Proving the utility of a stably replicating neo-chromosome for engineering pathways in Pichia pastoris
The annual market for therapeutic proteins is estimated at over £300 billion, of which in excess of £10 billion is spent on their manufacture using mammalian cells. This practice is not only expensive but also time-consuming and requires complex media and sophisticated production facilities. The use of a suitably engineered yeast alternative can save significant time and cost and increase the accessibility of treatments to patients.
However, some protein targets require complex post-translational modifications, such as glycosylation, to be suitably active or stable as human therapeutics. Organisms differ in the types of protein modifications they can perform. Yeast cells require extensive engineering before they can manufacture many of the post-translationally modified protein that are currently candidates for therapeutic applications.
Challenge
In a previous project, the team synthesised an extra chromosome, as a dedicated repository for extraneous DNA, and introduced it to the industrially important yeast, Pichia Pastoris. They demonstrated that this ‘neo’-chromosome was stably replicated over many cell divisions.
The team now wanted to find out if the neo-chromosome could facilitate the crucial engineering steps needed for microbial production of therapeutic proteins with the correct post-translational modifications. To achieve this, they planned to insert multiple genes into their neo-chromosome without destabilising it, and test whether these genes could be co-expressed to produce useful quantities of modified protein.
They also wanted to see if they could use the neo-chromosome as a source of auxiliary enzymes to support the folding and appropriate chemical modifications of target proteins produced from genes incorporated into the native chromosomes of P. pastoris using classical techniques.
Solution
With Innovation funding from IBioIC, Ingenza worked with Paul Barlow at University of Edinburgh.
Ingenza contributed background P. pastoris strains, technical advice and suggestions for applications of the technology, which enabled the Barlow group to introduce into cells the group’s latest versions of the neo-chromosome, and explore their potential. The structural and segregational stability of these molecules over numerous generations was confirmed using whole-genome sequencing that confirmed one copy of the neo-chromosome per cell.
Gene insertion into neo-chromosome was also proven and validated with a number of proteins. Finally, the neo-chromosome was used as a repository for a gene encoding a chaperone protein that boosted production of a glycoprotein from the host genome.
Outcome
This novel means of performing précise, reproducible genetic manipulation in Pichia is entirely new. It potentially offers a platform for a range of services that Ingenza could provide, including manufacture of a range of therapeutics or vaccines.
The technology developed could enable Ingenza to identify additional commercial opportunities to expand their current P. pastoris biologics manufacturing platform and attract new commercial end-user partnerships. The market potential for Ingenza to utilise P. pastoris based bio-manufacturing technologies as services, licensed processes or means to generate new equity partnerships is significant, likely £2-3M annually at this time with opportunities for significant future uplift.
