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Centrient Pharmaceuticals Netherlands B.v.

Roelof Ary Lans Bovenberg

Maarten Nieboer

Erik De Vroom

Richard Kerkman

Jan Metske Van Der Laan

Paul Klaassen

Alrik Pieter Los

Dirk Schipper

Adrianus Wilhelmus Hermanus Vollebregt

Johannis Lugtenburg

Marco Alexander Van Den Berg

Johannes Andries Roubos

René Verwaal

Bianca Elisabeth Maria Gielesen

Bernard Meijrink

Thibaut José Wenzel

Marcus Hans

Arnold Jacob Mattieu Driessen

Rémon Boer

Yvonne Irene Nygård

Carsten Pohl

Ulrike Maria Mueller

Jan Andries Kornelis Willem Kiel

Bart Willem Swinkels

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.

CRISPR-CAS system for a filamentous fungal host cell

The present invention describes a process for the production of an N-&#x3b1;-amino-hydroxyphenylacetyl or an N-&#x3b1;-aminophenylacetyl &#x3b2;-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 &#x3b2;-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. Also a host cell is provided capable of fermentatively producing &#x3b2;-lactam antibiotics with N-&#x3b1;-amino-hydroxyphenylacetyl or an N-&#x3b1;-aminophenylacetyl side chains.

Production of &#x3b2;-Lactam antibiotics

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.

Bi-directional selection markers with improved activity

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.

Process for preparing variant polynucleotides

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.

Method to localize expandase in the cytosol

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

Process for the fermentative production of deacylated cephalosporins

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

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.

Process for the preparation of cephalosporins using acremonium chrysogenum

Growing community of inventors

Rotterdam, Netherlands

Roelof Ary Lans Bovenberg