Abstract: The invention provides improved in vivo production of acylated cephalosporins with higher yield using expandase which is predominantly localised in the cytosol of the host cell.

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 discloses a process for the preparation of variant polynucleotides using a reassembly process of preferably blunt-ended restriction enzyme fragments prepared form a starting population of heterologous polynucleotides in the presence of a thermostable ligase.