Deregulation of methanol metabolism reverts transcriptional limitations of recombinant Pichia pastoris (Komagataella spp) with multiple expression cassettes under control of the AOX1 promoter. Issue 7 (17th February 2019)

• Record Type: Journal Article
• Authors: Cámara, Elena; Monforte, Sergi; Albiol, Joan; Ferrer, Pau
• Is Part Of: Biotechnology and bioengineering. Volume 116:Issue 7(2019)
• Page Start: 1710
• Page End: 1720
• Publication Date: 2019-02-17
• Subjects: AOX1 promoter -- heterologous gene dosage -- methanol metabolism -- Mit1 -- Mxr1 -- Pichia pastoris (Komagataella spp.) -- recombinant protein production; Biotechnology -- Periodicals; Bioengineering -- Periodicals; 660.6
• DOI: 10.1002/bit.26947
• Languages: English
• ISSNs: 0006-3592

Abstract

Abstract: 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. Abstract : 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, it can provoke a titration effect of key transcriptional factors such as Mxr1 and Mit1 regulating methanol metabolism. This causes a significant transcriptional downregulation of methanol metabolism and, consequently, a reduction in cell growth and protein production. Overexpression of one of these transcriptional factors was sufficient to reverse such transcriptional attenuation effect.