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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 E. coli, yeast and mammalian cells are the main cell
the biopharmaceutical industry. It is therefore desirable to factories for production of nearly all therapeutic proteins,
develop yeast platform strains for over-production of various with a distribution of 68% and 32% in the quantity of the
biopharmaceutical proteins, but this requires fundamental market needs in 2010 using microbial and mammalian
knowledge of the cellular machinery, especially the protein production, respectively [2]. Among these cell factories,
secretory pathway. Integrated analyses of multi-omics yeast is employed for production of a wide range of
datasets can provide comprehensive understanding of cellular proteins due to its ability to perform proper protein
function, and can enable systems biology-driven and processing and secrete recombinant proteins to the extra-
mathematical model-guided strain engineering. Rational cellular medium. Further advantages are that yeast can
engineering and introduction of trackable genetic modifications grow on inexpensive chemically defined media and there
using synthetic biology tools, coupled with high-throughput are well established fermentative technologies [3]. Yeast
screening are, however, also efficient approaches to relieve is used for production of about 1/6th of the biopharma-
bottlenecks hindering high-level protein production. Here we ceuticals approved for human use [2], but it plays a
review advances in systems biology and metabolic engineering particular dominant role for production of insulin analogs
of yeast for improving recombinant protein production. (bulk component of microbial production) and hepatitis
vaccines (Table 1, [4]).

A major limitation on using yeasts is its inappropriate


Addresses
glycosylation capabilities, but through engineering of the
1
Department of Biology and Biological Engineering, Chalmers University glycosylation and sialylation processes the native high-
of Technology, SE41296 Gothenburg, Sweden mannose type N-glycosylation can be abolished, and
2
Novo Nordisk Foundation Center for Biosustainability, Chalmers hereby confer yeasts the capability to produce recombi-
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 such as active antibodies [5–7]. This would promote
the use of yeasts going beyond the application in produc-
Corresponding author: Nielsen, Jens (nielsenj@chalmers.se) tion of relatively simple proteins (Table 2) and as tools for
*
Both authors contributed equally to this work. antibody assessment through surface display [8], laying
the pavement for a broader applications as cell factory for
Current Opinion in Biotechnology 2017, 48:77–84
diverse biopharmaceuticals.
This review comes from a themed issue on Pharmaceutical
biotechnology
Edited by Chu-Young Kim and Tiangang Liu
Improve the titer, rate and yield (TRY) through
rational metabolic engineering
For a complete overview see the Issue and the Editorial
Besides proper biological activity of the recombinant
Available online 11th April 2017 protein, the TRY of the yeast cell factory are the most
http://dx.doi.org/10.1016/j.copbio.2017.03.017 important for the biopharmaceutical industry. Secreted
0958-1669/ã 2017 Elsevier Ltd. All rights reserved. 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
Introduction secretory machinery may not be competent to handle a
With the development of genetic engineering, Genen- high flux of proteins that may require a specific post-
tech developed a process for production of recombinant translational modification. This can result in mis-sorting
human insulin by Escherichia coli, and this was marketed where the heterologous protein instead of being secreted
by Eli Lilly in 1982 as the first biopharmaceutical. Since is targeted to the vacuole for degradation, reducing pro-
then, biotechnology based pharmaceutical production has tein secretion (Figure 1). Optimization of heterologous
grown rapidly and formed a global biopharmaceuticals protein expression and metabolic engineering of the
market of USD176.9B in 2015 and an expected annual secretory pathway therefore needs to be rationally con-
growth rate of 8.6% till 2021 [1]. sidered in order to obtain maximum protein titers.

