Available online at www.sciencedirect.
com
ScienceDirect
Exploring the potential of Saccharomyces cerevisiae for
biopharmaceutical protein production
Guokun Wang1,*, Mingtao Huang1,2,* and Jens Nielsen1,2,3
Production of recombinant proteins by yeast plays a vital role in
the biopharmaceutical industry. It is therefore desirable to
develop yeast platform strains for over-production of various
biopharmaceutical proteins, but this requires fundamental
knowledge of the cellular machinery, especially the protein
secretory pathway. Integrated analyses of multi-omics
datasets can provide comprehensive understanding of cellular
function, and can enable systems biology-driven and
mathematical model-guided strain engineering. Rational
engineering and introduction of trackable genetic modifications
using synthetic biology tools, coupled with high-throughput
screening are, however, also efficient approaches to relieve
bottlenecks hindering high-level protein production. Here we
review advances in systems biology and metabolic engineering
of yeast for improving recombinant protein production.
Addresses
1
Department of Biology and Biological Engineering, Chalmers University
of Technology, SE41296 Gothenburg, Sweden
2
Novo Nordisk Foundation Center for Biosustainability, Chalmers
University of Technology, SE41296 Gothenburg, Sweden
3 nant proteins with humanized glycosylation patterns,
Novo Nordisk Foundation Center for Biosustainability, Technical
University of Denmark, DK2970 Hørsholm, Denmark
Corresponding author: Nielsen, Jens (nielsenj@chalmers.se)
*
Both authors contributed equally to this work.
Current Opinion in Biotechnology 2017, 48:77–84
This review comes from a themed issue on Pharmaceutical
biotechnology
Edited by Chu-Young Kim and Tiangang Liu
For a complete overview see the Issue and the Editorial
Available online 11th April 2017
http://dx.doi.org/10.1016/j.copbio.2017.03.017
0958-1669/ã 2017 Elsevier Ltd. All rights reserved.
Introduction
With the development of genetic engineering, Genen-
tech developed a process for production of recombinant
human insulin by Escherichia coli, and this was marketed
by Eli Lilly in 1982 as the first biopharmaceutical. Since
then, biotechnology based pharmaceutical production has
grown rapidly and formed a global biopharmaceuticals
market of USD176.9B in 2015 and an expected annual
growth rate of 8.6% till 2021 [1].
www.sciencedirect.com
E. coli, yeast and mammalian cells are the main cell
factories for production of nearly all therapeutic proteins,
with a distribution of 68% and 32% in the quantity of the
market needs in 2010 using microbial and mammalian
production, respectively [2]. Among these cell factories,
yeast is employed for production of a wide range of
proteins due to its ability to perform proper protein
processing and secrete recombinant proteins to the extra-
cellular medium. Further advantages are that yeast can
grow on inexpensive chemically defined media and there
are well established fermentative technologies [3]. Yeast
is used for production of about 1/6th of the biopharma-
ceuticals approved for human use [2], but it plays a
particular dominant role for production of insulin analogs
(bulk component of microbial production) and hepatitis
vaccines (Table 1, [4]).
A major limitation on using yeasts is its inappropriate
glycosylation capabilities, but through engineering of the
glycosylation and sialylation processes the native high-
mannose type N-glycosylation can be abolished, and
hereby confer yeasts the capability to produce recombi-
such as active antibodies [5–7]. This would promote
the use of yeasts going beyond the application in produc-
tion of relatively simple proteins (Table 2) and as tools for
antibody assessment through surface display [8], laying
the pavement for a broader applications as cell factory for
diverse biopharmaceuticals.
Improve the titer, rate and yield (TRY) through
rational metabolic engineering
Besides proper biological activity of the recombinant
protein, the TRY of the yeast cell factory are the most
important for the biopharmaceutical industry. Secreted
proteins are preferable for recombinant protein produc-
tion as this facilitates isolation and purification. The
secretory pathway, involving translocation, protein mod-
ification, and vesicle mediated transports, represents a
complex system which has tight quality control on the
destiny of processed proteins. However, the native
secretory machinery may not be competent to handle a
high flux of proteins that may require a specific post-
translational modification. This can result in mis-sorting
where the heterologous protein instead of being secreted
is targeted to the vacuole for degradation, reducing pro-
tein secretion (Figure 1). Optimization of heterologous
protein expression and metabolic engineering of the
secretory pathway therefore needs to be rationally con-
sidered in order to obtain maximum protein titers.
Current Opinion in Biotechnology 2017, 48:77–84
78 Pharmaceutical biotechnology

