DSM IP Assets B.V. 25440WO

IMPROVED CEPHALOSPORIN PRODUCTION

The present invention relates to the fermentative production of a cephalosporin in a microorganism. The filamentous fungus Penicillium chrysogenum is a natural producer of β-lactam antibiotics, such as penicillin-G. In the last decades, penicillin-G production capabilities were highly improved by classical strain improvement, resulting in powerful industrial production strains. Recently, by means of metabolic pathway engineering, several routes for cephalosporin production were successfully integrated in penicillin production strains, in particular Penicillium chrysogenum. In EP-A-0532341 , Penicillium chrysogenum has been transformed with the Streptomyces clavuligerus cefE gene encoding an expandase which enables the transformed strain to produce adipyl-7-ADCA (adipyl^-amino-S-methyl-S-cephem^-carboxylic acid) when cultured in the presence of the precursor adipic acid. In EP-A-0540210, in addition to the Streptomyces clavuligerus cefE gene, Penicillium chrysogenum has been transformed with a hydroxylase gene whose expression product converts the 3-methyl side chain of adipyl-7-ADCA to 3- hydroxymethyl, to give adipyl^-aminodeacetylcephalosporanic acid (adipyl-7-ADAC); in another example in EP-A-0540210 Penicillium chrysogenum, already transformed with genes encoding expandase and hydroxylase, is further transformed with an acetyltransferase gene whose expression product converts the 3-hydroxymethyl side chain to the 3-acetyloxymethyl side chain to give adipyl-7-ACA. In WO2004/106347, a strain of Penicillium chrysogenum has been transformed with genes encoding an expandase, a hydroxylase and an O-carbamoyl transferase enzyme resulting in adipyl-7- amino-3-carbamoyloxymethyl-3-cephem-4-carboxylic acid. Industrial strains of Penicillium chrysogenum have been selected for their capacity to produce and secrete large quantities of a variety of beta-lactam compounds, in particular penicillin G and penicillin V, into the culture medium. Various lines of evidence indicate that beta-lactam secretion is an active process, possibly via transport proteins. During the latter stages of such fermentation processes extremely high beta- lactam titers are found in fermentations on an industrial scale. WO01 /32904 discloses several ABC-transporter polypeptides that are involved in the secretion of penicillin G. Enhancing an ABC transporter activity resulted in an enhanced secretion of the beta- lactam compound.

The production of cephalosporin molecules in naturally non-cephalosporin recombinant production hosts such as the Penicillium strains discussed hereinbefore still imposes several problems. For example, the secretion level of the desired compounds is still relatively low and must be increased in order to be both technically and economically more attractive. The production rate of cephalosporins produced in P. chrysogenum will be limited due to the fact that the penicillin transporters, i.e. the ABC transporter polypeptides, will have a low affinity for the cephalosporin compound. It has been suggested in WO01/32904 that this could be overcome by cloning one or more homologous ABC transporter polypeptides, from natural cephalosporin producers such as Acremonium chrysogenum using the polynucleotides encoding the ABC-transporters disclosed in WO01/32904.

The filamentous fungus Acremonium chrysogenum is a natural producer of cephalosporins such as cephalosporin C. In a particular strain of this fungus, Acremonium chrysogenum C10, a CefT gene has been identified recently which encodes a putative efflux pump protein and, at the same time, significantly increases cephalosporin C production (Ullan et al. (2002) MoI. Genet. Genomics, 267, 673-683; Liras and Martin (2006) International Microbiology 9, 9-19). The nucleotide sequence of the CefT-gene has been deposited in the nucleotide database of the National Center for Biotechnology Information under accession number (locus) AJ487683. The NCBS can be accessed on the INTERNET at www.ncbi.nlm.nih.gov and the CefT gene nucleotide sequence at http://www.ncbi. nlm.nih.qov/entrez/viewer.fcqi?db=nucleotide&val=2121400 8. From the amino acid sequence of the protein encoded by the CefT, the authors concluded that it is a transmembrane protein since it has 12 transmembrane segments (TMS) and contains motifs A, B, C, D2 and G characteristic of the Drug:H+ antiporter 12 TMS group of the major facilitator superfamily. The CefT protein confers resistance to some toxic organic acids, including isovaleric acid and phenylacetic acid, as was concluded from the fact that these acids were toxic to an Acremonium strain in which the CefT was made inactive. Overexpression of the homologous CefT gene resulted in an improved cephalosporin C production in Acremonium chrysogenum. This was not the case when a truncated form of the CefT gene was used. However, targeted inactivation of the CefT gene did not affect the cephalosporin C production in Acremonium chrysogenum which suggests that there are redundant systems involved in cephalosporin export in Acremonium chrysogenum.

