15

Yeast in Biotechnology

Yeasts that belong to the fungal kingdom have been used for fermentation of food and beverages
since ancient times and to date are widely used for industrial production of chemicals,
pharmaceuticals, and proteins. In this concert, Saccharomyces cerevisiae still is probably the
unicellular eukaryotic organism most widely employed for these processes. Other systems that have
been developed and successfully used for biotechnical purposes in recent years are other yeasts,
such as Hansenuela polymorpha, Pichia pastoris, or Kluyveromyces lactis. All these organisms grow
relatively quickly and are highly adaptable to large-scale production. Major advantages are that they
do not produce endotoxin and that they are capable of glycosylating proteins up to a certain extent like
mammalian cells [Glick & Pasternak, 1998].

15.1

Classical Fermentation Procedures

For several centuries, S. cerevisiae has been used in the production of food and alcoholic beverages
by selecting natural isolates. With the birth of yeast genetics, it became feasible to improve strains of
bakers and brewers yeasts by random mutagenesis or classical breeding and genetic crossing of two
strains followed by screening for mutants exhibiting particular properties. Finally, recombinant DNA
technology has widened this field by allowing to manipulate pathways of interest in a more directed
approach [Hammond, 1999]. At this point, it seems compulsory to differentiate between industrial
procedures solely aimed at the mass production of compounds derived from fermentation, such as
bioethanol, and fermentation industries being interested in the production of qualified food products,
such as good beers or wines. While nobody would care as to how chemicals are manufactured from
yeast (and these are manifold), it is still a delicate matter which hazards might be connected to the use
of engineered yeast strains used in the production of consumables.

15.2

Fermentation, Food and Chemical Industries

For decades, yeast has been used for the production of savoury flavours, enzymes, pigments, or food
acidulants. To date, many novel developments are taking place.
Two reqirements have to be fulfilled before a yeast cell is turned into a suitable factory that produces
the desired compounds: (i) appropriate strains have to be generated by genetic engineering, i.e.
introduction of genes capable of synthesizing the component(s) of interest, expression of product in
suitable amounts and (normally) secretion into the growth medium; (ii) production facilities and growth
media have to be adapted to allow for optimal yield, a goal which can be reached by metabolic
engineering [Ostergaard et al., 2000].
By metabolic engineering it is possible to extend the substrate range of yeast. Strains modified by
introducing appropriate genes from other sources can consume starch, lactose, melibiose or xylose as
a carbon source in growth media. Metabolic engineering can bring about improvements in productivity
and yield, at the same time eliminating unwanted by-products in brewers, distillers and wine yeasts.
An enormous extension of product range has been reached by heterologous expression of foreign
genes. To this end, studies on the metabolomes of yeasts and fungi have to be performed in order to
obtain the necessary data for metabolic profiling [Smedsgaard & Nielsen, 2005].
Among the products of considerable commercial interest are many isoprenoids which can be used as
flavors, fragrances (e.g. limonene, nootkatone, menthol, camphor, cubebol), food colorants

(carotenoids) or pharmaceuticals (e.g. taxol, bisabolol, lycopene, artemisinin). Therefore, metabolic

engineering approaches have aimed at establishing S.cerevisiae as an efficient cell factory for the
production of a variety of isoprenoids [e.g., Kealey et al., 1998].
Some recent developments may illustrate that the potentials of yeast are by long are not exhausted. A
recent review [Veen & Lang, 2004] describes the production of novel high-value lipid compounds in
yeast. A major advantage in choosing yeast as an object for metabolic engineering is the fact that the
lipid pathways in this organism have been described in detail and are well characterized. The intrest is
focussed on the de novo production of three major families of lipid products. (1) sterols, providing
some previously known and some novel applications as examples of the lipid pathway enhancement
that occurs naturally in yeast, (2) the reconstitution of the biosynthetic pathway of steroid hormones
and (3) the biosynthesis of polyunsaturated fatty acids, leading to the biosynthesis of different omega3 and omega-6 fatty acids which do not occur naturally in yeast. Yeast could also be usedto produce
mammalian milk k-casein. This macropeptide has various biological activities and is used as a
functional food ingredient as well as a pharmaceutical compound [Kim, Y.J. et al., 2005].
When desired features or organisms have been identified, it is often desirable to manipulate and/or
transfer these features to other production hosts that are more suited for industrial production or
change the organism to one better suited for the production environment and increased yield.
It may be interesting to note that filamentous fungi are also used extensively in biotechnology as they
can produce a wide range of chemicals that are used as food ingredients, pharmaceuticals, enzymes,
and solvents. It must be born in mind, however, that besides the beneficial use of filamentous fungi as
factories, these micro-organisms are also involved in food spoilage and that many species are also
pathogens to plants, animals, and humans.

