Title of Invention

MUTANT EXPANDASES

Abstract The present invention relates to a mutant expandase that is a variant of a model polypeptide with expandase activity whereby the mutant expandase has an at least 2-fold improved in vitro expandase activity towards adipyl-6-APA in comparison with the model polypeptide with expandase activity.
Full Text MUTANT EXPANDASES
Field of the invention
The present invention relates to mutant expandase enzymes, polynucleotides
encoding such enzymes, to microorganisms transformed with said polynucleotides
encoding said mutant expandase enzymes and with the use of such mutant expandase
enzymes or such microorganisms in the ring expansion of 5-carboxypentanoyl-6-
aminopenicillanic acid (adipyl-6-APA = ad-6-APA).
Background of the invention
Beta-lactam antibiotics constitute the most important group of antibiotic
compounds with a long history of clinical use. Among this group, the prominent ones
are the penicillins and cephalosporins. Penicillins are naturally produced by various
filamentous fungi such as Penicillium (e.g. P. chrysogenum). Cephalosporins are
naturally produced by various microorganisms such as Acremonium (e.g. A.
chrysogenum) and Streptomyces (e.g. Streptomyces clavuligerus)
As a result of classical strain improvement techniques, the production levels of
the antibiotics in P. chrysogenum and A. chrysogenum have increased remarkably over
the past decades. With the increasing knowledge of the biosynthetic pathways leading
to penicillins and cephalosporins, and the advent of recombinant DMA technology, new
tools for the improvement of production strains have become available.
Most enzymes involved in p-lactam biosynthesis have been identified and their
corresponding genes have been cloned, as can be found in Ingolia and Queener, Med
Res Rev (1989) 9:245-264 (biosynthesis route and enzymes), and Aharonowitz,
Cohen, and Martin, Ann Rev Microbiol (1992) 46:461-495 (gene cloning).
The first two steps in the biosynthesis of penicillin in P. chrysogenum are the
condensation of the three amino acids L-5-amino-5-carboxypentanoic acid (L-ccaminoadipic
acid) (A), L-cystein (C) and L-valine (V) into the tripeptide LLD-ACV,
followed by cyclization of this tripeptide to form isopenicillin N. This compound contains
the typical (3-lactam structure.
The third step involves the replacement of the hydrophilic side chain of L-5-
amino-5-carboxypentanoic acid by a hydrophobic side chain by the action of the
enzyme acyltransferase (AT),
In EP-A-0448180 has been described that the enzymatic exchange reaction
mediated by AT takes place inside a cellular organelle, the microbody. The observation
that substantial quantities of deacetoxycephalosporin C (DAOC) can be formed by nonprecursed
P. chrysogenum transformants expressing deacetoxycephalosporin C
synthase (EC 1.14.20.1 - DAOCS, further indicated herein as expandase) implies the
presence of significant amounts of penicillin N, the natural substrate for expandase, in
P. chrysogenum (AM et al., J Antibiot (1995) 48:338-340). However, the D-a-aminoadipyl
side chains of DAOC cannot be easily removed.
Cephaiosporins are much more expensive than penicillins. One reason is that
some cephalosporins (e.g., cephalexin) are made from penicillins by a number of
chemical conversions. Another reason is that, so far, only cephaiosporins with a D-aamino-
adipyl side chain could be tormented. Cephalosporin C, by far the most
important starting material in this respect, is very soluble in water at any pH, thus
implying lengthy and costly isolation processes using cumbersome and expensive
column technology. Cephalosporin C obtained in this way has to be converted into
therapeutically used cephaiosporins by a number of chemical and enzymatic
conversions.
The methods currently favored in industry to prepare the intermediate 7-
amino-deacetoxycephaloporanic acid (7-ADCA) involve complex chemical steps
leading to the expansion and derivfltization of penicillin G. One of the necessary
chemical steps to produce 7-ADCA involves the expansion of the 5-membered
penicillin ring structure to a 6-membered Cephalosporin ring structure (see for instance
US 4,003,894). This complex chemical processing is both expensive and noxious to
the environment.
Consequently, there is a great desire to replace such chemical processes with
enzymatic reactions such as enzymatic catalysis, preferably during fermentation. A key
to the replacement of the chemical expansion process by a biological process is the
central enzyme in the Cephalosporin biosynthetic pathway, expandase.
The expandase enzyme from the bacterium Streptomyces clavuligerus (S.
clavuligerus) was found to carry out, in some cases, penicillin ring expansions. When
introduced into P. chrysogenum, it can convert the penicillin ring structure into the
Cephalosporin ring structure, as described in Cantwell et al., Proc R Soc Lond B (1992)
248:283-289. The expandase enzyme has been well characterized (EP-A-0366354)
both biochemical and functional, as has its corresponding gene. Both physical maps of
the cefE gene (the gene encoding the expandase enzyme of S. clavuligerus - EP-A-
0341892), DNA sequence and transformation studies in P. chrysogenum with cefE
have been described. The DNA and amino acid sequence of the S. clavuligerus
expandase enzyme are represented in SEQ ID NO 1.
Another source for an expandase enzyme is the bacterium Nocardia
lactamdurans (N. lactamdurans, formerly S. lactamdurans). Both the biochemical
properties of the enzyme and the DNA sequence of the gene encoding the enzyme
have been described - see Cortes et al., J Gen Microbiol (1987) 133:3165-3174 and
Coque et al., Mol Gen Genet (1993) 236:453-458, respectively. The DNA and amino
acid sequence of the N. lactamdurans expandase enzyme are represented in SEQ ID
NO 2.
Recently, novel expandase (cefE) genes and enzymes have been found in
Streptomyces jumonjinensis, Streptomyces ambofaciens and Streptomyces
chartreuses - see Hsu et al. (2004), Appl. and Environm. Microbiol. 70, 6257-6263.
Table 1 summarizes the amino acid sequence identities from several expandases and
shows that the sequence identities range from 67 to 85%, depending on the source of
the enzyme.
Table 1 : Amino acid sequence identities of several expandases (data taken from Hsu et al).
(Table Removed)
In the biosynthesis of cephatosporins, which takes mainly place in prokaryotic
cells, the deacetoxycephalosporin-C is subsequently converted to deacetylcephalosporin-
C by the enzyme deacetylcephalosporin C synthase also named
deacetoxycephalosporin-C hydroxylase or hydroxylase (EC 1.14.11.26 - DACS).
Genes encoding such hydroxylases are named cefF-genes (e.g. see Hsu et al.) The
expandase found in eukaryotic cells, e.g. Acremonium chrysogenurn can catalyze the
direct conversion of penicillin N to deacetoxycephalosporin-C due to possession of
both expandase and hydrolyase activity, hence the encoded gene is termed cefEF(see
Hsu et al.),
As defined herein, the term expandase relates to expandase enzymes (EC
1.14.20.1, encoded by cefE genes) as well as expandases also possessing in addition
the hydroxylase activity (EC 1.14.20.1 + EC 1.14.11.26 encoded by cefEF genes)
Since the expandase enzyme catalyses the expansion of the 5-membered
thiazolidine ring of penicillin N to the 6-membered dihydrothiazine ring of DAOC, this
enzyme would be of course a logical candidate to replace the ring expansion steps of
the chemical process. Unfortunately, the enzyme works on the penicillin N intermediate
of the cephalosporin biosynthetic pathway, but not or very inefficiently on the readily
available inexpensive penicillins as produced by P, chrysogenurn, like penicillin V or
penicillin G. Penicillin N is commercially not available and even when expanded, its Da-
amino-adipyl side chain cannot be easily removed by penicillin acylases.
It has been reported that the expandase enzyme is capable of expanding
penicillins with particular side chains to the corresponding 7-ADCA derivative. This
feature of the expandase has been exploited in the technology as disclosed in EP-A-
0532341, W095/04148 and W095/04149, In these disclosures the conventional
chemical in vitro conversion of penicillin G to 7-ADCA has been replaced by the in vivo
conversion of certain 6-aminopenicillanic acid (6-APA) derivatives in recombinant
Penidllium chrysogenurn strains transformed with an expandase gene.
More particularly, EP-A-0532341 teaches the in vivo use of the expandase
enzyme in P. chrysogenurn, in combination with a adipyl side chain (further referred to
as adipyl) as a feedstock, which is a substrate for the acyltransferase enzyme in P.
chrysogenurn. This leads to the formation of adipyl-6-APA, which is converted by an
expandase enzyme introduced into the P. chrysogenurn strain to yield adipyl-7-ADCA.
Finally, the removal of the adipyl side chain is described, yielding 7-ADCA as a final
product. Furthermore, EP-A-540210 teaches the similar production of adipyl-7-ADAC
and adipyl-7-ACA.
As an alternative approach, expansion of penicillin G using P. chrysogenurn
transformed with cefE has been proposed in EP0828850. Furthermore, in order to
increase the expansion of penicillin G the use of mutant cefE gene of S. clavuligerus