The CefT protein from Acremonium chrysogenum C10 does not belong to the family of ABC-transporters. Comparison of the amino acid sequence of the CefT protein from Acremonium chrysogenum C10 (Ullan et al (2002)) with any of the amino acid sequence of the ABC-transporters disclosed in WO01 /32904 reveals that the CefT protein does not have any homology with the ABC transporter proteins (i.e. <25%).

The term "CefT gene" relates to a gene encoding a CefT protein, i.e. a protein involved in the transport of a cephalosporin compound from inside the cephalosporin producing microorganism towards the outside of the cephalosporin producing microorganism. An example of such a CefT gene from the strain Acrymonium chrysogenum C10 can be found in Ullan et al (2002) which also discloses the amino acid sequence of the CefT protein encoded by the CefT-gene.

The term 'heterologous CefT gene' means that the CefT gene may be obtained from any suitable host microorganism and is used to transform a cephalosporin- producing microorganism, with the proviso that the suitable host microorganism from which the CefT gene is derived is not the same species as the cephalosporin-producing microorganism transformed with the CefT gene.

The term 'homologous CefT gene" means that CefT gene is obtained from and any suitable host microorganism and used to transform a cephalosporin-producing microorganism, proviso that the suitable host microorganism from which the CefT gene is derived is the same species as the cephalosporin-producing microorganism transformed with the CefT gene. Transformation of the strain Acrymonium chrysogenum C10 with a homologous CefT gene originating from the same strain Acrymonium chrysogenum C10 has been described by Ullan et al. (2002).

The term 'homology' refers to the identity of the sequence of a first gene, protein or enzyme with the sequence of a second gene, protein or enzyme. Throughout this description, the degree of homology is expressed as a percentage of identity with a reference gene, protein or enzyme. Determining these homologies can be performed by methods known to the skilled man, for instance by using the protein sequence of the reference protein as a "query sequence" to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using BLAST programs (version 2.2) using the default parameters of the respective program. See for instance http://www.ncbi.nlm.nih.gov

In a first aspect, the present invention provides a cephalosporin-producing microorganism characterized in that it has been transformed with a heterologous CefT gene

and whereby the transformed microorganism has an improved cephalosporin-producing capacity compared with the microorganism not being transformed with the heterologous CefT gene. An improved cephalosporin-producing capacity is defined herein as the ratio of the cephalosporin production capacity of the transformed microorganism compared to the cephalosporin production capacity of the microorganism not being transformed with the heterologous CefT gene being at least 1.2, more preferably at least 1.4, more preferably at least 1.6, more preferably at least 1.8, more preferably at least 2.0, more preferably at least 2.1 , more preferably at least 2.3, more preferably at least 2.5, more preferably at least 3.0, more preferably at least 5.0, more preferably at least 10. The cephalosporin-producing microorganism is preferably a microorganism that is by nature capable of producing beta-lactam compounds such as penicillins and cephalosporins and may be selected from the group consisting of Penicillium, Aspergillus, Streptomyces and Acremonium. In this group, several microorganisms possess the capacity to produce cephalosporins by nature such as Streptomyces and Acremenium or penicillins by nature such as Penicillium and Aspergillus. Other microorganisms, such as Penicillium as disclosed hereinbefore, may have acquired the capacity to produce cephalosporins by genetic engineering such as Penicillium chrysogenum which, after having being transformed with a gene encoding expandase, is capable of producing N-acylated derivatives of 7-ADCA or, when further being transformed with hydroxylase, a hydroxylase and an acetyltransferase or with hydroxylase and an acetyltransferase and a O-carbamoyl transferase is capable of producing N-acylated derivatives 7-ADAC, 7- ACA and 7-amino-3-carbamoyloxymethyl-3-cephem-4-carboxylic acid respectively.

Preferably the heterologous CefT gene may be derived from any suitable cephalosporin producing microorganism, but is preferably derived from Acremonium, more preferably from Acremonium chrysogenum, most preferably from Acremonium chrysogenum C10 as disclosed by Ullan et al (2002). For the purpose of the invention, also a homologue of a suitable heterologous CefT gene may be used, for instance a CefT gene encoding a CefT protein with more than 30% homology, preferably more than 40% homology, preferably more than 50% homology, preferably more than 60% homology, preferably more than 70% homology, preferably more than 80% homology, preferably more than 90% homology on protein basis when compared to the CefT protein from Acremonium chrysogenum C10 as disclosed by Ullan et al (2002).

