Genencor International, Inc.

Dsm IP Assets B.v.

Dsm N.v.

Dsm Sinochem Pharmaceuticals Netherlands B.v.

Richard Kerkman

Wilhelmus J Quax

Jan Metske Van Der Laan

Stephane J Jenne

Anke Krebber

Yong Hong Chen

Roelof Ary Lans Bovenberg

Cornelis P Broekhuizen

Harold Monro Moody

Dirk Schipper

Thomas Van Der Does

Claus Krebber

Hesselien Touw-Riel

Matthew Tobin

Charlene To La Ching

Xiaodong Gao

Van Den Marco Alexander Berg

Van Der Jan Metske Laan

Yan Chen

The present invention relates to a mutant prokaryotic penicillin G acylase derived from a wild-type penicillin G acylase characterized in that the mutant is having an amino acid substitution at one or more amino acid positions selected from the group consisting of amino acid positions A3, A77, A90, A144 A192, B24, B109, B148, B313, B460 and B488 according to the amino acid numbering of thepenicillin G acylase having the amino acid sequence depicted in SEQ ID No: 1.

Mutant penicillin G acylases

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

Heterologous protein secretion from Gram-positive bacteria, in particular from Bacilli has, with few exceptions, met with little success. Incompatibility of the heterologous proteins with the protein secretion machinery of the host is the main cause of this effect. This limiting factor for the production of heterologous proteins in commercially significant concentrations from is removed by overexpressing the protein FtsY or FtsY protein in combination with overexpression of other members of the bacterial signal recognition particle. Said gene(s) is(are) overexpressed in Bacillus host cells expressing a heterologous protein which then shows an increased amount of the heterologous protein secreted in the surrounding medium.

Secretion factors for gram-positive micoorganisms, genes encoding them and methods of using it

Secretion factors for gram-positive microorganisms genes encoding them and methods of using it

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

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Zandvoort, Netherlands

Richard Kerkman