Another empirical strategy starts with a contract research organization like VTU Technology taking a gene that a company wants expressed in high quantities as a secreted protein. The firm then designs a synthetic gene that fits the Pichia pastoris codon usage table, along with other parameters. The gene is then pool-cloned into the methylotrophic yeast, and transformants are screened in 96-well deep-well plates.
VTU has a library of P. pastoris alcohol oxidase 1 (AOX-1) promoters, which the company’s head of R&D, Roland Weis, Ph.D., thinks is probably the strongest native promoter known in the microbial world. The library was created by Professor Anton Glieder from Graz University of Technology and his colleagues by transcription factor binding site mining, producing variants that allow for a variety of expression patterns. These can be transformed into a set of hosts that, for example, co-express various auxiliary proteins.
The key is that different proteins have different ideal expression requirements. Some proteins give the best yield when they begin to be expressed earlier than they would with the wild-type AOX-1 promoter, while other genes need to be kept silent longer, explains Dr. Weis.
“There is often no real rationale behind it, and that means you have to screen different promoter types to see which type best fits the needs of the gene.”
Using this combinatorial approach, VTU can produce and screen thousands of clones in microscale. The best two or three clones are put directly into a small fermentor. “The one-liter fermentation nicely reflects what will come out later in higher-volume fermentations,” Dr. Weis says.
A typical yield may be in the 5–15 g/L of folded, post-translationally modified protein. “Pichia hardly secretes any other proteins, so there is hardly anything else in the culture supernatant of Pichia fermentation except your protein (if it works well),” he extols.
By cutting out intermediary steps like shaker flask cultures, the entire process can reportedly be completed in about six weeks.
For customers who don’t want to deal with the toxicity and safety concerns of using methanol, VTU has created an analogous AOX-1 promoter system driven by glycerol, Dr. Weis notes. These yeast strains are capable of yielding between 30–70% of the best methanol-responsive strains—“but always far beyond the titers that you can reach with competitors’ systems.”