Abstract: The present invention relates to the field of molecular biology and cell biology. More specifically, the present invention relates to a CRISPR-CAS system for a filamentous fungal host cell.

Abstract: The present invention relates to a method to improve the production of a secondary metabolite catalyzed by a non-ribosomal peptide synthetase comprising contacting in a eukaryotic host a eukaryotic non-ribosomal peptide synthetase with an MbtH-like protein. The present invention further relates to a composition comprising a eukaryotic non-ribosomal peptide synthetase that is not a hybrid and a prokaryotic MbtH and to a eukaryotic host cell comprising a non-ribosomal peptide synthetase and a polynucleotide allowing the expression of an MbtH-like protein.

Abstract: The present invention relates to the field of molecular biology and cell biology. More specifically, the present invention relates to a CRISPR-CAS system for a filamentous fungal host cell.

Abstract: The present invention relates to the preparation of ?-lactam antibodies comprising contacting 4-hydroxyphenylglycine of phenylglycine, cysteine and valine with a non-ribosomal peptide synthetase and subsequent cyclization using an isopenicillin N synthase in the presence of an MbtH-like protein and to a host cell equipped to perform such preparation.

Abstract: The present invention relates to the preparation of ?-lactam antibiotics comprising contacting 4-hydroxyphenylglycine or phenylglycine, cysteine and valine with a non-ribosomal peptide synthetase and subsequent cyclization using an isopenicillin N synthase in the presence of an MbtH-like protein and to a host cell equipped to perform such preparation.

Abstract: The present invention relates to a host cell deficient in an essential gene, comprising a vector, said vector comprising at least said essential gene and an autonomous replication sequence, wherein the host cell is a filamentous fungal cell. The invention also relates to a host cell deficient in an essential gene, comprising a vector, said vector comprising at least said essential gene and an autonomous replication sequence, wherein the host cell comprises a recombinant polynucleotide construct comprising a polynucleotide encoding a biological compound of interest or a compound involved in the synthesis of a biological compound of interest.

Abstract: The present invention describes a process for the production of an N-?-amino-hydroxyphenylacetyl or an N-?-aminophenylacetyl ?-lactam antibiotic comprising an IPNS-catalysed conversion of a precursor tripeptide hydroxyphenylglycyl-cysteinyl-valine (HpgCV) or phenylglycyl-cysteinyl-valine (PgCV), respectively, to the N-hydroxyphenylglycyl or the N-phenylglycyl ?-lactam antibiotic, respectively. The tripeptide HpgCV or the tripeptide PgCV may further be prepared by contacting the amino acids hydroxyphenylglycine (Hpg) or phenylglycine (Pg), cystein (C) and valine (V) with a non-ribosomal peptide synthetase (NRPS) to effect formation of the tripeptide HpgCV or the tripeptide PgCV, the NRPS comprising a first module M1 specific for Hpg or Pg, a second module M2 specific for C and a third module M3 specific for V An IPNS is further provided having an improved activity in this conversion, as well as an NRPS catalysing the formation of the tripeptides.

Abstract: The present invention discloses a polypeptide selected from the group consisting of: a polypeptide having an amino acid sequence according to SEQ ID NO 3, a polypeptide having an amino acid sequence according to SEQ ID NO 6, a polypeptide having an amino acid that is substantially homologous to the sequence of SEQ ID NO 3 and a polypeptide having an amino acid that is substantially homologous to the sequence of SEQ ID NO 6, the polypeptide displaying acetamidase activity and providing a reverse selection on fluoroacetamide with an efficiency of at least 95%, preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably 100%. The gene encoding the polypeptide of the invention is used as an efficient bi-directional selection marker in the construction of selection marker free strains, in particular for processes for the production of a compound of interest.

Abstract: The present invention describes a process for the production of an N-?-amino-hydroxyphenylacetyl or an N-?-aminophenylacetyl ?-lactam antibiotic comprising an IPNS-catalysed conversion of a precursor tripeptide hydroxyphenylglycyl-cysteinyl-valine (HpgCV) or phenylglycyl-cysteinyl-valine (PgCV), respectively, to the N-hydroxyphenylglycyl or the N-phenylglycyl ?-lactam antibiotic, respectively. The tripeptide HpgCV or the tripeptide PgCV may further be prepared by contacting the amino acids hydroxyphenylglycine (Hpg) or phenylglycine (Pg), cystein (C) and valine (V) with a non-ribosomal peptide synthetase (NRPS) to effect formation of the tripeptide HpgCV or the tripeptide PgCV, the NRPS comprising a first module M1 specific for Hpg or Pg, a second module M2 specific for C and a third module M3 specific for V An IPNS is further provided having an improved activity in this conversion, as well as an NRPS catalysing the formation of the tripeptides.

