WO2001085951A1

WO2001085951A1 - A modified expandase and uses thereof

Info

Publication number: WO2001085951A1
Application number: PCT/GB2001/002047
Authority: WO
Inventors: Robert Ian Johnson; Roger William Newbert
Original assignee: Acs Dobfar Uk Limited
Priority date: 2000-05-09
Filing date: 2001-05-09
Publication date: 2001-11-15
Also published as: AU2001252438A1; GB0011185D0

Abstract

Penicillin expandases modified to improve the ring-expanding activity for a substrate which is other than the natural substrate of the unmodified expandase and in particular expandases such as penicillin N expandase having increased specificity for a substrate, such as penicillin G.

Description

n iona pp ica ion o

PCT/GB 01/02047

A. CLASSIFICATION OF SUBJECT MATTER

IPC 7 C12N15/53 C12N15/80 C12N9/02 C12N1/15 C12P35/00

//(C12N1/15,C12R1:82)

According to International Patent Classification (IPC) or to both national classification and IPC

B. FIELDS SEARCHED

Minimum documentation searched (classification system followed by classification symbols)

IPC 7 C12N C12P

Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched

Electronic data base consulted during the international search (name of data base and where practical, search terms used)

EPO-Internal , PAJ , BIOSIS, WPI Data, CHEM ABS Data

C. DOCUMENTS CONSIDERED TO BE RELEVANT

Categor ⁰ Citation of document with indication where appropπate, of the relevant passages Relevant to claim No

WO 99 33994 A (BALDWIN JACK EDWARD ;HARL0S 1-9 KARL (GB); ROACH PETER L (GB); ISIS IN) 8 July 1999 (1999-07-08) cited in the appl ication page 6 , paragraph 2 page 7 , l ine 22 -page 9, l ine 4 cl aims 12-15

WO 97 20053 A (SUTHERLAND JOHN DAVID ;UNIV 1-9

OXFORD (GB); BOVENBERG R0EL0F ARY LANS)

5 June 1997 (1997-06-05) page 10, l i ne 3 - l i ne 21 ; cl aim 1 ; figure

1

V Further documents are listed in the continuation of box C Patent family members are listed in annex

' Special categories of cited documents

"T" later document published after the international filing date or pnonty date and not in conflict with the application but

Α" document defining the general state of the art which is not cited to understand the principle or theory underlying the considered to be of particular relevance invention

Ε" earlier document but published on or after the international "X" document of particular relevance, the claimed invention filing date cannot be considered novel or cannot be considered to

'L' document which may throw doubts on pnonty claιm(s) or involve an inventive step when the document is taken alone which is cited to establish the publication date of another "Y" document of particular relevance, the claimed invention citation or other special reason (as specified) cannot be considered to involve an inventive step when the

O" document referring to an oral disclosure use exhibition or document is combined with one or more other such docuother means ments, such combination being obvious to a person skilled

'P" document published prior to the international filing date but in the art laterthan the priority date claimed ^■&' document member of the same patent family

Date of the actual completion of the international search Date of mailing of the international search report

20 September 2001 02/10/2001

Name and mailing address of the ISA Authorized officer

European Patent Office, P B 5818 Patentlaan 2 NL-2280 HV Rιjswi|k Tel (+31-70) 3₄0-20 0, Tx 31 651 epo nl, Fax (+31-70) 3₄0-3016 Schmi tz, T

Form PCT/ISA/210 (second sheet) (July 19g≥) PCT/GB 01/02047

C.(Contiπuatlon) DOCUMENTS CONSIDERED TO BE RELEVANT

Category ' Citation of document, with indication.where appropriate, of the relevant passages Relevant to claim No.

SAMI M ET AL : "GLUTAMINE-330 IS NOT 1-5,7-9

ESSENTIAL FOR ACTIVITY IN ISOPENICILLIN N

SYNTHASE FROM ASPERGILLUS NIDULANS"

FEBS LETTERS , ELSEVIER SCIENCE PUBLISHERS ,

AMSTERDAM, NL , vol . 405, no. 2, 1997, pages 191-194,

XP002059797

ISSN: 0014-5793 the whol e document

LANGE THEO ET AL: "Molecular 1-5,7-9 characteri sation of gibberel l in

20-oxidases : Structure-functi on studies on recombi nant enzymes and chimaeri c protei ns . "

PHYSIOLOGIA PLANTARUM, vol . 100 , no. 3, 1997, pages 543-549 ,

XP001015744

ISSN: 0031-9317 the whole document

WO 9960102 A (BOVENBERG R0EL0F ARY LANS ;DSM NV (NL); NIEB0ER MAARTEN (NL)) 25 November 1999 (1999-11-25) page 8, line 1 -page 9, line 7

KOVACEVIC S ET AL: "CLONING

CHARACTERIZATION AND EXPRESSION IN

ESCHERICHIA-COLI OF THE

STREPTOMYCES-CLAVULIGERUS GENE ENCODING

DEACET0XYCEPHAL0SP0RIN C SYNTHETASE"

JOURNAL OF BACTERIOLOGY, vol. 171, no. 2, 1989, pages 754-760,

XP001015670

ISSN : 0021-9193 the whol e document

VALEGARD ET AL : "Structure of a cephal ospori n synthase"

NATURE , MACMILLAN JOURNALS LTD . LONDON ,

GB, vol. 394, no. 6695,

20 August 1998 (1998-08-20), pages

805-809, XP002095826

ISSN: 0028-0836 the whole document

-/--

Form PC17ISA/210 (continuation of second shaet) (July 1992) PCT/GB 01/02047

C(Continuation) DOCUMENTS CONSIDERED TO BE RELEVANT

Category ° Citation of document, with indication.where appropriate, of the relevant passages Relevant to claim No.

LEE HWEI-JEN ET AL: "Kinetic and crystal ! ographic studies on deacetoxycephalosporin C synthase

(DAOCS)."

JOURNAL OF MOLECULAR BIOLOGY, vol. 308, no. 5, 18 May 2001 (2001-05-18), pages 937-948, XP002174530

ISSN: 0022-2836 the whol e document

Form pcT/ISA/210 (continuation of second shaet) (July 1992)

* *\ m ^*3 Λ>p *2 Information on patent family members

PCT/GB 01/02047

Patent document Publication Patent family Publication cited in search report date member(s) date

WO 9933994 08-07-1999 AU 1772099 A 19-07-1999 CN 1284997 T 21-02-2001 EP 1047785 Al 02-11-2000 O 9933994 Al 08-07-1999 PL 341396 Al 09-04-2001

WO 9720053 05-06-1997 AU 1097297 A 19-06-1997 W0 9720053 A2 05-06-1997 EP 0863989 A2 16-09-1998 GB 2323361 A 23-09-1998 US 5919680 A 06-07-1999

WO 9960102 25-11-1999 AU 4144999 A 06-12-1999 CN 1301303 T 27-06-2001 W0 9960102 A2 25-11-1999 EP 1078069 A2 28-02-2001

Form PCT/ISA/210 (patent family annex) (July 1982) A MODIFIED EXPANDASE AND USES THEREOF

Field of the Invention

The present invention relates to a modified expandase and polynucleotides encoding the modified expandase and in particular to expandase such as penicillin N expandase having increased specificity for a substrate, such as penicillin G.

