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ACIB GmbH – Austrian Centre of Industrial Biotechnologywww.acib.at � [email protected] � Graz/Vienna/Innsbruck/Tulln � Austria
This work has been supported by the Austrian BMWFW, BMVIT, SFG, Standortagentur Tirol,Government of Lower Austria, and ZIT as well as Biocrates Life Sciences, Biomin, BoehringerIngelheim, Lonza, Sandoz, and VTU Technology through the Austrian FFG-COMET- Funding Program.
Synthetic biology toolbox and application for recombinant protein production in Pichia pastoris:
Golden Gate cloning and CRISPR/Cas9Roland Prielhofer1,2, Juanjo Barrero2, Stefanie Steuer1, Richard Zahrl1,2, Kristin Baumann1,2, Lina Heistinger1,
Dariusz Jarych1, Franz Zehetbauer1, Matthias Mattanovich1, Matthias Steiger1,2, Hans Marx2, Michael Sauer1,2, Diethard Mattanovich1,2, Brigitte Gasser1,2
1Department of Biotechnology, University of Natural Resources and Life Sciences Vienna, Austria2Austrian Centre of Industrial Biotechnology (ACIB), Vienna, Austria
IntroductionState-of-the-art strain engineering techniques for the protein production host Pichia pastoris include overexpression of homologous andheterologous genes, and deletion of host genes. For this purpose overexpression vectors and gene deletion methods such as the split markertechnique have been used so far.
Gene Disruptionssplit-marker cassette
The overexpression of multiple coding sequences (CDSs) is based on sequential transformation with expression vectors (EFs) with screening for best producing clones in between. • the same promoter & terminator is used for each CDS• very time consuming for multiple overexpression
conventional
CRISPR/Cas9
The 5‘homologous region (HR) and 3‘HR determine the removal of the sequence in between. The resistance cassette (e.g. KanMX) for subsequent selection is split into two separate cassettes, which if not recombined, are not functional• reduces the number of false positive clones • precise (to the base pair)• low number of transformants• multiple knock-outs in one transformation not possible• marker recycling required for multiple KOs
hierarchical GoldenGate system:
advanced
The gRNA expression had to be adapted for Pichia pastoris:
• S. cerevisiae SNR52 promoter• T7 promoter + T7 polymerase • P. pastoris PGAP promoter + self-splicing ribozymes
By increasing the amount of DNA per transformation, a
gene disruption efficiency of 90% was reached for eGFP.
The CRIPSR/Cas9 can now be successfully applied to the P. pastoris expression system, allowing multiple / simultaneous knock-outs. Episomal Cas9 and gDNA expression allow marker free gene disruptions.
conventional
advanced
pPUZZLE GoldenGate
The GoldenGate system allows us to integrate more expression factorsat the same time & in total as well as obtaining quicker results.• modular concept• variation of expression level through varied promoters• enables easy & fast screening of multiple factors possibly yielding
synergistic effects• up to 8 constructs per vector possible• strain stability verified
Multiple coding sequences (CDSs) are integrated in a single vector and introduced into P. pastoris in a single transformation step .
Gene Overexpressions
This work has been supported by the Austrian BMWFW, BMVIT, SFG, Standortagentur Tirol,Government of Lower Austria, and ZIT as well as Biocrates Life Sciences, Biomin, BoehringerIngelheim, Lonza, Sandoz, and VTU Technology through the Austrian FFG-COMET- Funding Program.
This work has been supported by the Austrian BMWFW, BMVIT, SFG, Standortagentur Tirol, Government of Lower Austria, and ZIT through the Austrian FFG-COMET-Funding Program.
Applica
tion
14 promoters with expression strength from 10-100% of PGAP
10 terminators with similar transcript stability (at least as strong as Cyc1TT)
4 integration loci:AOXTT, NTS, ENO1, RGI
4 Selection markers:kanMX, hphMX, natMX, ZeoR
Example: Effect of overexpression of 3 different enhancing factors on recombinant product yield and titers. Advanced method allows fast parallel screening of multiple promoter-gene combinations, leading to better results in shorter time.
GoldenGate – Combinatorial strain engineering
