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Pàgina inicial > Articles > Articles publicats > Deregulation of methanol metabolism reverts transcriptional limitations of recombinant Pichia pastoris (Komagataella spp) with multiple expression cassettes under control of the AOX1 promoter |
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 |
Preprint 40 p, 1.6 MB |