Background of the Invention

Penicillins and cephalosporins have long been used as antibiotics in the treatment of infectious diseases. Many semisynthetic derivatives based on these compounds have been tried and a large number of the compounds are in medical use. Typically, both penicillins and cephalosporins are produced by fermentation. The biosynthesis of penicillin and cephalosporins antibiotics in microorganisms requires the formation of the bicyclic nucleus of penicillin. Isopenicillin N synthase (TPNS) catalyses the reaction of a tripeptide δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine, (ACV) to form isopenicillin N.

A wide variety of organisms can produce antibiotics. Examples include Λspergillus, Streptomyces, Bacillus, Monospora, Cephalosporium, Penicillium and

Nocardia. Many of these organisms express additional enzymes that result in the conversion of isopenicillin N into a variety of different antibiotics such as penicillin N, penicillin G and penicillin V. Typically, penicillins are produced by fermentation. Cephalosporins may be produced by expansion of the 5-membered thiazolidine ring of penicillin to the 6-membered d ydrothiazine ring of cephalosporins. In particular, cephalosporins may be derived from 7-amino desacetoxycephalosporamc acid (7-ADCA). Ring expansion enzymes are expressed by a number of bacterial species. In particular, expandases have been shown to be expressed in Streptomyces clavuligerus and Streptomyces lactamdurans, and other species such as: Xanthomonas lactamgenus, Flavobacteήum sp., Flavobacterium chitinovorum, Streptomyces orgcmanensis, Nocardia lactamdurans, Streptomyces lipmanii, Sti'eptomycesjumonjinensis, Streptomyces wadayamensi, Streptomyces cattleya, Streptomyces lactamgens, Streptomyces fradiae, Streptomyces griseus, Streptomyces olivaceus and Streptomyces sp. Penicillin N expandase isolated from Streptomyces clavuligerus, also known as desacetoxycephalosporin C synthase (DAOCS) has been characterised and described in PCT publication No. WO

99/33994. Penicillin N expandase isolated from Streptomyces clavuligerus when expressed in Penicillium species has been shown to ring-expand penicillin G at low level.

Penicillin N is not commercially available. Even if penicillin N has been expanded to produce desacetoxycephalosporin C, the D-α-amino adipoyl side chains cannot be easily removed. Penicillin V or penicillin G can readily be produced by fermentation for example by P.chrysogenum. However, such penicillins are not efficiently converted by penicillin N expandase.

Summary of the Invention

The present invention provides a modified expandase having a modified or improved ring-expanding activity. In particular, the activity of the expandase for a substrate other than its natural substrate is increased. Preferably, the expandase is a penicillin N expandase which is modified to increase the activity on penicillin G or penicillin N as a substrate. In one aspect, the modification comprises deletion of the C terminus of penicillin expandase. Preferably, the modified penicillin expandase comprises: a) the amino acid sequence of SEQ ID NO: 2 which is modified or deleted at the C terminus to improve or modify the ring-expanding activity;

(b) a variant of SEQ ID NO : 2 having a modification or deletion at the C terminus to improve or modify the ring-expanding activity.

In preferred embodiments the modification comprises deletion of from 1 to 6 amino acids from the C terminus of SEQ TD NO: 2 or a variant thereof. The modified penicillin expandase preferably has improved ring expanding activity on penicillin G as a substrate. -3-

In an alternative aspect, the modified expandase comprises

(a) the amino acid sequence of SEQ ID NO: 2 in which leucine at position 158 is substituted by valine; or

(b) a variant of SEQ ID NO: 2 in which the amino acid at an equivalent position to leucine 158 of SEQ ID NO: 2 is substituted to increase the size of the side chain binding cleft of the expandase. In another aspect, the modified expandase comprises (a) the amino acid sequence of SEQ TD NO: 2 in which asparagine at position 304 is substituted by alanine; or (b) a variant of SEQ ID NO : 2 in which the amino acid at an equivalent position to asparagine 304 SEQ ID NO: 2 is substituted. In another aspect, the modified expandase comprises (a) the amino acid sequence of SEQ ID NO: 2 in which isoleucine at position 305 is substituted by leucine; (b) the amino acid sequence of SEQ TD NO: 2 in which isoleucine at position 305 is substituted by methionine;

(c) a variant of SEQ ID NO: 2 in which the amino acid residue at an equivalent position to isoleucine position 305 of SEQ TD NO: 2 is substituted by leucine; or (d) a variant of SEQ ID NO: 2 in which the amino acid residue at an equivalent position to isoleucine position 305 of SEQ TD NO: 2 is substituted by methionine.

In an alternative aspect, the modified expandase comprises

(a) the amino acid sequence of SEQ TD NO: 2 in which arginine at position 306 is substituted by leucine; or

(b) a variant of SEQ ID NO: 2 in which the amino acid at an equivalent position to arginine 306 of SEQ ID NO: 2 is substituted to increase the size of the side chain binding cleft of the expandase.

In another aspect, the modified expandase comprises (a) the amino acid sequence of SEQ TD NO : 2 in which residues 307-310 are deleted and replaced with an alanine residue; or -4-

(b) a variant of SEQ ID NO: 2 in which the amino acid residues at an equivalent position to residues 307-310 of SEQ TD NO: 2 are deleted and replaced with an alanine residue. The invention also provides a polynucleotide encoding a modified expandase according to the invention, an expression vector comprising a polynucleotide according to the invention and a host cell transformed with a polynucleotide or vector of the invention.

The invention further provides methods of ring-expanding penicillin, such as penicillin G, using a modified expandase of the invention. In another aspect, the invention provides methods of ring-expanding penicillin, such as penicillin G to produce phenylacetyl-7-ADCA or 7-ADCA.

Description of the Figures

Figure 1 is a sequence alignment between the expandase of SEQ ID NO: 2 and an expandase from Nocardia lactamdurans, hydroxylases and expandase/hydroxylase from Acremonium chrysogenum. SC stands for Streptomyces clavuligerus, NL for Nocardia lactamdurans, CEFE are expandases, CEFF are hydroxylases and CEFEF is the Acremonium enzyme.

Description of the Sequences

SEQ TD NO: 1 is the amino acid and encoding nucleic acid sequence for penicillin N expandase of Streptomyces clavuligerus.

SEQ ID NO: 2 is the amino acid sequence alone for penicillin N expandase of Streptomyces clavuligerus.

Detailed Description of the Invention

The present invention provides a modified penicillin expandase which shows increased or modified ring-expanding activity preferably on penicillin G as a substrate.

A modified penicillin expandase according to the invention may comprise an expandase derived from Streptomyces clavuligerus or an expandase derived from other bacterial or fungal species. Alternatively, related enzymes such as hydroxylase may be used. An expandase or hydroxylase for use in accordance with the invention may be one isolated from Streptomyces lactamdurans, and other species such as: Xanthomonas lactamgenus, Flavobacterium sp., Flavobacterium chitinovorum, Streptomyces organanensis, Nocardia lactamdurans, Streptomyces lipmanii, Streptomyces jumonjinensis, Streptomyces wadayamensi, Streptomyces cattleya,

Streptomyces lactamgens, Streptomyces jradiae, Sti'eptomyces griseus, Streptomyces olivaceus and Streptomyces sp and Acremonium chrysogenum.

