otic expression systems, providing a set of
advantages in comparison to bacterial and
mammalian production hosts. Among them
are the fast growth to high cell densities in
cheap media, fully sequenced and stable genomes, the easy genetic manipulation with developed toolbox sets, the ability of posttranslational modifications and protein secretion, as well as the microbiological safety
(GRAS status of some yeasts themselves and
their products, and the absence of pyrogens
and adventitious pathogens) (Roohvand et al.
2017). The lower specific productivity compared to mammalian hosts is usually compensated by high cell densities (up to 150 g/L dry
cell weight), which on the other side also
demands a higher fraction of energy and
resources directed into biomass formation
(Maccani et al. 2014).
Due to the dominance of monoclonal antibodies (which represent 52% of new approvals
in the last 3 years), products derived from
mammalian expression systems have more
than 65% share in sales in the biopharmaceutical markets (Walsh 2018). However, if comparing product amounts, of the 26.4 metric tons
pure active protein, 68% are produced in
microbial systems (Walsh 2014), and microbially produced insulin is the most profitable
non-antibody-based product on the market.
Yeasts are primarily used for production of
secreted proteins which enables easy purification of the product from the supernatant.
Among them mainly hormones, growth factors,
antibody fragments, and enzymes are currently
produced on an industrial scale. Additionally,
yeasts are excellent producers of vaccines such
as for Hepatitis B (containing recombinant
hepatitis B surface antigen) and Human papilloma virus (HPV) (Walsh 2018). While in the
early 2000s attempts to produce full length
monoclonal antibodies in yeast were initiating
extensive glycoengineering efforts, the recent
years saw the re-advent of yeast hosts for
novel (mostly non-glycosylated) targets such
as single chain variable antibody fragments
(scFv), camelid single domain antibodies
(vHH), and other binding scaffolds and antibody mimetics (Liu and Huang 2018; Spadiut
et al. 2014).
Glycosylation is an important topic, especially for biopharmaceutical products. Yeasts,
as eukaryotic organisms, are generally able to
perform these post-translational modifications, but their N-glycan structure is different
to the complex mammalian patterns. Yeasts
synthesize mannose-rich structures (depending on the species, these contain from 6 up to
200 mannose residues) that are lacking galactose and sialic acid residues, which are present
in human glycans (Gemmill and Trimble 1999).
This might have two effects: first, the three
dimensional structure of the proteins can be
impaired, which can hamper or disable their
function. The second issue is immunogenicity:
the human immune system may recognize
these patterns as “unfamiliar” and elicited
immune reactions can lead to severe shocks.
Interestingly, the terminal glycosidic bonds
seem to evoke the immune reaction. While in
S. cerevisiae, the terminal mannose residues
have alpha-1,3 connections (Romero et al.
1999; Nakajima and Ballou 1975), other yeasts
such as Pichia pastoris and Hansenula polymorpha (syn. Ogataea polymorpha) lack the
enzymes for this reaction and produce terminal alpha-1,2-glycosidic bonds instead, which
were shown to be less immunogenic to humans
(Song et al. 2007; Kim et al. 2004). During the
last decade, major cell engineering efforts were
directed toward glycoengineering of different
yeast species. After blocking the initial steps of
mannose chain elongation, N-glycans of the
GlcNAc2Man3 or GlcNacMan5 type could be
obtained in S. cerevisiae, P. pastoris, Yarrowia
lipolytica, and H. polymorpha thereby avoiding
high-mannose type N-glycans [reviewed by
Anyaogu and Mortensen (2015) and De Wachter et al. (2018)]. For P. pastoris, more
advanced cell engineering was pursued involving 17 gene deletions or overexpressions
resulting in glycoengineered strains that do
not only prevent high-mannosylation but
even allowed mammalian-like N-glycans containing terminal galactose and sialic acid
(Hamilton and Gerngross 2007). These made
the yeast-based production of highly glycosylated human proteins such as full length IgGs
and erythropoietin (EPO) possible (Ye et al.
2011; Hamilton et al. 2006).
