TY - JOUR
AU - Kang, Hyun Ah
AB - The production of recombinant therapeutic proteins is one of the fast-growing areas of molecular medicine and currently plays an important role in treatment of several diseases. Yeasts are unicellular eukaryotic microbial host cells that offer unique advantages in producing biopharmaceutical proteins. Yeasts are capable of robust growth on simple media, readily accommodate genetic modifications, and incorporate typical eukaryotic post-translational modifications. Saccharomyces cerevisiae is a traditional baker's yeast that has been used as a major host for the production of biopharmaceuticals; however, several nonconventional yeast species including Hansenula polymorpha, Pichia pastoris, and Yarrowia lipolytica have gained increasing attention as alternative hosts for the industrial production of recombinant proteins. In this review, we address the established and emerging genetic tools and host strains suitable for recombinant protein production in various yeast expression systems, particularly focusing on current efforts toward synthetic biology approaches in developing yeast cell factories for the production of therapeutic recombinant proteins. biopharmaceuticals, glycoengineering, recombinant protein, secretory pathway engineering, yeast cell factory INTRODUCTION Yeasts have been used for thousands of years in food and fermentation processes to produce alcoholic beverages and breads. In recent times, the industrial importance of yeasts has extended beyond its traditional use in fermentation into various healthcare sectors, such as in the production of therapeutic recombinant proteins. Yeast cells are particularly advantageous as hosts for biopharmaceutical production in that they are generally recognized as safe (GRAS) organisms. The traditional baker's yeast Saccharomyces cerevisiae is one of the best-characterized eukaryotes and most widely used host systems for biopharmaceutical production since the early days of genetic engineering and recombinant protein production (Martinez et al., 2012). During last decade, nonconventional yeast species including Hansenula polymorpha,Pichia pastoris,Yarrowia lipolytica,Schizosaccharomyces pombe, and Kluyveromyces lactis have been developed as alternative hosts for the production of recombinant proteins (Table 1). Table 1. General characteristics and genomic information of various yeasts developed as hosts for recombinant protein production. Yeast species  Attributes  References  Saccharomyces  Favorable public acceptance  Nevoigt (2008)   cerevisiae  GRAS status      The most well studied of simple eukaryotes      Amenable to both classical genetics and modern recombinant DNA techniques      Versatile vector systems (episomal, integrative, copy-number regulated) are available (Invitrogen)      A wide range of mutant strains      Well-established fermentation and downstream processing      Hypermannosylation with immunogenic terminal α-1,3-linked mannose residues  Camirand et al. (1991)    Genome sequencing: Reference strain S288C; 12 157 Kb (6273 ORFs); Accession number PRJNA128  Goffeau et al. (1996)  Pichia pastoris  GRAS status  Ahmad et al. (2014)    Tightly regulated, methanol-inducible AOX promoters      A Crabtree-negative yeast allowing for high dilution rates and high biomass yields in fermentation processes      Can grow rapidly on inexpensive media at high cell densities (up to 150 g DCW L−1)      Integrated vectors developed that help genetic stability of the recombinant elements, even in continuous and large-scale fermentation processes      Well-established commercial vector systems and host strains (Invitrogen)      A lesser extent of hypermannosylation compared to S. cerevisiae; No terminal α-1,3-linked mannose residues  Bretthauer (2003)    Genome sequencing: Reference strain GS115; 9216 Kb (5040 ORFs); Accession number PRJNA39439, PRJEA37871  De Schutter et al. (2009)  Hansenula  GRAS status  Gellissen et al. (2005)   polymorpha  Stringently regulated strong promoters (MOX,FMDH, etc.)      A Crabtree-negative yeast allowing for high dilution rates and high biomass yields in fermentation processes      Stable, multicopy integration of foreign DNA into chromosomal locations      Thermotolerant (growth up to 45 °C), resistant to heavy metals and oxidative stress      Can assimilate nitrates      A lesser extent of hypermannosylation compared to S. cerevisiae; No terminal α-1,3-linked mannose residues  Kang et al. (1998) and Kim et al. (2004)    Genome sequencing: Reference strain DL1; 9056 Kb (5325 ORFs); Accession number PRJNA60503  Ravin et al. (2013)  Yarrowia lipolytica  An oleaginous yeast, based on its ability to accumulate large amounts of lipids  Madzak et al. (2004)    GRAS status      Can grow in hydrophobic environments, that is able to metabolize triglycerides, fatty acids, n-alkanes, and n-paraffins as carbon sources for the bioremediation of environments contaminated with oil spills      Can secrete a variety of proteins via cotranslational translocation and efficient secretion signal recognition similar to higher eukaryotes      Availability of a commercial expression kit (YEASTERN BIOTECH CO., LTD.)      Salt tolerance      A lesser extent of hypermannosylation compared to S. cerevisiae; a lack of the immunogenic terminal α-1,3-mannose linkages  Song et al. (2007)    Genome sequencing: Reference strain CLIB122; 20 503 Kb (7042 ORFs); Accession number PRJNA12414  Dujon et al. (2004)  Schizosaccharomyces  A fission yeast, reflecting proliferation of higher eukaryotic cells  Takegawa et al.   pombe  Many cellular processes similar to those of higher eukaryotes, such as mRNA splicing, post-translational modification (including protein galactosylation), cell cycle control, etc.  (2009)    Transcription start site similar to that in higher eukaryotes      Expression vectors for high-level expression developed      Presence of galactose in both O- and N-linked glycans  Ballou et al. (1994)    Genome sequencing: Reference strain 972h-; 12 554 Kb (5364 ORFs); Accession number PRJNA127  Wood et al. (2002)  Kluyveromyces  GRAS status  van Ooyen et al.  lactis  A Crabtree-negative yeast allowing for high dilution rates and high biomass yields in fermentation processes  (2006)    Lactose-fermenting present in milk and whey, and the strong, lactose-inducible LAC4 promoter      Very high cell density (> 100 g DCW L−1)      Able to use both integrative and episomal expression vectors      An available easy-to-use reagent kit for K. lactis protein expression (New England Biolabs)      Terminal N-acetylglucosamine and no mannose phosphate  Lopez-Avalos et al. (2001)    Genome sequencing: Reference strain NRRL Y-1140; 10 689 Kb (5502 ORFs); Accession number PRJNA12377  Dujon et al. (2004)  Yeast species  Attributes  References  Saccharomyces  Favorable public acceptance  Nevoigt (2008)   cerevisiae  GRAS status      The most well studied of simple eukaryotes      Amenable to both classical genetics and modern recombinant DNA techniques      Versatile vector systems (episomal, integrative, copy-number regulated) are available (Invitrogen)      A wide range of mutant strains      Well-established fermentation and downstream processing      Hypermannosylation with immunogenic terminal α-1,3-linked mannose residues  Camirand et al. (1991)    Genome sequencing: Reference strain S288C; 12 157 Kb (6273 ORFs); Accession number PRJNA128  Goffeau et al. (1996)  Pichia pastoris  GRAS status  Ahmad et al. (2014)    Tightly regulated, methanol-inducible AOX promoters      A Crabtree-negative yeast allowing for high dilution rates and high biomass yields in fermentation processes      Can grow rapidly on inexpensive media at high cell densities (up to 150 g DCW L−1)      Integrated vectors developed that help genetic stability of the recombinant elements, even in continuous and large-scale fermentation processes      Well-established commercial vector systems and host strains (Invitrogen)      A lesser extent of hypermannosylation compared to S. cerevisiae; No terminal α-1,3-linked mannose residues  Bretthauer (2003)    Genome sequencing: Reference strain GS115; 9216 Kb (5040 ORFs); Accession number PRJNA39439, PRJEA37871  De Schutter et al. (2009)  Hansenula  GRAS status  Gellissen et al. (2005)   polymorpha  Stringently regulated strong promoters (MOX,FMDH, etc.)      A Crabtree-negative yeast allowing for high dilution rates and high biomass yields in fermentation processes      Stable, multicopy integration of foreign DNA into chromosomal locations      Thermotolerant (growth up to 45 °C), resistant to heavy metals and oxidative stress      Can assimilate nitrates      A lesser extent of hypermannosylation compared to S. cerevisiae; No terminal α-1,3-linked mannose residues  Kang et al. (1998) and Kim et al. (2004)    Genome sequencing: Reference strain DL1; 9056 Kb (5325 ORFs); Accession number PRJNA60503  Ravin et al. (2013)  Yarrowia lipolytica  An oleaginous yeast, based on its ability to accumulate large amounts of lipids  Madzak et al. (2004)    GRAS status      Can grow in hydrophobic environments, that is able to metabolize triglycerides, fatty acids, n-alkanes, and n-paraffins as carbon sources for the bioremediation of environments contaminated with oil spills      Can secrete a variety of proteins via cotranslational translocation and efficient secretion signal recognition similar to higher eukaryotes      Availability of a commercial expression kit (YEASTERN BIOTECH CO., LTD.)      Salt tolerance      A lesser extent of hypermannosylation compared to S. cerevisiae; a lack of the immunogenic terminal α-1,3-mannose linkages  Song et al. (2007)    Genome sequencing: Reference strain CLIB122; 20 503 Kb (7042 ORFs); Accession number PRJNA12414  Dujon et al. (2004)  Schizosaccharomyces  A fission yeast, reflecting proliferation of higher eukaryotic cells  Takegawa et al.   pombe  Many cellular processes similar to those of higher eukaryotes, such as mRNA splicing, post-translational modification (including protein galactosylation), cell cycle control, etc.  (2009)    Transcription start site similar to that in higher eukaryotes      Expression vectors for high-level expression developed      Presence of galactose in both O- and N-linked glycans  Ballou et al. (1994)    Genome sequencing: Reference strain 972h-; 12 554 Kb (5364 ORFs); Accession number PRJNA127  Wood et al. (2002)  Kluyveromyces  GRAS status  van Ooyen et al.  lactis  A Crabtree-negative yeast allowing for high dilution rates and high biomass yields in fermentation processes  (2006)    Lactose-fermenting present in milk and whey, and the strong, lactose-inducible LAC4 promoter      Very high cell density (> 100 g DCW L−1)      Able to use both integrative and episomal expression vectors      An available easy-to-use reagent kit for K. lactis protein expression (New England Biolabs)      Terminal N-acetylglucosamine and no mannose phosphate  Lopez-Avalos et al. (2001)    Genome sequencing: Reference strain NRRL Y-1140; 10 689 Kb (5502 ORFs); Accession number PRJNA12377  Dujon et al. (2004)  GRAS, generally regarded as safe. View Large Table 1. General characteristics and genomic information of various yeasts developed as hosts for recombinant protein production. Yeast species  Attributes  References  Saccharomyces  Favorable public acceptance  Nevoigt (2008)   cerevisiae  GRAS status      The most well studied of simple eukaryotes      Amenable to both classical genetics and modern recombinant DNA techniques      Versatile vector systems (episomal, integrative, copy-number regulated) are available (Invitrogen)      A wide range of mutant strains      Well-established fermentation and downstream processing      Hypermannosylation with immunogenic terminal α-1,3-linked mannose residues  Camirand et al. (1991)    Genome sequencing: Reference strain S288C; 12 157 Kb (6273 ORFs); Accession number PRJNA128  Goffeau et al. (1996)  Pichia pastoris  GRAS status  Ahmad et al. (2014)    Tightly regulated, methanol-inducible AOX promoters      A Crabtree-negative yeast allowing for high dilution rates and high biomass yields in fermentation processes      Can grow rapidly on inexpensive media at high cell densities (up to 150 g DCW L−1)      Integrated vectors developed that help genetic stability of the recombinant elements, even in continuous and large-scale fermentation processes      Well-established commercial vector systems and host strains (Invitrogen)      A lesser extent of hypermannosylation compared to S. cerevisiae; No terminal α-1,3-linked mannose residues  Bretthauer (2003)    Genome sequencing: Reference strain GS115; 9216 Kb (5040 ORFs); Accession number PRJNA39439, PRJEA37871  De Schutter et al. (2009)  Hansenula  GRAS status  Gellissen et al. (2005)   polymorpha  Stringently regulated strong promoters (MOX,FMDH, etc.)      