OPENPichia MoClo - parts and concept

OPENPichia MoClo kit

The OPENPichia modular cloning kit is a Golden Gate-based cloning kit, relying on the use of Type IIS restriction endonucleases. These enzymes act outside of their recognition sites, generating a set of distinct 4-nucleotide overhangs. By carefully choosing these overhangs to flank a DNA sequence of interest ('part'), a unidirectional, multi-insert, single-reaction plasmid construction method is obtained.

We are distributing a kit that contains the most commonly used parts for Pichia engineering (e.g. promoters, signal peptides, tags, terminators, selection markers) already flanked by the appropriate Type IIS recognition and cleavage sites. The current toolbox can be expanded by your sequences of interest using synthetic oligo's, PCR amplicons or entry vectors containing the relevant flanking sequences. More information can be found in the reference below.

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OPENPichia® MoClo kit

The OPENPichia plasmid set is a rapid and simple Golden Gate-based modular cloning system for the generation of single- or multi-gene expression constructs for Pichia pastoris (Komagataella phaffii). The complete set as well as the individual plasmids are available through the BCCM/GeneCorner Plasmid Collection, both for academics and for companies.

OPENPichia Plasmid Set at BCCM/GeneCorner Plasmid Collection

Claes K, Van Herpe D, Vanluchene R, Roels C, Van Moer B, Wyseure E, Vandewalle K, Eeckhaut H, Yilmaz S, Vanmarcke S, Çıtak E, Fijalkowska D, Grootaert H, Lonigro C, Meuris L, Michielsen G, Naessens J, van Schie L, De Rycke R, De Bruyne M, Borghgraef P, and Callewaert N. OPENPichia: licence-free Komagataella phaffii chassis strains and toolkit for protein expression. Nat microb (2024) doi: 10.1038/s41564-023-01574-w
https://www.nature.com/articles/s41564-023-01574-w

OPENPichia^® is a registered trademark of VIB vzw.

¹ Kurtzman, C. P. Description of Komagataella phaffii sp. nov. and the transfer of Pichia pseudopastoris to the methylotrophic yeast genus Komagataella. Int. J. Syst. Evol. 55, 973–976 (2005).
² Brady, J. R. et al. Comparative genome‐scale analysis of Pichia pastoris variants informs selection of an optimal base strain. Biotechnology and Bioengineering 117, 543–555 (2020).
³ OPENPichia: building a free-to-operate Komagataella phaffii protein expression toolkit | bioRxiv. https://www.biorxiv.org/content/10.1101/2022.12.13.519130v1.
⁴Heiss, S., Maurer, M., Hahn, R., Mattanovich, D. & Gasser, B. Identification and deletion of the major secreted protein of Pichia pastoris. Appl Microbiol Biotechnol 97, 1241–1249 (2013).