has been described. US 5,919,680, WO 98/02551, WO 99/33994, WO 01/85951 and
EP 1348759 all disclose mutant cefE genes allegedly coding for enzymes having a
higher expandase activity on penicillin G.
Nevertheless, these mutant expandases still do not provide for a commercially
attractive process for the production of cephalosporins.
Therefore, there is a need for further improving the process wherein
cephalosporins are prepared from expansion of adipyl-6-APA. This process is
characterized by an efficient removal of the side chain material from the cephalosporin
core material. One step to further improve this process is improvement of the
expandase activity on adipyl-6-APA.
Description of the Figures
1/3. A& B Amino acid alignment of improved expandase mutants; SCLAVEIW
represents amino acid sequence of expandase of S. clavuligerus; NOCAEIW
represents amino acid sequence of expandase of N. lactamdurans; For the
other sequences the notation is as follows: 309EIWIT represents amino acid
sequence of expandase of mutant expandase H309, and so son.
2/3. Schematic representation of vector plATWAn
3/3. HPLC analysis of expandase assay mixture according to Example 3
A. at t = 0 minutes
B. at t = 30 minutes
Detailed description of the invention
In a first aspect, the invention provides a mutant expandase that is a variant of
a model polypeptide with expandase activity. Preferably, the invention provides a
mutant expandase, whereby the mutant expandase has an at least 2-fold improved in
vitro expandase activity towards adipyl-6-APA in comparison with the model
polypeptide with expandase activity. The determination of the in vitro expandase
activity towards adipyl-6-APA is described in detail in the Materials and Methods
(Assay (1) - (3)). More preferably the in vitro expandase activity towards adipyl-6-APA
of the mutant expandase is improved at least 2.5-fold, more preferably at least 3-fold,
more preferably at least 4-fold, more preferably at least 5-fold, more preferably at least
6-fold, more preferably at least 7-fold, more preferably at least 8-fold, more preferably
at least 9-fold, more preferably at least 10-fold, more preferably at least 11-fold.
In some of the mutant expandase enzymes according to the invention parts of
the amino acid sequence of the expandase of S. clavuligerus have been replaced by
corresponding parts of the amino acid sequence of expandase of N. lactamdurans. The
amino acid sequence of the Ce/E gene of N. lactamdurans strain ATCC27382 is
represented in SEQ ID NO: 2.
In the description of these mutant expandases the amino acids are numbered
according to the numbering of the homologous parts of the amino acid sequence of the
expandase of S. clavuligerus. The hotnology of the expandase amino acid sequences
of S. clavuligerus and N. lactamdurans is to be derived from Figure 1 and Table 1,
With "altered or mutant expandase" in the context of the present invention is
meant any enzyme having expandase activity, which has not been obtained from a
natural source and for which the amino acid sequence differs from the complete amino
acid sequences of the natural expandase enzymes of S. clavuligerus and N.
lactamdurans.
The invention also provides a mutant expandase which is preferably being
modified at least at an amino acid position selected from group 1 consisting of positions
2, 18, 59, 73, 74, 89, 90, 99, 101, 105, 112, 113, 155, 170, 177, 209, 213, 217, 244,
249, 251, 277, 278, 280, 281, 284, 293, 300, 307 and 311 using the amino acid
position numbering of the amino acid sequence of the expandase enzyme encoded by
the cefE gene of Streptomyces clavuligerus. The nucleotide sequence of the cefE gene
of Streptomyces clavuligerus as well as the amino acid sequence encoded by said
cefE gene are depicted in SEQ ID NO: 1. More preferably, the mutant expandase is
modified at least at an amino acid position selected from group 2 consisting of positions
2,18,59,89,90,99, 101, 105, 112, 113,170,177,209,213,217,249,251,278, 280,
284 and 293. The mutant expandase may also have been modified at least at an amino
acid position selected from the group 1 which does not form part of group 2, together
with at least an amino acid position selected from the group 2.
A highly preferred mutant expandase is modified at least at amino acid
position 89, in combination with any of the amino acid positions of the expandase,
preferably with an amino acid position selected from group 1 or an amino acid position
selected from group 2 or an amino acid position selected from group 1 which does not
form part of group 2, together with at least an amino acid position selected from the
group 2.
Most preferably, the invention provides a mutant expandase whereby the
mutant expandase has an at least 2-fold improved in vitro expandase activity towards
adipyl-6-APA in comparison with the model polypeptide with expandase activity, more
preferably at least 2.5-fold, more preferably at least 3-fold, more preferably at least 4-
fold, more preferably at least 5-fold, more preferably at least 6-fold, more preferably at
least 7-fold, more preferably at least 8-fold, more preferably at least 9-fold, more
preferably at least 10-fold, more preferably at least 11-fold and whereby the mutant
expandase is preferably being modified at at least an amino acid position selected from
the group 1 defined above using the amino acid position numbering of the amino acid
sequence of the expandase enzyme encoded by the cefE gene of Streptomyces
clavuligerus. More preferably, the mutant expandase is modified at least at an amino
acid position selected from the group 2. The mutant expandase with the improved
expandase activity may also have been modified at least at an amino acid position
selected from the group 1 together with at least at an amino acid position selected from
the group 2,
A highly preferred mutant expandase with improved expandase activity is
modified at least at amino acid position 89, in combination with any of the amino acid
positions of the expandase, preferably with an amino acid position selected from group
1 or an amino acid position selected from group 2 or an amino acid position selected
from group 1 together with at least at an amino acid position selected from group 2.
The modification at an amino acid position may comprise a substitution by
another amino acid, selected from the group of 20 L-amino acids that occur in Nature -
see Table 2. Alternatively, the modification at an amino acid position may comprise a
deletion of the amino acid at said position. Furthermore, the modification at an amino
acid position may comprise a substitution of one or more amino acids at the C-terminal
or N-terminal side of said amino acid.
(Table Removed)
The model polypeptide with expandase activity as used in the present
invention is selected from the group consisting of a polypeptide with expandase
activity, preferably having an amino acid sequence according to SEQ ID NO: 1 or
having an amino acid sequence according to SEQ ID NO: 2 and polypeptides with
expandase activity having an amino acid sequence with a percentage identity with SEQ
ID NO: 1 of at least 70%, preferably at least 75%, more preferably at least 80%, more
preferably at least 85%, more preferably at least 90%, most preferably at least 95%, or
with SEQ ID NO: 2 of at least 70%, preferably at least 75%, more preferably at least
80%, more preferably at least 85%, more preferably at least 90%, most preferably at
least 95%, such as the expandase enzymes that are summarized in Table 1. Most
preferred as model polypeptide with expandase activity as used in the present
invention is a polypeptide with expandase activity, having the amino acid sequence
according to SEQ ID NO: 1 or having the amino acid sequence according to SEQ ID
NO: 2.
The present invention preferably provides mutant expandases that have
modifications at least at
o 2 or more amino acid positions selected from the group consisting of 2, 89, 277,
281 and 300, more preferably at positions 2+281 or 89+281 or 277+300.
o 3 or more amino acid positions selected from the group consisting of 2, 89, 281,
293 and 311, more preferably at positions 2+89+281 or 89+281+311 or
89+281+293.
o 4 or more amino acid positions selected from the group consisting of 2, 73, 89,
90, 217, 244, 277, 280, 281, 306, 307 and 311, more preferably at positions
2+277+280+281 or 2+281+307+311 or 73+89+281+311 or 89+217+281+311 or
89+244+281+311 or 89+281+307+311 or 277+281+306+311 or
89+281+306+311 or 90+281+306+311.
o 5 or more amino acid positions selected from the group consisting of 2, 73, 89,
90,155, 213, 249, 278, 281, 293, 300, 306, 307 and 311, more preferably at
positions 2+89+281+306+311 or 2+155+281+306+311 or 73+89+213+281+311
or 89+78+218+307+311, 89+281 +293+300+311 or 89+249+281 +307+311.
o 6 amino acid positions selected from the group consisting of
90+105+113+281+306+311.
o 7 or more amino acid positions selected from the group consisting of 2, 73, 89,
105,113, 155, 170,177, 251, 277, 280, 281, 293, 300, 306, 307 and 311, more
preferably 73+89+281+293+300+307+311 or
105+113+155+177+281 +306+311 or 155+177+277+280+281 +306+ 311.
o 8 or more amino acid positions selected from the group consisting of 2, 89, 90,
99,105,113,155,177, 277, 281, 307 and 311 more preferably at
