Evolution and Genetic Engineering - Bioprocess Engineering: Kinetics, Biosystems, Sustainability and Reactor Design - page 726

Further advantages include the capacity to do simple glycosylation of proteins and to
secrete proteins. However, S . cerevisiae tends to hyperglycosylate proteins, adding large
numbers ofmannose units. In some cases, the hyperglycosylated proteinmay even be inactive.
These organisms are also on the GRAS list, which simplifies regulatory approval and
makes yeast particularly well suited to production of food-related proteins. Generally, the
limitations on S . cerevisiae are the difficulties of achieving high-protein expression levels,
hyperglycosylation, and good excretion. Although the genetics of S . cerevisiae are better
known than for any other eukaryotic cell, the range of genetic systems is limited, and stable
high-protein expression levels are more difficult to achieve than in E. coli . Also, the normal
capacity of the secretion pathways in S . cerevisiae is limited and is a bottleneck on excreted
protein production, even when high expression levels are achieved.
The methylotrophic yeasts, P. pastoris and Hansenula polymorpha , are very attractive hosts
for some proteins. These yeasts can grow on methanol as a carbon energy source; methanol is
also an inducer for the AOX 1 promoter, which is typically used to control expression of the
target protein. Very high cell densities (e.g. up to 100 g/L) can be obtained. Due to high densi-
ties and, for some proteins, high expression levels, the volumetric productivities of these
cultures can be higher than with those E. coli . Protein folding and secretion are, also, often
better than those in E. coli . These yeasts make simple glycosylation and are less likely to
hyperglycosylate than S. cerevisiae . Like many host systems, their effectiveness is often a func-
tion of the target protein. The disadvantages of the methylotrophic yeast are due to the high
cell density and rate of metabolism, which creates high levels of metabolic heat that must be
removed and high oxygen demand. Effective induction of expression, while maintaining cell
activity, requires very good process control due to methanol's dual functions as growth
substrate and inducer. Furthermore, high levels of methanol are inhibitory (i.e. substrate inhi-
bition), which also demands good process control. Scale-up to large reactors often is very
challenging, since heat removal, oxygen supply, and process control are typically more diffi-
cult in large reactors with longer mixing times. Also, methanol is flammable and handling
large volumes of methanol is a safety concern. Nonetheless, these methylotrophic yeasts
are of increasing importance.
Fungi, such as Aspergillus nidulans and Trichoderma reesei , are also potentially important
hosts. They generally have greater intrinsic capacity for protein secretion than S . cerevisiae.
Their filamentous growth makes large-scale cultivation somewhat more difficult. However,
commercial enzyme production from these fungi is well established, and the scale-up prob-
lems have been addressed. The major limitation has been the construction of expression and
secretion systems that can produce as large amounts of extracellular heterologous proteins as
some of the native proteins. A better understanding of the secretion pathway and its interac-
tion with protein structure is critical for this system to reach its potential.
All these lower eukaryotic systems are inappropriate when complex glycosylation and
posttranslational modifications are necessary. In such cases, animal cell tissue culture has
been employed.
14.7.4. Mammalian Cells
Mammalian cell culture is the prime choice of host if the virtual authenticity of the product
protein is complete. Authenticity implies not only the correct arrangement of all amino acids