The amino acid sequence of penicillin N expandase from Streptomyces clavuligerus is set out in SEQ ID NO: 2. Variations in the sequence of SEQ ID NO: 2 may be present in expandase obtained from other isolates or strains oϊ Streptomyces clavuligerus. Penicillin expandase or hydroxylase from other Streptomyces clavuligerus strains or other bacterial species expressing expandase or hydroxylase can be isolated following standard cloning techniques, for example, using the polynucleotide sequence of SEQ ID NO: 1 or a fragment thereof as a probe. A polypeptide for use in connection with the present invention is one which has expandase activity, namely, the ability to catalyse ring expansion of a 5- membered tbiazolidine ring, for example, of a penicillin to a 6-membered ring, for example, characteristic of cephalosporins. Preferably, a polypeptide suitable for modification is one which has penicillin expandase activity prior to modification, and preferably penicillin N expandase activity.

An expandase in accordance with the present invention is modified such that the ring expanding activity is modified or increased. In particular the activity of the expandase for a substrate other than its natural substrate is increased. For example, the modification preferably enhances the activity of the enzyme such as penicillin N expandase for penicillin G or penicillin V as a substrate. The modified expandase in accordance with the invention may have enhanced catalytic activity or increased specificity for another substrate such as penicillin G. The substrate specificity or activity of an enzyme can be monitored in vitro or in vivo for example in accordance with the methods which are described in more detail below. In particular, assays may be carried out to monitor activity of the enzyme by monitoring for production of phenylacetyl-7-ADCA production from penicillin G. Control experiments using unmodified expandase can be carried out to establish whether a modified enzyme demonstrates improved substrate specificity for penicillin G, for example, by monitoring for higher levels of phenylacetyl-7-ADCA.

Preferably, penicillin expandase is modified by mutation or deletion within the C-terminal region of the polypeptide or through substitution within the active site of the enzyme. Preferably, the modified polypeptide comprises the amino acid sequence of SEQ ID NO: 2 which is modified or deleted at the C-terminus to alter or improve the ring expanding activity, for example to improve the specificity for penicillin G as a substrate or a variant sequence of SEQ TD NO: 2 having a similar modification.

C-Terminal Modification or Deletion

In one aspect, an expandase in accordance with the invention incorporates a modification at or within the C-terminal region of the polypeptide. For example, the modification may comprise deletion of one or more of the amino acid residues within the C-terminal region. The deletion may comprise deletion of 1, 2, 3, 4, 5, up to 6 amino acids preferably deletion of 1 to 4 amino acids from the very C-terminus of the polypeptide. The deletion may be within the C-terminal region such that the very C- terminal amino acid or acids are retained but that one or more amino acids N- terminal to the C-terminal amino acid are deleted within a region up to 20 amino acids more preferably up to 10 or 5 amino acids from the very C-terminus of the polypeptide.

In another embodiment, the modified expandase includes a deletion within or at C-terminus, for example, a deletion of 1, 2 or 3, up to 4, 5 or 6 amino acids from the C-terminal region with subsequent additional modification at the C-terminus to -7- incorporate an alternative preferred amino acid. Preferably, the modified expandase comprises deletion of 1 to 4 amino acids at the C-terminus of the polypeptide and addition of a new C-terminal amino acid. Preferably, a modified peptide incorporating a non-naturally occurring C-terminal amino acid will have an amino acid selected from alanine, lysine, phenylalanine and serine at the very C-terminus.

In more detail, with reference to the amino acid sequence of SEQ ID NO: 2, preferred modifications include deletion of the C-terminal amino acids from lysine (position 310), serine (position 309), threonine (position 308), arginine (position 307) or deletion from any one of these positions and replacement of one or more amino acids to form the modified C-terminus, said replacement amino acids being selected from alanine, lysine, phenylalanine or serine. For example the modification may comprise the deletion of residues 307-310 and the replacement of these residues with the amino acid alanine (ΔR307A mutant). Alternatively, the deletion may comprise a deletion within the C-terminal, for example, deletion of the amino acids at positions 307, 308, 309, 310 or any combination of these deletions while mamtaining the other

C-terminal amino acids. For example, the modification may comprise deletion of the amino acids at position 307 and 308 so that the C-terminus of the expandase enzyme from position 306 et seq. comprises arginine, serine, lysine, alanine.

Additionally, the invention relates to a variant of SEQ ID NO: 2 having an equivalent C-terminal modification to those described above. A variant of SEQ TD

NO: 2 may be a naturally occurring variant seen in alternative strains of Streptomyces clavuligerus or an expandase or hydroxylase enzyme expressed by an organism selected from Streptomyces lactamdurans, and other species such as: Xanthomonas lactamgenus, Flavobacterium sp., Flavobacterium chitinovorum, Streptomyces organanensis, Nocardia lactamdurans, Streptomyces lipmanii, Streptomyces jumonjinensis, Streptomyces wadayamensi, Streptomyces cattleya, Streptomyces lactamgens, Streptomyces fradiae, Streptomyces griseus, Streptomyces olivaceus, Streptomyces sp and Acremonium chrysogenum. A variant may also be a non- naturally occurring variant as described in more detail below. The deletions and modifications described above may be made at or within the C-terminus of such variant polypeptides. -8-

For all embodiments, a modified expandase demonstrates improved activity in the ring-expansion of a non natural substrate such as penicillin G compared to the corresponding amino acid sequence which does not incorporate the selected C- terminal modification or deletion.

Modification of Leucine at position 158

In an alternative aspect of the invention, an expandase polypeptide incorporates mutation of leucine at position 158 of SEQ ID NO: 2 to increase the size of the side-chain binding cleft. For example leucine may be substituted with valine.

Alternative substitutions include alanine or serine. In addition, the invention relates to a variant of SEQ TD NO: 2 having an equivalent modification. A variant of SEQ ID NO: 2 may be a naturally occurring variant seen in alternative strains of Streptomyces clavuligerus or an expandase enzyme expressed by an organism selected from Streptomyces lactamdurans, and other species such as: Xanthomonas lactamgenus, Flavobacterium sp., Flavobacterium chitinovorum, Streptomyces organanensis, Nocardia lactamdurans, Streptomyces lipmanii, Streptomyces jumonjinensis, Streptomyces wadayamensi, Streptomyces cattleya, Streptomyces lactamgens, Streptomyces fradiae, Streptomyces griseus, Streptomyces olivaceus, Streptomyces sp and Acremonium chrysogenum. A variant may also be a non- naturally occurring variant as described in more detail below. The equivalent amino acid to leucine as position 158 of SEQ TD NO: 2 can be identified by aligning a variant polypeptide with the sequence of SEQ TD NO: 2 and thus to identify the equivalent amino acid of any such variant to leucine at position 158 of SEQ TD NO: 2. The equivalent amino acid is modified to enlarge the side-chain binding cleft for example to incorporate an amino acid having similar characteristics (e.g. acidic, basic etc) but having a shorter chain length for example leucine may be substituted by valine or serine or alanine.

