EP2310489A1

EP2310489A1 - Adipoyl-7-adca producing strains

Info

Publication number: EP2310489A1
Application number: EP09781467A
Authority: EP
Inventors: Marco Alexander Van Den Berg; Roelof Ary Lans Bovenberg
Original assignee: DSM IP Assets BV
Current assignee: Centrient Pharmaceuticals Netherlands BV
Priority date: 2008-08-05
Filing date: 2009-08-04
Publication date: 2011-04-20
Also published as: CN102131917A; WO2010015625A1

Abstract

The present invention relates to a mutant microbial strain capable of producing an N-adipoylated β-lactam compound when cultured in a culture medium comprising adipic acid characterized in that the strain has an improved incorporation yield of the adipic acid from the culture medium into the N-adipoylated β-lactam compound compared to the non-mutant parent strain.

Description

ADIPOYL-7-ADCA PRODUCING STRAINS

Field of the invention

The present invention relates to N-adipoylated β-lactam compound producing strains, to a method for their construction as well to the identification and functionally inactivating of genes and enzymes involved in the degradation of adipic acid.

Background of the invention

Semi-synthetic β-lactam antibiotics (SSA's) are produced on an industrial scale starting from β-lactam intermediates such as 6-aminopenicillanic acid (6-APA), 7-amino- desacetoxy-cephalosporanic acid (7-ADCA), 7-aminocephalosporanic acid (7-ACA) and 7-amino-3-chloro-3-cephem-4-carboxylate (7-ACCA), 7-amino-3-[(Z/E)-1-propen-1-yl]-3- cephem-4-carboxylate (7-PACA), 7-aminodeacetylcephalosporanic acid (7-ADAC), 7- amino-3-carbamoyloxymethyl-3-cephem-4-carboxylic acid (7-ACCCA) and others. The first generation 7-ADCA product was derived from PenG whereby both the expansion of the 5-membered penem ring to the 6-membered cephem ring and the subsequent cleavage of the phenylacetic acid side chain of the phenylacetyl-7-ADCA were carried out using chemical reactions. The next generation 7-ADCA product was still obtained from PenG but after the chemical ring expansion, the phenylacetic acid side chain of the phenylacetyl-7-ADCA was cleaved off enzymatically using a suitable (penicillin) acylase. Other processes have been developed wherein also the ring expansion of PenG to phenylacetyl-7-ADCA is carried out in vitro using a suitable expandase enzyme, but these processes are of little industrial importance. The most recent and most elegant production process for 7-ADCA comprises the culturing of a Penicillium chrysogenum, transformed with and expressing a gene encoding a suitable expandase. This engineered Penicillium chrysogenum strain, when grown in the presence of adipic acid as the side chain precursor in the fermentation vessel, produces and excretes adipoyl-7-ADCA - see WO93/05158. In this production process, the adipoyl-7-ADCA is recovered from the fermentation broth, subjected to a suitable acylase to cleave off the adipic acid side chain after which the 7-ADCA thus obtained is further purified, crystallized and dried. Other side chains precursors have been disclosed in WO95/04148 (2-(carboxyethylthio)acetic acid and 3- (carboxymethylthio)-propionic acid), WO95/04149 (2-(carboxyethylthio)propionic acid), WO96/38580 (phenyl acetic acid) and WO98/048034 and WO98/048035 (various dicarboxylic acids). The expandase takes care of the expansion of the 5-membered ring of the various N-acylated penicillanic acids, thereby yielding the corresponding N-acylated desacetoxycephalosporanic acids.

However, in addition to being used as side chain precursor, adipate may be degraded and used as a carbon source in the primary metabolism. This was demonstrated by Robin et al. (Appl. Microbiol. Biotechnol (2001 ) 57, 357-362)) in batch cultures with sucrose as the primary carbon source. After depletion of the glucose and fructose (derived from the sucrose), the formation of adipoyl-6-APA and adipoyl-7-ADCA commenced and in this stage only up to 2% of the adipic acid was incorporated in the β-lactam compounds while the remainder was used as a carbon source. It was suggested by the authors that, due to the similarity between adipic acid and fatty acids, the adipic acid degradation was likely to occur by β-oxidation. In a later study, Thykaer et al. (Metabolic Engineering (2002), 4, 151-158), on the basis of a metabolic network analysis of an adipoyl-7-ADCA producing strain of Penicillium chrysogenum, arrived at the conclusion that the adipate degradation takes place in the microbodies (glyoxysomes) by β-oxidation enzymes and not in the cytosol or mitochondria. The nature of the β-oxidation pathway in (filamentous) fungi and yeast in general, and in Penicillium chrysogenum in particular, is still obscure in terms of the intracellular localization, the enzymes involved, as well as the role of the β-oxidation pathway in the total metabolism of the micro-organism.

In order to reduce the adipic acid degradation, it has been suggested by Thykaer et al. (2002) to delete the enzyme responsible for the adipate degradation, however, without specifying any of such enzymes to be a suitable candidate. However, until now, no direct and unequivocal evidence has been provided that adipic acid is indeed degraded via the β- oxidation pathway. Most likely, degradation of adipic acid may also other degradation mechanisms as well, e.g. the so-called benzoate degradation pathway (http://www.genome.ad.jp/dbget-bin/get _pathway?org_name=sma&mapno=00362). In this pathway, 3-oxoadipate is converted in two steps into succinyl-CoA and acetyl-CoA, the same products identified by the study of Thykaer et al. (2002). Adipate could very well be oxidised by enzymes of Penicillium chrysogenum and the obtained oxoadipate subsequently converted by this pathway into the central carbon metabolism pathways.

To prevent any loss of adipic acid through such a degradation pathway one has to block this pathway in the early steps, preferably the first step. However, both the suggested adipate degradation pathway as well as the β-lactam biosynthesis pathway starts with the same reaction, namely, the activation of adipic acid to adipoyl-CoA with a CoA-ligase. Also, both pathways are suggested to be located in the peroxisomes. Thus, by simply following the authors' suggestions and inhibiting or deleting the first enzyme of the β- oxidation pathway one most likely also inhibits β-lactam biosynthesis, which is even more unfavorable.

Detailed description of the invention

In a first aspect, the invention provides a method for the identification of one or more genes of a microbial strain capable of producing an N-adipoylated β-lactam compound and which encode one or more enzymes which are involved in the degradation of adipate and/or the β-oxidation of fatty acids, the method comprising the following steps: a. Selection of a nucleotide sequence of one or more known genes and/or the amino acid sequence of one or more known enzymes, optionally encoded by said genes, which are involved in the degradation of adipate and/or the β-oxidation of fatty acids. b. Using the selected sequence from step (a) as a probe in a BLAST search for the identification of homologous sequences among available nucleotide or amino acid sequences of the microbial strain capable of producing an N-adipoylated β- lactam compound.