15.3

Biopharmaceuticals from Health-Care Industries

Since the 1980s, recombinant biotherapeutics have been produced. The first recombinant protein
expressed in S. cerevisiae was human interferon [Hitzeman et al., 1981] and followed in 1982 by the
synthesis and assembly of the hepatitis B surface antigen (HBAg), the first genetically engineered
vaccine [Valenzuela et al., 1982]. To date HBAg is produced by different strategies in yeast and sold
under a variety of names (see Table 19). Probably the greatest value provided by S. cerevisiae
recombinant technology is through the production of human insulin. The production covers
approximately half of the insulin needed by the diabetics world-wide.
Insulin production may illustrate the strategies and improvements used in the yeast system, following
the authors own words. In 1986, Thim et al. [1986] reported a series of dibasic insulin precursors
including proinsulin was expressed and secreted from Saccharomyces cerevisiae. Recombinant
plasmids were constructed to encode fusion proteins consisting of a modified mating factor a l leader
sequence and an insulin precursor. The leader sequence serves to direct the fusion protein into the
secretory pathway of the cell and to expose it to the Lys-Arg processing enzyme system (i.e. Kex2p
endonuclease). The secreted peptides were purified from the fermentation broth and characterized.
Processing at one or both dibasic sequences was shown in proinsulin and in other insulin precursors
containing a short spacer peptide in place of the C peptide. In contrast, no processing was observed in
the absence of a spacer peptide in the insulin precursor molecule, e.g., B-Lys-Arg-A (where A and B
are the A and B chain of human proinsulin, respectively). This type of single-chain insulin precursors

isolated from such constructions could be enzymatically converted into insulin by treatment with
trypsin and carboxypeptidase B.
Table 15-1. Examples for biopharmaceuticals produced in S. cerevisiae.
Product group
Prokaryotic products
Surface antigens of
viruses

Product
Tetanus toxin fragment C
Streptokinase
Hepatitis B antigen

Foot and mouse disease

Influenza
Polio
Polyoma
Animal products

Human hormones

Hirudin
Porcine interferon
Interleukin
Trypsin inhibitor
Insulin

Parathyroid hormone
Growth hormone
Chorionic gonadotropin
Glucagon
Human growth factors
IGF1
NGF
EGF
CSF
GM-CSF
PDGF
TNF
Human blood proteins
Hemoglobin
Factors VIII and XIII
alpha-1-antitrypsin
Antithrombin III
Serum albumin
Enzymes and further
Urate oxidase
compounds
Transferrin
Hydrocortisone
Ergosterone
Polyketides
r, recombinant; rh, recombinant human.