Table 1

Yeast-derived biopharmaceuticals approved for human use

Protein Company System Therapeutic application

Blood factors
Human albumin (HA) Albumedix S. cerevisiae Stabilizer for the formulation of
biological drugs and vaccines
Factor XIII A-subunit Novo Nordisk S. cerevisiae Congenital factor XIII A-subunit
deficiency
Tissue plasminogen activator
Hirudin Bayer Healthcare S. cerevisiae Anticoagulation therapy for heparin
associated
Hirudin Canyon Pharmaceuticals S. cerevisiae Prevention of venous thrombosis
Truncated form of human plasmin ThromboGenics P. pastoris Symptomatic vitreomacular
adhesion/vitreomacular traction
Plasma kallikrein inhibitor Dyax P. pastoris Hereditary angioedema
Recombinant hormones
Insulin aspart/insulin detemir/insulin Novo Nordisk S. cerevisiae Diabetes mellitus
degludec
Growth hormone (GH)/biosimilar GH BioPartners S. cerevisiae Growth failure/growth hormone
deficiency
Glucagon-like peptide1 analog with Novo Nordisk S. cerevisiae Type 2 diabetes
attached fatty acid
Glucagon Novo Nordisk S. cerevisiae Hypoglycemia
Platelet derived growth factor-BB Raritan S. cerevisiae Lower extremity diabetic
neuropathiculcers
Recombinant vaccines
HBsAg Merck/GlaxoSmithKline/Sanofi S. cerevisiae/H. polymorpha Immunization against hepatitis B
Human papilloma virus vaccine Sanofi-Pasteur/Lyon/Merck S. cerevisiae Vaccination against diseases
caused by HPV
Recombinant enzymes
Urate oxidase Sanofi S. cerevisiae Hyperuricemia
Fusion protein
GLP-1 receptor agonist (GLP-1-HA) GlaxoSmithKline S. cerevisiae Type 2 diabetes

Data are adapted from Ref. [2] and newly collected information. Here listed are the recombinant therapeutic proteins approved before 2014.

For driving heterologous gene expression at various levels

containing heterologous protein-encoding genes. Expres-
and at distinct fermentation time-points, constitutive
sion of genes that repair auxotrophy using a truncated
strong glycolytic promoters (PGK1p, TPI1p, ADH1p)
promoter or using the protein fused with ubiquitin/N-
and inducible promoters (galactose inducible GAL1p,
degron tag remarkably reduce expression or function of
GAL7p, GAL10p; vitamin-sensitive THI11p) have been
the marker protein, and therefore drives the copy number
developed for Saccharomyces cerevisiae and Pichia pastoris
of the plasmid to a high level in order to sustain growth
[15,16]. In addition, a range of selection markers have
under selective synthetic condition [17]. Thus, the utili-
been employed or engineered for the purpose of main-
zation of heterologous triose-phosphate isomerase (TPI)
taining the stability and high copy number of plasmids

Table?2

Overview of some therapeutic proteins under development produced by yeast

Protein System Titer Localization a Refs.

Antithrombin III S. cerevisiae 320 mg/L In [9]
Human hemoglobin S. cerevisiae >7% of the total cell protein In [10]
Human serum albumin P. pastoris 10 g/L S [11]
Interleukin-2-HSA fusion protein P. pastoris 520 mg/L S [12]
Trastuzumab, anti-human HER II antibody P. pastoris 10 mg/L S [13]
Human erythropoietin with terminally sialylated biantennary N-glycan structures P. pastoris 50 mg/L S [14]
a
In: intracellular; S: secreted.

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 Biopharmaceutical protein production by yeast Wang, Huang and Nielsen 79

Figure 1

Building blocks & cofactors

Transcription Translation Folding & Mis- Vesicle

modification sorting mediated
transport

Building blocks
&
cofactors

Capacity handling specific proteins Metabolic/protein flux towards secreted target proteins Distinct target proteins

Current Opinion in Biotechnology

Constraints of the native cell machinery limiting production of distinct heterologous proteins.
Building blocks from cellular metabolism or environment need go through the protein synthesis (transcription and translation) and secretory
pathway (protein folding, modification, sorting and transport) to form the active secreted proteins. The incompetence of the native cell machinery
to handle precursors of matured proteins at specific processes (bottlenecks) hindered the efficient protein production, depending on the proteins’
characters, for example, amino acid composition, molecular weight, modifications essential for activity.