Enzymes involved in β-oxidation of fatty acids may be selected from the following groups:

• Group I: CoA ligase (EC 6.2.1.xx)

• Group II: acyl-CoA dehydrogenase (EC 1.3.99.xx) and acyl-CoA oxidase (EC 1.3.3.XX)

• Group III: enoyl-CoA hydratase (EC 4.2.1.17)

• Group IV: 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35)

• Group V: acetyl-CoA C-acyltransferase (thiolase - EC 2.3.1.16).

In a preferred embodiment, the selected sequence (probe) is a known nucleotide sequence of the gene or a known amino acid sequence of the enzyme optionally encoded by said gene and may be selected from (but is not limited to) the group consisting of the following genes and/or amino acid sequences (see Table 1 ). Table 1.

For the purpose of the present invention, the BLAST algorithm is used to identify homologous sequences (Altschul, et al., 1990, J. MoI. Biol. 215: 403-410). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (httjx//m^ The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLAST program uses as default a word length (W) of 1 1 , the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

In addition to the method of the invention described above, the genes encoding one or more enzymes which are involved in the incorporation of the adipic acid from the culture medium into the N-adipoylated β-lactam compound, may further be selected on the basis of their ratio of the transcript level of the gene when the parent strain is grown in a medium containing adipic acid with the transcript level of the gene when the parent strain is grown in a control medium without adipic acid (defined herein as "adipate/control" ratio). Preferably, such a gene has an "adipate/control" ratio of more than 1 , preferably ≥ 2, preferably ≥ 3, preferably ≥ 4, preferably ≥ 5, preferably ≥ 10, preferably ≥ 15, preferably ≥ 20, preferably ≥ 30, preferably ≥ 40, preferably more ≥ 50, preferably ≥ 60, preferably ≥ 70, preferably ≥ 80, most preferably ≥ 90. As a second control, the ratio of the transcript level of the gene when the parent strain is grown in a medium containing phenyl acetic acid with the transcript level of the gene when the parent strain is grown in a control medium without phenyl acetic acid (designated as "PAA/control") may be determined.

A third ratio may be calculated by dividing the "adipate/control" ratio by the "PAA/control" ratio thus yielding the "adipate/PAA" ratio. Preferably, the gene of the invention has an "adipate/PAA" ratio of ≥ 1 , preferably ≥ 2, preferably ≥ 3, preferably ≥ 4, preferably ≥ 5, preferably ≥ 10, preferably ≥ 15, preferably ≥ 20, preferably ≥ 30, preferably ≥ 40, preferably more ≥ 50, preferably ≥ 60, preferably ≥ 70, preferably ≥ 80, most preferably ≥ 90. Preferred genes combine a high "adipate/control" ratio with a low "PAA/control" ratio. For example, a gene with an "adipate/control" ratio of around 50 and a PAA/control ratio of around 10 (i.e. a adipate/PAA of 5) is preferred over a gene with an adipate/control ratio of around 50 and a PAA/control ratio of around 50 (i.e. a adipate/PAA of 1 ).

In a second aspect, the invention provides a mutant microbial strain derived from a parent microbial strain capable of producing an N-adipoylated β-lactam compound when cultured in a medium comprising adipic acid characterized in that the mutant microbial strain has an improved incorporation yield of the adipic acid from the culture medium into the N-adipoylated β-lactam compound. The incorporation yield is defined herein as the molar percentage of adipic acid incorporated in the N-adipoylated β-lactam compound relative to the total molar amount of adipic acid consumed. The total molar amount of adipic acid consumed is equal to the total molar amount of adipic acid added to the fermentation process minus the molar amount of adipic acid remaining after the fermentation process. The improved incorporation yield may be expressed as the relative improvement if the mutant microbial strain compared to the parent microbial strain. For instance, when the parent microbial strain has an incorporation yield of 5% as defined above and the mutant microbial strain of 6%, than the improved incorporation yield of the mutant is 20% (6/5^*100 - 100).

Preferably, the mutant strain of the invention has an improved incorporation yield of at least 5%, more preferably at least 7.5%, more preferably at least 10%, more preferably at least 15%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 100%,

The maximal improved incorporation yield that can be obtained is dependent on the actual incorporation yield of the parent microbial strain. When the parent microbial strain has an incorporation yield of 5% and 100% is the theoretical maximal incorporation yield, then the mutant microbial strain may have a maximal improved incorporation yield of 1900% (100/5^*100 - 100). Likewise, when the parent microbial strain has already an incorporation yield of 50% and 100% is the theoretical maximal incorporation yield, then the mutant microbial strain may have a maximal improved incorporation yield of 100% (100/50^*100 - 100).

In one embodiment, the invention provides a mutant microbial strain derived from a parent microbial strain wherein one or more genes of the mutant strain of the invention encoding one or more enzymes which are involved in one or more degradation pathways of adipic acid have been made functionally inactive. The gene of any enzyme involved in one or more degradation pathways of adipic acid may be made functionally inactive. Preferably, a functionally inactivated gene of any mutant microbial strain identified by using the method of the present invention and thus leading to an improved incorporation yield, may be combined with another functionally inactivated gene(s) from any other mutant microbial strain identified by using the method of the present invention which lead to an improved incorporation yield; the combination of two or more of these functionally inactivated genes may lead to even further improved incorporation yields. In a specific embodiment, three functionally inactivated genes may be combined with each other to obtain even further improved incorporation yields.

In one specific embodiment, the invention provides a mutant microbial strain derived from a parent microbial strain wherein one or more genes of the mutant strain of the present invention encoding one or more enzymes which are involved in one or more degradation pathways of adipic acid have been made functionally inactive. In addition, such a mutant microbial strain may contain one or more genes encoding one or more enzymes which are involved in the incorporation of the adipic acid from the culture medium into the N-adipoylated β-lactam compound, preferably a CoA-ligase and/or a CoA-transferase, which is/are over expressed in the mutant microbial strain of the present invention compared to the parent microbial strain in which said gene is not over expressed. The combination of functionally inactivated genes involved in the degradation of adipic acid and the overexpression of genes involved in the incorporation of adipic acid into the N-adipoylated β-lactam compound in one single microbial strain, leads to an even further improved incorporation yield. Preferably, genes encoding one or more enzymes which are involved in the incorporation of the adipic acid from the culture medium into the N-adipoylated β-lactam compound may be selected from the group consisting of genes encoding enzymes that are capable of catalyzing the conversion of the intracellular adipic acid into adipoyl-CoA such as CoA-ligase and CoA-transferase. Over expression of a gene is defined herein as the expression of the gene which results in an activity of the enzyme encoded by said gene in the mutant microbial strain being at least 1.5-fold the activity of the enzyme in the parent microbial; preferably the activity of said enzyme is at least 2-fold, more preferably at least 3-fold, more preferably at least 4- fold, more preferably at least 5-fold, even more preferably at least 10-fold and most preferably at least 20-fold the activity of the enzyme in the parent microbial.