2+90+99+105+113+281 +306+311 or 2+90+105+113+155+177+277+281
o 9 amino acid positions selected from the group consisting of
2+90+105+113+155+177+281+306+311.
o 10 amino acid positions selected from the group consisting of 2, 59, 90,101,
105,113,155,177, 209, 277, 281, 307 and 311 more preferably at
2+90+105+113+155+177+277+281 +306+311.
o 11 amino acid positions selected from the group consisting of 2, 90,105,113,
155,177,277,281,293,307,311.
The present invention relates to substitutions of certain of the amino acids of
expandase. Preferably such substitutions involve replacement of:
• histidine at position 18 according to SEQ ID NO 1 by cystein, serine, threonine,
asparagine, glutamine, tyrosine, lysine and arginine; more preferably serine,
threonine, asparagine, glutamine, lysine and arginine; most preferably arginine;
• methionine at position 74 according to SEQ ID NO 4 by threonine or by a
hydrophobic amino acid such as valine, leucine, isoleucine and phenylalanine or by
an amphiphilic amino acid such as tyrosine, tryptophan, histidine, glutamine and
asparagine, more preferably threonine or isoleucin;
• Threonine at position 89 according to SEQ ID NO 1 preferably by a charged amino
acid, preferably a negatively charged amino acid such as aspartic acid or glutamic
acid, most preferably a positively charged amino acid such as lysine, arginine or
hystidine; most preferred being lysine.
• serine at position 112 according to SEQ ID NO 1 preferably by threonine;
• histidine at position 244 according to SEQ ID NO 1 by glutamine or asparagine,
more preferably glutamine;
• aspartic acid at position 284 according to SEQ ID NO 1 and at position 285 in SEQ
ID NO 4 by an amino acid with a polar side chain such as serine, three-nine,
asparagine, glutamine, glutamic acid, histidine, lysine, arginine and tyrosine, more
preferably asparagine, glutamine, glutamic acid, lysine and arginine; most preferred
being asparagine
• glycine at position 300 according to SEQ ID NO 1 by an amino acid with a small
residue, such as alanine, serine, threonine, cystein, valine, isoleucine, leucine,
asparagine and aspartic acid, more preferably valine.
The invention furthermore provides mutant expandases as defined
hereinbefore that are variants of the group consisting of the wild type expandases of S.
clavuligerus, S. jumonjinensis, S. ambofaciens, S. chartreuses and N. lactamdurans.
Most preferred are mutant expandases as defined hereinbefore that are variants of the
wild type expandase of S. clavuligerus or N. lactamdurans. Most preferred are mutant
expandases as defined hereinbefore that are variants of the wild type expandase of S.
clavuligerus. Preferred mutant expandases that are mutants of the expandase of
Streptomyces olavuligems have modifications at least at
o 2 or more amino acid positions selected from the group consisting of D2, T89,
1277, C281 and G300.
o 3 or more amino acid positions selected from the group consisting of D2, T89,
C281,T293andA311
o 4 or more amino acid positions selected from the group consisting of D2, M73,
T89, N90, Y217, H244,1277, E280, C281, R306, R307 and A311
o 5 or more amino acid positions selected from the group consisting of D2, M73,
T89, N90, C155, T213, R249, A278, C281, T293, R306, R307 and A311
o 6 amino acid positions selected from the group consisting of
N90+T105+G113+C281+R306+A311.
o 7 or more amino acid positions selected from the group consisting of D2, M73,
T89, T105, G113, C155, H170, P177, D251, L277, E280, C281, T293, G300,
R306, R307 and A311
o 8 or more amino acid positions selected from the group consisting of D2, T89,
N90, M99, T105, G113, 0155, P177, L277, 0281, R306, R307 and A311.
o 9 amino acid positions selected from the group consisting of
D2+N90+T105+G113+0155+P177+0281+R306+A311.
o 10 amino acid positions selected from the group consisting of D2, S59, N90,
T105, G113, T105, G113, C155, P177, G209,1277, C281, R306, R307, A311.
o 11 amino acid positions selected from the group consisting of D2, N90, T105,
G113, C155, P177, L277, C281, R306, R307, A311.
Preferred modifications at the respective positions are D2N, D2H, D2Y, S59G,
M73H, M73I, T89A, T89K, T89V, N90W, IM90S, M99T, T105I, Y101F, G113D, C155W,
H170Y, P177L, G209A, T213A, Y217H, H244R, R249C, D251G, L277Q, L277T,
A278V, E280G, C281Y, T293K, G300S, R306deletion, R307deletion, R370T and
A311D. In this notation, the letter following the number represents the amino acid (one
letter code) present in the mutant expandase. The deletion at position R306 or R307
mean that the original arginine at position 306 or 307 is no longer present in the mutant
expandase. Highly preferred mutant expandases are the mutant expandases that are
summarized in Table 3.
Preferably, the mutant expandases provided by the present invention have an
improved expandase activity on adipyl-6-APA as defined herein before, the mutant
expandases provided by the present invention have likewise, an improved expandase
activity on Pen-G. Preferably, the mutant expandases provided by the present
invention have an improved expandase on both adipyl-6-APA as well as Pen-G. Mutant
expandases with an improved activity on Pen-G can be used advantageously in a
process for the production of phenylacetyl-7-ADCA as described further below.
The present invention also provides mutant expandases with a decreased or
even absent expandase activity with iso-penicillin N (iPN). Preferably the expandase
activity with either adipyl-6-APA or Pen-G is not affected, but more preferably the
expandase activity with either adipyl-6-APA or Pen-G or both is improved as defined
hereinbefore. The advantage of these more preferred mutant expandases is that the
decreased or even absent expandase activity with iso-penicillin N (iPN) results in less
byproduct in a fermentation process to produce ad-7-ADCA or phenylacetyl-7-ADCA.
Preferred mutant expandases have a decreased or even absent expandase
activity with iso-penicillin N (iPN) as a substrate optionally combined with an improved
expandase activity on ad-6-APA as a substrate and have been modified at position 89
according to the amino acid numbering of SEQ ID No 1. whereby the naturally
occurring amino acid has been replaced, preferably by a charged amino acid,
preferably a negatively charged amino acid such as aspartic acid or glutamic acid,
most preferably a positively charged amino acid such as lysine, arginine or hystidine;
most preferred being lysine.
A highly preferred mutant expandase is selected from the group consisting of
H101, H106, H111, H122, H127, H262, H301, H305, H308, H309, H401, H402, H403,
H501, H502, H503, H504, H505, H506, H507, H508, H601, H602, H603, H604, H605,
H606, H607, H608, H609, H650, H651, H652, H653, H654, H655, H656, H657, H658,
H659, H660, H661, H662, G601, G602, G603, G604, G605, G606, G607, G608, G609,
G610, G611, G613 and G614 (see Table 3).
In a second aspect, the invention provides a polynucleotide encoding the
mutant expandase of the present invention. The polynucleotide encoding the mutant
expandase according to the present invention can be any polynucleotide that encodes
the proper amino acid sequence according to the invention. This implies that it may
correspond to the ce/Egene of S. clavuligerus except for the nucleotides encoding the
modifications at the respective amino acid positions. Alternatively, it may correspond to
the cefE gene of S. clavuligerus except for the polynucleotide fragments encoding the
parts of the amino acid sequence corresponding with sequences of N. lactamdurans
expandase. Alternatively, the polynucleotide of the invention may comprise a coding
sequence in which the codon usage for the various amino acids deviates from the
codon usage in S. clavuligerus and/or in N. lactamdurans. For example, the codon
usage may be adapted to the codon usage of a particular host cell, which will or has
been transformed with the DNA fragment encoding the altered expandase.
In a third aspect, the invention provides an expression vector or expression
cassette comprising the polynucleotide of the invention as defined hereinbefore.
In a fourth aspect, the invention provides a transformed host cell, transformed
with the polynucleotide of the invention or the expression vector or expression cassette
of the invention. The transformed host cell may be used for the production of the
mutant expandase of the invention or the host cell may be used for the production of a
beta-lactam compound of interest.
Host cells for the production of the mutant expandase of the invention are
preferably host cells which are known in the art for their efficient protein or enzyme
production, either extracellular or intracellularly, for example microorganisms such as
fungi, yeast and bacteria. Examples of preferred host cells comprise, but are not limited
to, the following genera: Aspergillus (e.g. A. niger, A. oryzea), Penicillium (e.g. P.
emersonii, P. chrysogenum), Saccharomyces (e.g. S. cerevisiae), Kluyvemmyces (e.g.
K. lactis), Bacillus (e.g. B. subtilis, B. licheniformis, B. amyloliquefadens). Escherichia,
(E. coif), Streptomyces (e.g. S. clavuligerus).
Host cells for the production of a beta-lactam compound of interest are
preferably host cells that are known In the art for their efficient beta-lactam compound
