Pichia pastoris protein secretion: analysis of constraints optimisation and methods development

In recent years we have seen a significant increase in the number of biopharmaceutical products coming on to the market and in development. These are generally protein based and may consist of natural proteins such as insulin or therapeutic agents which exploit the affinity of antibodies to target a compound to a specific tissue. None of these biopharmaceuticals can readily be obtained from natural sources, so it is necessary to produce them as foreign (heterologous) proteins in cells which can be grown on a large scale. As well as producing large amounts of protein it is also important that it is of a consistent quality (ie a low degree of heterogeneity) to meet the approval of regulatory authorities. This may refer not only to the protein but also to the sugars which are added to it (glycosylation) in the process of export from the cell. To meet these needs a number of cell types have been developed for expression of heterologous proteins, including bacteria, yeasts, fungi, insect and animal cells. Yeasts are particularly useful as, like bacteria, their growth is simple, but they have the same basic machinery for protein export as animal cells. So unless a protein has a complex pattern of glycosylation, which would require production in animal cells, production in a yeast is an attractive option. The yeast Pichia pastoris has been developed into a very efficient production system, and there are reports of high heterologous protein yields from this system. However, some proteins do not express well and a common cause is that the protein gets stuck in the export pathway. This causes the cell to induce a stress response called the unfolded protein response (UPR), which can actually lead to degradation of the protein stuck in the system, so reducing the level of expression. (This response is actually found in all eukaryotic cells, but is more likely when the cells are expressing a foreign protein). While there are some characteristic features of the UPR they are quite laborious to investigate. So one of the principle aims of this project is to develop one or more 'reporter' systems, which indicate when the UPR is starting to be induced as a result of blocking the export pathway. Reporters need to have an easily measurable signal, such as absorbance or emission of light at a particular wavelength, or a characteristic pattern of signals arising from direct chemical analysis. Once we have found and developed the best reporter system, based on sensitivity and ease of analysis, we will demonstrate its use for monitoring and controlling the UPR in lab scale production systems and also for screening multiple small scale cultures to discover which induce or do not induce the UPR (and also the conditions under which it is induced). Furthermore, with a sensitive and easily measurable reporter system it should be possible to screen for variants of the secreted protein in which the UPR is not induced and, in this way, understand the features of a protein, which lead to induction of UPR. Even where there is no problem with the UPR, improvement in the rate of protein production would be useful. So we will also use a variety of techniques, some of which have only recently become available for this organism, to understand what might be limiting the secretion of well-secreted proteins. This might turn out also to be the effects of UPR, but may also be due to limitations in the capacity of parts of the system (eg biosynthesis of precursors), which could be improved by nutritional means or through metabolic engineering. Overall, the project aims to speed up process development from the point of targeting a useful product to production on a scale suitable for testing and commercialisation.

The methylotrophic yeast Pichia pastoris is an established expression platform for secreted and membrane proteins and is being modified to 'humanise' its glycosylation pathway. However, a number of secreted proteins do not express well in this host, partly because of inefficient trafficking through the ER, which leads to the induction of the unfolded protein response (UPR). Although the initial UPR expression of chaperones may be beneficial, it can ultimately result in reduced secretion, proteolysis and increased product heterogeneity. In this project we will undertake a global transcriptome and metabolic profile analysis of the UPR in P pastoris, (initially on chemostat cultures then validated in a typical fed batch regime) and use the information gained to evaluate different potential reporters for the UPR including GFP, and metabolic fingerprinting. The optimum reporter system, based on factors such as responsiveness (correlated with the protein induction profile) and sensitivity (this may depend on the scale and type of culture) will be used to explore the potential for development of an on-line UPR monitoring and control system as well as for screening of constructs on a small scale. Applications of the reporter linked to moderate/ high throughput screening will also be investigated, with the aim of devising a strategy to screen large numbers of variants to select for those with improved secretion. Even when there is no evidence of induction of UPR, the specific productivity of secreted protein production is moderate, and nothing is known about what limits productivity. Therefore, we intend to explore the physiological status of highly secreting cells using combined transcriptomic, metabolomic and flux analysis of a construct with good secretion. This should indicate whether productivity limits are due to the secretion apparatus or biosynthetic capacity. In principle, a similar surrogate reporter approach may be used to indicate secretion saturation.