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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]).
Table 1
Data are adapted from Ref. [2] and newly collected information. Here listed are the recombinant therapeutic proteins approved before 2014.
Table?2
Figure 1
Building blocks
&
cofactors
Capacity handling specific proteins Metabolic/protein flux towards secreted target proteins Distinct target proteins
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
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
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
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
6. Tiels P, Baranova E, Piens K, De Visscher C, Pynaert G, 21. Shi S, Liang Y, Zhang MM, Ang EL, Zhao H: A highly efficient
Nerinckx W, Stout J, Fudalej F, Hulpiau P, Tannler S et al.: A single-step, markerless strategy for multi-copy chromosomal
bacterial glycosidase enables mannose-6-phosphate integration of large biochemical pathways in Saccharomyces
modification and improved cellular uptake of yeast-produced cerevisiae. Metab. Eng. 2016, 33:19-27.
recombinant human lysosomal enzymes. Nat. Biotechnol. 2012,
30:1225-1231. 22. Mori A, Hara S, Sugahara T, Kojima T, Iwasaki Y, Kawarasaki Y,
Sahara T, Ohgiya S, Nakano H: Signal peptide optimization tool
7. Hamilton SR, Davidson RC, Sethuraman N, Nett JH, Jiang YW, for the secretion of recombinant protein from Saccharomyces
Rios S, Bobrowicz P, Stadheim TA, Li HJ, Choi BK et al.: cerevisiae. J. Biosci. Bioeng. 2015, 120:518-525.
Humanization of yeast to produce complex terminally
sialylated glycoproteins. Science 2006, 313:1441-1443. 23. Rakestraw JA, Sazinsky SL, Piatesi A, Antipov E, Wittrup KD:
Directed evolution of a secretory leader for the improved
8. Tillotson BJ, Cho YK, Shusta EV: Cells and cell lysates: a direct expression of heterologous proteins and full-length
approach for engineering antibodies against membrane antibodies in Saccharomyces cerevisiae. Biotechnol. Bioeng.
proteins using yeast surface display. Methods 2013, 60:27-37. 2009, 103:1192-1201.
9. Mallu MR, Vemula S, Ronda SR: Production, purification and 24. Nishiyama T, Sakai Y: Method for producing heterologous
characterization of recombinant human antithrombin III by protein using yeast with disruption of VPS gene. EP Patent
Saccharomyces cerevisiae. Electron. J. Biotechnol. 2016, 22: 2015, EP2684948 A1.
81-89.
25. Rodriguez-Limas WA, Tannenbaum V, Tyo KE: Blocking
10. Martinez JL, Liu L, Petranovic D, Nielsen J: Engineering the endocytotic mechanisms to improve heterologous protein
oxygen sensing regulation results in an enhanced titers in Saccharomyces cerevisiae. Biotechnol. Bioeng. 2015,
recombinant human hemoglobin production by 112:376-385.
Saccharomyces cerevisiae. Biotechnol. Bioeng. 2015, 112:
181-188. 26. Hou J, Osterlund T, Liu Z, Petranovic D, Nielsen J: Heat shock
response improves heterologous protein secretion in
11. Mallem M, Warburton S, Li F, Shandil I, Nylen A, Kim S, Jiang Y, Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 2013,
Meehl M, d’Anjou M, Stadheim TA et al.: Maximizing 97:3559-3568.
recombinant human serum albumin production in a Mut(s)
Pichia pastoris strain. Biotechnol. Prog. 2014, 30:1488-1496. 27. Gu L, Zhang J, Du G, Chen J: Multivariate modular engineering
of the protein secretory pathway for production of
12. Lei JY, Guan B, Li B, Duan ZY, Chen Y, Li HZ, Jin J: Expression, heterologous glucose oxidase in Pichia pastoris. Enzyme
purification and characterization of recombinant human Microb. Technol. 2015, 68:33-42.
interleukin-2-serum albumin (rhIL-2-HSA) fusion protein in
Pichia pastoris. Protein Expr. Purif. 2012, 84:154-160. 28. Tang H, Bao X, Shen Y, Song M, Wang S, Wang C, Hou J:
Engineering protein folding and translocation improves
13. Shibui T, Bando K, Misawa S: High-level secretory expression, heterologous protein secretion in Saccharomyces cerevisiae.
purification, and characterization of an anti-human Her II Biotechnol. Bioeng. 2015, 112:1872-1882.
monoclonal antibody, trastuzumab, in the methylotrophic This study demonstrates combinatorial strengthening of protein folding
yeast Pichia pastoris. Adv. Biosci. Biotechnol. 2013, 4:640-646. and translocation important for heterologous protein production in
S. cerevisiae.
14. Nett JH, Gomathinayagam S, Hamilton SR, Gong B, Davidson RC,
Du M, Hopkins D, Mitchell T, Mallem MR, Nylen A et al.: 29. Van Zyl JH, Den Haan R, Van Zyl WH: Overexpression of native
Optimization of erythropoietin production with controlled Saccharomyces cerevisiae ER-to-Golgi SNARE genes
glycosylation-PEGylated erythropoietin produced in increased heterologous cellulase secretion. Appl. Microbiol.
glycoengineered Pichia pastoris. J. Biotechnol. 2012, 157: Biotechnol. 2016, 100:505-518.