A "parent microbial strain" may be defined as a micro-organism capable of producing β-lactam compounds, preferably N-adipoylated β-lactam compounds. A "mutant microbial strain" may be defined as a strain derived from the parent microbial strain by genetic engineering or classical mutagenesis.

"Functionally inactive" is defined herein as the inactivation of a gene which results in a residual activity of the encoded enzyme of preferably less than 50%, more preferably less than 40%, more preferably less than 30%, more preferably less than 20%, more preferably less than 10%, more preferably less than 5%, more preferably less than 2%. "Inactivation of a gene" is defined herein as the modification of a gene in order to obtain a functionally inactive gene as defined hereinbefore. Methods for the modification of a gene in order to obtain a functionally inactive gene are known in the art and may include: inactivation of the gene by base pair mutation resulting in a(n early) stop or frame shift; mutation of one or more codons which encode one or more a critical amino acids (such as the catalytic triad for hydrolases); mutations in the gene resulting in mutations in the amino acid sequence of the enzyme which lead to a decreased half- life of the enzyme; modifying the mRNA molecule in such away that the mRNA half-life is decreased; insertion of a second sequence (i.e. a selection marker gene) disturbing the open reading frame; a partial or complete removal of the gene; removal/mutation of the promoter of the gene; using anti-sense DNA or comparable RNA inhibition methods to lower the effective amount of mRNA in the cell. Most preferably the gene has been made functionally inactive by deletion resulting in a total absence of the encoded polypeptide and hence enzyme activity.

In a preferred embodiment the invention provides a mutant microbial strain derived from a parent microbial strain wherein the genes encoding putative enzymes involved in β-oxidation of fatty acids have been made functionally inactive. These genes may be identified using the method of the invention which has been described hereinbefore using one or more of the probes of Table 1.

The N-adipoylated β-lactam compound produced by the microbial strain may be any N-adipoylated β-lactam wherein the β-lactam moiety is a penem or cephem. Preferred N-adipoylated β-lactam compounds are adipoyl-derivates of the intermediates listed before: 6-aminopenicillanic acid (6-APA), 7-amino-desacetoxy-cephalosporanic acid (7-ADCA), 7-aminocephalosporanic acid (7-ACA) and 7-amino-3-chloro-3-cephem- 4-carboxylate (7-ACCA), 7-amino-3-[(Z/E)-1-propen-1-yl]-3-cephem-4-carboxylate (7- PACA), 7-aminodeacetylcephalosporanic acid (7-ADAC), 7-amino-3- carbamoyloxymethyl-3-cephem-4-carboxylic acid (7-ACCCA) and others. Most preferred are N-adipoylated cephalosporins, most preferred is adipoyl-7-ADCA.

The microbial strain capable of producing an N-adipoylated β-lactam compound may be selected from the group consisting of a fungus, bacterium or yeast. Preferably the microbial strain of the present invention is a fungus, more preferably a filamentous fungus. A preferred filamentous fungus may be selected from the group consisting of Aspergillus, Acremonium, Trichoderma and Penicillium. More preferably the mutant microbial strain of the present invention belongs to the species Penicillium, most preferably Penicillium chrysogenum. A preferred bacterium may be selected from the groups consisting of Streptomyces, Nocardia, or Flavobacterium.

In a preferred embodiment, the mutant microbial strain of the present invention capable of producing an N-adipoylated β-lactam compound belongs to the species Penicillium, most preferably is Penicillium chrysogenum, which has been transformed with a gene encoding an expandase, preferably the Streptomyces clavuligerus cefE gene, which enables the strain to produce adipoyl-7-ADCA when cultured in the presence of the precursor adipic acid.

In another embodiment, the mutant microbial strain of the present invention capable of producing an N-adipoylated β-lactam compound belongs to the species Penicillium, most preferably is Penicillium chrysogenum, and in addition to an expandase gene, preferably the Streptomyces clavuligerus cefE gene, has been transformed with a hydroxylase gene, preferably the Streptomyces clavuligerus cefF gene, whose expression product converts the 3-methyl side chain of adipoyl-7-ADCA to 3- hydroxymethyl, to give adipoyl^-aminodeacetylcephalosporanic acid (adipoyl-7-ADAC).

In another embodiment, the mutant microbial strain of the present invention capable of producing an N-adipoylated β-lactam compound belongs to the species Penicillium, most preferably is Penicillium chrysogenum, has been transformed with a expandase/hydroxylase gene, preferably the Acremonium chrysogenum cefEF gene, whose expression product converts the 3-methyl side chain of adipoyl-7-ADCA to 3- hydroxymethyl, to give adipoyl-7-aminodeacetylcephalosporanic acid (adipoyl-7-ADAC).

In another embodiment the mutant microbial strain of the present invention capable of producing an N-adipoylated β-lactam compound belongs to the species Penicillium, most preferably is Penicillium chrysogenum, and in addition to the genes encoding expandase, preferably the Streptomyces clavuligerus cefE gene, and hydroxylase, preferably the Streptomyces clavuligerus cefF gene, is further transformed with an acetyltransferase gene, preferably the Streptomyces clavuligerus cefG gene, whose expression product (i.e. the acyltransferase) converts the 3-hydroxymethyl side chain to the 3-acetyloxymethyl side chain to give adipoyl-7-ACA.

In a further embodiment the mutant microbial strain of the present invention capable of producing an N-adipoylated β-lactam compound belongs to the species Penicillium, most preferably is Penicillium chrysogenum and has been transformed with genes encoding an expandase, preferably the Streptomyces clavuligerus cefE gene, a hydroxylase, preferably the Streptomyces clavuligerus cefF gene, and an O-carbamoyl transferase enzyme, preferably the Streptomyces clavuligerus cmcH gene, resulting in adipoyl-7-amino-3-carbamoyloxymethyl-3-cephem-4-carboxylic acid.

In another embodiment the mutant microbial strain of the present invention belongs to the species Penicillium, most preferably Penicillium chrysogenum, optionally transformed with one or more of the above mentioned genes encoding an expandase, a hydroxylase, an expandase/hydroxylase, an acetyltransferase and/or an O-carbamoyl transferase, and in addition is also transformed with genes encoding enzymes capable of increasing the activity of activation and/or incorporation of adipic acid into N- adipoylated β-lactam. Preferred examples of such modifications are (but not limited to): an increased transport of the adipic acid from the culture medium into the interior of the microbial cell, increased activation of adipate towards adipoyl-CoA and/or increased incorporation of the adipate side chain onto the β-lactam nucleus. The respective enzymes involved may be a transporter protein, a CoA-ligase or CoA-transferase and an acyl-CoA:6-aminopenicillanic acid acyltransferase respectively.