A Crabtree-negative yeast allowing for high dilution rates and high biomass yields in fermentation processes      Stable, multicopy integration of foreign DNA into chromosomal locations      Thermotolerant (growth up to 45 °C), resistant to heavy metals and oxidative stress      Can assimilate nitrates      A lesser extent of hypermannosylation compared to S. cerevisiae; No terminal α-1,3-linked mannose residues  Kang et al. (1998) and Kim et al. (2004)    Genome sequencing: Reference strain DL1; 9056 Kb (5325 ORFs); Accession number PRJNA60503  Ravin et al. (2013)  Yarrowia lipolytica  An oleaginous yeast, based on its ability to accumulate large amounts of lipids  Madzak et al. (2004)    GRAS status      Can grow in hydrophobic environments, that is able to metabolize triglycerides, fatty acids, n-alkanes, and n-paraffins as carbon sources for the bioremediation of environments contaminated with oil spills      Can secrete a variety of proteins via cotranslational translocation and efficient secretion signal recognition similar to higher eukaryotes      Availability of a commercial expression kit (YEASTERN BIOTECH CO., LTD.)      Salt tolerance      A lesser extent of hypermannosylation compared to S. cerevisiae; a lack of the immunogenic terminal α-1,3-mannose linkages  Song et al. (2007)    Genome sequencing: Reference strain CLIB122; 20 503 Kb (7042 ORFs); Accession number PRJNA12414  Dujon et al. (2004)  Schizosaccharomyces  A fission yeast, reflecting proliferation of higher eukaryotic cells  Takegawa et al.   pombe  Many cellular processes similar to those of higher eukaryotes, such as mRNA splicing, post-translational modification (including protein galactosylation), cell cycle control, etc.  (2009)    Transcription start site similar to that in higher eukaryotes      Expression vectors for high-level expression developed      Presence of galactose in both O- and N-linked glycans  Ballou et al. (1994)    Genome sequencing: Reference strain 972h-; 12 554 Kb (5364 ORFs); Accession number PRJNA127  Wood et al. (2002)  Kluyveromyces  GRAS status  van Ooyen et al.  lactis  A Crabtree-negative yeast allowing for high dilution rates and high biomass yields in fermentation processes  (2006)    Lactose-fermenting present in milk and whey, and the strong, lactose-inducible LAC4 promoter      Very high cell density (> 100 g DCW L−1)      Able to use both integrative and episomal expression vectors      An available easy-to-use reagent kit for K. lactis protein expression (New England Biolabs)      Terminal N-acetylglucosamine and no mannose phosphate  Lopez-Avalos et al. (2001)    Genome sequencing: Reference strain NRRL Y-1140; 10 689 Kb (5502 ORFs); Accession number PRJNA12377  Dujon et al. (2004)  Yeast species  Attributes  References  Saccharomyces  Favorable public acceptance  Nevoigt (2008)   cerevisiae  GRAS status      The most well studied of simple eukaryotes      Amenable to both classical genetics and modern recombinant DNA techniques      Versatile vector systems (episomal, integrative, copy-number regulated) are available (Invitrogen)      A wide range of mutant strains      Well-established fermentation and downstream processing      Hypermannosylation with immunogenic terminal α-1,3-linked mannose residues  Camirand et al. (1991)    Genome sequencing: Reference strain S288C; 12 157 Kb (6273 ORFs); Accession number PRJNA128  Goffeau et al. (1996)  Pichia pastoris  GRAS status  Ahmad et al. (2014)    Tightly regulated, methanol-inducible AOX promoters      A Crabtree-negative yeast allowing for high dilution rates and high biomass yields in fermentation processes      Can grow rapidly on inexpensive media at high cell densities (up to 150 g DCW L−1)      Integrated vectors developed that help genetic stability of the recombinant elements, even in continuous and large-scale fermentation processes      Well-established commercial vector systems and host strains (Invitrogen)      A lesser extent of hypermannosylation compared to S. cerevisiae; No terminal α-1,3-linked mannose residues  Bretthauer (2003)    Genome sequencing: Reference strain GS115; 9216 Kb (5040 ORFs); Accession number PRJNA39439, PRJEA37871  De Schutter et al. (2009)  Hansenula  GRAS status  Gellissen et al. (2005)   polymorpha  Stringently regulated strong promoters (MOX,FMDH, etc.)      A Crabtree-negative yeast allowing for high dilution rates and high biomass yields in fermentation processes      Stable, multicopy integration of foreign DNA into chromosomal locations      Thermotolerant (growth up to 45 °C), resistant to heavy metals and oxidative stress      Can assimilate nitrates      A lesser extent of hypermannosylation compared to S. cerevisiae; No terminal α-1,3-linked mannose residues  Kang et al. (1998) and Kim et al. (2004)    Genome sequencing: Reference strain DL1; 9056 Kb (5325 ORFs); Accession number PRJNA60503  Ravin et al. (2013)  Yarrowia lipolytica  An oleaginous yeast, based on its ability to accumulate large amounts of lipids  Madzak et al. (2004)    GRAS status      Can grow in hydrophobic environments, that is able to metabolize triglycerides, fatty acids, n-alkanes, and n-paraffins as carbon sources for the bioremediation of environments contaminated with oil spills      Can secrete a variety of proteins via cotranslational translocation and efficient secretion signal recognition similar to higher eukaryotes      Availability of a commercial expression kit (YEASTERN BIOTECH CO., LTD.)      Salt tolerance      A lesser extent of hypermannosylation compared to S. cerevisiae; a lack of the immunogenic terminal α-1,3-mannose linkages  Song et al. (2007)    Genome sequencing: Reference strain CLIB122; 20 503 Kb (7042 ORFs); Accession number PRJNA12414  Dujon et al. (2004)  Schizosaccharomyces  A fission yeast, reflecting proliferation of higher eukaryotic cells  Takegawa et al.   pombe  Many cellular processes similar to those of higher eukaryotes, such as mRNA splicing, post-translational modification (including protein galactosylation), cell cycle control, etc.  (2009)    Transcription start site similar to that in higher eukaryotes      Expression vectors for high-level expression developed      Presence of galactose in both O- and N-linked glycans  Ballou et al. (1994)    Genome sequencing: Reference strain 972h-; 12 554 Kb (5364 ORFs); Accession number PRJNA127  Wood et al. (2002)  Kluyveromyces  GRAS status  van Ooyen et al.  lactis  A Crabtree-negative yeast allowing for high dilution rates and high biomass yields in fermentation processes  (2006)    Lactose-fermenting present in milk and whey, and the strong, lactose-inducible LAC4 promoter      Very high cell density (> 100 g DCW L−1)      Able to use both integrative and episomal expression vectors      An available easy-to-use reagent kit for K. lactis protein expression (New England Biolabs)      Terminal N-acetylglucosamine and no mannose phosphate  Lopez-Avalos et al. (2001)    Genome sequencing: Reference strain NRRL Y-1140; 10 689 Kb (5502 ORFs); Accession number PRJNA12377  Dujon et al. (2004)  GRAS, generally regarded as safe. View Large Since the complete genomic sequence of S. cerevisiae was published as the first-sequenced eukaryotic genome in 1996 (Goffeau et al., 1996), the whole-genome sequences of several nonconventional yeast species are also currently available in public databases (Wood et al., 2002; Dujon et al., 2004; De Schutter et al., 2009; Ravin et al., 2013), facilitating functional genomics analysis based on various ‘omics’ techniques. Systems biology-based integrated functional genomics could provide a deeper, more holistic understanding of yeast biology, which can help identify potential limitations in protein production on a global scale. Furthermore, novel methods and tools in synthetic biology are under active development to facilitate rapid and efficient engineering of yeast by rational design, based on the information obtained from systems-level analyses. Thus, synthetic biology approaches are expected to accelerate the construction of ‘yeast factories’ designed for the production of recombinant proteins with improved yield and quality (Vogl et al., 2013). In this review, we address recent advances in yeast synthetic biology with respect to therapeutic recombinant protein production, particularly focusing on genetic tools and host strains suitable for secretory protein production in several yeast expression systems. YEAST SPECIES AS HOSTS FOR THE PRODUCTION OF THERAPEUTIC RECOMBINANT PROTEINS Pharmaceutical proteins comprise one of the fastest growing groups of molecular medicines and currently play important roles in the treatment of many diseases. The estimated market value of biopharmaceuticals, including recombinant therapeutic proteins, nucleic acid-based products, and engineered cell-based products, is c. 70–80 billion USD, with an annual growth rate of c. 7–15% (Goodman 2009; Walsh 2010). Saccharomyces cerevisiae,Escherichia coli, and mammalian cells are the most widely used host systems for biopharmaceutical protein production, accounting for 15%, 31%, and 43% of biopharmaceutical products developed, respectively (Berlec and Strukelj 2013). The main strength of E. coli is its capacity for fast and hardy growth in bioreactors using simple media, although producing eukaryotic proteins in E. coli often results in inclusion body formation and/or low specific yields. Mammalian cells are ideal for incorporating typical eukaryotic post-translation modifications, such as glycosylation (Demain and Vaishnav 2009; Walsh 2010b; Berlec and Strukelj 2013); however, the culture of mammalian cells is relatively slow, requires complex media, and is vulnerable to viral contaminations. As unicellular eukaryotic microbial host cells, yeast offers unique advantages in biopharmaceutical protein production. The use of yeasts enables an ideal combination of hardy growth on simple media in large-scale bioreactors with the capacity of the desired post-translational modifications and feasibility in genetic manipulations (Mattanovich et al., 2012). Dozens of pharmaceutical products produced in S. cerevisiae including vaccines and blood factors have been marketed since the first industrial production of recombinant human insulin in S. cerevisiae in 1987, several of which are blockbusters (Walsh 2010a, b). Some physiological characteristics of non-Saccharomyces yeast species, such as faster growth on cheap carbon sources and higher secretion capacity than S. cerevisiae, make them practical alternative hosts for