As before, a modified expandase polypeptide demonstrates improved capacity to ring expand a substrate such as penicillin G. .9.

Modification of Asparagine at position 304

In an alternative aspect of the invention, an expandase polypeptide incorporates mutation of asparagine at position 304 of SEQ ID NO: 2. For example asparagine may be substituted with alanine. Alternative substitutions may be made.

In addition, the invention relates to a variant of SEQ TD NO: 2 having an equivalent modification. A variant of SEQ TD NO: 2 may be a naturally occurring variant seen in alternative strains of Streptomyces clavuligerus or an expandase enzyme expressed by an organism selected from Streptomyces lactamdurans, and other species such as: Xanthomonas lactamgenus, Flavobacterium sp., Flavobacterium chitinovorum, Streptomyces organanensis, Nocardia lactamdurans, Streptomyces lipmanii, Streptomyces jumonjinensis, Streptomyces wadayamensi, Streptomyces cattleya, Streptomyces lactamgens, Streptomyces fradiae, Streptomyces griseus, Streptomyces olivaceus, Streptomyces sp and Acremonium chrysogenum. A variant may also be a non-naturally occurring variant as described in more detail below. The equivalent amino acid to asparagine as position 304 of SEQ TD NO: 2 can be identified by aligning a variant polypeptide with the sequence of SEQ ID NO: 2 and thus to identify the equivalent amino acid of any such variant to asparagine at position 304 of SEQ ID NO: 2. As before, a modified expandase polypeptide demonstrates improved capacity to ring expand a substrate such as penicillin G.

Modification of Isoleucine at position 305

In an alternative aspect of the invention, an expandase polypeptide incorporates mutation of asparagine at position 305 of SEQ TD NO: 2. For example isoleucine may be substituted with leucine or with melhionine. Alternative substitutions may be made. In addition, the invention relates to a variant of SEQ ID NO: 2 having an equivalent modification. A variant of SEQ ID NO: 2 may be a naturally occurring variant seen in alternative strains of Streptomyces clavuligerus or an expandase enzyme expressed by an organism selected from Streptomyces -10- lactamdurans, and other species such as: Xanthomonas lactamgenus, Flavobacterium sp., Flavobacterium chitinovorum, Streptomyces organanensis, Nocardia lactamdurans, Streptomyces lipmanii, Streptomyces jumonjinensis, Streptomyces wadayamensi, Streptomyces cattleya, Streptomyces lactamgens, Streptomyces jradiae, Streptomyces griseus, Streptomyces olivaceus, Streptomyces sp and

Acremonium chrysogenum. A variant may also be a non-naturally occurring variant as described in more detail below. The equivalent amino acid to isoleucine as position 305 of SEQ TD NO: 2 can be identified by aligning a variant polypeptide with the sequence of SEQ ID NO: 2 and thus to identify the equivalent amino acid of any such variant to isoleucine at position 305 of SEQ ID NO: 2.

As before, a modified expandase polypeptide demonstrates improved capacity to ring expand a substrate such as penicillin G.

Modification of Arginine at position 306

In an alternative aspect of the invention, an expandase polypeptide incorporates mutation of arginine at position 306 of SEQ ID NO: 2. For example arginine may be substituted with leucine. Alternative substitutions may be made. In addition, the invention relates to a variant of SEQ ID NO: 2 having an equivalent modification. A variant of SEQ ID NO: 2 may be a naturally occurring variant seen in alternative strains of Streptomyces clavuligerus or an expandase enzyme expressed by an organism selected from Streptomyces lactamdurans, and other species such as: Xanthomonas lactamgenus, Flavobacterium sp., Flavobacterium chitinovorum, Streptomyces organanensis, Nocardia lactamdurans, Streptomyces lipmanii, Streptomyces jumonjinensis, Streptomyces wadayamensi, Streptomyces cattleya,

Streptomyces lactamgens, Streptomyces fradiae, Streptomyces griseus, Streptomyces olivaceus, Streptomyces sp and Acremonium chrysogenum. A variant may also be a non-naturally occurring variant as described in more detail below. The equivalent amino acid to arginine as position 306 of SEQ TD NO: 2 can be identified by aligning a variant polypeptide with the sequence of SEQ ID NO: 2 and thus to identify the equivalent amino acid of any such variant to arginine at position 306 of SEQ TD NO: -11-

2. The equivalent amino acid is modified to enhance the specificity of the enzyme for a substrate other than it's natural substrate for example improved specificity for penicillin G.

A modified peptide in accordance with the present invention may incorporate one or more of the modifications described for example modification or deletion within the C-terminus or, modification of leucine at position 158. In a particularly preferred embodiment, a modified polypeptide in accordance with the invention incorporates modification or deletion of the C-terminus and substitution of valine for leucine at position 158.

As described above, a variant polypeptide having an amino acid sequence which varies from that of SEQ ID NO: 2 may be modified in accordance with the present invention. A variant for use in accordance with the invention is one having expandase activity. A modified variant in accordance with the invention is one which demonstrates an improved ability to ring expand a substrate such as penicillin G when compared to a variant sequence not so modified.

A variant of SEQ ID NO: 2 may be a naturally occurring variant which is expressed by another strain of Streptomyces . Such variants may be identified by looking for expandase activity in those strains which have a sequence which is highly conserved compared to SEQ ID NO: 2. Such proteins may be identified by analysis of the polynucleotide encoding such a protein isolated from an alternative strain of Streptomyces, for example, by carrying out the polymerase chain reaction using primers derived from portions of SEQ ID NO: 2.

Variants of SEQ TD NO: 2 include sequences which vary from SEQ TD NO: 2 but are not necessarily naturally occurring penicillin N expandase. Over the entire length of the amino acid sequence of SEQ ID NO: 2, a variant will preferably be at least 80% homologous to that sequence based on amino acid identity. More preferably, the polypeptide is at least 85% or 90% and more preferably at least 95%, 97% or 99% homologous to the amino acid sequence of SEQ ID NO: 2 over the entire sequence. There may be at least 80%, for example at least 85%, 90% or 95%, amino acid identity over a stretch of 40 or more, for example 60, 100 or 120 or more, -12- contiguous amino acids ("hard homology").

Amino acid substitutions may be made to the amino acid sequence of SEQ T_D NO: 1, for example from 1, 2 or 3 to 10, 20 or 30 substitutions. Conservative substitutions may be made, for example, according to the following table. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other:

One or more amino acid residues of the amino acid sequence of SEQ ID NO: 2 may alternatively or additionally be deleted. From 1, 2 or 3 to 10, 20 or 30 residues may be deleted, or more. Polypeptides of the invention also include fragments (c) of the above-mentioned sequences. Such fragments retain expandase activity. Fragments may be at least from 10, 12, 15 or 20 to 60, 100 or 200 amino acids in length.

Such fragments may be used to produce chimeric enzymes using portions of enzyme derived from other expandase polypeptides.