In yet another embodiment, the mutant microbial strain of the present invention belongs to the species Penicillium, most preferably Penicillium chrysogenum, optionally transformed with one or more of the above mentioned genes encoding an expandase, a hydroxylase, an expandase/hydroxylase, an acetyltransferase, an O-carbamoyl transferase and/or genes encoding enzymes capable of increasing the activity of activation and/or incorporation of adipic acid into N-adipoylated β-lactam, and in addition is also transformed with genes encoding enzymes capable of increasing the secretion of N-adipoylated β-lactam in to the medium. Preferred examples of such genes are (but not limited to) are the Acremonium chrysogenum cefT gene or homologs thereof and the Acremonium chrysogenum cefM gene or homologs thereof.ln a preferred embodiment, the invention provides a mutant microbial strain derived from a parent microbial strain capable of producing an N-adipoylated β-lactam compound when cultured in a medium comprising adipic acid, preferably a mutant Penicillium chrysogenum strain, wherein one or more of the genes encoding one or more of the enzymes involved in β-oxidation of fatty acids have been made functionally inactive whereby the inactivation of the gene is as defined hereinbefore. Highly preferred is a mutant microbial strain, preferably a mutant Penicillium chrysogenum strain, strain wherein one or more genes, encoding one or more enzymes of Group I, Group II, Group III, Group IV and/or Group V as defined hereinbefore, have been made functionally inactive whereby

• The gene encoding the enzyme of Group I may have a nucleotide sequence selected from the group consisting of SEQ ID NO 1 , SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 1 1 , SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 21 , SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25 as well as a nucleotide sequence which is substantially homologous to any of the sequences listed.

• The gene encoding the enzyme of Group Il may have a nucleotide sequence selected from the group consisting of SEQ ID No. 51 , SEQ ID No. 52, SEQ ID No. 53, SEQ ID No. 54, SEQ ID No. 55, SEQ ID No. 56, SEQ ID No. 57, SEQ ID No. 58, SEQ ID No. 59, SEQ ID No. 60, SEQ ID No. 61 , SEQ ID No. 62, SEQ ID No. 63, SEQ ID No. 64, SEQ ID No. 65, SEQ ID No. 66, SEQ ID No. 67, SEQ ID No. 68, SEQ ID No. 69, SEQ ID No. 135 as well as a nucleotide sequence which is substantially homologous to any of the sequences listed.

• The gene encoding the enzyme of Group III may have a nucleotide sequence selected from the group consisting of SEQ ID No. 89, SEQ ID No. 90, SEQ ID No. 91 , SEQ ID No. 92, SEQ ID No. 93, SEQ ID No. 94 as well as a nucleotide sequence which is substantially homologous to any of the sequences listed.

• The gene encoding the enzyme of Group IV may have a nucleotide sequence selected from the group consisting of SEQ ID No. 92, SEQ ID No. 93, SEQ ID No. 101 , SEQ ID No. 102, SEQ ID No. 103, SEQ ID No. 104, SEQ ID No. 105, SEQ ID No. 106, SEQ ID No. 107, SEQ ID No. 108 as well as a nucleotide sequence which is substantially homologous to any of the sequences listed.

• The gene encoding the enzyme of Group V may have a nucleotide sequence selected from the group consisting of SEQ ID No. 117, SEQ ID No. 118, SEQ ID No. 119, and SEQ ID No. 120 as well as a nucleotide sequence which is substantially homologous to any of the sequences listed.

It is defined herein that a sequence (sequence 1 ) is "substantially homologous" to another sequence (sequence 2) when sequence 1 possesses a degree of identity to sequence 2 of at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, still more preferably at least 96%, still more preferably at least 97%, still more preferably at least 98% and most preferably at least 99%. This definition of "substantially homologous" applies to nucleotide sequences as well as to amino acid sequences.

For the purpose of the present invention, the homology between two nucleotide sequences refers to the percentage of bases that are identical between the two sequences. DNA sequences related to the specified DNA sequences and obtained by degeneration of the genetic code are also part of the invention. Homologues may also encompass biologically active fragments of the full-length sequence. A substantially homologous polypeptide may encompass polymorphisms that may exist in cells from different populations or within a population due to natural allelic or intra-strain variation. A substantially homologous polypeptide may further be derived from a fungus other than the fungus where the specified amino acid and/or DNA sequence originates from, or may be encoded by an artificially designed and synthesized DNA sequence.

Substantially homologous polypeptides may contain only conservative substitutions of one or more amino acids of the specified amino acid sequences or substitutions, insertions or deletions of non-essential amino acids. Accordingly, a non-essential amino acid is a residue that can be altered in one of these sequences without substantially altering the biological function. For example, guidance concerning how to make pheno- typically silent amino acid substitutions is provided in Bowie, J. U. et ai, (Science 247:1306-1310 (1990)) wherein the authors indicate that there are two main approaches for studying the tolerance of an amino acid sequence to change. The first method relies on the process of evolution, in which mutations are either accepted or rejected by natural selection. The second approach uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene and selects or screens to identify sequences that maintain functionality. As the authors state, these studies have revealed that proteins are surprisingly tolerant of amino acid substitutions. The authors further indicate which changes are likely to be permissive at a certain position of the protein. For example, most buried amino acid residues require non-polar side chains, whereas few features of surface side chains are generally conserved. Other such phenotypically silent substitutions are described in Bowie et al, and the references cited therein.

The term "conservative substitution" is intended to mean that a substitution in which the amino acid residue is replaced with an amino acid residue having a similar side chain. These families are known in the art and include amino acids with basic side chains (e.g. lysine, arginine and histidine), acidic side chains (e.g. aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagines, glutamine, serine, threonine, tyrosine, cystein), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophane), branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine tryptophane, histidine).

In a third aspect, the invention provides a method for the construction of the mutant microbial strain of the invention comprising functionally inactivating a gene encoding an enzyme which is involved in the degradation of adipate and/or inactivating a gene encoding an enzyme which is involved in the β-oxidation of fatty acids. To delete such a gene in a parent microbial strain to obtain such a mutant microbial strain one can apply several methods. One approach is a temporary one using an anti-sense molecule or RNAi molecule (Kamath et al. 2003. Nature 421 :231-237). Another is using a regulatable promoter system, which can be switched off using external triggers like tetracycline (see Park and Morschhauser, 2005, Eukaryot Cell. 4:1328-1342). Yet another one is to apply a chemical inhibitor or a protein inhibitor or a physical inhibitor (see Tour et al. 2003. Nat Biotech 21 :1505-1508). The most preferred situation is to remove part of or the complete gene(s) encoding the adipate degradation activity. To obtain such a mutant one can apply state of the art methods like Single Cross-Over Recombination or Double Homologous Recombination. For this one needs to construct an integrative cloning vector that may integrate at the predetermined target locus in the chromosome of the host cell. In a preferred embodiment of the invention, the integrative cloning vector comprises a DNA fragment, which is homologous to a DNA sequence in a predetermined target locus in the genome of host cell for targeting the integration of the cloning vector to this predetermined locus. In order to promote targeted integration, the cloning vector is preferably linearized prior to transformation of the host cell. Linearization is preferably performed such that at least one but preferably either end of the cloning vector is flanked by sequences homologous to the target locus. The length of the homologous sequences flanking the target locus is preferably at least 0.1 kb, even preferably at least 0.2 kb, more preferably at least 0.5 kb, even more preferably at least 1 kb, most preferably at least 2 kb. The length that finally is best suitable in an experiment depends on the organism, the sequence and length of the target DNA.