biotechnological purposes. The biopharmaceutical products produced in various yeast species, which are on the market or in the final stages of development, are summarized in Table 2. The recombinant hepatitis B vaccine produced in the nonconventional yeast H. polymorpha has been commercialized (Seo et al., 2008). The H. polymorpha-produced hepatitis B virus S antigen (HbsAg) was found to be assembled into yeast-derived lipid membranes. Previous studies have indicated that this lipoprotein particle structure is essential for the antigenicity of the HBsAg (Rutgers et al., 1988). The methanol induction condition, which is routinely used for high-level production of recombinant proteins in the methylotrophic yeast H. polymorpha, is favorable condition for lipid membrane formation and thus advantageous to produce the recombinant hepatitis B vaccine with more desirable antigenicity. Another methylotrophic yeast P. pastoris was recently approved as a host for biopharmaceutical production. The first recombinant biopharmaceutical, a Kallikrein inhibitor (Kalbitor), developed in P. pastoris was approved by the FDA in 2009, and several others are undergoing evaluation in clinical trials (Walsh 2010a). Traditional approaches for the development of toolboxes for genetically engineering host strains have been applied in nonconventional yeasts to extend protein production capacity, improve product quality, and ease downstream processing (Krainer et al., 2012; Mattanovich et al., 2012; Saraya et al., 2014). Combining recent advances in molecular and genetic resources with the scale-up processes of high cell-density cultivation in various yeast species increases the diversity of choices for yeast expression systems in which recombinant protein production and optimization can be performed quickly and efficiently. Table 2. Commercial biopharmaceuticals produced by yeast. System  Protein  Brand name  Therapeutic area  Company  S. cerevisiae  Hepatitis (or plus other infectious disease) vaccines (I)  Comvax  H. influenzae type B and hepatitis B infection in infants  Merck      Recombivax  Hepatitis B        Euvax B    Sanofi Pasteur (France)      Engerix-B    GlaxoSmithKline (GSK)      Fendrix          Ambirix  Hepatitis A and B        Twinrix          Pediarix8  Various conditions inducing hepatitis B in children        Tritanrix-HB  Diphtheria, tetanus, pertussis, and hepatitis B        Infanrix Hep B          Infanrix-Penta  Diphtheria, tetanus, pertussis, polio, and hepatitis B        Infanrix-Hexa  Diphtheria, tetanus, pertussis, hepatitis B, polio, and H. influenzae type B        Hexavac    Aventis Pasteur      Procomvax  H. influenzae type B and hepatitis B        Primavax  Diphtheria, tetanus, and hepatitis B        HBVaxPro  Hepatitis B in children and adolescents  Aventis Pharma    Lepirudin (S)  Refludan  Heparin-induced thrombocytopenia type II  Hoechst Marion Rousse (USA), Behringwerke AG (Germany)    Desirudin (S)  Revasc  Venous thrombosis  Canyon Pharmaceuticals (UK)    Insulin (S)  Actrapid, Velosulin, Monotard, Insulatard, Protaphane, Mixtard, Actraphane, Ultratard  Diabetes mellitus  Novo Nordisk    Insulin aspart (S)  Novolog, Novolog FlexPen, Novolog Penfill, NovoRapid, NovoRapid Penfill, Novomix 30, Novolog mix 70/30        Insulin detemir (S)  Levemir, Levemir FlexPen        GLP-1 (S)  Victoza  Type 2 diabetes      Glucagon (S)  GlucaGen  Hypoglycemia        Glucagon    Eli Lilly    GM-CSF (S)  Leukine  Cancer, bone marrow transplant  Berlex Laboratories      Leucomax    Novartis    HGH (S)  Valtropin  Dwarfism, pituitary turner syndrome  Biopartners    PDGF (I)  Regranex  Lower extremity diabetic neuropathic ulcers  Ortho-McNeil Pharmaceutical (USA), Janssen-Cilag      GEM 125  Periodontal defects  Luitpold Pharmaceuticals (USA) BioMimetic Pharmaceuticals (USA)    HPV vaccine (I)  Gardasil  Cervical cancer caused by human papillomavirus (HPV)  Merck, Sanofi Pasteur, Merck Sharp and Dohme    Rasburicase (I)  Fasturtec, Elitex  Hyperuricemia  Sanofi-Synthelabo (France), Sanofi-Aventis (France)  P. pastoris  Ecallantide (I)  Kalbitor  Hereditary angioedema  Dyax (USA)    Insulin (S)  Insugen  Type 2 diabetes  Biocon (India)    Human serum albumin (S)  Medway  Blood volume expansion  Mitsubishi Tanabe Pharma (Japan)    Hepatitis vaccine (I)  Shanvac  Hepatitis B  Shantha/Sanofi (India)    IFN-α 2b (S)  Shanferon  Hepatitis C, cancer  Shantha/Sanofi (India)    Ocriplasmin (I)  Jetrea  Vitreomacular adhesion (VMA)  ThromboGenics (Belgium)    Anti-IL-6R Ab (I)  Nanobody ALX-0061  Rheumatoid arthritis  Ablynx (Belgium)    Anti-RSV Ab (S)  Nanobody ALX00171  Respiratory syncytial virus (RSV) infection  Ablynx (Belgium)    HB-EGF (I)  -  Treatment of interstitial cystitis/bladder pain syndrome (IC/BPS)  Trillium (Canada)    Collagen (I)  -  Medical research reagents/dermal filler  Fibrogen (USA)  H. polymorpha  HBV vaccine (I)  Hepavax-Gene  Hepatitis B  Rhein Biotech (Germany), Green Cross Vaccine Corp (Korea)  Y. lipolytica  Pancrelipase (S)  Creon, Ultresa, Viokase  Exocrine pancreatic insufficiency  Aptalis Pharma  System  Protein  Brand name  Therapeutic area  Company  S. cerevisiae  Hepatitis (or plus other infectious disease) vaccines (I)  Comvax  H. influenzae type B and hepatitis B infection in infants  Merck      Recombivax  Hepatitis B        Euvax B    Sanofi Pasteur (France)      Engerix-B    GlaxoSmithKline (GSK)      Fendrix          Ambirix  Hepatitis A and B        Twinrix          Pediarix8  Various conditions inducing hepatitis B in children        Tritanrix-HB  Diphtheria, tetanus, pertussis, and hepatitis B        Infanrix Hep B          Infanrix-Penta  Diphtheria, tetanus, pertussis, polio, and hepatitis B        Infanrix-Hexa  Diphtheria, tetanus, pertussis, hepatitis B, polio, and H. influenzae type B        Hexavac    Aventis Pasteur      Procomvax  H. influenzae type B and hepatitis B        Primavax  Diphtheria, tetanus, and hepatitis B        HBVaxPro  Hepatitis B in children and adolescents  Aventis Pharma    Lepirudin (S)  Refludan  Heparin-induced thrombocytopenia type II  Hoechst Marion Rousse (USA), Behringwerke AG (Germany)    Desirudin (S)  Revasc  Venous thrombosis  Canyon Pharmaceuticals (UK)    Insulin (S)  Actrapid, Velosulin, Monotard, Insulatard, Protaphane, Mixtard, Actraphane, Ultratard  Diabetes mellitus  Novo Nordisk    Insulin aspart (S)  Novolog, Novolog FlexPen, Novolog Penfill, NovoRapid, NovoRapid Penfill, Novomix 30, Novolog mix 70/30        Insulin detemir (S)  Levemir, Levemir FlexPen        GLP-1 (S)  Victoza  Type 2 diabetes      Glucagon (S)  GlucaGen  Hypoglycemia        Glucagon    Eli Lilly    GM-CSF (S)  Leukine  Cancer, bone marrow transplant  Berlex Laboratories      Leucomax    Novartis    HGH (S)  Valtropin  Dwarfism, pituitary turner syndrome  Biopartners    PDGF (I)  Regranex  Lower extremity diabetic neuropathic ulcers  Ortho-McNeil Pharmaceutical (USA), Janssen-Cilag      GEM 125  Periodontal defects  Luitpold Pharmaceuticals (USA) BioMimetic Pharmaceuticals (USA)    HPV vaccine (I)  Gardasil  Cervical cancer caused by human papillomavirus (HPV)  Merck, Sanofi Pasteur, Merck Sharp and Dohme    Rasburicase (I)  Fasturtec, Elitex  Hyperuricemia  Sanofi-Synthelabo (France), Sanofi-Aventis (France)  P. pastoris  Ecallantide (I)  Kalbitor  Hereditary angioedema  Dyax (USA)    Insulin (S)  Insugen  Type 2 diabetes  Biocon (India)    Human serum albumin (S)  Medway  Blood volume expansion  Mitsubishi Tanabe Pharma (Japan)    Hepatitis vaccine (I)  Shanvac  Hepatitis B  Shantha/Sanofi (India)    IFN-α 2b (S)  Shanferon  Hepatitis C, cancer  Shantha/Sanofi (India)    Ocriplasmin (I)  Jetrea  Vitreomacular adhesion (VMA)  ThromboGenics (Belgium)    Anti-IL-6R Ab (I)  Nanobody ALX-0061  Rheumatoid arthritis  Ablynx (Belgium)    Anti-RSV Ab (S)  Nanobody ALX00171  Respiratory syncytial virus (RSV) infection  Ablynx (Belgium)    HB-EGF (I)  -  Treatment of interstitial cystitis/bladder pain syndrome (IC/BPS)  Trillium (Canada)    Collagen (I)  -  Medical research reagents/dermal filler  Fibrogen (USA)  H. polymorpha  HBV vaccine (I)  Hepavax-Gene  Hepatitis B  Rhein Biotech (Germany), Green Cross Vaccine Corp (Korea)  Y. lipolytica  Pancrelipase (S)  Creon, Ultresa, Viokase  Exocrine pancreatic insufficiency  Aptalis Pharma  (I), intracellular expression; (S), secretory expression. View Large Table 2. Commercial biopharmaceuticals produced by yeast. System  Protein  Brand name  Therapeutic area  Company  S. cerevisiae  Hepatitis (or plus other infectious disease) vaccines (I)  Comvax  H. influenzae type B and hepatitis B infection in infants  Merck      Recombivax  Hepatitis B        Euvax B    Sanofi Pasteur (France)      Engerix-B    GlaxoSmithKline (GSK)      Fendrix          Ambirix  Hepatitis A and B        Twinrix          Pediarix8  Various conditions inducing hepatitis B in children        Tritanrix-HB  Diphtheria, tetanus, pertussis, and hepatitis B        Infanrix Hep B          Infanrix-Penta  Diphtheria, tetanus, pertussis, polio, and hepatitis B        Infanrix-Hexa  Diphtheria, tetanus, pertussis, hepatitis B, polio, and H. influenzae type B        Hexavac    Aventis Pasteur      Procomvax  H. influenzae type B and hepatitis B        Primavax  Diphtheria, tetanus, and hepatitis B        HBVaxPro  Hepatitis B in children and adolescents  Aventis Pharma    Lepirudin (S)  Refludan  Heparin-induced thrombocytopenia type II  Hoechst Marion Rousse (USA), Behringwerke AG (Germany)    Desirudin (S)  Revasc  Venous thrombosis  Canyon Pharmaceuticals (UK)    Insulin (S)  Actrapid, Velosulin, Monotard, Insulatard, Protaphane, Mixtard, Actraphane, Ultratard  Diabetes mellitus  Novo Nordisk    Insulin aspart (S)  Novolog, Novolog FlexPen, Novolog Penfill, NovoRapid, NovoRapid Penfill, Novomix 30, Novolog mix 70/30        Insulin detemir (S)  Levemir, Levemir FlexPen        GLP-1 (S)  Victoza  Type 2 diabetes      Glucagon (S)  GlucaGen  Hypoglycemia        Glucagon    Eli Lilly    GM-CSF (S)  Leukine  Cancer, bone marrow transplant  Berlex Laboratories      Leucomax    Novartis    HGH (S)  Valtropin  Dwarfism, pituitary turner syndrome  Biopartners    PDGF (I)  Regranex  Lower extremity diabetic neuropathic ulcers  Ortho-McNeil Pharmaceutical (USA), Janssen-Cilag      GEM 125  Periodontal defects  Luitpold Pharmaceuticals (USA) BioMimetic Pharmaceuticals (USA)    HPV vaccine (I)  Gardasil  Cervical cancer caused by human papillomavirus (HPV)  Merck, Sanofi Pasteur, Merck Sharp and Dohme    Rasburicase (I)  Fasturtec, Elitex  Hyperuricemia  Sanofi-Synthelabo (France), Sanofi-Aventis (France)  P. pastoris  Ecallantide (I)  Kalbitor  Hereditary angioedema  Dyax (USA)    Insulin (S)  Insugen  Type 2 diabetes  Biocon (India)    Human serum albumin (S)  Medway  Blood volume expansion  Mitsubishi Tanabe Pharma (Japan)    Hepatitis vaccine (I)  Shanvac  Hepatitis B  Shantha/Sanofi (India)    IFN-α 2b (S)  Shanferon  Hepatitis C, cancer  Shantha/Sanofi (India)    Ocriplasmin (I)  Jetrea  Vitreomacular adhesion (VMA)  ThromboGenics (Belgium)    Anti-IL-6R Ab (I)  Nanobody ALX-0061  Rheumatoid arthritis  Ablynx (Belgium)    Anti-RSV Ab (S)  Nanobody ALX00171  Respiratory syncytial virus (RSV) infection  Ablynx (Belgium)    HB-EGF (I)  -  Treatment of interstitial cystitis/bladder pain syndrome (IC/BPS)  Trillium (Canada)    Collagen (I)  -  Medical research reagents/dermal filler  Fibrogen (USA)  H. polymorpha  HBV vaccine (I)  Hepavax-Gene  Hepatitis B  Rhein Biotech (Germany), Green Cross Vaccine Corp (Korea)  Y. lipolytica  Pancrelipase (S)  Creon, Ultresa, Viokase  Exocrine pancreatic insufficiency  Aptalis Pharma  System  Protein  Brand name  Therapeutic area  Company  S. cerevisiae  Hepatitis (or plus other infectious disease) vaccines (I)  Comvax  H. influenzae type B and hepatitis B infection in infants  Merck      Recombivax  Hepatitis B        Euvax B    Sanofi Pasteur (France)      Engerix-B    GlaxoSmithKline (GSK)      Fendrix          Ambirix  Hepatitis A and B        Twinrix          Pediarix8  Various conditions inducing hepatitis B in children        Tritanrix-HB  Diphtheria, tetanus, pertussis, and hepatitis B        Infanrix Hep B          Infanrix-Penta  Diphtheria, tetanus, pertussis, polio, and hepatitis B        Infanrix-Hexa  Diphtheria, tetanus, pertussis, hepatitis B, polio, and H. influenzae type B        Hexavac    Aventis Pasteur      Procomvax  H. influenzae type B and hepatitis B        Primavax  Diphtheria, tetanus, and hepatitis B        HBVaxPro  Hepatitis B in children and adolescents  Aventis Pharma    Lepirudin (S)  Refludan  Heparin-induced thrombocytopenia type II  Hoechst Marion Rousse (USA), Behringwerke AG (Germany)    Desirudin (S)  Revasc  Venous thrombosis  Canyon Pharmaceuticals (UK)    Insulin (S)  Actrapid, Velosulin, Monotard, Insulatard, Protaphane, Mixtard, Actraphane, Ultratard  Diabetes mellitus  Novo Nordisk    Insulin aspart (S)  Novolog, Novolog FlexPen, Novolog Penfill, NovoRapid, NovoRapid Penfill, Novomix 30, Novolog mix 70/30        Insulin detemir (S)  Levemir, Levemir FlexPen        GLP-1 (S)  Victoza  Type 2 diabetes      Glucagon (S)  GlucaGen  Hypoglycemia        Glucagon    Eli Lilly    GM-CSF (S)  Leukine  Cancer, bone marrow transplant  Berlex Laboratories      Leucomax    Novartis    HGH (S)  Valtropin  Dwarfism, pituitary turner syndrome  Biopartners    PDGF (I)  Regranex  Lower extremity diabetic neuropathic ulcers  Ortho-McNeil Pharmaceutical (USA), Janssen-Cilag      GEM 125  Periodontal defects  Luitpold Pharmaceuticals (USA) BioMimetic Pharmaceuticals (USA)    HPV vaccine (I)  Gardasil  Cervical cancer caused by human papillomavirus (HPV)  Merck, Sanofi Pasteur, Merck Sharp and Dohme    Rasburicase (I)  Fasturtec, Elitex  Hyperuricemia  Sanofi-Synthelabo (France), Sanofi-Aventis (France)  P. pastoris  Ecallantide (I)  Kalbitor  Hereditary angioedema  Dyax (USA)    Insulin (S)  Insugen  Type 2 diabetes  Biocon (India)    Human serum albumin (S)  Medway  Blood volume expansion  Mitsubishi Tanabe Pharma (Japan)    Hepatitis vaccine (I)  Shanvac  Hepatitis B  Shantha/Sanofi (India)    IFN-α 2b (S)  Shanferon  Hepatitis C, cancer  Shantha/Sanofi (India)    Ocriplasmin (I)  Jetrea  Vitreomacular adhesion (VMA)  ThromboGenics (Belgium)    Anti-IL-6R Ab (I)  Nanobody ALX-0061  Rheumatoid arthritis  Ablynx (Belgium)    Anti-RSV Ab (S)  Nanobody ALX00171  Respiratory syncytial virus (RSV) infection  Ablynx (Belgium)    HB-EGF (I)  -  Treatment of interstitial cystitis/bladder pain syndrome (IC/BPS)  Trillium (Canada)    Collagen (I)  -  Medical research reagents/dermal filler  Fibrogen (USA)  H. polymorpha  HBV vaccine (I)  Hepavax-Gene  Hepatitis B  Rhein Biotech (Germany), Green Cross Vaccine Corp (Korea)  Y. lipolytica  Pancrelipase (S)  Creon, Ultresa, Viokase  Exocrine pancreatic insufficiency  Aptalis Pharma  (I), intracellular expression; (S), secretory expression. View Large TOOLBOXES FOR THE OPTIMIZED EXPRESSION OF RECOMBINANT PROTEINS A goal of synthetic biology is to use modular, well-characterized biological parts to construct novel genetic devices and complex cell-based systems following engineering principles. Thus, successful synthetic biological approaches for protein production require the development of synthetic promoters, codon-optimized synthetic genes, artificial transcription factors, and insertion expression cassettes as typical biological components and devices. These tools can target different levels in biosynthetic processes and allow multilevel modifications of yeast host strains to improve the quality and yield of recombinant proteins. Synthetic promoters for protein expression Efficient transcription is a critical step in controlling gene expression at the initial stage. Thus, well-characterized constitutive or inducible promoters with strong transcriptional activity are generally used to achieve overproduction of recombinant proteins. The well-characterized and strong constitutive TEF1 and GPD (TDH3) promoters have long been used to predominately direct high-level expression of heterologous genes in S. cerevisiae (Partow et al., 2010). It is noteworthy that in the case of secretory protein expression, strong overexpression might rather lead to lower secretion efficiency due to aggregation of misfolded proteins in the endoplasmic reticulum (ER), as reported in the expression of single-chain antibody fragments, insulin precursor, and α-amylase in S. cerevisiae (Shusta et al., 1998; Liu et al., 2012). Thus, a set of promoters with different extent of transcriptional activity would be useful to achieve optimal secretion expression. However, native promoters may not offer continual gradients of transcriptional strength, thereby limiting the ability to fine-tune gene expression. In recent years, significant efforts have been made to develop constitutive promoters exhibiting a wide range of transcriptional activities. For example, synthetic promoter libraries have been created through the application of randomized oligonucleotides (Jeppsson et al., 2003) and the random mutagenesis of promoter regions via error-prone PCR (Alper et al., 2005), extending the choice of available constitutive promoters in S. cerevisiae. More recently, a study on converting a constitutive promoter to a set of synthetic regulatable promoters with a tailor-made regulatory profile was reported (Blount et al., 2012). On the other hand, the constitutive promoter of the glyceraldehyde-3-phosphate dehydrogenase gene of P. pastoris has been also engineered by random mutagenesis to generate additional promoters with the potential for finely tuned gene expression or the generation of new regulatory circuits (Qin et al., 2011). Such diverse sets of constitutive promoters with varied activities and sequences will facilitate the investigation of optimal expression levels of heterologous genes in constructing yeast host strains with heterologous biosynthesis pathway, such as the humanized glycosylation pathway. Inducible promoter systems offer the advantage of controlling gene expression levels in response to the presence of specific inducer or repressor molecules (Shen et al., 2012). The most popular inducible promoters employed for recombinant protein production in S. cerevisiae are the endogenous GAL1 or GAL10 promoters. Although such native promoter systems provide well-defined, predictable expression profiles, a disadvantage of these promoters is that the inducer molecule, galactose, is consumed as a carbon source by yeast cells, which further complicates the control of gene expression. Moreover, galactose is a relatively expensive carbon source, further limiting the benefits of GAL promoters when producing recombinant therapeutic proteins on an industrial scale. A recent study using a modified GAL1 promoter with six Zif268 binding sites showed that this synthetic promoter, in the presence of an agonist β-estradiol that binds to and activates the transcriptional activity of estrogen receptor, was able to control gene expression levels with greater sensitivity in S. cerevisiae with an artificial transcription factor Z3EV. The Z3EV factor was constructed by fusion of the Zif268 DNA-binding domain containing three zinc fingers, the ligand binding domain of the human estrogen receptor, and viral protein 16 from herpes simplex virus (McIsaac et al., 2014). The synthetic promoter was shown to be induced by a nonhormonal small molecule recognized by the modified human estrogen receptor. The industrial potential of such synthetic GAL promoters should be evaluated in further application studies. In the methylotrophic yeast P. pastoris, the methanol-inducible AOX1 promoter (PAOX1) is the most frequently utilized strong and tightly controlled promoter. Because the use of toxic and inflammable methanol can pose a considerable safety risk in industrial settings, this promoter has been extensively studied to create synthetic variants with increased promoter strength and altered methanol-free regulation (Vogl and Glieder 2013). Based on an in silico analysis for putative conserved eukaryotic transcription factor binding sites within the PAOX1, the transcriptional activity of this promoter was rationally optimized by mutagenesis (Hartner et al., 2008; Xuan et al., 2009). In addition, it was shown that short semi-synthetic variants of PAOX1 constructed by fusing natural core promoter fragments with cis-acting elements had greater transcriptional activity than the full-length wild-type promoter (Ruth et al., 2010). Optimizing synthetic gene sequence for yeast expression All living organisms use synonymous codons with variable frequencies that encode specific amino acid residues, although different organisms use alternative redundant codons for a given amino acid with differing efficacy. Codon usage is important because target genes may possess codons that the desired host rarely uses due to a codon usage bias or that originate from organisms using noncanonical code, causing inefficient translation. The translational efficiency of heterologous genes can be improved by optimizing synonymous codon usage to better match that of the host organism. Such codon optimization has been established as a standardized tool for improving heterologous gene expression, particularly in the disciplines of synthetic biology and metabolic engineering (Gustafsson et al., 2004; Young and Alper 2010). While most commonly applied in prokaryotic systems, codon optimization has also been of considerable benefit to yeast expression systems. For example, codon optimization of the human papillomavirus type 58 L1 gene enhanced its expression in S. cerevisiae, increasing the yield of soluble L1 protein and the amount of purified product attainable for vaccine production (Kim and Kwag 2013). With improvements in the speed and cost of gene synthesis, codon-optimized target genes may be readily redesigned and chemically synthesized. A variety of additional tools and approaches have been developed for the redesign of target genes, including modification of translation initiation regions, alteration of mRNA structural elements, and the use of different codon biases (Gustafsson et al., 2004). In addition to codon optimization, the balance of A+T/G+C content has also been proposed as a major factor affecting translation efficiency. The expression of diphtheria toxin and human glucocerebrosidase in P. pastoris was improved by codon optimization and removal of an AT-rich region that caused early termination of transcription (Woo et al., 2002). A very recent study in P. pastoris further demonstrated the importance of codon optimization and balanced A+T/G+C content for the production of lysostaphin, a promising therapeutic agent for treating staphylococcal infections, particularly those caused by the methicillin-resistant Staphylococcus aureus (Zhao et al., 2014). The effect of the codon bias and consensus sequence (CACA) at the translation initiation site on the expression of human interferon alpha 2b (hIFN-α2b) was investigated in Y. lipolytica. Whereas codon optimization increased hIFN-α2b production by 11-fold, the additional insertion of a CACA sequence upstream of the initiation codon of the codon-optimized hIFN-α2b construct increased production by 16.5-fold, indicating that translational efficiency should be also considered as a key factor in heterologous protein expression levels (Gasmi et al., 2011). A recent study