One or more amino acids may be alternatively or additionally added to the polypeptides described above. An extension may be provided at the N-terminus or C-terminus of the amino acid sequence of SEQ ID NO: 2 or polypeptide variant or fragment thereof. The or each extension may be quite short, for example from 1 to 10 amino acids in length. Alternatively, the extension may be longer. A carrier protein may be fused to an amino acid sequence according to the invention. A fusion protein incorporating the polypeptides described above can thus be provided. -13-

Polypeptides of the invention may be in a substantially isolated form. It will be understood that the polypeptide may be mixed with carriers or diluents which will not interfere with the intended purpose of the polypeptide and still be regarded as substantially isolated. A polypeptide of the invention may also be in a substantially purified form, in which case it will generally comprise the polypeptide in a preparation in which more than 90%, e.g. 95%, 98% or 99%, by weight of the polypeptide in the preparation is a polypeptide of the invention.

Polypeptides of the invention may be modified for example by the addition of histidine residues to assist their identification or purification or by the addition of a signal sequence to promote their secretion from a cell where the polypeptide does not naturally contain such a sequence. It may be desirable to provide the polypeptides in a form suitable for attachment to a solid support. For example the polypeptides of the invention may be modified by the addition of a cysteine residue.

A polypeptide of the invention above may be labelled with a revealing label. The revealing label may be any suitable label which allows the polypeptide to be detected. Suitable labels include radioisotopes, e.g. ¹²⁵1, ³⁵S, enzymes, antibodies, polynucleotides and linkers such as biotin. Labelled polypeptides of the invention may be used in diagnostic procedures such as immunoassays in order to determine the amount of a polypeptide of the invention in a sample. The proteins and peptides of the invention may be made synthetically or by recombinant means. The amino acid sequence of proteins and polypeptides of the invention may be modified to include non-naturally occurring amino acids or to increase the stability of the compound. When the proteins or peptides are produced by synthetic means, such amino acids may be introduced during production. The proteins or peptides may also be modified following either synthetic or recombinant production.

The proteins or peptides of the invention may also be produced using D- amino acids. In such cases the amino acids will be linked in reverse sequence in the C to N orientation. This is conventional in the art for producing such proteins or peptides.

A number of side chain modifications are known in the art and may be made -14- to the side chains of the proteins or peptides of the present invention. Such modifications include, for example, modifications of amino acids by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH₄, amidination with methylacetimidate or acylation with acetic anhydride. The polypeptides of the invention may be introduced into a cell by in situ expression of the polypeptide from a recombinant expression vector. The expression vector optionally carries an inducible promoter to control the expression of the polypeptide.

Such cell culture systems in which polypeptides of the invention are expressed may be used in assay systems.

A polypeptide of the invention can be produced in large scale following purification by high pressure liquid chromatography (HPLC) or other techniques after recombinant expression as described below.

A polynucleotide of the invention typically is a contiguous sequence of nucleotides which is capable of hybridising selectively with the coding sequence of

SEQ ID NO: 1 or to the sequence complementary to that coding sequence. Polynucleotides of the invention include variants of the coding sequence of SEQ ID NO: 1 which encode the amino acid sequence of SEQ ID NO: 2. Such polynucleotides additionally incorporate one or more modification to encode a modified polypeptide as described in more detail above.

A polynucleotide for use in the invention and the coding sequence of SEQ ID NO: 1 can hybridize at a level significantly above background. Background hybridization may occur, for example, because of other cDNAs present in a cDNA library. The signal level generated by the interaction between a polynucleotide of the invention and the coding sequence of SEQ TD NO: 1 is typically at least 10 fold, preferably at least 100 fold, as intense as interactions between other polynucleotides and the coding sequence of SEQ TD NO: 1. The intensity of interaction may be measured, for example, by radiolabelling the probe, e.g. with ³²P. Selective hybridization is typically achieved using conditions of medium to high stringency (for example 0.03M sodium chloride and 0.003M sodium citrate at from about 50°C to about 60°C). -15-

A nucleotide sequence capable of selectively hybridizing to the DNA coding sequence of SEQ ID NO: 1 or to the sequence complementary to that coding sequence will be generally at least 80%, preferably at least 90% and more preferably at least 95%, homologous to the coding sequence of SEQ ID NO: 1 or its complement over a region of at least 20, preferably at least 30, for instance at least 40, 60 or 100 or more contiguous nucleotides or, indeed, over the full length of the coding sequence. Thus there may be at least 85%, at least 90% or at least 95% nucleotide identity over such regions.

Any combination of the above mentioned degrees of homology and minimum size may be used to define polynucleotides of the invention, with the more stringent combinations (i.e. higher homology over longer lengths) being preferred. Thus for example a polynucleotide which is at least 85% homologous over 25, preferably over 30, nucleotides forms one aspect of the invention, as does a polynucleotide which is at least 90% homologous over 40 nucleotides. For example the UWGCG Package provides the BESTFIT program which can be used to calculate homology (for example used on its default settings).

(Devereux et al (1984) Nucleic Acids Research 12, p387-395). The PILEUP and BLAST algorithms can be used to calculate homology or line up sequences (such as identifying equivalent or corresponding sequences (typically on their default settings), for example as described in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S, F et al (1990) J Mol Biol 215:403-10.

Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold (Altschul et al, supra). These initial neighbourhood word hits act as seeds for initiating searches to find HSP's containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extensions for the word hits in each direction are halted when: the -16- cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.

The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci.

USA 90: 5873-5787. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a sequence is considered similar to another sequence if the smallest sum probability in comparison of the first sequence to the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

Polynucleotides of the invention may comprise DNA or RNA. They may also be polynucleotides which include within them synthetic or modified nucleotides. A number of different types of modification to polynucleotides are known in the art.

These include methylphosphate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. For the purposes of the present invention, it is to be understood that the polynucleotides described herein may be modified by any method available in the art. Polynucleotides of the invention may be used to produce a primer, e.g a PCR primer, a primer for an alternative amplification reaction, a probe e.g. labelled with a revealing label by conventional means using radioactive or non-radioactive labels, or the polynucleotides may be cloned into vectors. Such primers, probes and other fragments will be at least 15, preferably at least 20, for example at least 25, 30 or 40 nucleotides in length, and are also encompassed by the term polynucleotides of the invention as used herein. -17-

Polynucleotides such as a DNA polynucleotide and primers according to the invention may be produced recombinantly, synthetically, or by any means available to those of skill in the art. They may also be cloned by standard techniques. The polynucleotides are typically provided in isolated and/or purified form, In general, primers will be produced by synthetic means, involving a step wise manufacture of the desired nucleic acid sequence one nucleotide at a time. Techniques for accomplishing this using automated techniques are readily available in the art.

Longer polynucleotides will generally be produced using recombinant means, for example using PCR (polymerase chain reaction) cloning techniques. This will involve making a pair of primers (e.g. of about 15-30 nucleotides) to a region of the expandase gene which it is desired to clone, bringing the primers into contact with DNA obtained from a suitable cell, performing a polymerase chain reaction under conditions which bring about amplification of the desired region, isolating the amplified fragment (e.g. by purifying the reaction mixture on an agarose gel) and recovering the amplified DNA. The primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable cloning vector.

Although in general the techniques mentioned herein are well known in the art, reference may be made in particular to Sambrook et al, 1989.