The efficiency of targeted integration of a nucleic acid construct into the genome of the host cell by homologous recombination, i.e. integration in a predetermined target locus, is preferably increased by augmented homologous recombination abilities of the host cell. Such phenotype of the cell preferably involves a deficient hdfA or hdfB gene as described in WO 05/95624. WO 05/95624 discloses a preferred method to obtain a filamentous fungal cell comprising increased efficiency of targeted integration by preventing non-homologous random integration of DNA fragments into the genome. The vector system may be a single vector or plasmid or two or more vectors or plasmids, which together contain the total DNA to be introduced into the genome of the host cell. Alternative methods using second and/or lethal selectable markers are described in WO2007115886 and WO20071 15887.

Fungal cells may be transformed by protoplast formation, protoplast transformation, and regeneration of the cell wall. Suitable procedures for transformation of fungal host cells are described in EP 238023 and Yelton et al. (1984. Proc. Nat. Acad. Sci. USA 81 :1470-1474). Suitable procedures for transformation of filamentous fungal host cells using Agrobacterium tumefaciens are described by de Groot MJ. et al. (1998. Nat. Biotechnol. 16:839-842. Erratum in: Nat. Biotechnol. 1998. 16:1074). Other methods like electroporation, described for Neurospora crassa, may also be applied.

Fungal cells are transfected using co-transformation, i.e. along with gene(s) of interest also a selectable marker gene is transformed. This can be either physically linked to the gene of interest (Ae. on a plasmid) or on a separate fragment. Following transfection transformants are screened for the presence of this selection marker gene and subsequently analyzed for the integration at the preferred predetermined genomic locus. A selectable marker is a product, which provides resistance against a biocide or virus, resistance to heavy metals, prototrophy to auxotrophs and the like. Useful selectable markers include, but are not limited to, amdS (acetamidase), argB (ornithine- carbamoyltransferase), bar (phosphinothricinacetyltransferase), hygB (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC or sutB (sulfate adenyltransferase), trpC (anthranilate synthase), ble (phleo- mycin resistance protein), as well as equivalents thereof. The most preferred situation is providing a DNA molecule comprising a first DNA fragment comprising a desired replacement sequence (i.e. the selection marker gene) flanked at its 5' and 3' sides by DNA sequences substantially homologous to sequences of the chromosomal DNA flanking the target sequence. Cells wherein the target sequence in the chromosomal DNA sequence is replaced by the desired replacement sequence can be selected by the presence of the selectable marker of the first DNA fragment. To increase the relative frequency of selecting the correct mutant microbial strain, a second DNA fragment comprising an expression cassette comprising a gene encoding a selection marker and regulatory sequences functional in the eukaryotic cell can be operably linked to the above described fragment (i.e. 5'-flank of target locus + selection marker gene + 3'flank of target locus) and cells wherein the target sequence in the chromosomal DNA sequence is replaced by the desired replacement sequence can be selected by the presence of the selectable marker of the first DNA fragment and the absence of the second selection marker gene. The 5'- and 3'-flanks of the target locus can be for example the promoter and terminator of a gene, or the 5'- and 3'-end of the gene, or any combination of these.

The example provided as an illustration of the method, incorporated in the present invention, uses the promoter of the gene as 5'-flank and the gene as the 3'-flank to insert a selection marker between the promoter and gene, thereby disturbing (i.e. inactivating) gene transcription. The gene sequences given above can be used to make similar gene deletions. The genes may be split in two, yielding a 5'-flank and a 3'-flank, but the gene may also be used to clone a larger piece of genomic DNA containing the promoter and terminator regions of the gene, which than can function as 5'-flank and a 3'-flanks.

In a fourth aspect, the invention provides a process for the production of an N- adipoylated β-lactam compound comprising culturing the mutant strain of the invention in a fermentation medium comprising adipic acid.

Figure 1 is a representation of the steps involved in deleting the Penicillium chrysogenum gene Pc20g07920. Legend: solid arrow, Pc20g07920 promoter; open arrow, Pc20g07920 ORF; hatched box, trpC terminator; dashed box, ccdA gene; solid box, lox site; crosses, recombination event; downwards arrows, subsequent steps in the procedure; REKR and KRAM, overlapping non-functional amdS selection marker fragments; REKRAM, functional amdS selection marker gene.

Numbers indicate the SEQ ID NO.'s of the oligonucleotides "tag" indicates the presence of a specific nucleotide sequence which can be used for mutant identification.

Figure 2 is a representation of the steps involved in confirming the actual deletion of the Penicillium chrysogenum gene Pc20g07920. Legend: solid arrow, Pc20g07920 promoter; open arrow, Pc20g07920 ORF; hatched box, trpC terminator; solid box, lox site; REKRAM, functional amdS selection marker gene. Numbers indicate the SEQ ID NO.'s of the oligonucleotides for the three PCR reactions indicated (see also table 7).

General Methods

BLAST algorithms are used to identify homologous sequences (Altschul, et al., 1990, J. MoI. Biol. 215: 403-410). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). 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 1 1 , the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=- 4, and a comparison of both strands.

Standard procedures were carried out as described elsewhere (Sambrook, J. et al. (1989), Molecular cloning: a laboratory manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York). DNA was amplified using the proofreading polymerases Turbo-Pfu-Polymerase (Stratagene, The Netherlands) or Phusion (Finnzymes), following the manufacturers protocol, while the verification of constructed strains and plasmids was achieved by using Taq polymerase. Restriction enzymes were from Invitrogen or New England Biolabs. For routine cloning, Escherichia coli strains Top10 and DH10B (Invitrogen) were employed. Verification of the constructed plasmids was carried out by restriction analysis and subsequent sequencing.

EXAMPLES

Example 1

Identification of Penicillium chrysogenum genes encoding putative adipate degrading and/or β-oxidation enzymes

1. CoA ligase (EC 6.2.1. xx)

Using probe A, probe B, probe C and probe D (see text), the genome of Penicillium chrysogenum strain Wisconsin54-1255 was searched and subsequent annotation revealed 25 genes encoding a putative CoA-ligase activity (EC 6.2.1.xx); see table 2. The percentages indicated are local homology scores obtained using the blastP algorithm.