production. Examples of preferred host cells comprise, but are not limited to, to the
following genera: Penicillium (e.g. P. chrysogenum), Acremonium (e.g. A.
chrysogenum), Streptomyces (e.g. S. clavuligerus), Nocardia (e.g. N. lactamdurans),
Lysobacter (e.g. L. lactamgenus) and Flavobacterium species.
In a fifth aspect, the invention provides a process for the production pf the
mutant expandase of the invention comprising cultivating the transformed host cell
according to the invention under conditions conducive to the production of the mutant
expandase and, optionally, recovering the mutant expandase. The recovered mutant
expandase may be used advantageously in an in vitro process to produce a desired
cephalosporin from a corresponding penicillin, for instance the recovered mutant
expandase may be used in a process to produce phenylacetyl-7-ADCA from Pen-G or
adipyl-7-ADCA from adipyl -6-APA.
In a sixth aspect, the invention provides a process for the production of a betalactam
compound of interest comprising cultivating the transformed host cell according
to the invention under conditions conducive to the production of the beta-lactam
compound of interest and, optionally, recovering the beta-lactam compound. Preferred
beta-lactam compounds belong to the group of cephaiosporins such as phenylacetyl-7-
ADCA, adipyl-7-ADCA, adipyl-7-ADAC and adipyl-7-ACA. In a preferred embodiment,
the invention provides a process for the production of phenylacetyl-7-ADCA or adipyl-7-
ADCA by cultivating a selected strain of Penicillium chrysogenum, that has been
transformed with a selected polynucleotide of the invention that encodes a mutant
expandase of the invention. For the production of phenylacetyl-7-ADCA, a mutant
expandase is selected that has a high improvement factor on Pen-G as a substrate.
For the production of adipyl-7-ADCA, a mutant expandase is selected that has a high
improvement factor on ad-6-APA as a substrate.
In another preferred embodiment, the invention provides a process for the
production of 7-ADCA comprising the process for the production of phenylacetyl-7-
ADCA or adipyl-7-ADCA as described herein before, followed by, after optional
purification of said 7-ADCA-derivatives, a process step in which the phenylacetyl or
adipyl side chain of phenylacetyl-7-ADCA and adipyl-7-ADCA respectively is cleaved
off thereby generating 7-ADCA and the liberated side chains acid. Said cleavage can
be obtained by chemical means or, more preferably, enzymatically using an acylase
enzyme. Suitable acylases for the cleavage of the adipyl side chain are obtainable from
various Pseudomonas species such as Pseudomonas SY-77 or Pseudomonas SE-83.
Suitable acylases fro the cleavage of the phenylacetyl side chains are the penicillin
acylases from Escherichia coli or Alcaligenes feacalis.
In a seventh aspect, the invention provides a process for the production of a
cephalosporin from a corresponding penicillin whereby the expansion of the 5-
membered thiazolidine ring of the penicillin to the 6-membered dihydrothiazine ring of
the cephalosporin occurs in an in vitro process, catalyzed by a mutant expandase of
the invention. In a preferred process, adipyl-6-APA is expanded to the corresponding
adipyl-7-ADCA by a mutant expandase of the invention, preferably a mutant
expandase that has a high improvement factor adipyl-6-APA as a substrate. In another
preferred process, Pen-G is expanded to the corresponding phenylacetyl-7-ADCA by a
mutant expandase of the invention, preferably a mutant expandase that has a high
improvement factor on Pen-G as a substrate.
The process may be followed by, after optional purification of said 7-ADCAderivatives,
a process step in which the side chains of adipyl-7-ADCA and
phenylacetyl-7-ADCA are cleaved off thereby generating 7-ADCA and the liberated
side chains acid - supra vide.
The desired end product 7-ADCA can be recovered according to methods
known in the art involving chemical or enzymatic cleavage of the side chain of adipyl-7-
ADCA or phenylacetyl-7-ADCA and optionally the resulting 7-ADCA can be further
purified and/or crystallized.
In an eight aspect, the invention provides a method to obtain the mutant
expandases of the invention whereby the process comprises the following steps:
1. Mutagenesis of a cloned gene encoding a model polypeptide with expandase
activity thus obtaining a collection of mutagenised genes encoding the mutant
expandases;
2. Expression of the collection of mutagenised genes encoding the mutant
expandases in a suitable host and screening the collection of mutant expandases
for an improved activity with a suitable substrate;
3. Optionally repeating steps 1 and 2 one or several times using either the gene
encoding the model polypeptide with expandase activity or one or more of the
mutagenised genes encoding mutant expandases with an improved activity on the
suitable substrate.
Cloning of the gene encoding a model polypeptide with expandase activity can
be carried out according to methods known in the art.
Preferred model polypeptides with expandase activity are selected from the
group consisting of a polypeptide with expandase activity obtainable from
Streptomyces davuligerus, preferably having an amino acid sequence according to
SEQ ID NO: 1 and polypeptides with expandase activity having an amino acid
sequence with a percentage identity with SEQ ID NO: 1 of at least 70%, preferably at
least 75%, more preferably at least 80%, more preferably at least 85%, more preferably
at least 90%, most preferably at least 95%, such as the expandase enzymes that are
summarized in Table 1.
Any mutagenesis technique can be employed which results in mutations over
the entire gene. Suitable techniques are error prone (EP) PCR (Polymerase Chain
Reaction) and/or by using saturated Mutation Primer PCR (sMPP) exactly according to
WO 03/010183.
Materials and Methods
7. General
Oligonucleotides were synthesized by Invitrogen (Carlsbad CA, US).
DNA sequencing was carried out by SEQLAB (Gb'ttingen, Germany) or by Baseclear
(Leiden, The Netherlands).
Restriction enzymes were purchased from Invitrogen.
The protein assays were carried out according to the method described by Bradford,
MM (1976) Anal Biochem. 72:248-54
Escherichia co//DH10B electromax competent cells were obtained from Invitrogen. The
protocol was delivered by the manufacturer,
SDS-PAGE electrophoresis was carried out in the system supplied by Invitrogen.
NuPAGE Novex Bis-Tris Gels (Invitrogen). SimplyBlue SafeStain Microwave protocol
Overview of used constructs
Plasmid
PBAD-MH-Zeo-MBP-ScEwt
PBAD-MH-Zeo-MBP-H127
PBAD-MH-Z80-MBP-H406
PBAD-MH-Zeo-MBP-H402
PBAD-MH-Zeo-MBP-H403
Description
Arabinose inducible E.coli expression construct with
maltose binding protein (MBP) fused to wild type
S.clavuligerus expandase. Zeocine marker
Idem but fused to H127 expandase (see also Table 3x)
Idem but fused to H406 expandase (see also Table 3x)
Idem but fused to H402 expandase (see also Table 3x)
Idem but fused to H403 expandase (see also Table 3x)
1. Cloning of the E. coli expression vectors
The fusion product is ligated in the pBAD/MH MBP-ScEwt Dest zeo vector by
an EcoRI/Sa/l digestion. This replaces the wild type expandase gene by the library
genes. To prevent significant numbers of wild type constructs, the ligation constructs
were digested with Sma\.
2. Construction of the libraries
Transformation of E. coll with the ligation mix yielded libraries of roughly
12,000-13,000 colonies. To test the quality of the libraries, a random set of colonies
was selected and the constructs were digested with restriction enzymes. This showed
that approximately 26% of the library revealed an aberrant pattern. Next, sequence
analysis was performed on 10 regular clones to determine mutation frequencies.
Sequences showed that both saturation and error prone mutations were present at a
satisfactory level
3. Expression and screening of mutant expandases
3.1. Screening of the cefE library for improved activity
E. coli TG1 cells containing the library A were plated and grown on agar
plates. Colonies are picked robotically into 96-wells microtiter plates containing liquid
medium (2YT, 30jig/ml chloramphenicol, 05% glucose), and grown to saturation by
shaking at 37°C. Cells are subcultured in 96 well megatiter plates; 1:40 inoculum into
2YT, 30 (ig/ml chloramphenicol and induced by 0.2 mM IPTG shaking at 28°C for 24 h.
Cells are pelleted and washed with an equal volume of buffer (5 mM morpholine
pH7.5). After spinning, the cells are resuspended in lysis mix for primary screening
Expandase assay (1)).
3.2. Primary screening in microtitre plates (MTP)
Growth and induction
The expandase E. coli library is plated on Luria-Bertani (LB) medium plates low
salt agar + 25 u.g/ml Zeocine and incubated overnight at 37 °C, Microtitre plates (MTP)
with 150 pi LB low salt medium and 25 u.g/ml Zeocine are inoculated from the plates
and incubated 36 hrs at 25 °C and 550 rpm.
From the MTP, 5 u.l is inoculated in deep well plate with 1 ml 2*TY (tryptone and
yeast extract) medium + 50 u.g/ml Zeocine + arabinose. To the remaining culture in the
MTP, 100 ul 50% glycerol is added and frozen at -80 °C as glycerol stock.