reported the use of a condition-specific codon optimization approach, based on codon usage bias generated using only genes induced under a given condition. The application of this delicate approach in S. cerevisiae showed that the synthetic CatA gene encoding Acinetobacter baylyi catechol 1,2-dioxygenase, optimized for stationary phase production, generated an approximate threefold increase in yield over a commercially optimized gene variant (Lanza et al., 2014). On the other hand, there are other challenges to optimize gene sequences by incorporating orthogonal pathways and potential synthetic biology solutions. For example, Hancock et al., reported the expansion of the genetic code of S. cerevisiae for incorporation of diverse unnatural amino acids via a pyrrolysyl-tRNA synthetase/tRNACUAPyl pair. The functional and orthogonal pyrrolysyl-tRNA synthetase/tRNACUAPyl pairs from Methanosarcina barkeri and M. mazei were evolved in E. coli and used to allow the site-specific incorporation of a variety of unnatural amino acids in S. cerevisiae. By this approach, an alkyne-containing amino acid, an important post-translationally modified amino acid and one of its analogs, a photocaged amino acid, and a photo-cross-linking amino acid were successfully incorporated into proteins in yeast (Hancock et al., 2010). Copy-number control tools Another important approach for manipulating heterologous gene expression in yeast involves controlling the gene copy number of a desired expression cassette. In S. cerevisiae, 2 μ-based plasmids are maintained at c. 5–30 copies per cell, while yeast centromeric plasmids containing an autonomously replicating origin paired with a yeast centromere are present at c. 1 copy per cell (Fang et al., 2011). High-copy plasmids facilitate strong gene expression, but can impose a heavy burden on cells resulting in increased instability of the constructs. Conversely, low-copy plasmids are more stable, but their use is limited by lower gene expression levels. Inserting an expression cassette in a target locus on a native yeast chromosome by homologous recombination is beneficial because it allows an expression cassette to be stably maintained even in the absence of selective pressure. High-level expression of heterologous proteins may be achieved by introducing DNA cassettes into multiple sites in yeast chromosomes, such as the yeast repeating ribosomal (Lopes et al., 1996), delta (Oliveira et al., 2007), and sigma element sequences (Kudla and Nicolas 1992). These multiple integration approaches coupled with the use of a deficient selection marker have been advanced as helpful technologies to modulate the copy number of an integrated DNA construct in S. cerevisiae and Y. lipolytica (Nicaud et al., 2002). Recently, ‘Reiterative Recombination’ was developed as an efficient platform technology in S. cerevisiae for the assembly of combinatorial libraries for simultaneous expression of several different proteins through continuous targeted integration and the use of exchangeable, replaceable selection markers (Wingler and Cornish 2011). However, it has become apparent that there is not always a linear correlation between expression cassette copy number and protein yield, particularly for the secretory production of recombinant proteins (Aw and Polizzi 2013). For example, a study involving secretory production of human serum albumin in H. polymorpha showed that the effect of gene dosage effect on expression was abolished using an optimized expression cassette. Thus, a single copy of the expression vector integrated into the MOX locus was sufficient for maximal expression of human serum albumin (Kang et al., 2001). Similarly, expression of human serum albumin in P. pastoris showed that increasing gene copy number did not result in higher production levels due to secretion saturation (Whyteside et al., 2011). Thus, using controllable multiple integration vectors would be a good strategy to introduce optimal copy numbers for high-level expression of recombinant proteins. Gene deletion and integration tools for host strain construction Efficient strategies for gene replacements and marker recycling are essential for synthetic biology applications involving knocking out genes and introducing multiple heterologous genes besides the expression cassettes. In addition to the counter-selection strategy using the URA3 marker that has been conventionally applied in yeast, more advanced approaches such as the Cre/loxP and Flp/FRT recombinase-based systems have been developed. These systems proceed via active excision of marker gene fragments to recycle markers for sequential rounds of deletions in several yeast species including S. cerevisiae (Park et al., 2011), H. polymorpha (Qian et al., 2009), P. pastoris (Pan et al., 2011; Naatsaari et al., 2012), and Y. lipolytica (Fickers et al., 2003). These strategies have been applied to the development of a conditional gene deletion system, in which the Cre or Flp recombinases are placed under the strict regulation of galactose- or methanol-inducible promoters to control target gene deletion processes. In eukaryotes, double-strand breaks in DNA can be repaired by two mechanisms that work independently but competitively: one is homologous recombination and the other is nonhomologous end-joining (NHEJ). Site-specific knockout and integration for construction of yeast strains rely on endogenous homologous recombination. Whereas in S. cerevisiae, homologous recombination is highly efficient, nonhomologous end-joining is the preferred pathway for repairing double-strand breaks in most other non-Saccharomyces yeasts. After deletion of KU70 or KU80 homolog-encoding proteins involved in nonhomologous end-joining, the frequency of homologous recombination was significantly enhanced in several yeast species including H. polymorpha,P. pastoris, and Y. lipolytica (de Jong et al., 2010; Naatsaari et al., 2012; Verbeke et al., 2013). Thus, these ku-deletion mutant strains are useful recipients for targeted gene manipulation when constructing useful host strains for the production of recombinant proteins in nonconventional yeast systems. In very recent, several cutting-edge technologies based on bacterial nucleic acid modification systems have made dramatic leaps in genome engineering in eukaryotic systems. Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) systems originate from immune system of bacteria and archaea by RNA-guided nuclease activity (Barrangou et al., 2007). The type II bacterial CRISPR/Cas9 system has been applied to S. cerevisiae for genome engineering. The increased homologous recombination efficiency and feasibility for site-specific mutation and allelic replacement were observed in S. cerevisiae, indicating that the CRISPR-Cas system has a great potential to become a valuable genome engineering tool in yeast (DiCarlo et al., 2013). Transcription activator-like effectors fused to the catalytic domain of endonucleases (TALENs) have been widely used for genome editing or engineering by generating site-specific double-strand breaks, which can be repaired through DNA repair mechanisms including NHEJ and homology-directed repair (HDR) (Gaj et al., 2013). A recent study presented the potential of homodimeric TALENs as a useful tool to simultaneously modify the yeast genome at multiple genomic loci in a site-specific manner (Aouida et al., 2014). HOST STRAIN ENGINEERING FOR SECRETORY PRODUCTION OF RECOMBINANT PROTEINS Engineering of protein secretion pathway Due to the similarities between yeast and animal cell secretion pathways, yeasts have been a preferred microbial host system for the secretory production of recombinant proteins. Particularly, yeasts secrete only a few endogenous proteins, so it is easy to purify the secreted recombinant protein. Secretory protein production requires multiple steps in the conversion of protein-coding DNA sequences to mature proteins, which is complex and involves many different stages in processing, including transcription, translation, translocation, post-translational modifications and protein folding, peptide cleavage and additional glycosylation, sorting, and secretion (Hou et al., 2012). Although yeast is a favorable platform for secretory production of recombinant proteins, several limiting steps, such as different glycosylation processes and proteolytic degradation, are often encountered during secretory production of heterologous proteins in yeast. Each step of the protein secretion pathway may be an engineered target for improving protein expression levels and quality (Fig. 1). Therefore, synthetic biology approaches represent excellent strategies for engineering yeast secretion pathways to satisfy commercial production requirements. Figure 1. View largeDownload slide Schematic representation of the yeast secretory pathway together with target genes for host strain engineering and enhanced production of heterologous proteins. Secretory proteins enter the endoplasmic reticulum (ER). The accumulation of misfolded proteins leads to the induction of the unfolded protein response (UPR) and ER-associated degradation (ERAD). The heat shock response (HSR) also induces upregulated expression of many genes encoding cytoplasmic or secretory protein-folding chaperones. Correctly folded proteins are transported to the Golgi for further processing including additional glycosylation. Proteins exiting the Golgi may be secreted extracellularly or targeted to vacuoles for storage or degradation. Alternatively, recombinant products are subjected to proteolytic degradation by several proteases located in cell compartments such as the Golgi, cytosol, mitochondria, and the cell wall. The functions of target genes are described in Table 3. Figure 1. View largeDownload slide Schematic representation of the yeast secretory pathway together with target genes for host strain engineering and enhanced production of heterologous proteins. Secretory proteins enter the endoplasmic reticulum (ER). The accumulation of misfolded proteins leads to the induction of the unfolded protein response (UPR) and ER-associated degradation (ERAD). The heat shock response (HSR) also induces upregulated expression of many genes encoding cytoplasmic or secretory protein-folding chaperones. Correctly folded proteins are transported to the Golgi for further processing including additional glycosylation. Proteins exiting the Golgi may be secreted extracellularly or targeted to vacuoles for storage or degradation. Alternatively, recombinant products are subjected to proteolytic degradation by several proteases located in cell compartments such as the Golgi, cytosol, mitochondria, and the cell wall. The functions of target genes are described in Table 3. The initial step of secretion is the transfer of a protein through the ER membrane into the secretion pathway. Secretion signal sequences are required to translocate proteins into the ER and thus the efficiency of signal sequences is one of critical factors in determining the final yield of secretory proteins. In general, the native prepro-leader sequences derived from yeast secretory proteins, such as S. cerevisiae alpha-mating factor, and their modified versions are exploited to facilitate secretory expression of heterologous proteins in yeast. It was reported that the synthetic prepro leaders of alpha-mating factor lacking consensus N-linked glycosylation sites directed secretion of correctly folded insulin precursor with a yield comparable to, or better than, the native leader in S. cerevisiae (Kjeldsen et al., 1998). Correct protein folding in the yeast ER is very important as it determines whether the protein enters the secretory pathway or whether