Understanding and improving the specific productivity of pichia pastoris recombinant protein secretion

The specific productivity of the majority of recombinant proteins secreted by Pichia pastoris is relatively poor. Identification of high producing recombinant strains is largely an empirical process with little understanding of the characteristics that lead to high productivity on scale up of culture conditions from shake flask scale to high cell density industrial fermentations. Current practice when expressing proteins using Pichia pastoris is to produce recombinants using integrating vectors leading to position independant expression. The most widely used system is based on the alcohol oxidase promoter (AOX) which requires derepression and then induction by methanol. Copy number of the target gene is an important variable but does not necessarily correlate with titre. Selection of the 'best' clone for scale up and manufacturing is complicated by the fact that clones achieving high secreted protein titre in shake flasks do not always achieve high titre in high cell density fermentations. This is also significantly influenced by the impact of methanol utilisation phenotype (mut+, mutS or mut-) of the host. Studies (limited to laboratory processes) have shown that poor expression in Pichia pastoris can in some cases be correlated with folding problems in the ER, which induces an unfolded protein response (UPR). BRIC project 06/18 (David Leak, Imperial College (IC)) is (1) developing metabolomic methods and applying these with transcriptomics to understand the underlying physiology of UPR stressed, non-stressed and non-expressing cultures and, (2) developing easily monitored reporters of UPR to allow ideal expression conditions to be determined on a small scale. This project will use industrially relevant proteins (induced with methanol and non-methanol) and protein models (low and high secretion titres) with an industrial fermentation platform to characterise and better understand the inter-play between the parameters described above and induction of the UPR (UPR tools developed by BRIC 06/18). Metabolomic information (based on extracellular (footprint) metabolites) will also be collected. The student will spend a significant amount of time working at Avecia: 2-3 model proteins, AOX recombinants in range of mut hosts, establishing copy number and titre in shake flask culture, establishing research cell banks of clones for further work (matrix of mut/copy number/titre), establishing fermentation performance of 4-6 clones in 15L fed-batch fermentations - including analysis for footprint metabolites and/or UPR linked GFP expression. The above work is envisaged to take 15 mo with the student spending an equal amount of time at IC (see below) and Avecia. The student will initially work at IC (ca.4 mo) becoming familiarised with the UPR reporter systems being developed and will making constructs (e.g. GFP in various mut backgrounds) necessary for integration into the work at Avecia. The first half (15-18 mo) will define a pattern/decision matrix. Depending on the outcome of this we will either: (1) Expand work with GFP, which provides us with a ready made system for the sorting of variants, making it ammenable for high-throughput analysis to get a more fundamental understanding of the causes of UPR. This coupled with the transcriptomic analysis being done at IC (BRIC 06/18) should point to some host features that can be developed further to improve secretion and protein titre. Or, (2) Investigate the possibility of online control of UPR, which would require taking one/more of the poorly expressing constructs and dissecting the expression conditions to establish the conditions at which specific productivity plateaus/declines and then then investigating whether we can develop a feedback control regime which keeps at this level. The second phase of the work will be done mainly at IC, but decisions as to which route to follow will involve all parties taking into account the timescale and academic needs of the student.