with a weakened function, Pot1p from Schizosaccharomyces
prombe, in a Tpi1p deficient S. cerevisiae strain increases
the plasmid copy number to sustain rapid growth on
glucose, adaptable even using rich medium [18]. This
system was introduced, and patented, by Novo Nordisk
for production of their first recombinant human insulin,
and has since then been used extensively for industrial
production of insulin and insulin analogs.
Multiple genome integration also serves as an ideal
approach for generating stable strains with high copy
numbers of heterologous genes. The utilization of engi-
neered markers described above in combination with a
non-transcribed space (NTS) and ribosomal DNA [19] or
long terminal repeats of Ty retrotransposons/delta
sequence [20,21] enabled multi-copy chromosomal
integration of the capsid protein of red-spotted grouper
necrosis virus, fluorescent protein and even whole bio-
chemical pathways.
Whereas expression is important for high-level protein
production, it is often more important to engineer the
protein secretory pathway. This was very well illustrated
in a comparative study of production of insulin and a
fungal a-amylase, where insulin production was found to
be expression limited whereas production of a-amylase
was optimal for low level expression where protein
synthesis is better coordinated with secretion [18].
Manipulation of the protein secretory pathway is there-
fore also important for enhancing protein production. The
leader signal peptide determines that the protein enter
the endoplasmic reticulum (ER) and the further targeting

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80 Pharmaceutical biotechnology
of intracellular transport. Alternation in the leader pep-
tide significantly affected the protein secretion of insulin
precursor, a-amylase and b-galactosidase in
S. cerevisiae [18,22], and sole optimization of the leader
peptide could improve the secretion of a single-chain
antibody fragment by 16-fold [23]. Heterologous proteins
can be mis-sorted to the vacuole with the help of vacuolar
sorting proteins (VPS), manipulation of which also served
as an effective strategy for productivity improvement of
heterologous protein: disruptions of VPS10 and PRB1
(vacuolar protease B) elevated the Fab production more
than twofold in P. pastoris [24]. Besides the engineering
on the intracellular protein transport direction, disruption
of the endocytosis complex on the plasma membrane
responsible for protein uptake from the extracellular
matrix also improved the heterologous protein titer in
S. cerevisiae [25].
Heterologous protein expression and secretion employ
the secretory pathway and hereby competes with endog-
enous proteins that have to be processed through this
pathway, and depending on requirements for post-trans-
lational modifications this may cause limitations in
protein secretion (Figure 1). Engineering of the protein
secretory pathway with the objective to overcome limita-
tions has therefore proven to be an efficient approach to
increase protein production. Single or combinatorial gene
overexpression of ER stress response components (Hsf1p,
Gcn4p) [26,27], protein disulfide isomerase (Pdi1p), chap-
erone (Kar2p), co-translocation components (Srp14p,
Srp54p) [28], ER-to-Golgi SNAREs (soluble N-ehyla-
maleimide-sensitive factor attachment receptor proteins)
(Bos1p, Bet1p, Sec22p, Sed5p) [29], regulator of Golgi-to-
cell membrane SNAREs (Sec1p) [30] have to some extent
contributed to improvements in heterologous protein
production.
Whereas engineering a specific secretory process may
improve the productivity of a given heterologous protein,
this engineering strategy may not generally improve
production of other proteins, as distinct secretory bottle-
necks exist for specific proteins (Figure 1). It is therefore
desirable to develop yeast cell platform strains that have
high capacity for protein secretion for a range of biophar-
maceutical proteins, even though specific optimization
may be further needed for each protein.
Systems biology guided improvements of
protein production
Although engineering the secretory pathway has proven
to be a direct and efficient approach to improve heterolo-
gous protein production, the limitation factors for protein
production are not only confined in the secretory path-
way, but also related to transcription and translation
capacity and efficiency, the central metabolic network
providing energy and building blocks, the redox
homeostasis and so on. Hence the construction of
Current Opinion in Biotechnology 2017, 48:77–84
efficient platform strains necessitates a comprehensive
understanding of the bottlenecks in the secretory machin-
ery, even the network of the whole cell factory, as this can
then form the basis for rational metabolic engineering
(design-build-test-learn cycles). Systems biology enables
analysis of the cellular behavior at system level by inte-
gration of large scale datasets (-omics) with mathematical
modeling. Using mathematical models for integrative
analysis it is possible to obtain a quantitative description
of cellular processes, and this can be used for rational
engineering of cell factories with predictable behavior
(Figure 2).
The rapid development of systems biology technologies
including sequencing and mass spectrometry has
advanced our understanding of the cellular machinery
at a quantitative level. Transcription and translation
processes are prerequisite activities for polypeptide syn-
thesis and subsequent protein processing and secretion,
and efficient polypeptide synthesis is therefore important
for high level production of heterologous proteins. Quan-
tifying the rate of transcription and translation processes
is thus of significance. Computational models of transla-
tion based on sequencing data, covering information of
ribosomes, tRNA and mRNA, conveyed that translation is
generally limited by initiation, and fast initiation or high
codon bias in a transgene increased its protein production
[31]. In addition, protein production in yeast cells might
be typically limited by the availability of free ribosomes
under specific conditions [31,32]. Ribosome profiling is an
approach that enables genome-wide analysis of mRNA
bound to ribosomes, hence reflecting actively translated
mRNAs; this approach enables calculation of the protein
synthesis rates and cellular resource distribution [33].
With the combination of this approach and mRNA abun-
dance analysis, slower translation in the beginning of
coding regions and codons matching rare tRNAs was
found to take place in S. cerevisiae [34].
Besides quantifying translation rate, gene expression and
protein synthesis in the cell can be quantitatively
reflected by transcriptome analysis and quantitative
proteomics. These kind of data helps to understand
the cellular response to perturbations such as alternation
in gene expression and environmental changes, and
hereby unravel the underlying mechanisms associated
with the cellular objective of maintaining homeostasis,
often referred to as biological robustness [35–38]. This
can also provide potential targets for strain engineering
[39].
Protein production is costly in terms of high demand for
energy- and building blocks, and heterologous protein
production competes for cellular resources and therefore
alters the intracellular metabolism, which often causes a
metabolic burden [40]. Therefore, efficient protein
production also requires optimization of cellular
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 Biopharmaceutical protein production by yeast Wang, Huang and Nielsen 81