Polynucleotides or primers of the invention may carry a revealing label. Suitable labels include radioisotopes such as ³²P or ³⁵S, enzyme labels, or other protein labels such as biotin. Such labels may be added to polynucleotides or primers of the invention and may be detected using techniques known per se. Polynucleotides of the invention can be incorporated into a recombinant replicable vector. The vector may be used to replicate the nucleic acid in a compatible host cell. Thus in a further embodiment, the invention provides a method of making polynucleotides of the invention by introducing a polynucleotide of the invention into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about replication of the vector. The vector may be recovered from the host cell. Suitable host cells are -18- described below in connection with expression vectors.

Preferably, a polynucleotide of the invention in a vector is operably linked to a control sequence which is capable of providing for the expression of the coding sequence by the host cell, i.e. the vector is an expression vector. Such expression vectors can be used to express the polypeptide of the invention.

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 control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. Multiple copies of the same or different modified expandase genes may be introduced into the vector.

Such vectors may be transformed into a suitable host cell to provide for expression of a polypeptide of the invention. Thus, a polypeptide according to the invention can be obtained by cultivating a host cell transformed or transfected with an expression vector as described above under conditions to provide for expression of the polypeptide, and recovering the expressed polypeptide. More preferably, such host cells may be used in the production of 7-ADCA.

The vectors may be for example, plasmid, virus or phage vectors provided with an origin of replication, optionally a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter. The vectors may contain one or more selectable marker genes, for example a tetracycline resistance gene.

Promoters and other expression regulation signals may be selected to be compatible with the host cell for which the expression vector is designed. Multiple copies of the same or different modified expandase gene in a single expression vector, or more than one expression vector each including a modified expandase gene which may be the same or different may be transformed into the host cell.

In a preferred aspect of the invention, the promoter sequence is a promoter sequence derived from an antibiotic-producing organism and in particular a fungal organism such as from Aspergillus, Penicillium. In a particularly preferred embodiment, a promoter sequence derived from Penicillium chrysogenum is used such as the ipnA promoter sequence, or other fungal promoters such as alcR,pacC or -19- gpd.

Host cells transformed (or transfected) with the polynucleotides or vectors for the replication and expression of polynucleotides of the invention will be chosen to be compatible with the said vector. Preferably, the host cells will be antibiotic- producing cells such as fungal cells for example Penicillium cells, Acremonium chrysogenum (Cephalosporium acremonium). Alternatively, they may be cells of bacterial origin such as E.coli, particularly, for the production in vitro of the modified polypeptide of the invention.

A modified enzyme in accordance with the invention is useful in the ring- expansion of penicillin G. Such ring-expansion may be carried out in vitro or in vivo. Such ring-expansion may be used as part of a process for the production of 7-

ADCA and derivatives thereof.

A modified enzyme in accordance with the invention may be used to ring expand semisynthetic penicillins to their cephalosporin form. For example, amoxycillin could be converted to cephadroxil and ampicillin could be converted to cephalexin. Alternatively, a modified enzyme of the invention can be used in the conversion 6-APA to 7-ADCA. An enzyme in accordance with the invention can be used to convert penicillin G to cephalosporin G or penicillin N to the corresponding cephalosporin. The enzymes can be used as part of a sequence of reactions or enzymes to produce compounds of interest such as 7-ACA. Alternatively, a modified enzyme such as a modified CefF or CefEF gene described above could be used to produce hydroxylated forms of the compounds described above.

An enzyme in accordance with the invention can be used in vitro, for example, bound to an immobile substrate. The enzyme can be immobilised through the addition of a binding sequence such as a His-tag or maltose binding site or by using a general immobiliser. The immobilised enzyme can then be used in the ring expansions and conversions described above.

In an alternative aspect of the invention, host cells are provided such as

Penicillium or Acremonium cells as described above, transformed with polynucleotide encoding a modified expandase for use in one or more of the conversions described above. -20-

In a particularly preferred aspect of the present invention, the modified enzyme is expressed in a host cell which is capable of producing penicillin G such that penicillin G within the host cell is converted to phenylacetyl-7-ADCA. Host cells may be selected which naturally produce penicillin G. Such host cells may be cultured in media which promote production of penicillin G. Such host cells may be cultured in media containing phenylacetic acid to drive the production of penicillin G. Such host cells may additionally be transformed with expression vectors encoding additional enzymes required for the production of penicillin G which may subsequently be ring-expanded using a modified enzyme in accordance with the present invention.

The activity of a modified enzyme in accordance with the invention may be monitored by carrying out assays in vitro or in vivo, that is within a host cell, to monitor for ring-expanding activity of the enzyme. Such assays may include monitoring for the production of phenylacetyl-7-ADCA from penicillin G either in vitro or using an organism which is capable of producing penicillin G such as

Penicillium chrysogenum.

The activity of the enzyme may, for example, be monitored by monitoring the conversion of a co-factor (2-oxoglutarate) to the oxidized product succinate. Alternatively, after reaction with the enzyme, any residual penicillin could be digested away using a penicillinase. The antibiotic effect of the remaining cephalosporin is assayed, for example, by looking at the kill zone of samples placed in wells cut into an agar plate seeded with an indicator organism. Alternatively, activity of transformants can be tested by growing in medium containing phenyl acetic acid and assaying for the production of cephalosporin G by HPLC. Expandases and host cells of the present invention, as described above, may be used in a method of ring expansion. Suitable methods may include methods of ring expanding a substrate, for example penicillin G, comprising contacting the substrate with an expandase of the present invention or comprising culturing a host cell of the invention under suitable conditions such that the substrate (e.g. penicillin G) produced by the host cell is ring expanded. Preferably the substrate is penicillin

G. Preferably the penicillin G is ring-expanded to produce phenylacetyl-7-ADCA. -21-

This phenylacetyl-7-ADCA may then be extracted. Phenylacetyl-7-ADCA may be directly extracted from the enzyme reaction medium or may be extracted from the fermentation broth resulting from the culture of a host cell of the invention. Extraction may be performed using any suitable method. For example, following production of phenylacetyl-7-ADCA at the fermentation stage, this product and/or any 7-ADCA produced by enzymation may be efficiently recovered from the medium by methods commonly known in the art such as simple solvent extraction methods.

For example, the fermentation broth may be filtered and an organic solvent immiscible with water added to the filtrate. The pH is then adjusted in order to extract the cephalosporin from the aqueous layer. Preferably the pH is adjusted to less than 4.5, more preferably between 4 and 1. In this way cephalosporin may be separated from any impurities present in the fermentation broth. Preferably a small volume of organic solvent is used, giving a concentrated solution of the cephalosporin, so achieving reduction of the volumetric flow rates. A second possibility is that the whole broth extraction procedure is carried out at a pH of 4 or lower.

Any solvent which does not interfere with the cephalosporin molecule may be used. Suitable solvents include, for example, butyl acetate, ethyl acetate, methyl isobutyl ketone, alcohols such as butanol. Preferably the solvent used is butylacetate.

