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Superior protein titers in half the fermentation time: Promoter and process engineering for the glucose‐regulated GTH1 promoter of Pichia pastoris
Author(s) -
Prielhofer Roland,
Reichinger Michaela,
Wagner Nina,
Claes Katrien,
Kiziak Christoph,
Gasser Brigitte,
Mattanovich Diethard
Publication year - 2018
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.26800
Subject(s) - pichia pastoris , promoter , bioprocess , yeast , gene , biology , synthetic biology , transcription factor , pichia , metabolic engineering , transcriptional regulation , titer , transcription (linguistics) , biochemistry , computational biology , genetics , gene expression , recombinant dna , paleontology , linguistics , philosophy , antibody
Protein production in Pichia pastoris is often based on the methanol‐inducible P AOX1 promoter which drives the expression of the target gene. The use of methanol has major drawbacks, so there is a demand for alternative promoters with good induction properties such as the glucose‐regulated P GTH1 promoter which we reported recently. To further increase its potential, we investigated its regulation in more details by the screening of promoter variants harboring deletions and mutations. Thereby we could identify the main regulatory region and important putative transcription factor binding sites of P GTH1 . Concluding from that, yeast metabolic regulators, monomeric Gal4‐class motifs, carbon source‐responsive elements, and yeast GC‐box proteins likely contribute to the regulation of the promoter. We engineered a P GTH1 variant with greatly enhanced induction properties compared with that of the wild‐type promoter. Based on that, a model‐based bioprocess design for high volumetric productivity in a limited time was developed for the P GTH1 variant, to employ a glucose fed‐batch strategy that clearly outperformed a classical methanol fed‐batch of a P AOX1 strain in terms of titer and process performance.