it is targeted for ER-associated degradation (ERAD). Misfolding of nascent peptides leads to a luminal burden that results in ER stress, which regulates the unfolded protein response (UPR) (Patil and Walter 2001; Payne et al., 2008). A recent study showed that imbalance of heterologous protein folding and disulfide bond formation rates generated runaway oxidative stress, which also leads to UPR activation (Tyo et al., 2012). Both UPR and ERAD govern the flux of proteins through the ER-to-Golgi apparatus for secretion. Several studies to manipulate the ER luminal environments have shown that enhanced secretion capacities of target proteins can be obtained through overexpression of protein-folding factors (chaperones) or redox enzymes in the ER, including SIL1,LHS1,JEM1,SCJ1,KAR2,ERO1, and PDI1 in S. cerevisiae (Smith et al., 2004; Payne et al., 2008), P. pastoris (Zhang et al., 2006; Gasser et al., 2007), and K. lactis (Lodi et al., 2005). The overexpression of Hac1p, a transcriptional activator in the UPR response, led to expression of a set of ER chaperones, improving secretory production of some heterologous proteins in S. cerevisiae (Valkonen et al., 2003) and P. pastoris (Guerfal et al., 2010). However, depending on the target protein, coexpression of Hac1p could instead decrease heterologous expression or exert no effect on expression levels, indicating the effect of Hac1 overexpression needs to be evaluated on a case-by-case basis. Overexpression of Sly1p and Sec1p, involved in vesicle trafficking from ER-to-Golgi and Golgi-to-plasma membrane, respectively, improved the secretion of heterologous human insulin precursor and α-amylase proteins in S. cerevisiae, along with the secretion of an endogenous invertase protein (Hou et al., 2012). These results strongly suggest that upregulating protein trafficking can improve protein secretion. On the other hand, activation of heat shock factor 1, a primary heat shock response (HSR) transcription factor, can also induce upregulated expression of many genes encoding cytoplasmic or secretory protein, folding chaperones that facilitate protein folding and prevent the accumulation of misfolded or aggregated proteins. A very recent study on the overexpression of a constitutively activated mutant of heat shock factor 1 in S. cerevisiae showed improved heterologous protein secretion by constitutively activating HSR (Hou et al., 2013). Yeasts express numerous proteases, many of which are either localized to the secretory pathway or pass through it en route to the cell surface. Some recombinant proteins are subjected to aberrant proteolytic cleavage by proteases during the secretion process, which not only reduces the final yield of proteins but also affects the quality of recombinant proteins. In this case, construction of protease-deficient strains with multiple gene deletions could be employed as an efficient strategy to solve the protein cleavage problem. For example, a multiple-yapsin-deficient mutant strain of S. cerevisiae lacking the YPS1,YPS2,YPS3,YPS6, and YPS7 genes showed diminished cleavage of the recombinant parathyroid hormone protein at high cell-density cultivation during fed-batch fermentation (Cho et al., 2010). Yapsin-deficient strains have been developed in other yeast species, including H. polymorpha and P. pastoris, demonstrating that aspartyl proteases localized at the cell surface are principally responsible for aberrant proteolytic cleavage of secretory recombinant proteins (Sohn et al., 2010; Wu et al., 2013). In addition, reducing major intracellular protease activities by the disruption of vacuolar protease genes, such as PEP4 and PRB1, also enhanced the secretory production of human interferon-β by c. 10-fold in S. cerevisiae (Tomimoto et al., 2013). Yeast strain engineering strategies have been extended from single target gene manipulation to the deletion and/or overexpression of multiple genes involved in various processes. As a representative example, the simultaneous modification of several targets through the deletion of YPS1 (previously named YAP3) and HSP150 and overexpression of PDI1 was applied to engineer-enhanced production of recombinant human transferrin (rTf) with improved yield and quality in S. cerevisiae (Finnis et al., 2010). Overexpression of PDI1 improves correct protein production by assisting protein folding, and deletion of YPS1 diminishes target protein degradation. A secretory protein Hsp150p is generally copurified with other recombinant proteins. Therefore, deletion of HSP150 facilitates purification in downstream processing by eliminating a potential contaminant. Indeed, the final production strain, which was chemically mutagenized and further modified with the YAP3 and HSP150 deletions and PDI1 overexpression, showed nearly 50-fold increased production of rTf compared to the original progenitor strain. A similar approach involving multiple combined genetic manipulations has also been applied toward the improved production of human growth hormone (hGH) in the fission yeast S. pombe. Approximately 30-fold enhanced secretion of hGH was achieved through the deletion of seven obstructive proteases. However, intracellular retention of secretory hGH was found in the resultant multiprotease-deficient strains. As a solution to increase hGH secretion efficiency, further deletions of vacuolar protein sorting genes were made and the deletion of VPS10, involved in vacuolar protein sorting, was found to lead to an approximately twofold increase in hGH secretion (Idiris et al., 2010). These studies clearly demonstrate the possible application of secretory-pathway modification and multiple protease deletion for improving heterologous protein secretion from yeast. Some other case studies on secretion pathway engineering, which are not mentioned in this review, are summarized in Table 3. Table 3. Target genes modified in strain engineering for enhancement of secretory protein production. Category  System  Gene  Protein  Biological function  References  Protein folding,  Sc  SIL1  Cochaperone, NEF  Protein translocation into the ER  Payne et al. (2008)  quality control    LHS1  Cochaperone  Polypeptide translocation and folding        JEM1  DnaJ-like cochaperone  Chaperone binding        SCJ1  HSP70-type cochaperone  Chaperone binding        HSF1  Heat shock transcription factor  Response to highly diverse stresses  Hou et al. (2012a, b, 2013)     Hp  CNE1  ER chaperone calnexin  Folding and quality control of glycoproteins  Klabunde et al. (2007)    Pp  YDJ1  Type I HSP40 cochaperone  Regulation of HSP90 and HSP70 functions  Zhang et al. (2001, 2006)      SSA1 SSA4  Cochaperone, HSP70 family  Protein foldingSRP-dependent protein-membrane targeting and translocation  Zhang et al. (2001, 2006)Gasser et al. (2007)      SSE1    Unfolded protein binding      Sc, Pp  HAC1  bZIP transcription factor  UPR regulation  Valkonen et al. (2003) and Gasser et al. (2007)      KAR2  HSP70 chaperones, BiP  Protein folding, UPR regulation  Harmsen et al. (1996) and Zhang et al. (2001, 2006)    Kl, Pp  ERO1  Thiol oxidase  Oxidative protein folding  Lodi et al. (2005) and Gasser et al. (2007)    Sc, Kl, Pp  PDI1  Protein disulfide isomerase  Disulfide bond formation of proteins  Smith et al. (2004), Lodi et al. (2005) and Zhang et al. (2001, 2006)  Degradation  Sc  CYM1  Cytosolic metalloprotease  Mitochondrial lysine-specific metalloprotease  Jonson et al. (2004)      CPY1  Carboxypeptidase Y  Vacuolar serine-type C-terminal exopeptidase  Van Den Hazel et al. (1996)    Sc,Pp  KEX2  Proprotein convertase  Serine-type calcium-dependent serine protease  Zhang et al. (2001, 2006) and Werten and de Wolf (2005)    Pp,Hp  YPS1  Yapsin-1  Aspartic-type endopeptidase  Werten and de Wolf (2005), Cho et al. (2010) and Choi et al. (2003)    Sc, Kl  UBI4  Ubiquitin  Ubiquitin-26S proteasome system  Chen et al. (1994) and Bao and Fukuhara (2001)    Sc, Pp, Cb  PEP4  Proteinase A  Vacuolar aspartyl protease  Kang et al. (1998, 2000, 2001), Komeda et al. (2002) and Wu et al. (2013)    Sc, Sp,Cb  PRB1  Proteinase B  Vacuolar serine-type endopeptidase  Kang et al. (1998, 2000, 2001), Komeda et al. (2002) and Idiris et al. (2010)    Sp  ATG4  Cysteine protease  Cytosolic cysteine-type endopeptidase  Idiris et al. (2010)      OMA1  Metalloendopeptidase  Mitochondrial membrane-bound metalloendopeptidase        FMA2  Methionine aminopeptidase  Cytosolic aminopeptidase specific to N-terminal methionine        PPP20  Aminopeptidase  Vacuolar/cytosolic aminopeptidase    Trafficking  Sc  SEC1  Sm-like protein  SNARE binding, docking of exocytic vesicles  Hou et al. (2012, b, 2013)       MON2  SEC7 family, GEF  Endocytosis, Golgi-to-endosome transport, vacuole integrity  Kanjou et al. (2007)      PMR1  Ca2+/Mn2+ ATPase  Calcium-dependent protein sorting and processing  Harmsen et al. (1996)      SSO1, SSO2  Plasma membrane t-SNARE  Fusion of secretory vesicles at the plasma membrane  Ruohonen et al. (1997)    Pp  BFR2  90S preribosome  ER-to-Golgi transport, rRNA processing  Gasser et al. (2007)      BMH2  14-3-3 protein  Exocytosis, vesicle transport, and Ras/MAPK signaling      CUP5  Vacuolar membrane ATPase  Hydrogen ion transmembrane transport        KIN2  Serine/threonine protein kinase  Exocytosis regulation        SEC63  Sec63 complex subunit  Cytosol-to-ER transport  Zhang et al. (2001, 2006)    Sc,Sp  Vsp10  Vacuolar protein sorting receptor  Protein targeting to vacuole, vacuolar transport  Zhang et al. (2001, 2006) and Idiris et al. (2010)  Glycosylation  Sc,Kl  MNN1  α-1,3-mannosyltransferase  Addition of α-1,3-mannose to N-/O-linked oligosaccharides  Nakanishi-Shindo et al. (1993) and Liu et al. (2009, 2012)    Sc  MNN4  Positive regulator of Mnn6p  Mannosylphosphorylation of N-linked oligosaccharides  Chiba et al. (1998)      ALG11  α-1,2-mannosyltransferase  N-linked oligosaccharide assembly  Parsaie Nasab et al. (2013)    Sp  GMS1  UDP-galactose transporter  Transport of UDP-galactose into the lumen of Golgi apparatus  Ohashi and Takegawa (2010)      OMH1  O-glycoside α-1,2-mannosyltransferase  Extending α-1,2-linked mannose in the O-glycan pathway  Ikeda et al. (2009)    Sc, Pp, Hp  ALG3  α-1,3-mannosyltransferase  Synthesis of dolichol-linked oligosaccharide for N-glycosylation  Nakanishi-Shindo et al. (1993), Hamilton and Gerngross (2007) and Cheon et al. (2012)    Sc, Pp, Kl Sp, Hp, Yl  OCH1  α-1,6-mannosyltransferase  Outer chain elongation of N-linked oligosaccharides  Nakanishi-Shindo et al. (1993), Hamilton and Gerngross (2007), Song et al. (2007), Liu et al. (2009, 2012), Ohashi and Takegawa (2010) and Cheon et al. (2012)  Category  System  Gene  Protein  Biological function  References  Protein folding,  Sc  SIL1  Cochaperone, NEF  Protein translocation into the ER  Payne et al. (2008)  quality control    LHS1  Cochaperone  Polypeptide translocation and folding        JEM1  DnaJ-like cochaperone  Chaperone binding        SCJ1  HSP70-type cochaperone  Chaperone binding        HSF1  Heat shock transcription factor  Response to highly diverse stresses  Hou et al. (2012a, b, 2013)     Hp  CNE1  ER chaperone calnexin  Folding and quality control of glycoproteins  Klabunde et al. (2007)    Pp  YDJ1  Type I HSP40 cochaperone  Regulation of HSP90 and HSP70 functions  Zhang et al. (2001, 2006)      SSA1 SSA4  Cochaperone, HSP70 family  Protein foldingSRP-dependent protein-membrane targeting and translocation  Zhang et al. (2001, 2006)Gasser et al. (2007)      SSE1    Unfolded protein binding      Sc, Pp  HAC1  bZIP transcription factor  UPR regulation  Valkonen et al. (2003) and Gasser et al. (2007)      KAR2  HSP70 chaperones, BiP  Protein folding, UPR regulation  Harmsen et