be changed. To provide penicillin with a more polar side chain, such as glutaryl-6APA or adipoyl-6APA, AT needs to bind preferably linear aliphatic side chains with a polar head group. No binding of an aromatic ring is required. Therefore the substrate binding groove, which in the wild type AT reaction has to accommodate both the aromatic phenylacetyl moiety and the alpha-L-aminoadipoyl moiety, may now be more narrow because the aliphatic side chains tolerate a more narrow groove. The

specificity can be changed in favor of aliphatic side chains by tailoring the width of the groove by substitution of W120, F122, Y166 and/or A168.

In silico, A168 was replaced by valine. The energy of the phenylacetyl side chain and the alpha-L-amino adipoyl side chain is measured for wild type and A168V mutant. Results are given below in Table 5

Table 5

( ) indicates energy of the acyl side chain after energy minimization of the mutation

Energy minimizations and energy calculations were done with Accelrys

Discover™ software. For the steepest-descent and conjugate gradient energy minimization steps a distance dependent dielectric constant was applied and charges were included, while Morse potential and cross-terms were switched off. All geometry optimisation operations were performed with the consistent valence force field in the Discover program from Accelrys Inc. The programs were run under IRIX 6.3 on an Octane Silicon Graphics station.

The results in Table 5 indicate that the mutation A168V is quite unfavorable with respect to the phenylacetyl side chain as the interaction energy gets more unfavorable by about 321 kcal/mole without further adaptation and 1 kcal/mole more unfavourable after relaxation, while the mutation has little effect on the energy of the α-L-amino adipoyl side chain and even small favorable effect after relaxation. As a consequence the mutation is a good candidate for shifting specificity from aromatic towards aliphatic side chains such as the adipoyl side chain.

EXAMPLE 4

Preparation of mutant AT polypeptides

Construction ofpHARK) encoding AT:MBP fusion protein

Recombinant E. coli XL1-Blue containing a plasmid, pHARIO encoding an AT:maltose binding protein fusion (AT:MBP), was used in this study. Plasmid pHARIO was constructed from pMAT4 [Gene 1993, 132, 199-206]. The E. coli malE gene from pMAL-c2 (New England Biolabs, U.S.A.) was amplified by PCR using a forward primer designed with a SamHI site (underlined) [5'-tcatcatcatggatccaaaatcgaagaaggtaaa-3', SEQ ID NO: 4] and a reverse primer including a H/ndlll site (underlined) after the malE gene stop codon [5'-gtcgtcgtcgtcaagctttcaagtctgggcgtctttcag-3', SEQ ID NO: 5], and the obtained fragment was inserted between the SamHI site and H/ndlll sites of pHAR6 (constructed by change of the stop codon sequence of penDE in pMAT4 into an Xba\ site by Kunkel mutagenesis [Proc. Natl. Acad. ScL U S A. 1985, 82, 488-492] resulting in pHAR9. To create an in-frame penDE-malE fusion, linker oligonucleotides (sense sequence including part of an Xba\ site and a Sacl site (both underlined) [5'- ctagatcgagctcgaacaacaacaacaataacaataacaacaacg-3', SEQ ID NO: 6] and anti-sense sequence including part of a SamHI site (underlined) [5'- gatccgttgttgttattgttattgttgttgttgttcgagctcgat-3', SEQ ID NO: 7]) were inserted between the Xba\ and SamHI sites of pHAR9 resulting in pHARIO which directs the synthesis of AT: M BP fusion protein.

Oligonucleotide site-directed mutagenesis

AT mutants were constructed in pHARIO by PCR mutagenesis using the QuickChange® Site-Directed Mutagenesis Kit (Stratagene, U.S.A.) according to the manufacturer's protocol with the following oligonucleotide primer pairs.

Preparation of soluble protein from E. coli

1) Cells transformed with pHAR10, or a mutant derivative thereof, and were grown at 28 ⁰ C in 2xTY medium containing 30μg/ml chloramphenicol, and after 2.5h cultivation, 0.1mM isopropyl-b-D-thiogalactopyranoside (IPTG) was added. After overnight growth, cells were harvested by centrifugation in 50ml polypropylene tubes (30mins 5,500rpm, 4°C). Cell pellets were frozen at -20 ⁰ C until needed. Freezing also improved the efficiency of lysis.

2) The cell pellet was resuspended in 5ml of ice-cold lysis buffer (5OmM Tris HCI, pl-17.5, 8.7% (v/v) glycerol, 0.1 %( v/v) Triton X-100) by pipetting or vortexing. Prior to vortexing, loosening of the pellet with a sterile wire greatly aided resuspension.

3) The tube containing the cell suspension was placed in an ice-water bath and sonicated using a Soniprep 150 ultrasonic disintegrator. Ten cycles of 10s sonication followed by 20s cooling time was usually sufficient for complete lysis.