A preferred embodiment of the present invention is a Penicillium strain, preferably Penicillium chrysogenum, that has acquired the capacity to produce an N-

substituted 7-ADCA derivative by genetic engineering as a result of its transformation with a gene encoding an expandase - for example as disclosed in EP-A-0532341 - and in addition has been transformed with a heterologous CefT gene, preferably with a CefT gene from Acremonium, more preferably with a CefT gene from Acremonium chrysogenum; most preferred is the CefT gene from Acremonium chrysogenum C10 encoding the CefT protein with the amino acid sequence as has been disclosed in Ullan et al (2002). Another preferred embodiment of the present invention is a Penicillium strain, preferably Penicillium chrysogenum, that has acquired the capacity to produce a N-acylated 7-ADAC derivative, by genetic engineering as a result of its transformation with a gene encoding an expandase and a hydroxylase as disclosed in EP-A-0540210 and in addition has been transformed with a heterologous CefT gene, preferably with a CefT gene from Acremonium, more preferably with a CefT gene from Acremonium chrysogenum.; most preferred is the CefT gene from Acremonium chrysogenum C10 encoding the CefT protein with the amino acid sequence as has been disclosed in Ullan et al (2002). Another preferred embodiment of the present invention is a Penicillium strain, preferably Penicillium chrysogenum, that has acquired the capacity to produce a N-acylated 7-ACA derivative, by genetic engineering as a result of its transformation with a gene encoding an expandase and a hydroxylase and a acyltransferase as disclosed in EP-A-0540210 and in addition has been transformed with a heterologous CefT gene, preferably with a CefT gene from Acremonium, more preferably with a CefT gene from Acremonium chrysogenum; most preferred is the CefT gene from Acremonium chrysogenum C10 encoding the CefT protein with the amino acid sequence as has been disclosed in Ullan et al (2002). Another preferred embodiment of the present invention is a Penicillium strain, preferably Penicillium chrysogenum, that has acquired the capacity to produce a N-acylated 7-amino-3-carbamoyloxymethyl-3-cephem-4-carboxylic acid derivative, by genetic engineering as a result of its transformation with a gene encoding an expandase, a hydroxylase and an O-carbamoyl transferase as disclosed in WO2004/106347and in addition has been transformed with a heterologous CefT gene, preferably with a CefT gene from Acremonium, more preferably with a CefT gene from Acremonium chrysogenum; most preferred is the CefT gene from Acremonium chrysogenum C10 encoding the CefT protein with the amino acid sequence as has been disclosed in Ullan et al (2002).

In a second aspect, the present invention provides a method for the construction of the cephalosporin-producing microorganism according to the invention which

comprises the steps of isolating the heterologous CefT gene from a suitable host microorganism and transforming the cephalosporin-producing microorganism with the isolated CefT gene with the proviso that the host microorganism is not the same species as the cephalosporin-producing microorganism. In a preferred embodiment, the present invention provides a method for the construction of the cephalosporin-producing microorganism according to the invention which comprises the steps of isolating the heterologous CefT gene from Acremonium, preferably Acremonium chrysogenum, most preferably Acremonium chrysogenum C10 as described by Ullan et al (2002) and transforming the cephalosporin-producing microorganism, preferably Penicillium, more preferably Penicillium chrysogenum transformed with a gene encoding an expandase and optionally one or more of the genes encoding a hydroxylase, acyltransferase and O- carbamoyl transferase thus providing the Penicillium strain with the capacity to produce 7-ADCA, 7-ADAC, 7-ACA and N-acylated 7-amino-3-carbamoyloxymethyl-3-cephem-4- carboxylic acid as described before, with the isolated CefT gene. In a third aspect the invention provides a process for the production of a cephalosporin comprising culturing the corresponding cephalosporin-producing microorganism of the invention according to methods known in the art. Preferred embodiments of the invention are processes for the production of N-acylated derivatives of 7-ADCA, 7-ACA, 7-ADCA and 7-amino-3-carbamoyloxymethyl-3-cephem-4-carboxylic acid. A preferred embodiment of the invention is a process for the production of the adipyl derivate of 7-ADCA, 7-ACA, 7-ADCA or 7-amino-3-carbamoyloxymethyl-3- cephem-4-carboxylic acid using fermentation processes as disclosed in EP-A-0532341 , EP-A-0540210 and WO2004/106347.

EXAMPLES

General materials and methods

Standard procedures were carried out as described in Sambrook, J. et al. (1989), Molecular cloning: a laboratory manual, 2 ^nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. DNA was amplified using the proofreading polymerases Turbo-Pfu-Polymerase or Herculase (Stratagene, The Netherlands), 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 (Seqlab GmbH, Goettingen, Germany).

The filamentous fungus Penicillium chrysogenum Wisconsin 54-1255 (ATCC 28089) was used as a host for genetic modification and production strain construction.