13 Yeast Cell Factories
329
advantages in comparison to bacterial and
mammalian production hosts. Among them
are the fast growth to high cell densities in
cheap media, fully sequenced and stable genomes, the easy genetic manipulation with developed toolbox sets, the ability of posttranslational modifications and protein secretion, as well as the microbiological safety
(GRAS status of some yeasts themselves and
their products, and the absence of pyrogens
and adventitious pathogens) (Roohvand et al.
2017). The lower specific productivity compared to mammalian hosts is usually compensated by high cell densities (up to 150 g/L dry
cell weight), which on the other side also
demands a higher fraction of energy and
resources directed into biomass formation
(Maccani et al. 2014).
Due to the dominance of monoclonal antibodies (which represent 52% of new approvals
in the last 3 years), products derived from
mammalian expression systems have more
than 65% share in sales in the biopharmaceutical markets (Walsh 2018). However, if comparing product amounts, of the 26.4 metric tons
pure active protein, 68% are produced in
microbial systems (Walsh 2014), and microbially produced insulin is the most profitable
non-antibody-based product on the market.
Yeasts are primarily used for production of
secreted proteins which enables easy purification of the product from the supernatant.
Among them mainly hormones, growth factors,
antibody fragments, and enzymes are currently
produced on an industrial scale. Additionally,
yeasts are excellent producers of vaccines such
as for Hepatitis B (containing recombinant
hepatitis B surface antigen) and Human papilloma virus (HPV) (Walsh 2018). While in the
early 2000s attempts to produce full length
monoclonal antibodies in yeast were initiating
extensive glycoengineering efforts, the recent
years saw the re-advent of yeast hosts for
novel (mostly non-glycosylated) targets such
as single chain variable antibody fragments
(scFv), camelid single domain antibodies
(vHH), and other binding scaffolds and antibody mimetics (Liu and Huang 2018; Spadiut
et al. 2014).
Glycosylation is an important topic, especially for biopharmaceutical products. Yeasts,
as eukaryotic organisms, are generally able to
perform these post-translational modifications, but their N-glycan structure is different
to the complex mammalian patterns. Yeasts
synthesize mannose-rich structures (depending on the species, these contain from 6 up to
200 mannose residues) that are lacking galactose and sialic acid residues, which are present
in human glycans (Gemmill and Trimble 1999).
This might have two effects: first, the three
dimensional structure of the proteins can be
impaired, which can hamper or disable their
function. The second issue is immunogenicity:
the human immune system may recognize
these patterns as “unfamiliar” and elicited
immune reactions can lead to severe shocks.
Interestingly, the terminal glycosidic bonds
seem to evoke the immune reaction. While in
S. cerevisiae, the terminal mannose residues
have alpha-1,3 connections (Romero et al.
1999; Nakajima and Ballou 1975), other yeasts
such as Pichia pastoris and Hansenula polymorpha (syn. Ogataea polymorpha) lack the
enzymes for this reaction and produce terminal alpha-1,2-glycosidic bonds instead, which
were shown to be less immunogenic to humans
(Song et al. 2007; Kim et al. 2004). During the
last decade, major cell engineering efforts were
directed toward glycoengineering of different
yeast species. After blocking the initial steps of
mannose chain elongation, N-glycans of the
GlcNAc2Man3 or GlcNacMan5 type could be
obtained in S. cerevisiae, P. pastoris, Yarrowia
lipolytica, and H. polymorpha thereby avoiding
high-mannose type N-glycans [reviewed by
Anyaogu and Mortensen (2015) and De Wachter et al. (2018)]. For P. pastoris, more
advanced cell engineering was pursued involving 17 gene deletions or overexpressions
resulting in glycoengineered strains that do
not only prevent high-mannosylation but
even allowed mammalian-like N-glycans containing terminal galactose and sialic acid
(Hamilton and Gerngross 2007). These made
the yeast-based production of highly glycosylated human proteins such as full length IgGs
and erythropoietin (EPO) possible (Ye et al.
2011; Hamilton et al. 2006).
13 Yeast Cell Factories
329