The deep well plate is incubated for 30 hrs at 25°C and 550 rpm. The deep well
plate cultures are centrifuged at 2750 rpm (Heraeus multifuge 4 kr.) for 10 min. The
supernatant is discarded and the pellet is frozen at -20 "C.
Cell free extract (CFE)
The frozen pellets obtained in the previous section are thawed in 200u.l
extraction buffer (50 mM Tris/HCI pH=7.5; 5 mM DTT; 0.1 mg/ml DNAse; 5 mM
MgSCyH20 and 0.5 mg/ml lysozyme). To resuspend pellets, the plates are shaken.
The plates are incubated for 30 minutes at room temperature and centrifuged for 10
minutes at 2750 rpm.
3.3 Secondary screening in shake flasks
Growth and induction
Selected colonies are inoculated from plate in 10 ml LB low salt + Zeocin 25
ug/ml and incubated overnight at 37°C, 280 rpm. The grown culture is inoculated in
100 ml LB low salt with 25 ug/ml Zeocine at an optical density (600 nm) between 0.010
and 0.050 (Biochrom Ultraspec 2000). After growth at 37°C, 280 rpm, the cells are
harvested at an optical density at 600 nm between 0.4-0.6. Then, arabinose is added
(final concentration 0.2%) and induced overnight at 27°C at 220 rpm.
4. Expandase assays
4.1 Expandase assay (1) - Screening assay (primary assay):
Expandase assay buffer: 5 mM morpholine buffer pH 7,5 and cofactor mix
(final concentrations 50 jiM ATP, 1 mM DTT, 60 u.M FeS04, 2,7 mM ascorbate), 500
|iM ad-6-APA, 1/10th volume of E. coli cell lysate and 2.4 mM a-KG. Each cofactor was
made in buffer pH 7.5, which was critical for activity.
The expandase reaction was found to be linear for up to 3 hours with respect
to adipyi-7ADCA formation/detection. The optimal temperature for the in vitro
expandase reaction was determined to be approximately 11°C, probably due to
instability of the enzyme or production at higher temperatures. Nevertheless, actual
screening was performed at a temperature between room temperature and 25°C,
which is more reflective of the desired reaction condition temperature.
Screening of the family shuffled libraries for improved 7-ADCA formation was
determined by Flow Injection Analysis-MS (FIA-MS, negative mode, Mass Transition
[M-H]'341>295). Final analysis: LC column: using solvent conditions between 0.1%
formic acid in 40% MeOH to 100% MeOH. Two channels (two mass transitions [M-H~])
are used to monitor for each reaction product. Samples are analyzed after 30 minutes
of reaction.
4.2 Expandase assay (2)- Secondary exoandase assay
Expandase assay buffer: 5 mM morpholine buffer pH 7,5 and cofactor mix
(final concentrations: 50 \iM ATP, 1 mM DTT, 60 \iM FeS04, 2,7 mM ascorbate), 2 mM
adipyl-6-APA, 1/10tn volume of £ co//cell lysate and 2.4 mM a-ketoglutarate. Each
cofactor was made in buffer pH 7.5, which was critical for activity. The conversion was
performed at 29°C and samples were taken between 0 and 30 minutes. The reaction
was stopped with 60% (v/v) acetonitrile final concentration. The formed adipyl-7-ADCA
was analysed on HPLC. (Mobile phase: 10% acetonitrile in 10 mM potassium
phosphate buffer pH 3.0) Column: X-Terra™ RP18 3.5u.. 3.9x100 mm detected at 214
and 254 nm (254 nm to analyse expanded antibiotics). HPLC data analysis: software:
Waters Millennium32® 3.20.
4.3 Expandase assay (3) - Expandase assay
A total ot 300 uj of a reaction mixture consisting of 50 mM Tris/HCI pH 7.5; 1
mM DTT; 2.7 mM Ascorbate; 0,05 mM ATP; 24 mM a-ketoglutarate; 0.06 mM
FeS04.7H20; 4 mM adipyl-6-amino-penicillanic acid (ad-6-APA) was added to 75 uJ
CFE and incubated at 29^ for the desired time. Adding 60 uJ maleic acid with 10 g/l
EDTA stopped the reaction. The formation of adipyl-7-amino-desacetoxycephalosporinic
acid (ad-7-ADCA) was detected using 1H-NMR.
The adipyl-6-APA was obtained from 6-APA and adipic acid catalyzed by penicillin
acylase (EC 3.5.1.11). Alternatively, adipyl-6-APA can be obtained by culturing for
instance a suitable Penicillium chrysogenum strain in the presence of adipic acid as
precursor.
EXAMPLES
Example 1
Cloning of wild type expandase
The cefE gene was PCR cloned from S. clavuligerus genomic DMA and
ligated in GFP-fusion vectors, the so-called pCK series, and used to transform E. coli
TG1 (Amersham Pharmacia Biotech, Inc. NJ USA). Several colonies from the plated
transformations were selected and checked for an active clone. Several active clones
were identified. Clone '778' appeared to be a wild type gene but interestingly, one other
clone, clone "779", revealed strongly improved activity on adipyl-6-APA expansion. The
product of clone "779" was sequenced and revealed three amino acid mutations
(H18R, D284N and G300V). The adipyl-7-ADCA production of the clones analysed by
Flow Injection Analysis-Mass Spectrometry (FIA/MS) showed that the Sc-cefE mutant
"779" was superior to the wild-type parents. Measurement of GFP fluorescence
suggests that the improvement of the expandase enzyme product of clone "779" is not
due to improved expression of cefE therein. Hence, it was concluded that the
improvement of the expandase enzyme product of clone "779" is an improvement of
the activity as a result of the amino acid substitutions.
pCK derived GFP fusion vectors
Fusion tag vector series (called pCK series) containing a His tag fused to the
3' end of expandase with green fluorescent protein (GFP) fusion at the 3' end of the
HIS tag were constructed. The GFP containing vectors enable high throughput
normalisation of expandase activity data in subsequent assays with respect to protein
concentration. The vectors contain the p15A origin of replication (low copy number),
and a lac promoter that is repressed in the presence of glucose.
Example 2
Family shuffling and Screening of wild-type N. lactamdurans and
S. clavuligerus cefE genes - Library A
The expandase genes were shuffled according to the process described in
EP752008. Expandase mutant libraries were made using the GFP-fusion tag vectors
(pCK). Only the Ce/E ORF was shuffled, while other sequences where not changed.
After moving the shuffled pool of expandases into the pCK vectors, one library set
(Library A) was made using the two parental wild type expandase genes (S.
clavuligerus and N. lactamdurans) plus clone "779".
Initial plating of a sample of the library showed that the libraries contained at
least 70% green fluorescent colonies. Due to this, it was not considered necessary to
pre-screen out non-fluorescent colonies before proceeding to the first FIA/MS screens.
DNA sequencing of randomly picked colonies form library set A indicated that
all parents were represented in the library, so this library was selected for initial
screening.
720 clones from library A were screened and 14 clones (mutant expandases
H101, H102, H104, H106, H108, H111, H112, H114, H115, H117, H120, H122, H125,
H127) were retested and the preliminary results showed an expandase activity
improvement compared to the S. clavuligerus expandase (clone "778") as judged by in
vitro expandase assay (1). These clones were chosen for further testing. Normalization
of the expandase activity with respect to expandase-GFP concentration (i.e. correcting
for differences in the expandase protein concentration) confirmed that these clones
were improved in specific activity.
Example 3
Family shuffling of wild-type N. lactamdurans and S. clavuligerus cefE genes.
Library B
Library B was constructed shuffling wild type N. lactamdurans and S.
clavuligerus cefE as parent genes, The "779" hit sequence was not used in
construction of this library. This expandase library showed to have a significant level of
inactive or very poorly active clones in the library. In order to remove inactive clones
from further testing, a FACS-based pre-screen was used for the library B screening to
improve the percentage of active clones that enter the expandase assays.
After FAGS sorting based on their fluorescence by GFP, selected cells were recovered
under repressing conditions. DNA prepared from these cultures was used to transform
coll TG1. These expandase constructs used are also fusion proteins with GFP as
described for library A.
70-80 percent of the library was GFP-active. In total, 2200 clones from this
library were then screened using the in vitro expandase assay and FIA-MS detection of
adipyl-7-ADCA formation.
Fourteen possible hits were selected (H200 series). Four of these hits,
amongst them H262, were confirmed through retesting procedure including
^transformation and in vitro testing and analysis MS.
Example 4
Construction of chimeric expandase sequences using shuffled expandases
Library C
Using traditional molecular biological techniques and bioinformatics tools, the
hit sequences from the two above described libraries (libraries A and B) were
recombined, resulting in library C (H300 series). Three resulting chimeras were tested
for activity, amongst them expandase H301.
Six remaining clones (amongst them: H305, H307, H308, H309) were not
tested as GFP fusion product nor analysed by FIA-MS. These clones were directly
recloned in the pSJ vector series fused with maltose binding protein (MBP) upstream of
the CefcL mutant genes and analysed, after the In vitro assay, on HPLC.
Example 5
Recjoning of genes encoding improved expandases using pSJ based vector series and
enzymatic activity of the improved expandases
pSJO.8 was used as cloning vector for the expandase libraries. This plasmid