Commercial name
Ambirix (combination vaccine containing rHBsAg)
HBVAXPRO (rHBsAg)
Twinrix (adult and pediatric forms in EU; combination vaccine
containing rHBsAg)
Infanrix-Hexa (combination vaccine containing rHBsAg)
Hexavac (combination vaccine containing rHBsAG)
Primavax (combination vaccine containing r HBsAg]
Comvax (combination vaccine containing HbsAg]
Recombivax (r HBsAg)
Gardasil (quadrivalent human papillomavirus (HPV) recombinant
vaccine; contains major caspid proteins from four HPV types)

r Hirudin as anticoagulant

Insulin detemir, long-acting rh insulin analog

rh insulin formulated as short-, intermediate- or long-acting product)
Insulin aspart, short-acting rh insulin analog
Valtropin (somatropin, rh GH)
Glucagen (rh glucagon)

Regranex (rh PDGF)

Recombumin
Fasturtec (Elitex in US) (rasburicase; r urate oxidase
Scaffold for protein engineering

One year later, Thim et al. [1987] reported: A yeast expression plasmid encoding a mini-proinsulin
molecule was constructed and transformed into Saccharomyces cerevisiae. The plasmid encoded the
sequence: B-Arg-Arg-Leu-Gln-Lys-Arg-A, in which B represents the B-chain (30 amino acid residues)
and A represents the A-chain (21 amino acid residues) of human insulin. The secreted peptides were
shown to be a mixture of human insulin and des(B-30)human insulin. Thus, correct disulphide bridges
can be established in proinsulin-like molecules devoid of a normal C-peptide region. Furthermore, the
specificity of the yeast processing enzymes is so similar to the proinsulin converting enzymes in the
human pancreatic beta-cell that it allows the processing of the mini-proinsulin to insulin.In 2000,
Kjeldsen [2000] described a further development of the insulin secretory expression system in S.
cerevisiae and its subsequent optimisation. Expression of a cDNA encoding a proinsulin-like molecule
with deletion of threonine(B30) as a fusion protein with the S. cerevisiae alpha-factor prepro-peptide
(leader), followed either by replacement of the human proinsulin C-peptide with a small C-peptide (e.g.
AAK), or by direct fusion of lysine(B29) to glycine(A1), results in the efficient secretion of folded single-

chain proinsulin-like molecules to the culture supernatant. The secreted single-chain insulin precursor
can then be purified and subsequently converted to human insulin by tryptic transpeptidation in
organic aqueous medium in the presence of a threonine ester. The leader confers secretory
competence to the insulin precursor, and the Kex2 endoprotease, specific for dibasic sites, cleaves the
leader-insulin precursor fusion protein in the late secretory pathway and the folded insulin precursor is
secreted to the culture supernatant. However, the Kex2 endoprotease processing of the pro-peptideinsulin precursor fusion protein is incomplete and a significant part of the pro-peptide-insulin precursor
fusion protein is secreted to the culture supernatant in a hyperglycosylated form. A spacer peptide
localised between the leader and the insulin precursor has been developed to optimise Kex2
endoprotease processing and insulin precursor fermentation yield.
To upgrade and optimize the production of insulin and other biopharmaceuticals, high-intensity feedbatch fermentation processes for the production of pharmaceutical-grade proteins in S.cerevisiae have
been developed using automatic feed control, operating to a working volume of 8,000 l. (e.g.,
biopharmaceuticals.novozymes.com).
Extensive strain development and process development were undertaken to establish S.cerevisiae as
an alternative, attractive system for the commercial production of novel biotherapeutics, free from
animal material. It may be mentioned here that the majority of biotherapeutics are produced in bacteria
(E. coli) or in animal cells, such as CHO cells; for a complete list, see [Walsh, 2006].
Recently, gene manipulation techniques established the making of recombinant human serum albumin
(rHSA) or mutants with desirable therapeutic properties and for various protein fusion products. For
example, rHSA can serve as a carrier for synthetic heme protein, thus reversibly carrying oxygen.
Preferential albumin uptake by tumor cells appears applicable for albumin-anticancer drug conjugate
formulation. Furthermore, drug targeting can be achieved by incorporating drugs into albumin
microspheres whereas targeting to liver can be achieved by conjugating drugs with galactosylated or
mannosylated albumin. Microspheres and nanoparticles derived from rHSA can be used for drug
delivery or diagnostic purposes,when loaded with radioisotopes.
Promising results were obtained by in vivo implantation of cells that are encapsulated in HSA-alginate
coated beads and express albumin fusion proteins. Cationized albumin loaded with chimeric peptides
may be used to transport and deliver drugs via receptor mediated transcytosis through the blood brain
barrier. Albumin microbubbles bearing particular genes and at the same time containing ultrasound
contrast agents could be used to non-invasive delivery of gene material after destruction of the
particles by ultrasound. The first product authorized for manufacturing use in human therapeutics is a
commercially available recombinant human albumin, Recombumin. It has been approved for the
production of childhood vaccines for measles, mumps, and rubella.
Similarly interesting is the production of a soluble and active transferrin receptor-targeting single-chain
antibody (OX26 scFv) using recombinant yeast [Hackel et al., 2006]. Yeast-produced scFv has the
potential of targeting and delivery of small molecules, proteins, or drug carriers across the bloodbrain
barrier.