The cephalosporin may then be back extracted with water at a pH of between 4 and 10. The cephalosporin is recovered into a small volume of water, preferably a smaller volume than the volume of solvent used earlier. The recovery may be carried out at a temperature of between 0 and 50 °C, and preferably at ambient temperature. These extraction stages may be repeated to improve purity. Additionally purity may be improved by isolation of the phenylacetyl-7-ADCA as a solid as the free acid or as an inorganic salt, for example as the potassium, sodium or calcium salt. In this case the phenylacetyl-7-ADCA may be redissolved in aqueous solution between pH 4 and pH 10 in order to carry out any subsequent enzymation to 7-ADCA. The aqueous cephalosporin solution from this resuspension, or obtained by an alternative extraction method, may then be treated with a suitable enzyme in order to -22- remove the phenylacetyl side chain and obtain 7-ADCA. Suitable enzymes for this include penicillin G acylases (also known as penicillin amidases).

Preferably an immobilised enzyme is used, so that the enzyme may be used repeatedly. Methods for the preparation of such particles and the immobilisation of enzymes have been described extensively in the art.

The pH of the aqueous solution should be chosen in order to minimise the degradation of cephalosporin and optimise the desired conversion with the enzyme. A suitable pH may easily be determined by the skilled person, and will be dependent on, for example, the particular enzyme used. A suitable pH may be, for example, between 4 and 9.

The enzyme is added to the aqueous cephalosporin solution while maintaining the pH at an appropriate level by, for example, adding an inorganic base, such as potassium hydroxide solution, or applying a cation exchange resin. When the reaction is completed the immobilised enzyme may be removed by filtration. Alternatively, the enzyme may be immobilised and applied in a fixed or fluidised bed column, of the enzyme may be used in solution with the products being removed by membrane filtration.

The reaction mixture may then be acidified in the presence of an organic solvent immiscible with water. Preferably the pH is adjusted to about 0.1 to 1.5. The layers are then separated and the pH of the aqueous layer adjusted to between 2 and 5. The crystalline 7-ADCA may then be filtered off.

The deacylation may also be carried out chemically. Suitable methods would be known to the skilled person. For example, chemical deacylation may be carried out via the formation of an imino-chloride side chain, by adding phosphorus pentachloride at a temperature of lower than 10 ° C and subsequently adding isobutanol at ambient temperature or lower.

7-Amino desacetoxycephalosporamc acid (7-ADCA) produced by the methods of the present invention is a starting point for the production of a range of semi-synthetic cephalosporins, most notably cephalexin.

Examples -23-

Materials and methods

Strains, media and growth conditions

Penicillium chrysogenum strains A and B. Escherichia coli strains for genetic manipulation and protein expression were XL 1-blue (Stratagene) JM109(DE3) (Promega). Solid medium for growth of P. chrysogenum transformants was glycerol-molasses (GM) as detailed by Smith etal (1989), supplemented where necessary with phleomycin (Cayla) to a concentration of 50μg/ml. Standard solid and liquid media were used for the growth of Kcoli, supplemented where necessary with phleomycin and kanamycin to concentrations of 5μg/ml and 50 μg/ml repectively. Nucleic acids

E.coli expression vector pHLl consisting of the 1086bp NdeVBamTTT fragment of pNM88 (Morgan et al 1994) cloned into corresponding sites within the pET24a (Promega) multiple cloning site. E. coli sub cloning vector pUC 19 (Promega)

Fungal expression vector pUT1040 (Cayla) carrying the Trichoderma reesei cbhl promoter and terminator for expression of the gene of interest, and the Streptomyces hindustanus bleomycin resistance gene for selection of transformants in both bacterial and fungal hosts. Mutagenesis

In vitro mutagenesis was carried out using the Quickchange site-directed mutagenesis system (Stratagene) and the Unique Site Elimination (U.S.E.) System (Pharmacia) or by Kunkel mutagenesis (Kunkel (1985) Proc. Natl. Acad Sci 82, 488- 492; Kunkel et al (1987) Meth. Enzymol. 154, 367-382). Mutant proteins were purified essentially as reported in Lloyd et al (1999) J. Mol. Biol. 287, 943-960. The intended identity of the mutants was confirmed by sequencing. Purification

Purification of the N-terminal His-tagged expandase protein was carried out by Immobilised Metal Affinity Chromatography using Ni-NTA agarose (Qiagen). Isolation and manipulation of DNA

Plasmid DNA for mutagenesis, sequence analysis and transformation was -24- prepared using the Wizard midiprep system (Promega) and the Qiaquick spin-column system (Qiagen). Restriction digests, ligations and PCR amplifications were carried out using enzymes from Promega and Kramel Biotech.

Experimental details

Construction of expression vectors Modification of pHLl.

Examination of the S. clavuligerus expandase crystal structure revealed that the N-terminus lies on the surface of the enzyme, well away from the putative active site, and is therefore unlikely to affect activity if modified. Bacterial expression plasmid pHLl, containing the Streptomyces clavuligerus cejE gene, was linearised with Ndeϊ. Two complementary oligonucleotides were annealed by heating to 70 °C for 15 minutes and allowing to cool to room temperature. The resulting linker, encoding a hexa-His sequence followed by an R-G-S thrombin cleavage site, was ligated into pHLl using Ndel half sites created by overlaps at both ends of the linker.

The resulting construct was designated pTAG24. Translated linker sequence: M-P-I-H-H-H-H-H-H-R-G-S-H Development of fungal expression vectors.

The two Ndel sites in pUT1014 (Cayla) were sequentially destroyed by Ndel digestion, Klenow mediated overlap fill-in, followed by blunt-end ligation. The

895bp ipnA promoter sequence was amplified from P. chrysogenum strain A using primers carrying restriction sites in 5' extensions to permit replacement of the HindlTTJBamHT cbhl promoter fragment in pUTl 014. During PCR amplification of the ipnA promoter, the ACCATG sequence at the ipnA translation start site was mutated to introduce a CATATG N<iel restriction site and allow in frame ligation of the cefβ gene. The 1086bp Ndel/BamRT fragment of pHLl containing the cefE gene was then inserted to generate the completed plasmid, designated pNSla. On sequencing, a single base mutation was identified at position -107 from the ATG translation start site (TACACTT compared to TACTCTT of the published sequence). Since this sequence was amplified from a high titre production strain of

P. chrysogenum, it is considered possible that this mutation was introduced during -25- rando mutagenesis and selected as a result of beneficial effects on ipnA transcription. The vector was therefore retained for expression studies.

Amplification and subcloning of the ipnA promoter sequence were repeated using template DNA from the distantly related P .chrysogenum strain B. The amplified fragment was subcloned into the BamΗT/HindTTl sites of pUC 19 using corresponding sites carried on the 5' extensions of the oligonucleotide primers. The promoter sequence was then liberated by a HindXTTJNdel digest, and used to replace the promoter sequence in pVSla. The resulting plasmid was designated pNSlb. Using the methodology detailed above, the acvA gene promoter was also amplified from P. chrysogenum strain B and subcloned; first as a HindTTXlBamYTT fragment into pUC19, and from there as a HindTTXJNdel fragment into pVSla, replacing the ipnA promoter sequence. During amplification of the cvA promoter, the GACATG sequence at the αcvA translation start site was mutated to introduce a CATATGN- restriction site and allow in frame ligation of the cefE gene. Expression and purification pTAG24, and derivatives carrying sequence-verified mutations, were used to transform Ecoli JM109(DE3) competent cells, and the expandase protein expressed according to the protocols laid down for the pET expression system (Promega). Following induction with IPTG, the expandase was expressed for 3 hours at 30° C, thus avoiding insolubility associated with expression of this protein at 37°C.