198-206.
30. Hou J, Tyo K, Liu Z, Petranovic D, Nielsen J: Engineering of
15. Landes N, Gasser B, Vorauer-Uhl K, Lhota G, Mattanovich D, vesicle trafficking improves heterologous protein secretion in
Maurer M: The vitamin-sensitive promoter PTHI11 enables Saccharomyces cerevisiae. Metab. Eng. 2012, 14:120-127.
pre-defined autonomous induction of recombinant protein
production in Pichia pastoris. Biotechnol. Bioeng. 2016, 31. Shah P, Ding Y, Niemczyk M, Kudla G, Plotkin JB: Rate-limiting
113:2633-2643. steps in yeast protein translation. Cell 2013, 153:1589-1601.
The authors establish a detailed computational model based on
16. Hou J, Tyo KE, Liu Z, Petranovic D, Nielsen J: Metabolic sequencing data and identify the fast initiation or high codon bias
engineering of recombinant protein secretion by important for protein yield of a transgene in S. cerevisiae.
Saccharomyces cerevisiae. FEMS Yeast Res. 2012, 12:491-510.
32. Kafri M, Metzl-Raz E, Jona G, Barkai N: The cost of protein
17. Chen Y, Partow S, Scalcinati G, Siewers V, Nielsen J: Enhancing production. Cell Rep. 2016, 14:22-31.
the copy number of episomal plasmids in Saccharomyces
cerevisiae for improved protein production. FEMS Yeast Res. 33. Li GW, Burkhardt D, Gross C, Weissman JS: Quantifying
2012, 12:598-607. absolute protein synthesis rates reveals principles underlying
allocation of cellular resources. Cell 2014, 157:624-635.
18. Liu Z, Tyo KE, Martinez JL, Petranovic D, Nielsen J: Different
expression systems for production of recombinant proteins in 34. Weinberg DE, Shah P, Eichhorn SW, Hussmann JA, Plotkin JB,
Saccharomyces cerevisiae. Biotechnol. Bioeng. 2012, 109: Bartel DP: Improved ribosome-footprint and mRNA
1259-1268. measurements provide insights into dynamics and regulation
This study systematically evaluates factors influencing recombinant of yeast translation. Cell Rep 2016, 14:1787-1799.
protein secretion, including protein type, expression vector and leader This study provides improved ribosome-footprint profiles and mRNA
peptide, and demonstrates a generally applicable strategy for initial abundances confirming the slow translation of the beginning of coding
optimization of key factors for rational engineering cell factory for novel regions and codons matching rare tRNAs in S. cerevisiae.
proteins.
35. Liu Z, Osterlund T, Hou J, Petranovic D, Nielsen J: Anaerobic
19. Moon HY, Lee DW, Sim GH, Kim HJ, Hwang JY, Kwon MG, alpha-amylase production and secretion with fumarate as the
Kang BK, Kim JM, Kang HA: A new set of rDNA-NTS-based final electron acceptor in Saccharomyces cerevisiae. Appl.
multiple integrative cassettes for the development of Environ. Microbiol. 2013, 79:2962-2967.
antibiotic-marker-free recombinant yeasts. J. Biotechnol.
2016, 233:190-199. 36. Sims AH, Gent ME, Lanthaler K, Dunn-Coleman NS, Oliver SG,
Robson GD: Transcriptome analysis of recombinant protein
20. Maury J, Germann SM, Baallal Jacobsen SA, Jensen NB, secretion by Aspergillus nidulans and the unfolded-protein
Kildegaard KR, Herrgard MJ, Schneider K, Koza A, Forster J, response in vivo. Appl. Environ. Microbiol. 2005, 71:2737-2747.
Nielsen J et al.: EasyCloneMulti: a set of vectors for
simultaneous and multiple genomic integrations in 37. Liu Z, Hou J, Martinez JL, Petranovic D, Nielsen J: Correlation of
Saccharomyces cerevisiae. PLoS One 2016, 11:e0150394. cell growth and heterologous protein production by
Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 2013, accuracy in the positive target prediction demonstrates the promising
97:8955-8962. application of model guided strain development.
