Method for generating site-specific mutations in filamentous fungi

A method of providing site-specifically mutated variant polypeptides, the method comprising the steps of:

• a) providing a methylated template autosomal filamentous fungal replicating double-stranded circular DNA vector comprising a parent polynucleotide encoding a parent polypeptide;
• b) providing a pair of end-to-end non-overlapping PCR primers directed to the parent polynucleotide, wherein at least one primer is mutagenic;
• c) performing a PCR amplification of the template vector with the pair of PCR primers to generate full-length vector mutated PCR fragments;
• d) removing the template vector with a suitable methylation-specific nuclease;
• e) circularizing the mutated PCR fragments by self-ligation;
• and
• f) transforming the circularized mutated PCR fragments directly into a filamentous fungal host cell to express the variant polypeptides,
• wherein either the PCR primers are phosphorylated prior to the PCR amplification, or the PCR fragments are phosphorylated before or during the self-ligation step to allow end-to-end ligation of the primers to circularize the mutated PCR fragments.