Yeast systems have been a staple for producing large amounts of proteins for industrial and biopharmaceutical use for many
years. Yeast can be grown to very high cell mass densities in well-defined medium. Recombinant proteins in yeast can be over-expressed
so the product is secreted from the cell and available for recovery in the fermentation solution. Proteins secreted by yeasts
are heavily glycosylated at consensus glycosylation sites. Thus, expression of recombinant proteins in yeast systems historically
has been confined to proteins where post-translations glycosylation patterns do not affect the function of proteins. Several
yeast expression systems are used for recombinant protein expression, including Sacharomyces, Scizosacchromyces pombe, Pichia pastoris and Hansanuela polymorpha.
In this article,* we discuss some of the technologies and aspects associated with biomanufacturing of therapeutics in yeast
systems and highlight some techniques that are used in either basic research or clinical manufacturing, but are not typically
utilized for both.
Yeast Expression Vectors
The expression of human proteins in prokaryotes has limitations in that prokaryotes do not have compartmentalized secretion
system like eukaryotes and hence post-translational modifications like glycosylation do not occur. In cases like antibodies,
these limiations also affect the proper folding of the proteins and hence their applicability. As a result, these proteins
have to be made in higher eukaryotic mammalian cell systems. But mammalian systems are expensive and may render a very low
yield. As a result, alternative systems using lower eukaryotes like yeasts Saccharomyces
cerevisiae, Schizosaccharomyces pombe, and methylotropic yeasts like Pichia pastoris, and Pichia methanolica have gained importance. These systems do not glycosylate the same way as that of human glycosylation. For example, S. cerevisiae produces high mannose structures, and has been useful in producing properly folded active and soluble multi-subunit proteins.
As in any prokaryote, yeast expression systems also require an origin of replication or integration, a strong promoter, and
a selection marker. Expression of recombinant proteins in S. cerevisiae can be done using three types of vectors: integration vectors (YIp), episomal plasmids (YEp), and centromeric plasmids (YCp).
YIp Vectors. The YIp integrative vectors are vectors that do not replicate autonomously, but integrate into the genome at low frequencies
by homologous recombination. Integration of circular plasmid DNA by homologous recombination leads to a copy of the vector
sequence flanked by two direct copies of the yeast sequence. Typically, YIp vectors integrate as a single copy. However, methods
to integrate multiple copies and stable cell lines with up to 15-20 copies of recombinant gene integrations have been developed
for over-expressing specific genes.2 YIp plasmids with two yeast segments, such as YFG1 and the URA3 marker, have the potential to integrate at either of the
genomic loci, whereas vectors containing repetitive DNA sequences, such as Ty elements or rDNA, can integrate at any of the
multiple sites within genome.2
YEp Vectors. The YEp yeast episomal plasmid vectors replicate autonomously because of the presence of a segment of the yeast 2 μm plasmid
that serves as an origin of replication (2 μm ori). The 2 μm ori is responsible for the high copy-number and high frequency
of transformation of YEp vectors. Most YEp plasmids are relatively unstable and even under conditions of selective growth,
only 60 to 95 percent of the cells retain the YEp plasmid. The copy number of most YEp plasmids ranges from 10 to 40 copies
per cell. Although this system is used for small scale expression studies, the use of YEp vectors in large-scale manufacturing
is not advisable.
YCp Vectors. YCp yeast centromere plasmid vectors are autonomously replicating vectors containing centromere sequences (CEN), and autonomously
replicating sequences (ARS). The YCp vectors are typically present at very low copy numbers from 1 to 3 per cell. These vectors
are also relatively unstable and not very useful in high level expression but are used as regular cloning vectors (e.g., pYC2,