lin in P. pastoris in 2003 (Meehl and Stadheim
2014). In 2018 the glargine biosimilar “Semglee” produced in P. pastoris was approved by
the EU authorities.
B. Pichia pastoris
Among the section of methylotrophic yeasts,
one of the most prominent representatives
Pichia pastoris (Komagataella spp.) is a widespread host for protein production. As these
yeasts can utilize methanol as the sole carbon
source, they were first used for single cell protein production for animal feed by Philips
Petroleum (later ConocoPhilips), since the
price for methanol derived from oil refining
was very cheap. After the rise of the feedstock
prices during the late 1970s, single cell protein
production was not profitable any longer and P.
pastoris was “set free” (Wegner 1990) to the
scientific community for research purposes
and further developed as a protein production
system in the 1980s. The most important reason
for its relevance in protein production is the
ability to utilize methanol as sole carbon
source. The genes that are responsible for this
metabolic pathway provide a set of strong
inducible and tightly regulated promoters
(alcohol oxidase AOX promoter system) that
are frequently utilized for overexpression of
recombinant proteins. Besides this, in the
recent years some alternatives to the AOX
inducible promoter systems (rhamnoseinducible, glucose-limit induced P GTH1 etc.)
were developed to circumvent fire-hazard and
toxicity issues when using methanol in industrial scale (Ahmad et al. 2014; Yang and Zhang
2018). Also the availability of commercial cloning kits that provide beginners of this host a full
guide from cloning to protein production
helped to propagate the application of P. pastoris. A unique feature of P. pastoris is the
GlycoFi-technology owned by Merck & Co., a
cell engineering technology that enables manufacturers to produce human-like biantennary
N-glycosylated proteins, especially necessary
for antibody production. For bioprocess optimization, P. pastoris has some important features. It can grow to very high cell densities up
to 100 g/L and more (Shay et al. 1987), and it
secretes only a comparably small number of
host cell proteins (Huang et al. 2011) into the
culture supernatant. Therefore downstream
processing is facilitated.
The most important industrial biopharmaceutical produced in P. pastoris is Insulin glargine (Basalog by Biocon Ltd.—India). Also
Hepatitis B vaccine and Interferon alpha 2b
are produced in this system (Shantha Biotechnics—India). In 2018, Semglee, a biosimilar to
insulin glargine Lantus, produced in P. pastoris
was approved by the EU authorities for the
treatment of Diabetes mellitus. Also Kalbitor
(INN: ecallantide), a plasma kallikrein inhibitor
for the treatment of hereditary angioedema, is
produced in this system. It was the first ever P.
pastoris-produced drug approved for human
treatment in 2009. A remarkable fact is that
despite the technical development by GlycoFi
which already enabled the production of glycoproteins with humanized N-glycosylation
structures, not a single P. pastoris-produced
antibody is on the biopharmaceutical market.
However, some exciting studies were published
in the last years, where nanobodies (single
domain antibodies lacking N-glycosylation)
were produced in high product titers [>10 g/
L, Schotte et al. (2016)]. There are also some P.
pastoris-derived products in the sector of
industrial enzymes that play an important
part: Quantum is an animal feed additive containing recombinant phytase approved by the
FDA Center of Veterinary Medicine (CVM) in
2008. Furthermore, carboxypeptidase, lipase,
and phospholipase C are also produced in P.
pastoris.
C. Hansenula polymorpha
Another important cell factory system for the
production of recombinant proteins among the
group of methylotrophic yeasts is Hansenula
polymorpha (syn. Ogataea polymorpha). Apart
from its methylotrophy, it has some other specifically interesting characteristics. First it is
able to assimilate nitrate (Siverio 2002), which
makes it one of just a few yeast species with this
ability. Furthermore, it shows an extraordinary
13 Yeast Cell Factories
331
2014). In 2018 the glargine biosimilar “Semglee” produced in P. pastoris was approved by
the EU authorities.
B. Pichia pastoris
Among the section of methylotrophic yeasts,
one of the most prominent representatives
Pichia pastoris (Komagataella spp.) is a widespread host for protein production. As these
yeasts can utilize methanol as the sole carbon
source, they were first used for single cell protein production for animal feed by Philips
Petroleum (later ConocoPhilips), since the
price for methanol derived from oil refining
was very cheap. After the rise of the feedstock
prices during the late 1970s, single cell protein
production was not profitable any longer and P.
pastoris was “set free” (Wegner 1990) to the
scientific community for research purposes
and further developed as a protein production
system in the 1980s. The most important reason
for its relevance in protein production is the
ability to utilize methanol as sole carbon
source. The genes that are responsible for this
metabolic pathway provide a set of strong
inducible and tightly regulated promoters
(alcohol oxidase AOX promoter system) that
are frequently utilized for overexpression of
recombinant proteins. Besides this, in the
recent years some alternatives to the AOX
inducible promoter systems (rhamnoseinducible, glucose-limit induced P GTH1 etc.)
were developed to circumvent fire-hazard and
toxicity issues when using methanol in industrial scale (Ahmad et al. 2014; Yang and Zhang
2018). Also the availability of commercial cloning kits that provide beginners of this host a full
guide from cloning to protein production
helped to propagate the application of P. pastoris. A unique feature of P. pastoris is the
GlycoFi-technology owned by Merck & Co., a
cell engineering technology that enables manufacturers to produce human-like biantennary
N-glycosylated proteins, especially necessary
for antibody production. For bioprocess optimization, P. pastoris has some important features. It can grow to very high cell densities up
to 100 g/L and more (Shay et al. 1987), and it
secretes only a comparably small number of
host cell proteins (Huang et al. 2011) into the
culture supernatant. Therefore downstream
processing is facilitated.
The most important industrial biopharmaceutical produced in P. pastoris is Insulin glargine (Basalog by Biocon Ltd.—India). Also
Hepatitis B vaccine and Interferon alpha 2b
are produced in this system (Shantha Biotechnics—India). In 2018, Semglee, a biosimilar to
insulin glargine Lantus, produced in P. pastoris
was approved by the EU authorities for the
treatment of Diabetes mellitus. Also Kalbitor
(INN: ecallantide), a plasma kallikrein inhibitor
for the treatment of hereditary angioedema, is
produced in this system. It was the first ever P.
pastoris-produced drug approved for human
treatment in 2009. A remarkable fact is that
despite the technical development by GlycoFi
which already enabled the production of glycoproteins with humanized N-glycosylation
structures, not a single P. pastoris-produced
antibody is on the biopharmaceutical market.
However, some exciting studies were published
in the last years, where nanobodies (single
domain antibodies lacking N-glycosylation)
were produced in high product titers [>10 g/
L, Schotte et al. (2016)]. There are also some P.
pastoris-derived products in the sector of
industrial enzymes that play an important
part: Quantum is an animal feed additive containing recombinant phytase approved by the
FDA Center of Veterinary Medicine (CVM) in
2008. Furthermore, carboxypeptidase, lipase,
and phospholipase C are also produced in P.
pastoris.
C. Hansenula polymorpha
Another important cell factory system for the
production of recombinant proteins among the
group of methylotrophic yeasts is Hansenula
polymorpha (syn. Ogataea polymorpha). Apart
from its methylotrophy, it has some other specifically interesting characteristics. First it is
able to assimilate nitrate (Siverio 2002), which
makes it one of just a few yeast species with this
ability. Furthermore, it shows an extraordinary
13 Yeast Cell Factories
331