2. Acyl-CoA dehydrogenase (EC 1.3.99.xx) or acyl-CoA oxidase (EC 1.3.3.xx)

Using probe E and probe F (see text), the genome of Penicillium chrysogenum strain Wisconsin54-1255 was searched and subsequent annotation revealed 19 genes encoding a putative acyl-CoA dehydrogenase (EC 1.3.99.xx) and/or acyl-CoA oxidases (1.3.3.6) - see Table 3. The percentages indicated are local homology (=similarity) scores obtained using the blastP algorithm.

3. Enoyl-CoA hydratase (EC 4.2.1.17)

Using probe G, probe H, probe I, probe J and probe K (see text), the genome of Penicillium chrysogenum strain Wisconsin54-1255 was searched and subsequent annotation revealed 6 genes encoding a putative enoyl-CoA hydratase (EC 4.2.1.17) - see Table 4. The percentages indicated are local homology (=similarity) scores obtained using the blastP algorithm.

4. 3-Hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35)

Using probe L and probe M (see text), the genome of Penicillium chrysogenum strain Wisconsin54-1255 was searched and subsequent annotation revealed 10 genes encoding a putative 3-Hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) - see Table 5. The percentages indicated are local homology (=similarity) scores obtained using the blastP algorithm.

5. Acetyl-CoA C-acyltransferase (EC 2.3.1.16)

Using probe N, the genome of Penicillium chrysogenum strain Wisconsin54-1255 was searched and subsequent annotation revealed 4 genes encoding a putative 3- Acetyl- CoA C-acyltransferase (EC 2.3.1.16) - see Table 6. The percentages indicated are local homology (=similarity) scores obtained using the blastP algorithm.

Example 2

MicroArray analyses of Penicillium chrysogenum genes encoding putative adipate degrading enzymes

To identify which of the 65 identified in Example 1 are suitable candidates for modification to prevent or lower the adipate degradation, but not disturb the side-chain activation for β-lactam synthesis, a MicroArray study was performed. Hereto, the "adipate/control" ratio, "PAA/control" ratio and the "adipate/PAA" ratio of each of the genes were determined as described below.

The P. chrysogenum genome sequence was used to prepare a proprietary DNA microarray, using the Affymetrix Custom GeneChip program (Affymetrix, Inc., Santa Clara, CA): GeneChip, DSM_PENa520255F. P. chrysogenum strains with and without the Streptomyces clavuligerus cefE gene encoding expandase, were inoculated 100 ml shake flasks with 25 ml of β-lactam production medium (as described in US20020039758), with either 10 g/l adipate, 3 g/l phenylacetic acid (PAA) or |o precursor (control). The cultures were incubated at 25 C with 280 rotations per minute. After 90 hours the total broth was sampled and rapidly cooled. The cells were washed and the cell pellet was frozen in liquid nitrogen. The cells were subsequently disrupted by grinding the frozen pellet with a pestle and mortar, and RNA was isolated using Trizol. The quality and amount of total RNA was routinely checked on a Bioanalyzer (Agilent). Twenty microgram of total RNA was used for a standard cDNA synthesis and labeling reaction (according to Affymetrix instructions). Hybridisation of the GeneChips^® was performed according to the suppliers instructions (Affymetrix, Santa Clara, USA) Hybridized arrays were scanned and analyzed using the Affymetrix GeneChip^®Operating Software (GCOS, Affymetrix, Santa Clara, USA). All experiments were done in triplicate and the average of the three measurements was taken as the relative level of transcript. Tables 2-5 show the various ratios for the 65 genes identified in Example 1. Nine of the 65 genes have an "adipate/PAA" ratio ≥4. Fourteen of the 65 genes have an "adipate/PAA" ratio ≥3. The transcription of such genes can be modified to limit or prevent the degradation of adipate.

Table 2. Transcript levels of Penicillium chrysogenum genes encoding an enzyme with a putative CoA ligase activity (EC 6.2.1.xx).

CD

- = no detectable transcripts under the conditions studied

Table 3. Transcript levels of Penicillium chrysogenum genes encoding an enzyme with a putative acyl-CoA oxidases (EC 1.3.3.xx).

K) o

- = no detectable transcripts under the conditions studied

Table 4. Transcript levels of Penicillium chrysogenum genes encoding a putative enoyl-CoA hydratase (EC 4.2.1.17)

- = no detectable transcripts under the conditions studied

Table 5. Transcript levels of Penicillium chrysogenum genes encoding a putative 3-Hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) K)

- = no detectable transcripts under the conditions studied

Table 6. Transcript levels of Penicillium chrysogenum genes encoding a putative acetyl-CoA C-acyltransferase (EC 2.3.1.16)

- = no detectable transcripts under the conditions studied to to

Example 3

Deletion of Penicillium chrysogenum gene Pc20g07920 encoding a putative adipate catabolising enzyme

The gene Pc20g07920 encoding a putative acyl-CoA dehydrogenase (EC 1.3.99), was identified as a gene with an "adipate/control" ratio of 6.1 and an "adipate/PAA" ratio of 4.1 (see Table 3). In order to prevent the transcription of this gene a selection marker gene was inserted between the promoter and the open reading frame (ORF). To this end the promoter and the ORF were PCR amplified using the oligonucleotides SEQ ID NO. 125 plus 126 and SEQ ID NO. 127 plus 128, respectively. Phusion Hot-Start Polymerase (Finnzymes) was used to amplify the fragments. Both fragments are 1500 basepairs (bp) in length (SEQ ID NO. 129 and SEQ ID NO. 130) and contain a 14 bp tail suitable for the so-called STABY cloning method (Eurogentec).

From the standard STABY vector, pSTC1.3, two derivatives were obtained. One, pSTamdSL, was used for cloning the PCR amplified Pc20g07920 promoter. The other, pSTamdSR, was used for cloning the PCR amplified Pc20g07920 terminator. pSTamdSL (SEQ ID NO. 137) was constructed by insertion of an inactive part of the amdS selectionmarker gene (see for example the PgpdA-amdS cassette of pHELY-A1 in WO04106347) by PCR amplification of the last 2/3 of the gene {amdS) and cloning it in the Hind\\\-BamH\ sites of pSTC1.3. pSTamdSR (SEQ ID NO. 138) was constructed by insertion of another inactive part of the amdS selectionmarker gene (see for example the PgpdA-amdS cassette of pHELY-A1 in WO 04106347) by PCR amplification of the PgpdA promoter and the first 2/3 of the gene wherein the EcoRV sites where removed and cloning it in the Hindlll-Pmel sites of pSTC1.3. Also, a strong terminator was inserted in front of the PgpdA-amdS; the trpC terminator was PCR amplified and introduced via the Sbf\-Not\ sites of the PgpdA-amdS fragment. Both vectors do contain an overlapping but non-functional fragment of the fungal selectionmarker gene amdS, encoding acetamidase and allowing recipient cells that recombine the two fragments into a functional selectionmarker to grow on agar media with acetamide as the sole nitrogen source (EP 635,574; WO 9706261 ; Tilburn et al., 1983, Gene 26: 205-221 ). The PCR fragments were ligated into the vectors overnight using T4 ligase (Invitrogen) at 16 C, according to the STABY-protocol (Eurogentec) and transformed to chemically competent CYS21 cells (Eurogentec). Ampicillin resistant clones were isolated and used to PCR amplify the cloned fragments fused to the non-functional amdS fragments (see Fig. 1 ). This was done using the oligonucleotides SEQ ID NO. 131 and 132. The thus obtained PCR fragments (SEQ ID NO. 133 and SEQ ID NO. 134) were combined and used to transform a P. chrysogenum strain with the hdfA gene deleted (WO05095624). In this strain the non-homologous end-joining pathway is disturbed and therefore the random integration of DNA is drastically reduced. And as the combined PCR fragments themselves should recombine also to form a functional amdS selection marker gene (i.e. the so-called bipartite or split-marker method), correct targeted integrants should undergo a triple homologous recombination event (see Fig. 1 ).