was constructed by replacing the expandase gene (as Ndel-Nsil fragment) by a small
oligonucleotide introducing a A/of/ restriction site. S. davuligerus cefE was cloned as
Nde\-Nsi\ fragment in pSJOS, resulting in pSJOS.
pSJ05 contained an additional Nde\ site just upstream of the Nsi\ site. This
site was removed to prevent possible interference with further cloning of the shuffled
genes in the Nde\-Nsi\ sites, resulting in pSJ11. Next, maltose binding protein (MBP)
was fused to the 5' end of cefE resulting in pSJ10. A plasmid containing MBP-cefE
fusion genes for Nocardia lactambdurans (pSJ01) was also constructed. DNA
sequence analysis confirmed all sequences.
Improved clones from the A, B and C library were recloned from the pCKvectors
into the pSJ10 vector (MBP-cefE behind the tac promoter). This was done by
amplifying the improved expandases by PCR. The forward (5'-end) primer introduces a
A/del and the reverse (3'-0nd) primer introduces a Nsil site. After digestion with Ndeand Nsil the hit was cloned in the E. coli vector (pSJ10) and in the Penicillium
chrysogenum vector (plATWAn- Figure 2/3) Table 2.
Finally, a few clones of each were sequenced and one of each, containing the
verified correct sequence, was used for further analysis.
The DNA and amino acid sequences of the best expandase hits are included
in the sequence listing - see Table 2 for an overview.
Induction of expression
E. coli NM554 was grown overnight on 2xTY medium with chloramphenicol
(30 ug/ml) at 37°C. The cells were diluted to an optical density at 540 nm (OD540) of
0.010-0.020 in fresh 2xTY with chloramphenicol (30 ng/ml). When cells reached an
optical density at 540 nm of 0.4-0.6, IPTG was added (final concentration 0.5 mM) and
expression was induced overnight at 27°C.
The cells were washed and resuspended in extraction buffer (5 mM DTT, 50
mM Tris/HCI pH 7.5 with lysozym) and sonicated (on ice water). After centrifugation the
supernatant is used for the secondary expandase assay (2).
Expression of the expandase in the cell free extract was determined by SDS-PAGE.
Table 2. H100, H200 and H300 series expandase hits, their mutation(s). their E. coli
and Penidllium expression vectors and their expandase activity (assay 2).
Expandase
(Table Removed)
Specific activity (nmol 7-ADCA.min'1.mg~1 protein)
I.F. = Improvement factor; equal the ratio of the specific activity of the mutant
expandase and the specific activity of the expandase from S. clavuligeris
Example 6
Expression of expandase H122 and H127..in Penicillium chrvsoaenum
Expandase variants H122 and H127 were cloned into vectors for Penicillium
chrysogenum expression (plAT series plasmids, Table 2), Shuffled expandase genes
cloned into a plasmid vector with an E. coli replicon and selectable marker. A promoter
compatible with expression in P. chrysogenum, i.e., the IPNS promoter, was used to
express the expandase gene. Additionally a selectable marker for Penicillium
chrysogenum is present (amdS).
The expression constructs were obtained by digesting the expression
construct, plAT127 (expandase H127) and plAT122 (expandase H122), with A/of/. A
high producing Penicillium chrysogenum is co-transformed with the linear fragments
together with the amdS expression cassette (H/nc/lll digest).
By precursing with adipate, adipyl -7-ADCA production can be tested in strains
expressing active expandase genes.
The cephalosporin production of the strain expression expandase H127 was
compared to the strain expressing wild type expandase in batch regime with lactose as
carbon source. The adipyl-7-ADCA production is increased between 10-50% and the
total p-lactam production was increased by 0-30%, depending on the lactose and the
adipate concentration.
Additionally, expandase H122 was expressed in Penicillium chrysogenum.
Comparison of the cephalosporin production with the strain expressing wild type
expandase showed that the adipyl -7-ADCA production is increased between 0 to 50%
and the total beta-lactam production varied between -20 to 0% depending on the
lactose and the adipate concentration in the media.
Example 7
1st generation improved expandases (H400 series).
The first generation expandase library was constructed by performing error
prone (EP) PCR (Polymerase Chain Reaction) on wild type Streptomyces clavuligerus
CefE gene (SEQ ID No.1), After screening this library for improved conversion of ad-6-
APA to ad-7-ADCA, 3 different mutant genes were selected. The mutants exhibited an
improved ad-6-APA expansion activity up to 2.5-fold (H401, H402, H403: see Table 3).
Example 8
2nd generation ImprQved expandases (H500 series)
The second-generation expandase library was constructed by using saturated
Mutation Primer PCR (sMPP) exactly according to WO 03/010183. The selected
mutant genes from the first generation (H401, H402 and H403) were used as templates
for the construction of the 2nd generation library.
The sMPP was performed by using Taq polymerase, thereby introducing
additional mutations (random) and increasing the variation of the library. Designed
primers annealed at the mutation positions and were saturated at the mutations found
in the 1st generation expandase hits. Additionally, a universal forward and reverse
primer was designed in which an Ndel and Nsil site were introduced respectively. This
facilitated cloning into Penicillium expression vector for testing of the mutant
expandases in vivo.
The library was grown and expression of the expandase was induced as
described in the materials and methods section. The formation of ad-7-ADCA was
determined using NMR as described in the materials and methods section.
Approximately 150 improved mutant expandases were selected and retested for
their ad-6-APA expandase activity. Based upon the results of this retest, 17 mutants
were selected for final tests in shake flasks and analysis by HPLC as described in the
Materials and Methods.
In total, 15 of the 17 selected hits showed a significant improved expandase
activity with ad-6-APA as a substrate. To exclude the false positive selection of
expression mutants, protein levels were quantified on SDS-PAGE. This confirmed that
the improved activity was not due to stronger expression of expandase in E. coll, but
that the improvement could be attributed to an improved specific activity of the
expandase mutant.
In total, 8 mutant expandases were identified which exhibited an improvement
factor of their ad-6-APA expandase up to 4,5-fold (compared to the wild type
expandase of S. clavuligerus - SEQ ID No 1.). These mutants are designated H501 -
H508 (see Table 3).
Example 9
3rd generation of improved expandases (H600 series)
The 8 mutants obtained in the 2nd generation Example 2 (H501 - H508) were
used as templates for the construction of the 3rd generation expandase library. This
library was constructed in the same way as the 2nd generation expandase library.
Additionally, the SKA signaling sequence at the C-terminus of the expandases was
changed to SKD, causing expression of expandase solely in the cytosol when
expressed in Penicillium.
The screening of this library yielded 22 different expandase mutant genes that
were significantly improved between 5-fold and 11-fold compared to S. clavuligerus
expandase (Table 3, H600 series).
Screening of the same library for a second time yielded 13 additional mutant
expandases (G601 - G611 and G613 - G614; see Table 3)
Example 10
Activity of improved mutant expandases with iso-penicillin N (iPN) and Penicillin-G
(Pen-G) as a substrate.
The activity of several mutant expandases was measured using iPN and Pen-
G as a substrate. The assay with iPN as a substrate was carried out as described in
the materials and methods section except that ad-6-APA was replaced by the same
concentration of iPN (4 mM). The assay with Pen-G as a substrate was carried out as
described in the materials and methods section except that ad-6-APA was replaced by
7 mM Pen-G. Table 3 summarizes the activities of the various expandase mutants for
iPN and Pen-G.
Whereas most of the expandase mutants tested exhibited a 5-fold
improvement of their expandase activity with iPN, mutants that carry the T89K mutation
lost virtually all activity towards iPN.
The activity towards Pen-G of the various expandase mutants tested was
either the same as the wild type expandase (improvement factor is 1) or improved up to
8-fold. The data further show that there is no correlation whatsoever between the
improvement factors obtained for a single mutant with ad-6-APA and Pen-G as a
substrate. The ratio between the respective improvement factors for ad-6-APA and
Pen-G vary in a range from 0.1 (e.g. H654) to 1,2 (e.g. H503).
Table 3. Mutant expandases and their expandase activity toward ad-6-APA, Pen-G and
iPN. Each mutant is identified by its code; the mutations are introduced in the model
expandase from S. clavuligerus (SEQ ID NO 1). The expandase activity is expressed
as improvement factor compared to the model expandase from S. clavuligerus
(expandase activity = 1 by definition) according to the in vitro expandase assays
described in the Materials and Methods and Example 4.
(Table Removed)
Table 3 shows that mutant expandases have been obtained which have improvement
factors in the range of 1.5 to 10.6-fold,