38. Tyo KE, Liu Z, Petranovic D, Nielsen J: Imbalance of 46. Irani ZA, Kerkhoven EJ, Shojaosadati SA, Nielsen J:
heterologous protein folding and disulfide bond formation Genome-scale metabolic model of Pichia pastoris with native
rates yields runaway oxidative stress. BMC Biol. 2012, 10:16. and humanized glycosylation of recombinant proteins.
Biotechnol. Bioeng. 2016, 113:961-969.
39. Gasser B, Sauer M, Maurer M, Stadlmayr G, Mattanovich D:
Transcriptomics-based identification of novel factors 47. Feizi A, Osterlund T, Petranovic D, Bordel S, Nielsen J: Genome-
enhancing heterologous protein secretion in yeasts. Appl. scale modeling of the protein secretory machinery in yeast.
Environ. Microbiol. 2007, 73:6499-6507. PLoS One 2013, 8:e63284.
The authors construct a genome-scale model for protein secretion in
40. Klein T, Niklas J, Heinzle E: Engineering the supply chain for S. cerevisiae using a bottom-up approach. This model covers the entire
protein production/secretion in yeasts and mammalian cells. secretory process to provide a more detailed comprehensive view of the
J. Ind. Microbiol. Biotechnol. 2015, 42:453-464. secretory machinery.
An excellent review on optimization of supply chain for building blocks
and energy for protein production. 48. Caspeta L, Chen Y, Ghiaci P, Feizi A, Buskov S, Hallstrom BM,
Petranovic D, Nielsen J: Altered sterol composition renders
41. Li S, Jendresen CB, Grunberger A, Ronda C, Jensen SI, Noack S, yeast thermotolerant. Science 2014, 346:75-78.
Nielsen AT: Enhanced protein and biochemical production
using CRISPRi-based growth switches. Metab. Eng. 2016, 49. Liu Z, Liu L, Osterlund T, Hou J, Huang M, Fagerberg L,
38:274-284. Petranovic D, Uhlen M, Nielsen J: Improved production of a
heterologous amylase in Saccharomyces cerevisiae by
42. Nocon J, Steiger M, Mairinger T, Hohlweg J, Russmayer H, inverse metabolic engineering. Appl. Environ. Microbiol. 2014,
Hann S, Gasser B, Mattanovich D: Increasing pentose 80:5542-5550.
phosphate pathway flux enhances recombinant protein
production in Pichia pastoris. Appl. Microbiol. Biotechnol. 2016, 50. Sjostrom SL, Bai YP, Huang MT, Liu ZH, Nielsen J, Joensson HN,
100:5955-5963. Svahn HA: High-throughput screening for industrial enzyme
The authors combinatorially overexpress genes involved in pentose production hosts by droplet microfluidics. Lab Chip 2014,
phosphate pathway (PPP), enhancing the PPP flux ratio and recombinant 14:806-813.
hSOD production in P. pastoris.
51. Huang M, Bai Y, Sjostrom SL, Hallstrom BM, Liu Z, Petranovic D,
43. Chong WP, Reddy SG, Yusufi FN, Lee DY, Wong NS, Heng CK, Uhlen M, Joensson HN, Andersson-Svahn H, Nielsen J:
Yap MG, Ho YS: Metabolomics-driven approach for the Microfluidic screening and whole-genome sequencing
improvement of Chinese hamster ovary cell growth: identifies mutations associated with improved protein
overexpression of malate dehydrogenase II. J. Biotechnol. secretion by yeast. Proc. Natl. Acad. Sci. U. S. A. 2015, 112:
2010, 147:116-121. E4689-4696.
The authors isolate yeast mutant strains with significantly improved
44. Blazier AS, Papin JA: Integration of expression data in amylase production through microfluidic screening and systematical
genome-scale metabolic network reconstructions. Front. analysis of mutations and biological processes associated with improved
Physiol. 2012, 3:299. protein secretion.
45. Nocon J, Steiger MG, Pfeffer M, Sohn SB, Kim TY, Maurer M, 52. Jakociunas T, Jensen MK, Keasling JD: CRISPR/Cas9 advances
Russmayer H, Pflugl S, Ask M, Haberhauer-Troyer C et al.: Model engineering of microbial cell factories. Metab. Eng. 2016,
based engineering of Pichia pastoris central metabolism 34:44-59.
enhances recombinant protein production. Metab. Eng. 2014,
24:129-138. 53. Si T, Luo Y, Bao Z, Zhao H: RNAi-assisted genome evolution in
The authors utilized genome scale metabolic modeling to predict targets Saccharomyces cerevisiae for complex phenotype
for enhancing recombinant protein production in P. pastoris. A high engineering. ACS Synth. Biol. 2015, 4:283-291.