More than 20 transformants were obtained on acetamide containing agar (EP 635,574; WO97/06261 ) and these were subsequently transferred to a second acetamide selection plate to induce sporulation. The thus obtained strains are tested on β-lactam production media (US20020039758) with adipate or PAA as a side-chain precursor, compared to the control situation without any side-chain precursor.

The gene deletion method applied can be used for each gene identified by the present invention, individually or in combinations, to obtain the best mutant microbial strain with the highest incorporation of the adipic acid from the culture medium into the N-adipoylated β-lactam compound.

Mutant microbial strains with a lower adipate degradation can also be identified by comparing the growth of such mutant microbial strain with the parent microbial strain on adipate as a carbon source for biomass formation. In this case the lactose and the glucose in the P. chrysogenum medium described in US20020039758 should be replaced by 0.0-1.0 g/L glucose and 1-80 g/L of adipate. Mutant microbial strains with slower or no growth have a decreased adipate degradation and therefore might have increased incorporation of the adipic acid from the culture medium into the N-adipoylated β-lactam compound.

Example 4

Deletion of Penicillium chrysogenum genes Pc20g01800 and Pc20g15640 encoding a putative adipate catabolising enzymes

The same methodology as described in example 3 was followed for genes Pc20g01800 and Pc20g15640, both having an adipate/PAA ratio ≥3, respectively 3.0 and 6.4. The specific gene fragments (promoter and ORF) to be cloned were obtained by PCR amplification using the oligonucleotides SEQ ID NO. 139 plus SEQ ID NO. 140 for the specific fragments of gene Pc20g01800 and SEQ ID NO. 141 plus SEQ ID NO. 142 for the specific fragments of gene Pc20g 16540, respectively. These fragments were STABY cloned and the fragments for Penicillium chrysogenum transformation were obtained as in example 3.

For both gene deletions several acetamide consuming transformants were obtained.

Example 5

The deletion of Penicillium chrysogenum genes Pc20g07920, Pc20g01800 and Pc20g15640 are all correct

The mutants obtained in examples 3 and 4 were colony purified and used for further characterisation: verification of the actual gene deletion by PCR.

To isolate chromosomal DNA of the correct quality (i.e. enabling PCR amplification up to 9 kb) spores of the three isolated mutants were used to inoculate 3 ml of medium in a 24-well MTP plate and grown for 2-3 days at 550 rpm, 25°C and 80% humidity. Cells are washed and protoplasted using standard buffers (see Swinkels, B.W., Selten, G. C. M., Bakhuis, J. G., Bovenberg, R.A.L., Vollebregt, A.W. 1997. The use of homologous amdS genes as selectable markers. WO9706261 ). Protoplastation was done for 2 hours at 37°C, using Glucanex at 10 mg/ml in the 24-well plates. Cells (protoplasts and remaining mycelium) are washed again and DNA was isolated using the Puragen DNA isolation kit (Gentra) according to the suppliers' instructions. The DNA was air-dried and dissolved in 100 ul water. PCR reactions were performed in a final 50 μl, with the following composition:

5 μl DNA template (5x diluted from sample preparation)

21.5 μl water 10 μl GC buffer (Finnzymes)

1 μl dNTP (stocksolution of 1 OmM)

2 μl DMSO

5 μl Forward oligonucleotide (stocksolution of 2uM)

5 μl Reverse oligonucleotide (stocksolution of 2uM) 0.5 μl Phusion Polymerase (Finnzymes)

To verify the correct deletion 3 PCR reactions are performed (see Fig. 2). The first PCR reaction is to confirm the correct integration at the left flanking, using for the three different loci three specific forward reverse oligonucleotides (see table 7), which in the case of gene Pc20g02720 is the oligonucleotide of SEQ ID NO 143 and as reverse primer the oligonucleotide of SEQ ID NO 144; the former being specific for this gene locus and choosen just upfront of the fragment used for gene targeting and the latter annealing in the amdS selectionmarker, which can be used to verify all individual gene mutations. The second PCR reaction is to confirm the correct integration at right flanking, using for the locus of gene Pc20g02720 the specific reverse oligonucleotide of SEQ ID NO 146 and the forward oligonucleotide of SEQ ID NO 145; the former being specific for this gene locus and choosen just downstream of the fragment used for gene targeting and the latter annealing in the amdS selectionmarker, which can be used to verify all individual gene mutations. The third PCR reaction is to confirm the absence of the WT fragment and the correct integration at the locus of gene; for this one can combine the two locus specific oligonucleotides of the first two PCR reactions, i.e. in the case of locus of gene Pc20g02720 the forward oligonucleotide of SEQ ID NO 143 and the reverse oligonucleotide of SEQ ID NO 146; if the gene targeting is correct this yields a much larger band than in the case of the WT (see table 2).

The PCR amplification is performed in a Tetrad machine of Biorad using the following program:

Step 1 : 30 sec at 98°C Step 2: 10 sec at 98°C

Step 3: 30 sec at 55°C Step 4: 1.5-4.5 min at 72°C

(the actual extention time is set by using 0.5 min/kb to be amplified) Step 5: Repeat steps 2-4 for 35 cycles Step 6: 10min at 72°C

Table 7. Expected and observed PCR fragments in the three isolated mutants for the gene loci Pcg20g07920, Pc20g01800 and Pc20g16540 (see also Fig. 2).

The results depicted in Table 7 clearly demonstrate that mutants Pc20g07920 and Pc20g01800 are correctly targeted and no WT gene is present. The same procedure can be applied to all the mutants identified and obtained according to the present invention; in all cases the oligonucleotides of SEQ ID NO 144 and 145 stay the same, but the other oligonucleotides are gene specific.

Example 6

Deletion of Penicillium chrysogenum genes Pc20g07920, Pc20g01800 and Pc20g15640 lead to an increased incorporation yield of the adipic acid from the culture medium into the N-adipoylated β-lactam compound compared to the non- mutant parent strain.