CLAIMS
1. A mutant expandase that is a variant of a model polypeptide with expandase
activity whereby the mutant expandase has an at least 2-fold improved in vitro
expandase activity towards adipyl-6-APA in comparison with the model
polypeptide with expandase activity.
2. A mutant expandase that is a variant of a model polypeptide with expandase
activity whereby the mutant expandase has been modified at at least an amino
acid position selected from group 1 consisting of positions 2, 18, 59, 73, 74, 89,
90, 99, 101, 105, 112, 113, 155, 170, 177, 209, 213, 217, 244, 249, 251, 277,
278, 280, 281, 284, 293, 300, 307 and 311 using the amino acid position
numbering of the amino acid sequence of the expandase enzyme encoded by
the cefE gene of Slreptomyces clavuligerus (SEQ ID NO: 1).
3. A mutant expandase according to claim 2 whereby the mutant expandase has
been modified at at least an amino acid position selected from the group 2
consisting of positions 2, 18, 59, 89, 90, 99, 101, 105, 112, 113, 170, 177, 209,
213, 217, 249, 251, 278, 280, 284 and 293, optionally in combination with at
least an amino acid position selected from the group 1 and which does not
belong to group 2.
4. A mutant expandase according to claim 1 whereby the mutant expandase has
been modified at at least an amino acid position selected from group 1 as
defined in claim 2.
5. A mutant expandase according to claim 4 whereby the mutant expandase has
been modified at at least an amino acid position selected from group 2 as
defined in claim 3 optionally in combination with at least an amino acid position
selected from the group 1 and which does not belong to group 2.
6. A mutant expandase according to anyone of the preceding claims wherein the
model peptide is the expandase of Streptomyces clavuligerus encoded by the
cefE gene as depicted in SEQ ID No. 1 or wherein model peptide is the
expandase of Nocardia lactamdurans encoded by the cefE gene as depicted in
SEQ ID No. 2
7. A mutant expandase having an at least 2-fold improved in vitro expandase
activity towards adipyl-6-APA in comparison with the expandase from
Streptomyces clavuligerus and which is selected from the group consisting of
H101, H106, H111, H122, H127, H262, H301, H305, H308, H309, H402, H501,
H502, H503, H504, H505, H506, H507, H508, H601, H602, H603, H604, H605,
H606, H607, H608, H609, H650, H651, H652, H653, H654, H655, H656, H657,
H658, H659, H660, H661, H662, G601, G602, G603, G604, G605, G606,
G607, G608, G609, G610, G611, G613 and G614.
8. A polynucleotide encoding the mutant expandase of anyone of the preceding
claims,
9. An expression vector or cassette comprising the polynucleotide of claim 8.
10. A host cell transformed with the polynucleotide of claim 8 or the vector or
cassette of claim 9.
11. A method of producing the mutant expandase of claims 1-7 comprising
cultivating a host cell according to claim 10 under conditions conducive to the
production of the mutant expandase and, optionally, recovering the polypeptide.
12. A method of producing a (J-lactam compound of interest comprising cultivating a
host cell according to claim 10 under conditions conducive to the production of
the p-lactam compound and, optionally, recovering the (3-lactam compound,
13. A method according to claim 12, further comprising N-deacylating the p-lactam
compound to produce an N-deacylated (3-lactam compound.
14. A method to obtain the mutant expandases according to anyone of claims 1-7,
whereby the method comprises the following steps:
a. Mutagenesis of a cloned gene encoding a model polypeptide with
expandase activity, preferably cefE encoding the expandase from S.
clavuligerus, thus obtaining a collection of mutagenised genes encoding
the mutant expandases;
b. Expression of the collection of mutagenised genes encoding the mutant
expandases in a suitable host and screening the collection of mutant
expandases for an improved activity with a suitable substrate, preferably
ad-6-APA;
c. Optionally repeating steps 1 and 2 one or several times using thereby
mutagenising the gene encoding the model polypeptide with expandase
activity and/or one or more of the mutagenised genes encoding mutant
expandases with an improved activity on the suitable substrate.
15. Use of the mutant expandase polypeptide according to claim 1 to 7 in the
expansion of the penam ring into a ceph-3-em ring.
16. Use of the mutant expandase polypeptide according to claim 1 to 7 in the
expansion of the penam ring of adipyl-6-APA into a ceph-3-em ring.
17. Use according to claim 15 in the production of adipyl-7-ADCA, adipyl-7-ADAC
or adipyl-7-ACA.
18. An adipyl-6-APA producing microorganism transformed with a DNA fragment
encoding an altered expandase polypeptide according to claim 1 to 7.
19. Method for the fermentative production of adipyl-7-ADCA comprising
a. Culturing a microorganism capable of producing adipyl-6-APA and
transformed with a DNA fragment encoding an altered expandase
according to claim 1 to 3 under adipyl-6-APA-producing conditions
b. In situ converting the adipyl-6-APA into adipyl-7-ADCA using the altered
expandase; and
c. Isolating the desired adipyl-7-ADCA.
20. Method for the production of 7-ADCA comprising
a. Culturing a microorganism capable of producing adipyl-6-APA and
transformed with a DNA fragment encoding an altered expandase
according to claim 1 to 3 under adipyl-6-APA-producing conditions
b. In situ converting the adipyl-6-APA into adipyl-7-ADCA using the altered
expandase;
c. Isolating the desired adipyl-7-ADCA.
d. Removal of the adipyl side chain to yield 7-ADCA; and
e. Recovery of 7-ADCA.

