Year-end Sale % OFF 
Abstract
The methanol-regulated alcohol oxidase promoter (PAOX1) of Pichia pastoris is one of the strongest promoters for heterologous gene expression in this methylotrophic yeast. Although increasing gene dosage is one of the most common strategies to increase recombinant protein productivities, the increase of gene dosage of Rhizopus oryzae lipase (ROL) in P. pastoris has been previously shown to reduce cell growth, lipase production and substrate consumption in high-copy strains. To better assess that physiological response, transcriptomics analysis was performed of a subset of strains with 1 to 15 ROL copies. The macroscopic physiological parameters confirm that growth yield and carbon uptake rate are gene dosage dependent, and were supported by the transcriptomic data, showing the impact of increased dosage of AOX1 promoter-regulated expression cassettes on P. pastoris physiology under steady methanolic growth conditions. Remarkably, increased number of cassettes led to transcription attenuation of the methanol metabolism and peroxisome biogenesis in P. pastoris, concomitant with reduced secretion levels of the heterologous product. Moreover, our data also point to a block in ROL mRNA translation in the higher ROL-copies constructs, while the low productivities of multi-copy strains under steady growth conditions do not appear to be directly related to UPR and ERAD induction.
Highlights
Protein degradation (ERAD) pathway were analysed[20,21,22]
By comparing different growth parameters (Table 1), a clear physiological impact could be observed as the Rhizopus oryzae lipase gene (ROL) dosage increases, consistent with the previously reported results[16]
reactive oxygen species (ROS) levels were insignificant and no extracellular formaldehyde accumulation was detected under steady growth conditions of multi-copy strains, further supporting that residual methanol concentration were below toxicity levels under such conditions[16]
Summary
Protein degradation (ERAD) pathway were analysed[20,21,22]. there is increasing evidence that increased recombinant gene dosage leads to a metabolic burden effect at the level of central carbon metabolism, pointing at a limitation in the carbon and energy supply[23,24]. In a previous study[16], a series of strains harbouring different copy numbers of a Rhizopus oryzae lipase gene (ROL) under control of the PAOX1 was constructed and characterised, showing decreased product and biomass yields, as well as altered substrate specific consumption rates in the multi-copy strains in relation to the single copy strain. In the present study we performed global transcriptomic analyses of a set of multi-copy strains to gain better insight into the impact of heterologous gene (ROL) dosage on the metabolic network of P. pastoris under methylotrophic conditions. To minimize effects of other variables (e.g. specific growth rate) these analyses were carried out in continuous cultures under steady state conditions using a mixture of glycerol and methanol as carbon source to allow induction of ROL expression. Rol production was monitored both at transcriptional (Digital Droplet PCR) and translational (intra/extracellular enzyme activity and Western Blot analysis) level
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