al. (1996) and Zhang et al. (2001, 2006)    Kl, Pp  ERO1  Thiol oxidase  Oxidative protein folding  Lodi et al. (2005) and Gasser et al. (2007)    Sc, Kl, Pp  PDI1  Protein disulfide isomerase  Disulfide bond formation of proteins  Smith et al. (2004), Lodi et al. (2005) and Zhang et al. (2001, 2006)  Degradation  Sc  CYM1  Cytosolic metalloprotease  Mitochondrial lysine-specific metalloprotease  Jonson et al. (2004)      CPY1  Carboxypeptidase Y  Vacuolar serine-type C-terminal exopeptidase  Van Den Hazel et al. (1996)    Sc,Pp  KEX2  Proprotein convertase  Serine-type calcium-dependent serine protease  Zhang et al. (2001, 2006) and Werten and de Wolf (2005)    Pp,Hp  YPS1  Yapsin-1  Aspartic-type endopeptidase  Werten and de Wolf (2005), Cho et al. (2010) and Choi et al. (2003)    Sc, Kl  UBI4  Ubiquitin  Ubiquitin-26S proteasome system  Chen et al. (1994) and Bao and Fukuhara (2001)    Sc, Pp, Cb  PEP4  Proteinase A  Vacuolar aspartyl protease  Kang et al. (1998, 2000, 2001), Komeda et al. (2002) and Wu et al. (2013)    Sc, Sp,Cb  PRB1  Proteinase B  Vacuolar serine-type endopeptidase  Kang et al. (1998, 2000, 2001), Komeda et al. (2002) and Idiris et al. (2010)    Sp  ATG4  Cysteine protease  Cytosolic cysteine-type endopeptidase  Idiris et al. (2010)      OMA1  Metalloendopeptidase  Mitochondrial membrane-bound metalloendopeptidase        FMA2  Methionine aminopeptidase  Cytosolic aminopeptidase specific to N-terminal methionine        PPP20  Aminopeptidase  Vacuolar/cytosolic aminopeptidase    Trafficking  Sc  SEC1  Sm-like protein  SNARE binding, docking of exocytic vesicles  Hou et al. (2012, b, 2013)       MON2  SEC7 family, GEF  Endocytosis, Golgi-to-endosome transport, vacuole integrity  Kanjou et al. (2007)      PMR1  Ca2+/Mn2+ ATPase  Calcium-dependent protein sorting and processing  Harmsen et al. (1996)      SSO1, SSO2  Plasma membrane t-SNARE  Fusion of secretory vesicles at the plasma membrane  Ruohonen et al. (1997)    Pp  BFR2  90S preribosome  ER-to-Golgi transport, rRNA processing  Gasser et al. (2007)      BMH2  14-3-3 protein  Exocytosis, vesicle transport, and Ras/MAPK signaling      CUP5  Vacuolar membrane ATPase  Hydrogen ion transmembrane transport        KIN2  Serine/threonine protein kinase  Exocytosis regulation        SEC63  Sec63 complex subunit  Cytosol-to-ER transport  Zhang et al. (2001, 2006)    Sc,Sp  Vsp10  Vacuolar protein sorting receptor  Protein targeting to vacuole, vacuolar transport  Zhang et al. (2001, 2006) and Idiris et al. (2010)  Glycosylation  Sc,Kl  MNN1  α-1,3-mannosyltransferase  Addition of α-1,3-mannose to N-/O-linked oligosaccharides  Nakanishi-Shindo et al. (1993) and Liu et al. (2009, 2012)    Sc  MNN4  Positive regulator of Mnn6p  Mannosylphosphorylation of N-linked oligosaccharides  Chiba et al. (1998)      ALG11  α-1,2-mannosyltransferase  N-linked oligosaccharide assembly  Parsaie Nasab et al. (2013)    Sp  GMS1  UDP-galactose transporter  Transport of UDP-galactose into the lumen of Golgi apparatus  Ohashi and Takegawa (2010)      OMH1  O-glycoside α-1,2-mannosyltransferase  Extending α-1,2-linked mannose in the O-glycan pathway  Ikeda et al. (2009)    Sc, Pp, Hp  ALG3  α-1,3-mannosyltransferase  Synthesis of dolichol-linked oligosaccharide for N-glycosylation  Nakanishi-Shindo et al. (1993), Hamilton and Gerngross (2007) and Cheon et al. (2012)    Sc, Pp, Kl Sp, Hp, Yl  OCH1  α-1,6-mannosyltransferase  Outer chain elongation of N-linked oligosaccharides  Nakanishi-Shindo et al. (1993), Hamilton and Gerngross (2007), Song et al. (2007), Liu et al. (2009, 2012), Ohashi and Takegawa (2010) and Cheon et al. (2012)  Sc, S. cerevisiae;Pp, P. pastoris;Sp, S. pombe;Kl, K. lactis;Cb, C. boidinii;Hp, H. polymorpha;Yl, Y. lipolytica. View Large Table 3. Target genes modified in strain engineering for enhancement of secretory protein production. Category  System  Gene  Protein  Biological function  References  Protein folding,  Sc  SIL1  Cochaperone, NEF  Protein translocation into the ER  Payne et al. (2008)  quality control    LHS1  Cochaperone  Polypeptide translocation and folding        JEM1  DnaJ-like cochaperone  Chaperone binding        SCJ1  HSP70-type cochaperone  Chaperone binding        HSF1  Heat shock transcription factor  Response to highly diverse stresses  Hou et al. (2012a, b, 2013)     Hp  CNE1  ER chaperone calnexin  Folding and quality control of glycoproteins  Klabunde et al. (2007)    Pp  YDJ1  Type I HSP40 cochaperone  Regulation of HSP90 and HSP70 functions  Zhang et al. (2001, 2006)      SSA1 SSA4  Cochaperone, HSP70 family  Protein foldingSRP-dependent protein-membrane targeting and translocation  Zhang et al. (2001, 2006)Gasser et al. (2007)      SSE1    Unfolded protein binding      Sc, Pp  HAC1  bZIP transcription factor  UPR regulation  Valkonen et al. (2003) and Gasser et al. (2007)      KAR2  HSP70 chaperones, BiP  Protein folding, UPR regulation  Harmsen et al. (1996) and Zhang et al. (2001, 2006)    Kl, Pp  ERO1  Thiol oxidase  Oxidative protein folding  Lodi et al. (2005) and Gasser et al. (2007)    Sc, Kl, Pp  PDI1  Protein disulfide isomerase  Disulfide bond formation of proteins  Smith et al. (2004), Lodi et al. (2005) and Zhang et al. (2001, 2006)  Degradation  Sc  CYM1  Cytosolic metalloprotease  Mitochondrial lysine-specific metalloprotease  Jonson et al. (2004)      CPY1  Carboxypeptidase Y  Vacuolar serine-type C-terminal exopeptidase  Van Den Hazel et al. (1996)    Sc,Pp  KEX2  Proprotein convertase  Serine-type calcium-dependent serine protease  Zhang et al. (2001, 2006) and Werten and de Wolf (2005)    Pp,Hp  YPS1  Yapsin-1  Aspartic-type endopeptidase  Werten and de Wolf (2005), Cho et al. (2010) and Choi et al. (2003)    Sc, Kl  UBI4  Ubiquitin  Ubiquitin-26S proteasome system  Chen et al. (1994) and Bao and Fukuhara (2001)    Sc, Pp, Cb  PEP4  Proteinase A  Vacuolar aspartyl protease  Kang et al. (1998, 2000, 2001), Komeda et al. (2002) and Wu et al. (2013)    Sc, Sp,Cb  PRB1  Proteinase B  Vacuolar serine-type endopeptidase  Kang et al. (1998, 2000, 2001), Komeda et al. (2002) and Idiris et al. (2010)    Sp  ATG4  Cysteine protease  Cytosolic cysteine-type endopeptidase  Idiris et al. (2010)      OMA1  Metalloendopeptidase  Mitochondrial membrane-bound metalloendopeptidase        FMA2  Methionine aminopeptidase  Cytosolic aminopeptidase specific to N-terminal methionine        PPP20  Aminopeptidase  Vacuolar/cytosolic aminopeptidase    Trafficking  Sc  SEC1  Sm-like protein  SNARE binding, docking of exocytic vesicles  Hou et al. (2012, b, 2013)       MON2  SEC7 family, GEF  Endocytosis, Golgi-to-endosome transport, vacuole integrity  Kanjou et al. (2007)      PMR1  Ca2+/Mn2+ ATPase  Calcium-dependent protein sorting and processing  Harmsen et al. (1996)      SSO1, SSO2  Plasma membrane t-SNARE  Fusion of secretory vesicles at the plasma membrane  Ruohonen et al. (1997)    Pp  BFR2  90S preribosome  ER-to-Golgi transport, rRNA processing  Gasser et al. (2007)      BMH2  14-3-3 protein  Exocytosis, vesicle transport, and Ras/MAPK signaling      CUP5  Vacuolar membrane ATPase  Hydrogen ion transmembrane transport        KIN2  Serine/threonine protein kinase  Exocytosis regulation        SEC63  Sec63 complex subunit  Cytosol-to-ER transport  Zhang et al. (2001, 2006)    Sc,Sp  Vsp10  Vacuolar protein sorting receptor  Protein targeting to vacuole, vacuolar transport  Zhang et al. (2001, 2006) and Idiris et al. (2010)  Glycosylation  Sc,Kl  MNN1  α-1,3-mannosyltransferase  Addition of α-1,3-mannose to N-/O-linked oligosaccharides  Nakanishi-Shindo et al. (1993) and Liu et al. (2009, 2012)    Sc  MNN4  Positive regulator of Mnn6p  Mannosylphosphorylation of N-linked oligosaccharides  Chiba et al. (1998)      ALG11  α-1,2-mannosyltransferase  N-linked oligosaccharide assembly  Parsaie Nasab et al. (2013)    Sp  GMS1  UDP-galactose transporter  Transport of UDP-galactose into the lumen of Golgi apparatus  Ohashi and Takegawa (2010)      OMH1  O-glycoside α-1,2-mannosyltransferase  Extending α-1,2-linked mannose in the O-glycan pathway  Ikeda et al. (2009)    Sc, Pp, Hp  ALG3  α-1,3-mannosyltransferase  Synthesis of dolichol-linked oligosaccharide for N-glycosylation  Nakanishi-Shindo et al. (1993), Hamilton and Gerngross (2007) and Cheon et al. (2012)    Sc, Pp, Kl Sp, Hp, Yl  OCH1  α-1,6-mannosyltransferase  Outer chain elongation of N-linked oligosaccharides  Nakanishi-Shindo et al. (1993), Hamilton and Gerngross (2007), Song et al. (2007), Liu et al. (2009, 2012), Ohashi and Takegawa (2010) and Cheon et al. (2012)  Category  System  Gene  Protein  Biological function  References  Protein folding,  Sc  SIL1  Cochaperone, NEF  Protein translocation into the ER  Payne et al. (2008)  quality control    LHS1  Cochaperone  Polypeptide translocation and folding        JEM1  DnaJ-like cochaperone  Chaperone binding        SCJ1  HSP70-type cochaperone  Chaperone binding        HSF1  Heat shock transcription factor  Response to highly diverse stresses  Hou et al. (2012a, b, 2013)     Hp  CNE1  ER chaperone calnexin  Folding and quality control of glycoproteins  Klabunde et al. (2007)    Pp  YDJ1  Type I HSP40 cochaperone  Regulation of HSP90 and HSP70 functions  Zhang et al. (2001, 2006)      SSA1 SSA4  Cochaperone, HSP70 family  Protein foldingSRP-dependent protein-membrane targeting and translocation  Zhang et al. (2001, 2006)Gasser et al. (2007)      SSE1    Unfolded protein binding      Sc, Pp  HAC1  bZIP transcription factor  UPR regulation  Valkonen et al. (2003) and Gasser et al. (2007)      KAR2  HSP70 chaperones, BiP  Protein folding, UPR regulation  Harmsen et al. (1996) and Zhang et al. (2001, 2006)    Kl, Pp  ERO1  Thiol oxidase  Oxidative protein folding  Lodi et al. (2005) and Gasser et al. (2007)    Sc, Kl, Pp  PDI1  Protein disulfide isomerase  Disulfide bond formation of proteins  Smith et al. (2004), Lodi et al. (2005) and Zhang et al. (2001, 2006)  Degradation  Sc  CYM1  Cytosolic metalloprotease  Mitochondrial lysine-specific metalloprotease  Jonson et al. (2004)      CPY1  Carboxypeptidase Y  Vacuolar serine-type C-terminal exopeptidase  Van Den Hazel et al. (1996)    Sc,Pp  KEX2  Proprotein convertase  Serine-type calcium-dependent serine protease  Zhang et al. (2001, 2006) and Werten and de Wolf (2005)    Pp,Hp  YPS1  Yapsin-1  Aspartic-type endopeptidase  Werten and de Wolf (2005), Cho et al. (2010) and Choi et al. (2003)    Sc, Kl  UBI4  Ubiquitin  Ubiquitin-26S proteasome system  Chen et al. (1994) and Bao and Fukuhara (2001)    Sc, Pp, Cb  PEP4  Proteinase A  Vacuolar aspartyl protease  Kang et al. (1998, 2000, 2001), Komeda et al. (2002) and Wu et al. (2013)    Sc, Sp,Cb  PRB1  Proteinase B  Vacuolar serine-type endopeptidase  Kang et al. (1998, 2000, 2001), Komeda et al. (2002) and Idiris et al. (2010)    Sp  ATG4  Cysteine protease  Cytosolic cysteine-type endopeptidase  Idiris et al. (2010)      OMA1  Metalloendopeptidase  Mitochondrial membrane-bound metalloendopeptidase        FMA2  Methionine aminopeptidase  Cytosolic aminopeptidase specific to N-terminal methionine        PPP20  Aminopeptidase  Vacuolar/cytosolic aminopeptidase    Trafficking  Sc  SEC1  Sm-like protein  SNARE binding, docking of exocytic vesicles  Hou et al. (2012, b, 2013)       MON2  SEC7 family, GEF  Endocytosis, Golgi-to-endosome transport, vacuole integrity  Kanjou et al. (2007)      PMR1  Ca2+/Mn2+ ATPase  Calcium-dependent protein sorting and processing  Harmsen et al. (1996)      SSO1, SSO2  Plasma membrane t-SNARE  Fusion of secretory vesicles at the plasma membrane  Ruohonen et al. (1997)    Pp  BFR2  90S preribosome  ER-to-Golgi transport, rRNA processing  Gasser et al. (2007)      BMH2  14-3-3 protein  Exocytosis, vesicle transport, and