Method for preparing polypeptide variants

The present invention relates to methods for preparing variants of a nucleotide sequence, comprising: (a) introducing into a population of filamentous fungal host cells: (i) one or more circular plasmids comprising a DNA sequence and a plasmid replicator mediating autonomous replication, wherein the one or more circularized plasmids are linearized by digestion of the DNA sequence and removal of a portion of the DNA sequence; and (ii) a library of DNA fragments comprising one or more mutations of the DNA sequence, wherein the fragments comprise at least two regions, one or more regions which are homologous to the 5' region or the 3' region of the gap in the linearized DNA sequence and/or plasmid sequence and one or more second regions which are homologous to the 5' region or the 3' region of the DNA fragments of the library; wherein the linearized plasmids and the DNA fragments recombine by in vivo recombination to produce a plurality of autonomously replicating plasmids comprising one or more variants of the DNA sequence; (b) cultivating the population of recombinant filamentous fungal cells in a medium suitable for growth; and (c) screening the population of recombinant filamentous fungal cells for variants of the DNA sequence contained on one or more autonomously replicating circularized plasmids.