Example 1 Construction of the CefT expression cassette for transformation in Penicillium chrysogenum

Escherichia coli DH5a was used as host strain for high frequency transformation, plasmid DNA amplification (Sambrook, 1989) and plasmid construction using the

Multisite Gateway ^® Three-Fragment Vector Construction Kit of Invitrogen with different vectors. Vector pDONR™ P4-P1 R was used for cloning of the promotor region of 920bp upstream of the pcbC gene. The attB4F site was added by Polymerase Chain Reaction

(PCR) using primer attB4-F-IPNS and the attBI R site was added with primer attB1-R- IPNS. The amplified PCR product was cloned into pDONR™ P4-P1 R using BP clonase creating pDONR™ P4-P1 R-IPNS promotor. Vector pDONR™ 201 gateway vector was used to clone the CefT gene of Acremonium chrysogenum (acCefT). The attBI F site was added by PCR using primer attBI F-AcCefT and the attB2R site was added using attB2R-AcCefT-minus-Stop. The amplified AcCefT gene was cloned into pDONR™ 201 using BP clonase yielding pDONR™ 201-AcCefT. The stop codon of the AcCefT was removed to enable fusions with a His-tag or GFP-tag at the carboxyl terminal site of the gene. To add the attB2F site to the terminator region of 551 bp downstream of the penDE gene for cloning in the pDONR™ P2R-P3 primer attB2F-His8x-Tat was used. Together with primer attB3R-Tat the terminator region was cloned and a HIS-tag with a stop codon was added to create vector pDONR™ P2-P3R-HIS-Tat. To obtain a carboxyl-terminal fusion with GFP primers attB2R-GFP and attB3R-Tat were used to create vector pDONR™ P2-P3R-GFP-Tat.

Combining these pDONR™ vectors in the pDEST™ R4-R3 in the LR reaction of the Multisite Gateway ^® Three-Fragment Vector Construction Kit a destination vector was made with respectively the PcbC promotor, AcCefT gene, His- or GFP-tag and the terminator of the PenDE gene.

Table 1. Primers used for Gateway ^® cloning. Nucleotides in bold represent the att recombination sites of the Gateway ^® system, in italic represent nucleotides to maintain the frame between the HIS-tag, the GFP-tag and AcCefT gene and underlined nucleotides represent the 8 amino acids of the HIS-tag.

Example 2

Transformation of Penicillium chrysogenum ATCC 28089 CefE/CefF/CmcH (construction described in WO2004106347) with the Ce/T expression cassette designed in Example 1

For the transformation experiments of Penicillium chrysogenum ATCC 28089

CefE/CefF/CmcH a strain was used that produced circa 0.3 g/L adipyl-7-amino-3- carbamoyloxymethyl-3-cephem-4-carboxylic acid. Protoplasts of were made and isolated and transformation (Alvarez, 1987) was done by co-transformation of the pDEST™ P4- R3 vector with pcbC promotor, CefT gene and penDE terminator and the AMDS-gene used as selection marker on plates with acetamide as sole Nitrogen source. Total RNA of transformants was isolated using Trizol ^® (Invitrogen) and positive transformants were determined with RT-PCR beads (Amersham) using a AcCefT specific primer (AcCefT

F1 ; δ'-TCGATTCGTACCAGCACCAGGC-S') and a attB2 - HIS-tag primer (5'- TGGTGATGGTGATGGTGGACCACTT-S') to obtain a PCR fragment of 384bp.

Example 3

Cultivation of Penicillium chrysogenum ATCC 28089 CefE/CefF/CmcH Ce/Tand measurement of adipyl-7-amino-3-carbamoyloxymethyl-3-cephem-4-carboxylic acid secretion

Mycelium of 5 CefT transformants was inoculated in a 50 ml. shake flask in mineral medium containing (g/L): glucose (5); lactose (80); urea (4.5); (NhU) ₂ SO ₄ (1.1 ); Na ₂ SO ₄ (2.9); KH ₂ PO ₄ (5.2); K ₂ HPO ₄ (4.8) and 10 mL/L of a trace element solution A containing citric acid (150); FeSO ₄ .7H ₂ O (15); MgSO ₄ .7H ₂ O (150); H ₃ BO ₃ (0.0075); CuSO ₄ .5H ₂ O (0.24); CoSO ₄ JH ₂ O (0.375); ZnSO ₄ .7H ₂ O (5); MnSO ₄ -H ₂ O (2.28); CaCI ₂ .2H ₂ O (0.99); 3 g/L adipic acid; pH before sterilization 6.5. After 7 days growth at 25°C, 280 rpm, cells were pelleted and the supernatant was analyzed using NMR for the formation of cephalosporins. See table 2 for the results. The data clearly show the positive effect of CefT gene integration in the genomes of P. chrysogenum on cephalosporin production.

Table 2: Concentration of adipyl-7-amino-3-carbamoyloxymethyl-3-cephem-4-carboxylic acid in shake flask cultures of the CefT gene transformants of Penicillium chrysogenum ATCC 28089 CefE/CefF/CmcH.