Deregulation of methanol metabolism reverts transcriptional limitations of recombinant Pichia pastoris (Komagataella spp) with multiple expression cassettes under control of the AOX1 promoter
Artikel i vetenskaplig tidskrift, 2019

The methanol-regulated alcohol oxidase promoter (P AOX1 ) of Pichia pastoris (syn. Komagataella spp.) is one of the strongest promoters for heterologous gene expression. Although increasing the gene dosage is a common strategy to improve recombinant protein productivities, P. pastoris strains harboring more than two copies of a Rhizopus oryzae lipase gene (ROL) have previously shown a decrease in cell growth, lipase production, and substrate consumption, as well as a significant transcriptional downregulation of methanol metabolism. This pointed to a potential titration effect of key transcriptional factors methanol expression regulator 1 (Mxr1) and methanol-induced transcription factor (Mit1) regulating methanol metabolism caused by the insertion of multiple expression vectors. To prove this hypothesis, a set of strains carrying one and four copies of ROL (1C and 4C, respectively) were engineered to coexpress one or two copies of MXR1*, coding for an Mxr1 variant insensitive to repression by 14-3-3 regulatory proteins, or one copy of MIT1. Small-scale cultures revealed that growth, Rol productivity, and methanol consumption were improved in the 4C-MXR1* and 4C-MIT1, strains growing on methanol as a sole carbon source, whereas only a slight increase in productivity was observed for re-engineered 1C strains. We further verified the improved performance of these strains in glycerol-/methanol-limited chemostat cultures.




heterologous gene dosage

Pichia pastoris (Komagataella spp.)


AOX1 promoter


recombinant protein production


methanol metabolism




Elena Cámara

Universitat Autonoma de Barcelona (UAB)

Sergi Monforte

Universitat Autonoma de Barcelona (UAB)

Joan Albiol

Universitat Autonoma de Barcelona (UAB)

Pau Ferrer

Universitat Autonoma de Barcelona (UAB)

Luxembourg Institute of Science and Technology

Biotechnology and Bioengineering

0006-3592 (ISSN) 1097-0290 (eISSN)

Vol. 3 February



Medicinsk bioteknologi (med inriktning mot cellbiologi (inklusive stamcellsbiologi), molekylärbiologi, mikrobiologi, biokemi eller biofarmaci)




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