Documents:

2828-delnp-2007-Abstract-(27-05-2013).pdf

2828-delnp-2007-abstract.pdf

2828-delnp-2007-Assignment-(09-08-2012).pdf

2828-delnp-2007-Claims-(27-05-2013).pdf

2828-delnp-2007-claims.pdf

2828-delnp-2007-Correspondence Others-(02-08-2013).pdf

2828-delnp-2007-Correspondence Others-(09-08-2012).pdf

2828-delnp-2007-Correspondence Others-(20-12-2012).pdf

2828-delnp-2007-Correspondence-Others-(27-05-2013).pdf

2828-delnp-2007-correspondence-others.pdf

2828-delnp-2007-description (complete).pdf

2828-delnp-2007-Drawings-(27-05-2013).pdf

2828-delnp-2007-drawings.pdf

2828-delnp-2007-Form-1-(09-08-2012).pdf

2828-delnp-2007-form-1.pdf

2828-delnp-2007-Form-18-(25-08-2008).pdf

2828-delnp-2007-Form-2-(09-08-2012).pdf

2828-delnp-2007-form-2.pdf

2828-delnp-2007-Form-3-(02-08-2013).pdf

2828-delnp-2007-Form-3-(20-12-2012).pdf

2828-delnp-2007-form-3.pdf

2828-delnp-2007-form-5.pdf

2828-delnp-2007-GPA-(09-08-2012).pdf

2828-delnp-2007-gpa.pdf

2828-delnp-2007-pct-304.pdf


Patent Number 257847
Indian Patent Application Number 2828/DELNP/2007
PG Journal Number 46/2013
Publication Date 15-Nov-2013
Grant Date 12-Nov-2013
Date of Filing 16-Apr-2007
Name of Patentee DSM SINOCHEM PHARMACEUTICALS NETHERLANDS B.V.
Applicant Address Alexander Fleminlaan 1, 2613 AX DELFT, THE NETHERLANDS
Inventors:
# Inventor's Name Inventor's Address
1 RAAMSDONK, LOURINA MADELEINE KNOBBELZWAANSINGEL 15, NL-2496 LN DEN HAAG, THE NETHERLANDS
2 JENNE, STEPHANE J. 859 CABOT LANE, FOSTER CITY, CALIFORNIA 94404, USA
3 KREBBER, CLAUS 3500 LOUIS ROAD, PALO ALTO, CALIFORNIA 94303, USA
4 SCHIPPER, DICK OOSTSINGEL 205, NL-2612 HL DELFT, THE NETHERLANDS
5 KERKMAN, RICHARD KONINGINNEWEG 12, NL-2496 LN DEN ZANDVOORT, THE NETHERLANDS
6 BOVENBERG, ROELOF ARY LANS KRALINGSE PLASLAAN 10, NL-3062 DA ROTTERDAM, THE NETHERLANDS
7 CHEN, YAN 1765 BOWERS AVENUE, SANTA CLARA, CALIFORNIA 95051, USA
8 CHEN, YONG HONG 828 VEGA CIRCLE, FOSTER CITY, CALIFORNIA 94404, USA
9 GAO, XIAODONG 1005-M BRYANT WAY, SUNNYVALE, CALIFORNIA 94087, USA
10 KREBBER, ANKE 3500 LOUIS ROAD, PALO ALTO, CALIFORNIA 94303, USA
11 TO LA CHING, CHARLENE 4987 KENSON DRIVE, SAN JOSE, CALIFORNIA 95124, USA
12 TOBIN, MATTHEW 2072 EATON AVENUE, SAN CARLOS, CALIFORNIA 94070, USA
PCT International Classification Number C12N 9/02
PCT International Application Number PCT/EP2005/055148
PCT International Filing date 2005-10-11
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 04105022.0 2004-10-13 EUROPEAN UNION
2 05106347.7 2005-07-12 EUROPEAN UNION