The first new tool these Inventors have developed is a recombinase-based method of genome engineering. This technology utilizes strains of Pichia pastoris in which an attP sequence has been inserted into a defined location in the Pichia pastoris genome. The two vector components of this tool are a recombinase vector, which encodes an integrase enzyme, and a transfer vector containing the DNA sequence of the protein to be produced and a flanking attB sequence. When the recombinase vector and transfer vector are electroporated into Pichia pastoris strains containing an attP “landing site” the recombinase enzyme catalyzes the sequence-specific integration of the transfer vector at this site. This is a more efficient than current Pichia pastoris genome engineering techniques because all transformants contain the desired integration, integration copy number is controlled, very large constructs can easily be integrated, and the integration is scarless.
The second new tool is a technique to rapidly induce high levels of recombinant protein expression in Pichia pastoris. Currently, the major commercially utilized inducible promoter for Pichia pastoris is methanol inducible. However, this is not easily scaled and there is a significant level of background expression without induction. This technology utilizes a zinc-finger (ZF) DNA binding domain fused to a fragment of the human estrogen receptor and an effector domain that enhances transcription. Under normal growth conditions, this fusion protein is sequestered in the cytoplasm. When beta-estradiol is added to the Pichia pastoris culture, the fusion protein translocates to the nucleus, where the ZF DNA binding domain binds specifically to activate expression of a recombinant protein of interest. This is an improvement over traditional protein expression systems because the inducible system decouples culture growth and product accumulation, which allows initial biomass gain without the potential of product accumulation slowing growth or affecting culture viability. Additionally, this system is both safer and more sensitive than traditional methanol inducible systems.