Inspired by the successful work of converting S. cerevisiae into a microorganism capable of

synthesizing hydrocortisone, a 27-carbon molecule, from ethanol, a 2-carbon molecule, Dumas et al.
[2006] provided an overview of the potential of yeast as a recombinant organism in the 21st century.

15.4

Biomedical Research

Today, the use of yeast is undergoing a "rebirth" in both fundamental and applied research. Indeed,
advances in yeast technology have paved the way for a variety of new genome-wide screening
approaches. Experimental strategies using yeast aim to unravel disease-related molecular events and
to discover novel medicinal compounds. The impact of yeast as an experimental tool for diseaserelated studies has been summarized recently [Mager & Winderickx, 2005] and the use of yeast in
high-throughput screenings for pharmacological purposes has been evaluated. A promising approach
is the so-called approach of humanized yeast systems.
Yeast also paved the way for the systematic analysis of large and complex genomes by serving as a
test bed for novel experimental approaches and technologies, tools that are fast becoming the
standard in drug discovery research, and yeast has improved our understanding and facilitated the
molecular analysis of many disease genes. As a non-pathogenic model of fungal pathogens it can be
used to identify antifungal targets suitable for drug development and to elucidate mechanisms of
action of antifungal agents [Ma, 2001].

15.5

Cell-surface Display - Environmental Technologies

For some time, yeast research has been devoted to the construction of so-called arming yeasts, using
cell-surface engineering systems that will allow to display biocatalysts in the form of enzymes,
functional proteins, antibodies, and combinatorial protein libraries [Ueda & Tanaka, 2000; Kondo &
Ueda, 2004]. Among the many advantages of the system, in which proteins are genetically displayed
on the cell surface, are easy reproduction of the biocatalysts and easy separation of product from
catalyst. One such system was tailored by coupling the gene encoding the target protein with the
secretion signal to the gene encoding the C-terminal half of -agglutinin and containing the putative
glycosylphosphatidylinositol (GPI) anchor attachment signal sequence. Rhizopus oryzae
glucoamylase, Bacillus stearothermophilus -amylase, Aspergillus aculeatus -amylase, CMcellulase, and most promising, Rhizopus oryzae lipase (ROL) were coupled to -agglutinin and shown
to be active in appropriate constructs. Cells surface- engineered with glucoamylase or -amylase
could grow on starch as sole carbon source. Cells armed with CM-cellulase and/or -amylase were
capable of using cellulosic material as carbon sources. A second arming system was developed
[Matsumoto et al., 2002] by using the FLO1 gene encoding a lectin-like cell-wall protein (Flo1p) in
Saccharomyces cerevisiae. Flo1p is composed of several domains: the secretion signal domain, the
flocculation functional domain, the GPI anchor attachment signal domain, and the membraneanchoring domain. In the experiments part of the 3-region of the FLO1 gene was fused to a pro
sequence from Rhizopus oryzae lipase (rProROL), which has its active site near the C terminus. This
recombinant lipase is displayed on the cell surface and is capable of biodiesel (methylesters
synthesized from natural triglycerides, FAME) production in a solvent-free and water-containing
system.

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