The methylotrophic yeast Pichia pastoris is a popular expression platform for the production of heterologous proteins, has been implemented in over 1500 laboratories and used to produce over 70 marketed proteins. However, despite high volumetric productivities achievable in fermentor culture, and the methanol inducible AOX1 promoter, which can be manipulated to drive high levels of gene expression, P. pastoris suffers from low levels of cell specific productivity for secreted recombinant proteins. This has been attributed to bottlenecks in the secretory pathway, leading to product loss through the activation of ER stress responses, as well detrimental effects caused during cell growth on methanol. In addition a large disparity in protein production is observable within supposedly homogenous clonal populations raised from single cell lines, known as clonal variation, requiring extensive screening of transformants to find adequate producers.The widespread use of zeocin, a known mutagen, as a selective agent was initially evaluated as a potential contributing factor to clonal variation. A study comprising data for the production of 4 different recombinant proteins from clonal samples raised in the presence of selection revealed that growth on zeocin does not exacerbate clonal variation and that selection itself appears to have a stabilising effect on the distribution of productivity phenotypes within certain populations. Variation in a range of native factors was also explored for a recombinant strain secreting Penicillium funiculosum glucose oxidase, including transgene transcription, transcription of the UPR marker HAC1 and growth rate in culture. Whilst considerable variation was observed within samples for each of the factors tested, only a weak correlation between growth and productivity could be found, suggesting the involvement of other, currently unknown, factors that contribute to clonal variation.The homologue of the yeast transcription factor – OPI1 was deleted in P. pastoris as a strategy to increase productivity, as its removal in S. cerevisiae yields mutants with expanded ERs and improved secretion of recombinant proteins. However the loss of OPI1 was shown to be detrimental to protein secretion, resulting in reduced productivities and an increased activation of the UPR. In a final study, 3 strains centered on the concept of uncoupling the activity of the Mut pathway transcription factor Mit1 from its transcriptional regulation were developed as a strategy to reduce glucose repression of the AOX1 promoter. Each strain displayed alleviation of glucose repression to different degrees, with the least repressed strain - NGmit1; producing 20% more secreted recombinant protein than the native host during microexpression trials with a mixed feed of methanol and glucose.
|Date of Award||1 Sep 2016|
|Sponsors||Biotechnology and Biological Sciences Research Council & Fujifilm Diosynth Biotechnologies|
|Supervisor||David Leak (Supervisor), Paul Whitley (Advisor) & Ian Hodgson (Supervisor)|