Although the yeast quota of the recombinant protein market is dominated by products
of the “conventional” yeast S. cerevisiae, there
are many other—so-called non-conventional—
yeast species that provide interesting features
for heterologous protein production (Meehl
and Stadheim 2014). In this chapter, we summarize the characteristics and application of
different yeast species and their potential for
protein production with a focus on the industrial and biopharmaceutical use.
A. Saccharomyces cerevisiae
Baker’s yeast S. cerevisiae is one of the most
prominent and important model organisms for
cell and molecular biology and consequently
also highly used for recombinant protein production (Duina et al. 2014). Thus, most marketed yeast-derived therapeutic proteins are
produced in S. cerevisiae, as the production
processes are well established and authoritiesapproved. As a respiro-fermentative (so-called
Crabtree positive) yeast S. cerevisiae predominantely ferments a surplus of glucose to ethanol
even in the presence of oxygen. Therefore special requirements are necessary for the cultivation in the bioprocess, namely, a glucose
limited feed, mostly as a fed batch cultivation.
Efficient production of insulin (see Sect. IV.
A.1) and related biopharmaceuticals has been a
major driving force in the last decades of the
previous century to improve S. cerevisiae as a
protein production host, including efficient
promoter and stable vector systems like the
promoter of alcohol dehydrogenase ADH2, the
galactose-inducible GAL promoter or the constitutive heterologous TPI1-vector system (Stepie ´n et al. 1983; Hitzeman et al. 1981; Liu et al.
2012), improvement of secretion leaders (Kjeldsen et al. 1997; Kjeldsen 2000), and optimization of bioreactor cultivation (Diers et al. 1991;
Mendoza-Vega et al. 1994; Vasavada 1995). Pioneering work in S. cerevisiae also elucidated the
function of genes in the secretory pathway
[reviewed by Feyder et al. (2015)] and led to
the identification of cellular bottlenecks during
protein secretion (Mori 2015) as well as strategies to overcome them [reviewed by Hou et al.
(2012) and Zahrl et al. (2019)]. Other products
produced by S. cerevisiae include the hormones
glucagon, human growth hormone, interferon,
and subunit vaccines such as Hepatitis B surface antigen and HPV vaccines (Wang et al.
2017). Nowadays strategies for improving protein production with S. cerevisiae are mainly
driven by the high demand of cellulosic and
lignocellulosic enzymes needed for the production of second-generation biofuels (Tang et al.
2017; Xu et al. 2014; Kitagawa et al. 2011; Van
Zyl et al. 2016) aiming for high-level secretion
or surface display of these enzymes.
1. Insulin: The First Marketed Recombinant
Protein
Insulin was the first fully sequenced protein (by
Sanger in 1955) and also the first licensed drug
produced using recombinant DNA technology
in 1982. Today recombinant human insulin is
mainly produced either by Escherichia coli
(insoluble production as inclusion bodies and
refolding) or by the yeast S. cerevisiae [secretion of soluble insulin precursor, Baeshen et al.
(2014)]. In insulin precursor (IP), the naturally
35 amino acids long C-peptide is exchanged for
a mini-C-peptide comprising of Ala-Ala-Lys,
which links the insulin B-chain (29 amino
acids) to the A-chain (21 amino acids). Secreted
and purified IP is then converted to human
insulin by tryptic transpeptidation (Kjeldsen
2000). The global insulin market (insulin and
insulin analogs) was US$22 billion in 2017
(Walsh 2018), with an annual sales increase of
more than 10% due to the increasing worldwide
prevalence of diabetes (Wirtz 2016). Yeastbased production of insulin was first achieved
in 1986 and is now accounting for more than
half of market share. Novo Nordisk sells S.
cerevisiae derived insulin to more than 100
countries worldwide and has 44% value share
and 52% volume share. Additionally the Indian
biomanufacturers Biocon and Wockhardt produce insulin and insulin-derivatives such as
glargine in yeast hosts. While Novo Nordisk
started in 1991 to produce human insulin in
the S. cerevisiae expression system, Biocon
was the first company to produce human insu330
B. Schmelzer et al.
of the “conventional” yeast S. cerevisiae, there
are many other—so-called non-conventional—
yeast species that provide interesting features
for heterologous protein production (Meehl
and Stadheim 2014). In this chapter, we summarize the characteristics and application of
different yeast species and their potential for
protein production with a focus on the industrial and biopharmaceutical use.