After incubation, the cells were harvested by centrifugation and resuspended in 10ml of extraction buffer per 100ml starting culture. The extraction buffer was 50mM Tris-HCl, pH7.5, 20mM imidazole, 300mM ΝaCl, ImM bensamidine-HCl, ImM PMSF, and 1 μM leupeptin. The cells were then lysed by sonication; three 30 second bursts, amplitude 5, at 30 second intervals. Cellular debris was removed by centrifugation, and the clear lysate passed down a column containing 1ml Νi-ΝTA agarose (Qiagen), pre-equilibrated with 50mM Tris-HCl pH7.5, 20mM imidazole, 300mM ΝaCl (column buffer). The matrix-protein complex was washed twice with 3ml column buffer, and eluted in 1ml 50mM Tris-HCl pH7.5, 200mM imidazole. Purified fractions were dialysed against 2L 50mM Tris-HCl pH7.5 for 15 hours at

4°C to remove the imidazole which would otherwise affect in vitro ring-expansion

Claims

-26- reactions.

For use in Experiment 5, purified DNA and the pET24a vector were digested with Stw I and BamH I (10 units) at 37° C for 2 hours and repurified. Digested DNA (1 :5 ratio), 10 x T4 ligase buffer (1 μl) and T4 DNA ligase (0.5 μl, 10 units) in 10 μl were incubated at 22 °C overnight. The ligated DNA (5 μl) was transformed into E. coli DH5a competent cells (100 μl) and plated onto a LB agar containing 100 μl/ml kanamycin sulfate or ampicillin, and incubated at 37 °C overnight. The purified plasmid was analyzed by restriction enzyme digests and DNA sequencing. In vitro ring expansion assays Protein concentrations were determined using a spectrophotometric assay

(COB AS BIO, Roche Products) and diluted to a concentration of 0.5mg/ml. A co- factor cocktail, prepared from x5 stock solutions was 50mM Tris-HCl pH7.5 containing lOOmM (NH₄)₂SO₄, 2mM L-ascorbic acid, lOmM α-ketoglutarate, 4mM FeSO₄. All x5 stock solutions were prepared in 50mM Tris-HCl pH7.5, except FeSO₄ which was dissolved in ultra-pure water and added last to the cocktail mix.

50μl of cofactor cocktail was added to each 20μl of enzyme solution, and pre- incubated for 2 minutes before adding 20μl Tris-HCl pH7.5. Reactions were started by the addition of lOμl lOOmM potassium penicillin G in 50mM Tri-HCl pH7.5. Reactions were carried out for 20 minutes at 30°C at lOOOrpm on a Vibrax shaker, and were quenched by the addition of lOOμl of methanol and lOOμl ultra-pure water.

Phenylacetyl-7-ADCA concentration was assayed by HPLC analysis using a 25mM ammonium acetate mobile phase in 15% v/v methanol. Samples were run at 1 ml/minute through a 300 A Hypersil C4 column with a guard filter, and detection carried out at a wavelength of 250nm. In Experiment 5, recombinant E. coli cells were fermented, and the enzymes purified, analyzed, and assayed for G-7-ADCA as described, except that a 0.1-0.3 M NaCl gradient over 800 ml was used to elute the Q-sapharose anion-exchange column. (Lloyd et al, 1999). Results 1. Active site mutations

A mutation, LI 58V was introduced by site-directed mutagenesis, and, after -27- expression and purification, the activities of the mutant enzymes were assessed by HPLC assay as detailed above. Since a second protein of approximately 45 kDa co- purifies with the expandase enzyme, samples of the protein dilutions assayed were analysed by SDS-PAGE. Using a BioRad imaging densitometer, protein concentration comparisons were used to normalise the assay results with respect to the wild-type enzyme control. Analysis demonstrated LI 58V to have an activity 217% of that of the wild-type enzyme. L158A, however, displayed on activity only 20% of that of the wild-type enzyme.

In a separate experiment, mutations of N304A and R306L were shown to have activities of 141% and 98% respectively compared to wild type. The specificity of R306L for penicillin G compared to wild type was increased.

2. C-terminal mutations

Analysis of the S.clavuligerus expandase crystal structure revealed a trimeric arrangement in which the C-terminal residues from one DAOCS molecule interact with the active site of a neighbouring molecule (Lloyd et al 1999). Sequential deletions of amino-acid residues from the C-terminus were therefore made, and the corresponding proteins expressed and assayed as detailed above. In vitro activities observed, normalised with respect to the wild-type enzyme, were: ΔK310 167% ΔS309 181%

ΔT308 186%

3. In vitro analysis of double mutants

Double mutants carrying LI 58V and each of the above C-terminal deletion mutations have been generated and in the cases of L158V/ΔS309 and L158V/ΔT308 appear to display synergistic increases in expandase activity with penicillin G as the substrate

L158V/ΔS309 475%

L158V/ΔT308 465%

4. Further mutation of C-terminal deletion mutants Given the above data, it would seem reasonable to assume that for each of the

C-terminal truncations, activity will not be optimal and may depend on the nature of -28- the terminal amino acid. For this reason the following mutants have been generated and are undergoing in vitro activity assays:

ΔK310A

ΔK310L

ΔK310F

ΔK310D

ΔK310S

5. Effects of mutations on activity

In a separate experiment, the relative specific activity of C-terminal deleted mutants compared with wild-type enzyme was measured for penicillin N and penicillin G by radioassay and by HPLC assay (Table 1). The specific activity of wild-type enzyme with penicillin N as substrate was 18 nmol/mg/min (HPLC) and 21 nmol/mg/min (radioassay), and with penicillin G was 86 nmol/min/mg (HPLC) and 128 nmol/min/mg (radioassay). Circular dichroism analyses indicated the mutants to have similar secondary structure elements to the wild-type enzyme.

Table 1

Relative activity (%)

PeniciHJ LuN Penicillin G

Proteins C-terminal sequence Radioassay HPLC Radioassay HPLC assay assay

Wild-type IGGNYVNIRRTSKA 100 100 100 100

ΔG300 IGG - <5 - <5

ΔR307A IGGNYVNIRA 67 72 84 103

ΔK310 IGGNYVNIRRTSK 162 256 136 166

After truncation of eleven residues (ΔG300) the resulting mutant was (almost) totally inactive with penicillin N.

The results indicated that there is a significant difference in the effect of C- terminal truncations on the way in which the enzyme catalyzes penicillin N oxidation compared to penicillin G oxidation. -29-

6. In vivo expression studies

The L158V mutation has been introduced into pVSla, pVSlb and pVSlc both on its own, and in combination with the C-terminal deletions detailed above. Transformation of P. chrysogenum strain A has demonstrated expression and activity of the wild-type expandase from pVSla, pVSlb and pVSlc. Titres of phenylacetyl- 7-ADCA are variable due to ectopic integration of the transforming plasmids. However, a generally higher titre has been observed in pVSlb/L158V and p VS 1 cL 158 V transformants .