Supporting Information

PaHNL5 random mutagenesis library creation in Pichia pastoris The Pahnl5/L1Q/A111G gene with the secretion signal sequence of the Saccharomyces cerevisiae alpha mating factor was cleaved out of the expression vector pHILD2PaHNL5a/ L1Q/A111G[1,2] and ligated into pGAPZA[3] to shorten breeding cycles in directed evolution due to constitutive HNL expression. The resulting plasmid pGAPZAPaHNL5a/L1Q/A111G was applied as template DNA for the following PCR reactions to produce linear expression cassettes for direct transformation of Pichia pastoris. 1. Error prone PCR (PCRI) [4] The error prone PCR was performed in 100 µL with 5 U of HotStar Taq DNA polymeras