4) Cell debris, insoluble protein and unlysed cells were pelleted by centrifugation (30mins, 5,500 rpm, 4°C). Precipitation of DNA was achieved by addition of protamine sulphate to 0.5% (w/v) before centrifugation of DNA.

5) The protein solution was kept on ice or frozen at -80°C until needed.

Purification of AT: MBP fusion proteins by affinity chromatography

Purifications were ideally performed at 4°C in the cold room, however, benchtop purifications at room temperature were regularly performed using ice-cold buffers with no apparent loss of yield or activity.

1) Columns were frequently prepared from syringe bodies, the end of the column was plugged with glass wool.

2) For a typical purification of 10-20 mg fusion protein, 10ml of 50% (w/v) amylose resin slurry (New England Biolabs) was pipetted into the column and allowed to settle by gravity, forming a bed of 5ml volume.

3) Once the storage buffer had drained from the column, the resin was equilibrated with 8 bed volumes of column buffer (5OmM Tris HCI pH 7.4, 5mM DTT).

4) The soluble protein preparation was diluted in column buffer to a final volume of 6 bed volumes and applied to the top of the column. To ensure saturation of the amylose resin, the flow-through was collected and reapplied to the column.

5) The resin was washed with 8 bed volumes of column buffer to remove unbound protein and small molecules.

6) The AT:MBP fusion protein was eluted from the column with elution buffer (5OmM Tris HCI pH7.4, 5mM DTT, 1OmM maltose) and collected in 1 ml fractions. The protein concentration of the fractions was determined by Bradford assay.

7) Protein solutions were aliquotted into 0.5ml micro centrifuge tubes and frozen and stored at -80 ⁰ C until needed.

EXAMPLE 5

Activity of mutant AT polypeptides

Preparation of E.coli ESS bioassay plates

Plates were prepared according to the following protocol: 1) Glycerol freeze aliquots (300μl) of E.coli ESS were used to inoculateiOOml of

2xTY that was then incubated at 37°C, 250rpm until OD _60O = 2. This usually took about 8h.

2) A 20ml aliquot of this bacterial suspension was then added to 20OmL of sterile 2xTY agar medium maintained in a liquid state at ~45°C, and the bacteria evenly distributed by swirling

3) Aliquots (18ml) of the bacterial suspension in molten agar were pipetted into Standard 87mm disposable plastic Petri dishes.

4) Once solidified, the plates were stored at 4°C and used within two weeks.

5) Holes (diameter 7mm) were made in the agar with a cork borer immediately before use.

Bioassay of acyl-coenzyme A:6-APA acyltransferase (AAT) activity

Bioassay provided a simple method of determining the AT activity of crude extracts and purified protein samples. It was possible to quantify enzymic activity by plotting eg. penicillin G concentration against diameter of zone of inhibition of growth. Typically, 0.1-30μg of protein solution was bioassayed.

1) A buffer/ substrate/ cofactor concentrate was prepared containing 0.4mM 6- APA (or other beta-lactam nucleus), 2.5 rtiM phenylacetyl-coenzyme A, 5mM DTT, 5OmM Hepes pH7.4 in a final volume of 40μl_.

2) To start the reaction, 0.1-30μg of protein solution, diluted in 5OmM Hepes pH7.4 buffer, if necessary, was added to the buffer/ substrate/ cofactor concentrate and mixed gently.

3) The reaction mix was incubated at 27°C for 1 h and the reaction stopped by addition of an equal volume of ice-cold methanol. The sample was mixed by vortexing and centrifuged at 14,000rpm, for 5mins to remove the precipitated protein.

4) The reaction mixture (80μl) was added to 7mm diameter wells in E.coli ESS bioassay plates which were then incubated at 37°C overnight.

AA T reactions for reverse phase HPL C analysis

The standard AAT reaction mix was as follows: 5OmM Hepes pH7.4, 5mM DTT, 5mM Phenylacetyl-CoA, 0.5mM 7-ADCA (or other beta-lactam nucleus) and protein in a final volume of 60μl_. Typically, 30μg of protein was added and the reaction was incubated for 2h at 27°C and then quenched by addition of equal volume of ice-cold methanol. The reactions were left on ice for 5mins to precipitate the protein, which was then pelleted by brief centrifugation at 14,000rpm. Samples were then analysed by reverse-phase HPLC.

Reverse-phase HPLC analysis of AAT reactions

Reverse-phase analytical HPLC was performed using a Gilson HPLC system with a Waters Spherisorb® S 5 ODS2 4.6x250mm column. The mobile phase consisted of two buffers: buffer A was 0.1 M NaH ₂ PO ₄ and buffer B was a mixture of 50% buffer A and 50% acetonitrile.

HPLC mobile phase conditions

Retention times

Activity data for acylation various beta-lactam nuclei

G-7-ADCA stands for CefG or phenylacetyl-7ADCAG-7-ADCCA stands for phenylacetyl-7-amino-3-carbamoyldeacetylcephalosporin C