Figure 2

Fluxomics

Modeling
Fluxes
Genomics Transcriptomics Proteomics Metabolomics
O O
N O

H
O
OH H C C O=C
3
HN NH2 OH
O OH

O
OH
N NH2
H HO
DNA mRNA Proteins Metabolites

Mutagenesis Rational
Evolution engineering

Trackable
perturbations

Substrates Substrates Substrates

Synthetic biology tools
Current Opinion in Biotechnology

Systems biology- and metabolic engineering-aided potential exploring of yeast cell factory for protein production.
Rational engineering, mutagenesis, directed evolution and screening of strains with trackable perturbations are approaches generating yeast
strains with improved protein productivity. Mutant/evolved/screened strains, engineered strains and wild-type strain can be comprehensively
analyzed by the integration of different-omics datasets to reveal underlying mechanisms and principles. These knowledge helps to understand the
cellular networks deeper thus enhance the accuracy accordingly of model-guided strain engineering from a system level. Meanwhile, key nodes or
pathways can be identified for a new round of rational/semi-rational engineering of strains for protein production.
Numbers in red color indicate the potential bottlenecks: (1) energy and building blocks, (2) transcription, (3) translation, (4) protein folding, (5) post-
modification, (6) trafficking.

metabolism to ensure sufficient supply of energy and
precursors, as well as enabling a balanced transfer of these
to the target proteins. Manipulation of cellular growth rate
[37,41], redirecting the metabolic flux [42] and engineer-
ing the oxygen sensing pathway [10] were proven to
stimulate the productivity of heterologous proteins, dem-
onstrating the significance of optimizing energy metabo-
lism. Metabolome and fluxome analysis, quantitatively
reflects metabolism and can provide information about
how the cell responds to different cellular perturbations.
Furthermore, it also helps to discern potential bottle-
necks that need to be overcome for improving protein
production [43].
Mathematical model guided strain
engineering
Comprehensive understanding of cellular functions and
accurate phenotype prediction requires integration of
multi-level information and computational tools provide
support for the management and analysis of large-scale-
omics datasets. Mathematical modeling can give more
precise descriptions about cellular behavior with integra-
tion of different-omics data [44]; hence, this approach can
be utilized to unravel the underlying mechanism and
predict the phenotype of a modified biological system
(e.g., improved heterologous protein production) for
rational engineering. Through engineering of the targets
predicted by genome-scale metabolic modeling, the
production of human copper/zinc superoxide dismutase
(hSOD) was improved in P. pastoris by modifying the
metabolic fluxes [45]. In another study a genome-scale
metabolic model for P. pastoris was expanded to describe
protein glycosylation, and hereby it was possible to
simulate the impact of glycosylation on the yield of
different proteins [46]. Feizi et al. reported a detailed
model that was reconstructed for the protein secretory