A. Saccharomyces cerevisiae
Baker’s yeast S. cerevisiae is one of the most
prominent and important model organisms for
cell and molecular biology and consequently
also highly used for recombinant protein production (Duina et al. 2014). Thus, most marketed yeast-derived therapeutic proteins are
produced in S. cerevisiae, as the production
processes are well established and authoritiesapproved. As a respiro-fermentative (so-called
Crabtree positive) yeast S. cerevisiae predominantely ferments a surplus of glucose to ethanol
even in the presence of oxygen. Therefore special requirements are necessary for the cultivation in the bioprocess, namely, a glucose
limited feed, mostly as a fed batch cultivation.
Efficient production of insulin (see Sect. IV.
A.1) and related biopharmaceuticals has been a
major driving force in the last decades of the
previous century to improve S. cerevisiae as a
protein production host, including efficient
promoter and stable vector systems like the
promoter of alcohol dehydrogenase ADH2, the
galactose-inducible GAL promoter or the constitutive heterologous TPI1-vector system (Stepie ´n et al. 1983; Hitzeman et al. 1981; Liu et al.
2012), improvement of secretion leaders (Kjeldsen et al. 1997; Kjeldsen 2000), and optimization of bioreactor cultivation (Diers et al. 1991;
Mendoza-Vega et al. 1994; Vasavada 1995). Pioneering work in S. cerevisiae also elucidated the
function of genes in the secretory pathway
[reviewed by Feyder et al. (2015)] and led to
the identification of cellular bottlenecks during
protein secretion (Mori 2015) as well as strategies to overcome them [reviewed by Hou et al.
(2012) and Zahrl et al. (2019)]. Other products
produced by S. cerevisiae include the hormones
glucagon, human growth hormone, interferon,
and subunit vaccines such as Hepatitis B surface antigen and HPV vaccines (Wang et al.
2017). Nowadays strategies for improving protein production with S. cerevisiae are mainly
driven by the high demand of cellulosic and
lignocellulosic enzymes needed for the production of second-generation biofuels (Tang et al.
2017; Xu et al. 2014; Kitagawa et al. 2011; Van
Zyl et al. 2016) aiming for high-level secretion
or surface display of these enzymes.
1. Insulin: The First Marketed Recombinant
Protein
Insulin was the first fully sequenced protein (by
Sanger in 1955) and also the first licensed drug
produced using recombinant DNA technology
in 1982. Today recombinant human insulin is
mainly produced either by Escherichia coli
(insoluble production as inclusion bodies and
refolding) or by the yeast S. cerevisiae [secretion of soluble insulin precursor, Baeshen et al.
(2014)]. In insulin precursor (IP), the naturally
35 amino acids long C-peptide is exchanged for
a mini-C-peptide comprising of Ala-Ala-Lys,
which links the insulin B-chain (29 amino
acids) to the A-chain (21 amino acids). Secreted
and purified IP is then converted to human
insulin by tryptic transpeptidation (Kjeldsen
2000). The global insulin market (insulin and
insulin analogs) was US$22 billion in 2017
(Walsh 2018), with an annual sales increase of
more than 10% due to the increasing worldwide
prevalence of diabetes (Wirtz 2016). Yeastbased production of insulin was first achieved
in 1986 and is now accounting for more than
half of market share. Novo Nordisk sells S.
cerevisiae derived insulin to more than 100
countries worldwide and has 44% value share
and 52% volume share. Additionally the Indian
biomanufacturers Biocon and Wockhardt produce insulin and insulin-derivatives such as
glargine in yeast hosts. While Novo Nordisk
started in 1991 to produce human insulin in
the S. cerevisiae expression system, Biocon
was the first company to produce human insu330
B. Schmelzer et al.