The spores of thus proven correct mutants of loci Pc20g07920 and Pc20g01800 were inoculated in 25 ml medium as described in example 2 with adipic acid as side chain precursor, in 100 ml shake flasks and incubated for 168 hours at 25 ⁰C. and 280 rpm. As a control strain, P. chrysogenum strain DS17690 (S917), deposited at the Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands on April 15, 2008 with deposition number CBS 122850, was inoculated and grown in the same way. Subsequently, the cells were removed by centrifugation and 1 ml of the supernatant was used for NMR analysis. Quantitative ¹H NMR experiments were performed at 600 MHz on a Bruker Avance 600 spectrometer. To a known quantity of filtrate, a known quantity of internal standard (for example maleic acid), dissolved in phosphate buffer was added prior to lyophilisation. The residue was dissolved in D₂O and measured at 300⁰K. The delay between scans (30 s) was more than 5 times T₁ of all compounds, so the ratio between the integrals of the compounds of interest and the integral of the internal standard is an exact measure for the quantity of the penicillins, intermediates (6-APA and lsopenicillin N), degradation products (8-HPA), remaining sugar and remaining side- chain (adipate). Table 8. Relative incorporation yields of the adipic acid from the culture medium into N- adipoylated β-lactam compounds for the mutant microbial strains (i.e. the deletion mutants as com ared to the non-mutant arent strain.

Compared to the DS17690 strain, the incorporation yields of the adipic acid from the culture medium into the N-adipoylated β-lactam compounds for the mutant microbial strains are significantly increased.

Claims

1. A method for the identification of one or more genes of a microbial strain capable of producing an N-adipoylated β-lactam compound and which encode one or more enzymes which are involved in the degradation of adipate and/or the β-oxidation of fatty acids, the method comprising the following steps: a. Selection of a nucleotide sequence of one or more known genes and/or the amino acid sequence of one or more known enzymes, optionally encoded by said genes, which are involved in the degradation of adipate and/or the β- oxidation of fatty acids. b. Using the selected sequence from step (a) as a probe in a BLAST search for the identification of homologous sequences among available nucleotide or amino acid sequences of the microbial strain capable of producing an N- adipoylated β-lactam compound.

2. A method according to claim 1 wherein the enzyme involved in β-oxidation of fatty acids is selected from the following groups: a. Group I: CoA ligase (EC 6.2.1. xx) b. Group II: acyl-CoA dehydrogenase (EC 1.3.99.xx) and acyl-CoA oxidase (EC 1.3.3.XX) c. Group III: enoyl-CoA hydratase (EC 4.2.1.17) d. Group IV: 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) e. Group V: acetyl-CoA C-acyl transferase (thiolase - EC 2.3.1.16).

3. A method according to any of the preceding claims wherein the genes encoding one or more enzymes which are involved in the incorporation of the adipic acid from the culture medium into the N-adipoylated β-lactam compound have a "adipate/control" ratio of more than 1 , whereby the "adipate/control" ratio is the ratio of the transcript level of the gene when the parent strain is grown in a medium containing adipic acid with the transcript level of the gene when the parent strain is grown in a control medium without adipic acid.

4. A mutant microbial strain capable of producing an N-adipoylated β-lactam compound when cultured in a culture medium comprising adipic acid characterized in that the strain has an improved incorporation yield of the adipic acid from the culture medium into the N-adipoylated β-lactam compound compared to the non-mutant parent strain.

5. The strain of claim 4 characterized in that the improved adipate incorporation yield is at least 5%.

6. The strain of any of the preceding claims wherein a gene encoding an enzyme which is involved in the degradation of adipic acid and which may be identified by the method of any of claim 1-3, is functionally inactive.

7. The strain of any of the preceding claims wherein the enzyme which is involved in the degradation of adipic acid is selected from the group consisting of CoA ligase (EC 6.2.1. xx), acyl-CoA dehydrogenase (EC 1.3.99.xx) and acyl-CoA oxidase (EC 1.3.3.xx), enoyl-CoA hydratase (EC 4.2.1.17), 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35), acetyl-CoA C-acyl transferase (thiolase - EC 2.3.1.16).

8. The strain of any of the preceding claims wherein the gene encoding the enzyme which is involved in the degradation of adipic acid is selected from the group consisting of SEQ ID NO 1 , SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 1 1 , SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 21 , SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID No. 51 , SEQ ID No. 52, SEQ ID No. 53, SEQ ID No. 54, SEQ ID No. 55, SEQ ID No. 56, SEQ ID No. 57, SEQ ID No. 58, SEQ ID No. 59, SEQ ID No. 60, SEQ ID No. 61 , SEQ ID No. 62, SEQ ID No. 63, SEQ ID No. 64, SEQ ID No. 65, SEQ ID No. 66, SEQ ID No. 67, SEQ ID No. 68, SEQ ID No. 69, SEQ ID No. 89, SEQ ID No. 90, SEQ ID No. 91 , SEQ ID No. 92, SEQ ID No. 93, SEQ ID No. 94, SEQ ID No. 92, SEQ ID No. 93, SEQ ID No. 101 , SEQ ID No. 102, SEQ ID No. 103, SEQ ID No. 104, SEQ ID No. 105, SEQ ID No. 106, SEQ ID No. 107, SEQ ID No. 108, SEQ ID No. 117, SEQ ID No. 1 18, SEQ ID No. 119, SEQ ID No. 120, SEQ ID No. 135 as well as a nucleotide sequence which is substantially homologous to any of the sequences listed.

9. The strain of any of the preceding claims wherein the N-adipoylated β-lactam compound is selected from the group consisting of adipoyl-6-APA, adipoyl-7-ADCA, adipoyl-7-ACA, adipoyl-7-ACCA, adipoyl-7-PACA, adipoyl-7-ADAC, adipoyl-7-ACCCA

10. The strain of any of the preceding claims wherein the strain is a fungus, bacterium or yeast.

11. The strain of any of the preceding claims wherein the fungus belongs to the species Penicillium.

12. The strain of any of the preceding claims wherein the fungus is Penicillium chrysogenum, preferably transformed with and expressing a gene encoding an expandase or an expandase/hydroxylase.

13. A method for the construction of the mutant strain as defined in claims 1-9 comprising functionally inactivating a gene encoding an enzyme which is involved in the degradation of adipic acid.

14. A method according to claim 13 wherein the enzyme which is involved in the degradation of adipic acid is selected from the group consisting of CoA-ligase, acyl-CoA dehydrogenase, acyl-oxidase, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase and acetyl-CoA C-acyltransferase (thiolase) and the gene is selected from the group as defined in claim 5.

15. A process for the production of an N-adipoylated β-lactam compound comprising culturing the strain as defined in any of claims 1-9 in a fermentation medium comprising adipic acid.