thermotolerance up to 50
C (Wijeyaratne et al.
1986) which is quite unusual for yeasts. The
ability to induce methanol pathway promoters
such as P MOX in carbon source (e.g., glucose)
limit without the presence of methanol (in contrast to other methylotrophic yeasts) is a
unique feature of the H. polymorpha expression
system that makes it safe to use in industrial
bioprocesses. Recent advances regarding
genetic tools and bioprocess engineering of
the H. polymorpha platform are summarized
by Manfra ˜o-Netto et al. (2019), while Yoo
et al. (2019) provides guidelines on vectors
and strains that are suitable for production of
secretory recombinant proteins.
The most prominent commercial product
made by H. polymorpha is HEPLISAV-B, a
recombinant hepatitis B vaccine launched by
Dynavax Technologies (USA) in 2017. Hexacima, a multi-component vaccine containing
recombinant Hepatitis B antigens is also manufactured in H. polymorpha. Wockhardt Ltd.
(India) launched Asia’s first human recombinant insulin (Wosulin) in 2003, which is produced in H. polymorpha.
D. Yarrowia lipolytica
Yarrowia lipolytica is a yeast species with the
special ability to utilize higher hydrocarbons,
especially long-chain fatty acids. It also shows a
strong morphological dimorphism, as it can
grow from ovoid to hyphae form depending
on the present environmental conditions.
While the morphological status has a yet unresolved impact on the ability of Y. lipolytica to
produce metabolic products such as citric acid
and polyols (Timoumi et al. 2018), some
researchers hypothesized that the mechanisms
involved in dimorphic transition positively
influence the protein secretion behavior of
this yeast (Celin ´ska and Nicaud 2019). On the
other hand, dimorphism poses a challenge for
establishing efficient bioprocesses for recombinant secretory protein production (Vandermies
and Fickers 2019). Based on its natural habitats
(cheese, oil etc.), it secretes large amounts of
lipases and proteases for the retrieval of nutrients for growth. Many Y. lipolytica-derived products (citric acid, erythritol, eicosapentaenoic
acid) received GRAS status in the last decade
(Groenewald et al. 2014). Therefore, it can
unhesitatingly be utilized in food and feed products. For expression of recombinant genes
either the constitutive TEF promoter or inducible hybrid promoters are used (reviewed, e.g.,
by Madzak 2018). The most important recombinant products are mainly industrial enzymes
for applications in food and feed supplements,
cosmetics, detergents, textiles and paper industries, biofuels, and waste stream depollution
(Madzak 2015).
E. Kluyveromyces lactis
Kluyveromyces lactis, formerly classified as
Saccharomyces lactis, is known for its ability
to assimilate milk sugar (lactose). It can be
isolated from milk, butter, yoghurt, cheese,
and other fermented dairy products. Since K.
lactis is present in several dairy products, it is
generally recognized as safe (GRAS) by the FDA
authorities playing an important role for the
production of food and feed ingredients. Due
to the production of the pigment pulcherrimin,
the color of the colonies appears cream to pink.
For the expression of heterologous genes,
the availability of a strong and tightly regulated
promoter is of major importance. The major
promoter used in K. lactis is the promoter associated with the beta-galactosidase LAC4 gene
that is well repressed by low concentrations of
glucose (Swinkels et al. 1993). An overview
about genetic tools for recombinant protein
production as well as bioprocessing strategies
was given by Spohner et al. (2016). In terms of
industrial applications, the ability to grow on
inexpensive substrates, such as cheese whey, is
a basis of economic competitiveness (Ornelas
et al. 2007). The naturally secreted betagalactosidase (lactase) is an important industrial product of K. lactis cultivation, since it is
used for the production of lactose-free milk
products to a large extent. Furthermore, the
expression of heterologous chymosins plays a
major role for cheese production that nowadays
replaced the use of rennet from slaughtered
animals (van Ooyen et al. 2006).