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

www.sciencedirect.com Current Opinion in Biotechnology 2017, 48:77–84


80 Pharmaceutical biotechnology

of intracellular transport. Alternation in the leader pep- efficient platform strains necessitates a comprehensive
tide significantly affected the protein secretion of insulin understanding of the bottlenecks in the secretory machin-
precursor, a-amylase and b-galactosidase in ery, even the network of the whole cell factory, as this can
S. cerevisiae [18,22], and sole optimization of the leader then form the basis for rational metabolic engineering
peptide could improve the secretion of a single-chain (design-build-test-learn cycles). Systems biology enables
antibody fragment by 16-fold [23]. Heterologous proteins analysis of the cellular behavior at system level by inte-
can be mis-sorted to the vacuole with the help of vacuolar gration of large scale datasets (-omics) with mathematical
sorting proteins (VPS), manipulation of which also served modeling. Using mathematical models for integrative
as an effective strategy for productivity improvement of analysis it is possible to obtain a quantitative description
heterologous protein: disruptions of VPS10 and PRB1 of cellular processes, and this can be used for rational
(vacuolar protease B) elevated the Fab production more engineering of cell factories with predictable behavior
than twofold in P. pastoris [24]. Besides the engineering (Figure 2).
on the intracellular protein transport direction, disruption
of the endocytosis complex on the plasma membrane The rapid development of systems biology technologies
responsible for protein uptake from the extracellular including sequencing and mass spectrometry has
matrix also improved the heterologous protein titer in advanced our understanding of the cellular machinery
S. cerevisiae [25]. at a quantitative level. Transcription and translation
processes are prerequisite activities for polypeptide syn-
Heterologous protein expression and secretion employ thesis and subsequent protein processing and secretion,
the secretory pathway and hereby competes with endog- and efficient polypeptide synthesis is therefore important
enous proteins that have to be processed through this for high level production of heterologous proteins. Quan-
pathway, and depending on requirements for post-trans- tifying the rate of transcription and translation processes
lational modifications this may cause limitations in is thus of significance. Computational models of transla-
protein secretion (Figure 1). Engineering of the protein tion based on sequencing data, covering information of
secretory pathway with the objective to overcome limita- ribosomes, tRNA and mRNA, conveyed that translation is
tions has therefore proven to be an efficient approach to generally limited by initiation, and fast initiation or high
increase protein production. Single or combinatorial gene codon bias in a transgene increased its protein production
overexpression of ER stress response components (Hsf1p, [31]. In addition, protein production in yeast cells might
Gcn4p) [26,27], protein disulfide isomerase (Pdi1p), chap- be typically limited by the availability of free ribosomes
erone (Kar2p), co-translocation components (Srp14p, under specific conditions [31,32]. Ribosome profiling is an
Srp54p) [28], ER-to-Golgi SNAREs (soluble N-ehyla- approach that enables genome-wide analysis of mRNA
maleimide-sensitive factor attachment receptor proteins) bound to ribosomes, hence reflecting actively translated
(Bos1p, Bet1p, Sec22p, Sed5p) [29], regulator of Golgi-to- mRNAs; this approach enables calculation of the protein
cell membrane SNAREs (Sec1p) [30] have to some extent synthesis rates and cellular resource distribution [33].
contributed to improvements in heterologous protein With the combination of this approach and mRNA abun-
production. dance analysis, slower translation in the beginning of
coding regions and codons matching rare tRNAs was
Whereas engineering a specific secretory process may found to take place in S. cerevisiae [34].
improve the productivity of a given heterologous protein,
this engineering strategy may not generally improve Besides quantifying translation rate, gene expression and
production of other proteins, as distinct secretory bottle- protein synthesis in the cell can be quantitatively
necks exist for specific proteins (Figure 1). It is therefore reflected by transcriptome analysis and quantitative
desirable to develop yeast cell platform strains that have proteomics. These kind of data helps to understand
high capacity for protein secretion for a range of biophar- the cellular response to perturbations such as alternation
maceutical proteins, even though specific optimization in gene expression and environmental changes, and
may be further needed for each protein. hereby unravel the underlying mechanisms associated
with the cellular objective of maintaining homeostasis,
Systems biology guided improvements of often referred to as biological robustness [35–38]. This
protein production can also provide potential targets for strain engineering
Although engineering the secretory pathway has proven [39].
to be a direct and efficient approach to improve heterolo-
gous protein production, the limitation factors for protein Protein production is costly in terms of high demand for
production are not only confined in the secretory path- energy- and building blocks, and heterologous protein
way, but also related to transcription and translation production competes for cellular resources and therefore
capacity and efficiency, the central metabolic network alters the intracellular metabolism, which often causes a
providing energy and building blocks, the redox metabolic burden [40]. Therefore, efficient protein
homeostasis and so on. Hence the construction of 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 multi-level information and computational tools provide
precursors, as well as enabling a balanced transfer of these support for the management and analysis of large-scale-
to the target proteins. Manipulation of cellular growth rate omics datasets. Mathematical modeling can give more
[37,41], redirecting the metabolic flux [42] and engineer- precise descriptions about cellular behavior with integra-
ing the oxygen sensing pathway [10] were proven to tion of different-omics data [44]; hence, this approach can
stimulate the productivity of heterologous proteins, dem- be utilized to unravel the underlying mechanism and
onstrating the significance of optimizing energy metabo- predict the phenotype of a modified biological system
lism. Metabolome and fluxome analysis, quantitatively (e.g., improved heterologous protein production) for
reflects metabolism and can provide information about rational engineering. Through engineering of the targets
how the cell responds to different cellular perturbations. predicted by genome-scale metabolic modeling, the
Furthermore, it also helps to discern potential bottle- production of human copper/zinc superoxide dismutase
necks that need to be overcome for improving protein (hSOD) was improved in P. pastoris by modifying the
production [43]. metabolic fluxes [45]. In another study a genome-scale
metabolic model for P. pastoris was expanded to describe
Mathematical model guided strain protein glycosylation, and hereby it was possible to
engineering simulate the impact of glycosylation on the yield of
Comprehensive understanding of cellular functions and different proteins [46]. Feizi et al. reported a detailed
accurate phenotype prediction requires integration of model that was reconstructed for the protein secretory

