Deregulation of methanol metabolism reverts transcriptional limitations of recombinant Pichia pastoris (Komagataella spp) with multiple expression cassettes under control of the AOX1 promoter
Cámara, Elena (Universitat Autònoma de Barcelona. Departament d'Enginyeria Química, Biològica i Ambiental)
Monforte, Sergi (Universitat Autònoma de Barcelona. Departament d'Enginyeria Química, Biològica i Ambiental)
Albiol i Sala, Joan (Universitat Autònoma de Barcelona. Departament d'Enginyeria Química, Biològica i Ambiental)
Ferrer, Pau (Universitat Autònoma de Barcelona. Departament d'Enginyeria Química, Biològica i Ambiental)

Data:	2019
Resum:	The methanol-regulated alcohol oxidase promoter (PAOX1) 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.
Ajuts:	Agencia Estatal de Investigación CTQ2013-4239-R Agencia Estatal de Investigación CTQ2016-74959-R Agència de Gestió d'Ajuts Universitaris i de Recerca 2014/SGR-452
Drets:	Tots els drets reservats.
Llengua:	Anglès
Document:	Article ; recerca ; Versió sotmesa a revisió
Matèria:	Pichia pastoris (Komagataella spp.) ; Mxr1 ; Mit1 ; AOX1 promoter ; Methanol metabolism ; Heterologous gene dosage ; Recombinant protein production
Publicat a:	Biotechnology and Bioengineering, Vol. 116, Issue 7 (July 2019) , p. 1710-1720, ISSN 1097-0290

DOI: 10.1002/bit.26947
PMID: 30712270

Preprint 40 p, 1.6 MB

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