332
B. Schmelzer et al.
C (Wijeyaratne et al.
1986) which is quite unusual for yeasts. The
ability to induce methanol pathway promoters
such as P MOX in carbon source (e.g., glucose)
limit without the presence of methanol (in contrast to other methylotrophic yeasts) is a
unique feature of the H. polymorpha expression
system that makes it safe to use in industrial
bioprocesses. Recent advances regarding
genetic tools and bioprocess engineering of
the H. polymorpha platform are summarized
by Manfra ˜o-Netto et al. (2019), while Yoo
et al. (2019) provides guidelines on vectors
and strains that are suitable for production of
secretory recombinant proteins.
The most prominent commercial product
made by H. polymorpha is HEPLISAV-B, a
recombinant hepatitis B vaccine launched by
Dynavax Technologies (USA) in 2017. Hexacima, a multi-component vaccine containing
recombinant Hepatitis B antigens is also manufactured in H. polymorpha. Wockhardt Ltd.
(India) launched Asia’s first human recombinant insulin (Wosulin) in 2003, which is produced in H. polymorpha.
D. Yarrowia lipolytica
Yarrowia lipolytica is a yeast species with the
special ability to utilize higher hydrocarbons,
especially long-chain fatty acids. It also shows a
strong morphological dimorphism, as it can
grow from ovoid to hyphae form depending
on the present environmental conditions.
While the morphological status has a yet unresolved impact on the ability of Y. lipolytica to
produce metabolic products such as citric acid
and polyols (Timoumi et al. 2018), some
researchers hypothesized that the mechanisms
involved in dimorphic transition positively
influence the protein secretion behavior of
this yeast (Celin ´ska and Nicaud 2019). On the
other hand, dimorphism poses a challenge for
establishing efficient bioprocesses for recombinant secretory protein production (Vandermies
and Fickers 2019). Based on its natural habitats
(cheese, oil etc.), it secretes large amounts of
lipases and proteases for the retrieval of nutrients for growth. Many Y. lipolytica-derived products (citric acid, erythritol, eicosapentaenoic
acid) received GRAS status in the last decade
(Groenewald et al. 2014). Therefore, it can
unhesitatingly be utilized in food and feed products. For expression of recombinant genes
either the constitutive TEF promoter or inducible hybrid promoters are used (reviewed, e.g.,
by Madzak 2018). The most important recombinant products are mainly industrial enzymes
for applications in food and feed supplements,
cosmetics, detergents, textiles and paper industries, biofuels, and waste stream depollution
(Madzak 2015).
E. Kluyveromyces lactis
Kluyveromyces lactis, formerly classified as
Saccharomyces lactis, is known for its ability
to assimilate milk sugar (lactose). It can be
isolated from milk, butter, yoghurt, cheese,
and other fermented dairy products. Since K.
lactis is present in several dairy products, it is
generally recognized as safe (GRAS) by the FDA
authorities playing an important role for the
production of food and feed ingredients. Due
to the production of the pigment pulcherrimin,
the color of the colonies appears cream to pink.
For the expression of heterologous genes,
the availability of a strong and tightly regulated
promoter is of major importance. The major
promoter used in K. lactis is the promoter associated with the beta-galactosidase LAC4 gene
that is well repressed by low concentrations of
glucose (Swinkels et al. 1993). An overview
about genetic tools for recombinant protein
production as well as bioprocessing strategies
was given by Spohner et al. (2016). In terms of
industrial applications, the ability to grow on
inexpensive substrates, such as cheese whey, is
a basis of economic competitiveness (Ornelas
et al. 2007). The naturally secreted betagalactosidase (lactase) is an important industrial product of K. lactis cultivation, since it is
used for the production of lactose-free milk
products to a large extent. Furthermore, the
expression of heterologous chymosins plays a
major role for cheese production that nowadays
replaced the use of rennet from slaughtered
animals (van Ooyen et al. 2006).
332
B. Schmelzer et al.