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82 Pharmaceutical biotechnology
pathway in S. cerevisiae [47] and even though this model
could not be used for simulation of protein secretion it
represent the first reconstruction of this complex pathway
for a eukaryal cell. Furthermore, as the secretory process
remarkably affects the final yield and bioactivity of the
protein product, such models can aid in gaining under-
standing of the cellular activities related to protein secre-
tion, and as an essential part of the whole yeast model it
may contribute to the accuracy of predicting limitations in
secreting heterologous proteins by yeast.
High-throughput screening towards strains
with improved protein production capability
A genome scale model managing all cellular information
would have great application in design of cell factories
with improved protein secretion. However, the accuracy
of predicting cellular behavior at this stage is still chal-
lenging, though prediction has proven effective based on
analysis at specific levels (e.g., metabolic flux). The main
reason for this is that we still lack much knowledge about
many cellular processes. Introducing controlled pertur-
bation is therefore often used to get new insight into the
function of biological processes. Here high-throughput
sequencing coupling with adaptive laboratory evolution
can be used to rapidly identify causal mutations under-
lying specific phenotypes [48]. Identified target genes
can then potentially be used as targets for inverse
metabolic engineering and enable the attainment of
strains carrying specific mutations associated with the
desired phenotype.
This concept is also applicable for characterizing strains
with improved protein production [49], but was somehow
hampered by the throughput of identifying mutants,
since growth-coupled enrichment was not feasible. With
the establishment of high throughput microfluidic droplet
screening, mutants with higher protein yield could be
readily discerned [50] and analyzed via whole-genome
sequencing [51]. Information obtained from the
mutants helped to perceive the complex cellular network
related with protein production, and the identified targets
are promising for strain engineering to improve protein
production (Figure 2, [51]).
Developments in the field of genome editing and multi-
plexing made it possible to readily introduce trackable
perturbations to the yeast cell, for example, using
CRISPR-based interference [52] and RNA interference
[53]. As there is direct causality with the phenotype, such
perturbations are advantageous for rapidly identifying
functional targets compared with mutagenesis, screening
and genome sequencing. The functional perturbations
confirmed through high-throughput screening and -omics
analysis would definitely contribute to the rational strain
engineering cycle and hereby enable engineering yeast
strains with much improved protein production
(Figure 2).
Current Opinion in Biotechnology 2017, 48:77–84
Conclusion
Even though there has been much progress in improving
heterologous protein production by yeast it is still neces-
sary to obtain more fundamental insight into the complex
cellular machinery especially the protein secretory path-
way. For this systems biology analysis may allow to obtain
a holistic overview and a more comprehensive under-
standing of the cellular machinery, and hereby enable the
construction of yeast platform strains that can be used for
high-level production of a range of biopharmaceutical
proteins. Furthermore, based on such detailed under-
standing it will be possible to further engineer such a
cell factory platform using rational metabolic engineering
to relieve the bottlenecks hindering efficient secretion of
specific proteins.
With the help of mathematical modeling, systems biology
analysis also make it possible to identify key nodes or
pathways that are important for driving high-level protein
production. Furthermore, introducing trackable perturba-
tions (irrational or semi-rational build) and high-through-
put screening (directed test) can assist in identifying
targets for strain engineering and perceive the native
complex cellular network through the system-level anal-
ysis (learn). Hereby identified metabolic engineering
targets can be easily applied towards host optimization
by means of powerful genetic tools developed through
synthetic biology. It is hereby expected that it will be
possible in the future to significantly improve the pro-
duction capacity for biopharmaceutical proteins by yeast.
Acknowledgements
We would like to thank Prof. Dina Petranovic for suggestions and
comments on this paper. We acknowledge funding from the Novo Nordisk
Foundation, the Knut and Alice Wallenberg Foundation and FORMAS.
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