www.sciencedirect.com Current Opinion in Biotechnology 2017, 48:77–84


82 Pharmaceutical biotechnology

pathway in S. cerevisiae [47] and even though this model Conclusion


could not be used for simulation of protein secretion it Even though there has been much progress in improving
represent the first reconstruction of this complex pathway heterologous protein production by yeast it is still neces-
for a eukaryal cell. Furthermore, as the secretory process sary to obtain more fundamental insight into the complex
remarkably affects the final yield and bioactivity of the cellular machinery especially the protein secretory path-
protein product, such models can aid in gaining under- way. For this systems biology analysis may allow to obtain
standing of the cellular activities related to protein secre- a holistic overview and a more comprehensive under-
tion, and as an essential part of the whole yeast model it standing of the cellular machinery, and hereby enable the
may contribute to the accuracy of predicting limitations in construction of yeast platform strains that can be used for
secreting heterologous proteins by yeast. high-level production of a range of biopharmaceutical
proteins. Furthermore, based on such detailed under-
High-throughput screening towards strains standing it will be possible to further engineer such a
with improved protein production capability cell factory platform using rational metabolic engineering
A genome scale model managing all cellular information to relieve the bottlenecks hindering efficient secretion of
would have great application in design of cell factories specific proteins.
with improved protein secretion. However, the accuracy
of predicting cellular behavior at this stage is still chal- With the help of mathematical modeling, systems biology
lenging, though prediction has proven effective based on analysis also make it possible to identify key nodes or
analysis at specific levels (e.g., metabolic flux). The main pathways that are important for driving high-level protein
reason for this is that we still lack much knowledge about production. Furthermore, introducing trackable perturba-
many cellular processes. Introducing controlled pertur- tions (irrational or semi-rational build) and high-through-
bation is therefore often used to get new insight into the put screening (directed test) can assist in identifying
function of biological processes. Here high-throughput targets for strain engineering and perceive the native
sequencing coupling with adaptive laboratory evolution complex cellular network through the system-level anal-
can be used to rapidly identify causal mutations under- ysis (learn). Hereby identified metabolic engineering
lying specific phenotypes [48]. Identified target genes targets can be easily applied towards host optimization
can then potentially be used as targets for inverse by means of powerful genetic tools developed through
metabolic engineering and enable the attainment of synthetic biology. It is hereby expected that it will be
strains carrying specific mutations associated with the possible in the future to significantly improve the pro-
desired phenotype. duction capacity for biopharmaceutical proteins by yeast.

This concept is also applicable for characterizing strains Acknowledgements


with improved protein production [49], but was somehow
We would like to thank Prof. Dina Petranovic for suggestions and
hampered by the throughput of identifying mutants, comments on this paper. We acknowledge funding from the Novo Nordisk
since growth-coupled enrichment was not feasible. With Foundation, the Knut and Alice Wallenberg Foundation and FORMAS.
the establishment of high throughput microfluidic droplet
screening, mutants with higher protein yield could be References and recommended reading
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sequencing [51]. Information obtained from the have been highlighted as:
mutants helped to perceive the complex cellular network  of special interest
related with protein production, and the identified targets  of outstanding interest
are promising for strain engineering to improve protein
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84 Pharmaceutical biotechnology

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