Abstract: The present invention discloses a polypeptide selected from the group consisting of: a polypeptide having an amino acid sequence according to SEQ ID NO 3, a polypeptide having an amino acid sequence according to SEQ ID NO 6, a polypeptide having an amino acid that is substantially homologous to the sequence of SEQ ID NO 3 and a polypeptide having an amino acid that is substantially homologous to the sequence of SEQ ID NO 6, the polypeptide displaying acetamidase activity and providing a reverse selection on fluoroacetamide with an efficiency of at least 95%, preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, most preferably 100%. The gene encoding the polypeptide of the invention is used as an efficient bi-directional selection marker in the construction of selection marker free strains, in particular for processes for the production of a compound of interest.

Abstract: The present invention provides a recombinant Penicillium chrysogenum strain characterized in that a gene selected from the list consisting of penDE, pcbAB and pcbC is inactivated and the use of such a strain for the preparation of a compound of interest.

Abstract: The invention relates to a process for the biosynthetic production of 4-amino-4-deoxychorismate (ADC) performed fermentatively in vivo with a 4-amino-4-deoxychorismate synthase, preferably a PabAB bipartite protein (which may be a fusion protein), at an increased level of activity, thereby obtaining a broth comprising ADC and 4-amino-4-deoxyprephenate (ADP), that are recovered. The invention also relates to a further process of converting the ADP into p-aminophenylalanine. The invention, moreover relates to biosynthetic production of [3R,4R]-4-amino-3-hydroxycyclohexa-1,5-diene-1-carboxylic acid (3,4-CHA), by concerted action of such 4-amino-4-deoxychorismate synthase and of an enzyme capable of converting isochorismate into [5S,6S]-5,6 dihydroxycyclohexa-1,3-diene-1-carboxylic acid (2,3-CHD), preferably a phenazine biosynthesis protein PhzD, including recovery of 3,4-CHA. The invention also relates to expression vectors and host cells for use in any of such processes.

Abstract: The present invention discloses a process for the preparation of variant polynucleotides using a combination of mutagenesis of a starting population of polynucleotides and recombination of the mutated polynucleotides. The process comprises the steps of subjecting a population of polynucleotides to (a series of) two (or more) separate PCR's comprising a first PCR with a forward mutation-specific primer for a position to be mutated and a reverse universal primer and a second PCR with a forward universal primer and a reverse mutation-specific primer for a position to be mutated. The products of the (series of) two (or more) PCR's are assembled by a polymerase, preferably in one tube.

Abstract: The present invention describes the transformation of a microorganism that does not naturally produce a ?-lactam compound with polynucleotides involved in the biosynthesis of ?-lactam compounds and the use of such transformed microorganisms in the production of ?-lactam compounds or in the identification of genes or factors involved in the synthesis of a ?-lactam compound.

Abstract: The invention provides DNA, heterologous host cells capable of transcribing, translating or expressing said DNA and methods employing such host cells and cultures thereof for an improved in vivo production of acylated cephalosporins with higher yield. According to the invention, a host cell is provided comprising an enzyme having expandase activity which is predominantly localized in the cytosol (as opposed to localized mainly in or with the peroxisomes or microbodies) of the host cell.

Abstract: The present invention discloses a process for the production of N-deacylated cephalosporin compounds via the fermentative production of their 7-acylated counterparts.

Abstract: The present invention relates to filamentous fungi that comprise in their genomes at least two substantially homologous DNA domains which are suitable for integration of one or more copies of a recombinant DNA molecule and wherein at least two of these DNA domains comprise an integrated copy of a recombinant DNA molecule. The invention also relates to methods for preparing such filamentous fungi and for further multiplying the DNA domains with integrated recombinant DNA molecules through gene conversion or amplification.

Abstract: The present invention disclosures a process for the production of N-deacylated cephalosporin compounds via the fermentative production of their 7-acylated counterparts.

Abstract: An improved process for production of an N-acylated cephalosporin is described. The process utilizes an Acremonium strain which has been modified to express an acyl transferase of a filamentous fungus and is selected for production of the N-acylated cephalosporin at a level which is equal to or higher than the production of the corresponding &agr;-aminoadipyl-7-cephalosporins in the strain.

Abstract: The present invention discloses a process for the production of N-deacylated cephalosporin compounds via the fermentative production of their 7-acylated counterparts.