Ras/MAPK signaling      CUP5  Vacuolar membrane ATPase  Hydrogen ion transmembrane transport        KIN2  Serine/threonine protein kinase  Exocytosis regulation        SEC63  Sec63 complex subunit  Cytosol-to-ER transport  Zhang et al. (2001, 2006)    Sc,Sp  Vsp10  Vacuolar protein sorting receptor  Protein targeting to vacuole, vacuolar transport  Zhang et al. (2001, 2006) and Idiris et al. (2010)  Glycosylation  Sc,Kl  MNN1  α-1,3-mannosyltransferase  Addition of α-1,3-mannose to N-/O-linked oligosaccharides  Nakanishi-Shindo et al. (1993) and Liu et al. (2009, 2012)    Sc  MNN4  Positive regulator of Mnn6p  Mannosylphosphorylation of N-linked oligosaccharides  Chiba et al. (1998)      ALG11  α-1,2-mannosyltransferase  N-linked oligosaccharide assembly  Parsaie Nasab et al. (2013)    Sp  GMS1  UDP-galactose transporter  Transport of UDP-galactose into the lumen of Golgi apparatus  Ohashi and Takegawa (2010)      OMH1  O-glycoside α-1,2-mannosyltransferase  Extending α-1,2-linked mannose in the O-glycan pathway  Ikeda et al. (2009)    Sc, Pp, Hp  ALG3  α-1,3-mannosyltransferase  Synthesis of dolichol-linked oligosaccharide for N-glycosylation  Nakanishi-Shindo et al. (1993), Hamilton and Gerngross (2007) and Cheon et al. (2012)    Sc, Pp, Kl Sp, Hp, Yl  OCH1  α-1,6-mannosyltransferase  Outer chain elongation of N-linked oligosaccharides  Nakanishi-Shindo et al. (1993), Hamilton and Gerngross (2007), Song et al. (2007), Liu et al. (2009, 2012), Ohashi and Takegawa (2010) and Cheon et al. (2012)  Sc, S. cerevisiae;Pp, P. pastoris;Sp, S. pombe;Kl, K. lactis;Cb, C. boidinii;Hp, H. polymorpha;Yl, Y. lipolytica. View Large Synthetic biology for yeast glycoengineering Most proteins with therapeutic applications are produced with post-translational modifications, which affect their function and stability. Among various post-translational modifications, glycosylation is the most common and at the same time the most complex modification (Walsh 2010b). Although yeasts can perform N- and O-glycosylation of proteins, the final glycosylation patterns of yeasts significantly differ from those of humans. High-mannose-type glycans and terminal α-1,3-mannose linkages associated with glycoproteins produced from S. cerevisiae are known to cause short serum half-lives or even immunogenicity of therapeutic proteins (De Pourcq et al., 2010; Walsh 2010b). Thus, several efforts have been made toward the development of intelligent yeast strains devoid of yeast-specific glycans and the ability to synthesize human-type glycans (Fig. 2). The pioneering work by Dr. Jigami's group demonstrated the high potential of yeast glycoengineering to produce recombinant proteins with human-type glycans. A glycoengineered S. cerevisiae strain was constructed by deletion of the OCH1 gene that is responsible for the initiation of yeast-specific outer chain biosynthesis and subsequent introduction of an Aspergillus saitoi α-1,2 mannosidase in the ER for mannose trimming. This yeast strain was shown to produce human compatible Man5GlcNAc2 glycan (Chiba et al., 1998). Intensive attempts have been continued to implement humanized glycosylation pathways in various nonconventional yeast species, in which most N-linked glycans are much less hypermannosylated and lack hyperimmunogenic terminal α-1,3-linked mannose residues compared to those of S. cerevisiae (Hamilton and Gerngross 2007; De Pourcq et al., 2010; Cheon et al., 2012). Deletion of the OCH1 gene has been carried out in various nonconventional yeast species, such as H. polymorpha, P. pastoris, S. pombe, K. lactis, and Y. lipolytica, and is considered as an initiation step required for humanized glycoengineering (Hamilton and Gerngross 2007; Song et al., 2007; Liu et al., 2009; Ohashi and Takegawa 2010; Cheon et al., 2012). Figure 2. View largeDownload slide Schematic representation of engineering strategies of N-glycosylation pathways in yeasts. (a) N-glycosylation pathways in mammals and S. cerevisiae. Lipid-linked glycan assembly in the ER is a highly conserved process. The core oligosaccharide Man8GlcNAc2 is made in the ER and transferred to the Golgi, where further glycan processing diverges between humans and yeast. In humans, mannosidase (Mns) enzymes trim three mannose residues, leaving a Man5GlcNAc2 structure, which is modified to complex glycans through β-1,2-N-acetylglucosaminyltransferase I (GnT-I), MnsII, GnT-II, galactosyltransferase (GalT), and sialyltransferase (SAT). In contrast, N-glycans of S. cerevisiae are further extended by attachment of only mannose residues in the Golgi apparatus. (b) Engineering of the N-glycosylation pathway for the production of human complex-type N-glycans in several yeast species. The deletion of the OCH1 gene blocks outer chain elongation in Golgi apparatus. Introduction of heterologous α-1,2-mannosidase (α-1,2-Mns) into the och1 deletion mutant is required for mannose trimming to generate the Man5GlcNAc2 structure, which can be further utilized as the substrate for the GnT-I, MnsII, and GnT-II. On the other hand, a Man3GlcNAc2 structure is generated in the och1alg3 double-mutant-expressing α-1,2-Mns or directly in alg3alg11 double-mutant strains without heterologous expression of an α-1,2-Mns. The N-linked trimannosyl core glycan can serve as a substrate for GnT-I and GnT-II, giving rise to the human-hybrid/complex-type N-glycan in yeast cells. Sc, S. cerevisiae;Pp, P. pastoris;Sp, S. pombe;Kl, K. lactis;Hp, H. polymorpha; Yl, Y. lipolytica. Figure 2. View largeDownload slide Schematic representation of engineering strategies of N-glycosylation pathways in yeasts. (a) N-glycosylation pathways in mammals and S. cerevisiae. Lipid-linked glycan assembly in the ER is a highly conserved process. The core oligosaccharide Man8GlcNAc2 is made in the ER and transferred to the Golgi, where further glycan processing diverges between humans and yeast. In humans, mannosidase (Mns) enzymes trim three mannose residues, leaving a Man5GlcNAc2 structure, which is modified to complex glycans through β-1,2-N-acetylglucosaminyltransferase I (GnT-I), MnsII, GnT-II, galactosyltransferase (GalT), and sialyltransferase (SAT). In contrast, N-glycans of S. cerevisiae are further extended by attachment of only mannose residues in the Golgi apparatus. (b) Engineering of the N-glycosylation pathway for the production of human complex-type N-glycans in several yeast species. The deletion of the OCH1 gene blocks outer chain elongation in Golgi apparatus. Introduction of heterologous α-1,2-mannosidase (α-1,2-Mns) into the och1 deletion mutant is required for mannose trimming to generate the Man5GlcNAc2 structure, which can be further utilized as the substrate for the GnT-I, MnsII, and GnT-II. On the other hand, a Man3GlcNAc2 structure is generated in the och1alg3 double-mutant-expressing α-1,2-Mns or directly in alg3alg11 double-mutant strains without heterologous expression of an α-1,2-Mns. The N-linked trimannosyl core glycan can serve as a substrate for GnT-I and GnT-II, giving rise to the human-hybrid/complex-type N-glycan in yeast cells. Sc, S. cerevisiae;Pp, P. pastoris;Sp, S. pombe;Kl, K. lactis;Hp, H. polymorpha; Yl, Y. lipolytica. To achieve complex-type humanized glycosylation, further introductions of additional glycosyltransferases and glycosidases involved in human glycosylation pathways should be made in yeast. Besides simple expression of these genes, precise spatial positioning along the ER-Golgi secretory pathway is crucial for a successful assembly of the foreign glycosylation pathway. For functional targeted expression at the yeast Golgi membrane, semi-synthetic glycosyltransferase and glycosidase libraries composed of several combinations of C-terminal catalytic domains and transmembrane fragments were constructed, and these combinatorial libraries combined with high-throughput screening were used to screen P. pastoris-engineered strains harboring optimal foreign glycosylation pathways (Choi et al., 2003). Finally, the production of humanized glycoproteins with terminal sialic acids in P. pastoris was achieved by the introduction of an additional nine synthetic genes enabling sialic acid biosynthesis in yeast (Hamilton et al., 2006). An alternative strategy for reconstructing the biosynthetic pathway of initial lipid-linked oligosaccharide synthesis, which removes the requirement of mannosidase II, was developed based on deletion of the ALG3 gene coding for dolichyl-phosphate-mannose (Dol-P-Man)-dependent α-1,3 mannosyltransferase in P. pastoris and H. polymorpha (Davidson et al., 2004; Cheon et al., 2012). In a recent study, a novel synthetic N-glycosylation pathway generated by the ALG3 and ALG11 double deletion in S. cerevisiae was reported to produce glycoproteins containing a trimannosyl core glycan as a major portion of humanized N-glycan structures (Parsaie Nasab et al., 2013). Moreover, the overexpression of protozoan oligosaccharyltransferases, which possess altered specificities for the oligosaccharide and also for the protein acceptor site, was proposed as a promising tool to improve glycosylation site occupancy in the glycoengineered yeast strains, which ensures the production of glycoproteins fully glycosylated with homogeneous N-glycans (Piirainen et al., 2014). In addition to the N-glycosylation engineering, the glycoengineering of yeasts has been targeted toward O-glycosylation, which is characterized by relatively shorter glycan structures. Unlike the O-glycan of yeasts, the O-linked glycan structure of mammalian systems is elongated by mucin-type glycosylation, which includes an N-acetylgalactosamine (GalNAc) conjugated to the Ser/Thr residue (Hanisch 2001). This sugar is subsequently extended with other sugars, including N-acetylglucosamine (GlcNAc), galactose, fucose, and sialic acid. The other mammalian O-glycosylation is described as α-dystroglycan type (Lommel and Strahl 2009), which initiates with a mannose conjugated to the Ser/Thr residue and is elongated with GlcNAc, galactose, and sialic acid residues. Some higher eukaryotic glycan structures also have glucuronic acid or xylose residue, which is particularly important to linking proteoglycans to proteins or the biosynthesis of various types of glycosaminoglycans (Wilson 2004), respectively. Modified O-glycosylation pathways have been established in S. cerevisiae to produce mammalian-type glycosylation patterns by heterologous expression of several foreign genes involved in O-glycosylation of higher eukaryotes. The human GDP-fucose transporter and human O-FucT-1 genes were expressed in S. cerevisiae to produce GDP-fucose from intrinsic GDP-mannose in the yeast cytosol, resulting in production of an O-fucosylated epidermal growth factor domain (Chigira et al., 2008). The artificial S. cerevisiae strain was constructed by introducing UDP-glucose dehydrogenase and UDP-glucuronic acid decarboxylase derived from Arabidopsis thaliana, which is capable of converting UDP-glucose to UDP-xylose through the two-step process (Oka and Jigami 2006). Recently, production of mucin-type glycoproteins in this baker's yeast was succeeded through genetic engineering to introduce various heterologous enzymes, absent in yeast, such as ppGalNAc-T1, core1 β1–3GalT, UDP-Gal/GalNAc transporter, and UDP-Gal/GalNAc 4-epimerase (Amano et al., 2008). Moreover, human-like sialylated glycans, similar to α-dystroglycan-type glycoforms, were created in P. pastoris by the introduction of the enzymes α-1,2-mannosidase and β-1,2-N-acetylglucosaminyltransferase 1 into the engineered P. pastoris strain harboring a heterologous sialic biosynthesis pathway (Hamilton et al., 2013). Perspectives of yeast factories for recombinant protein production Yeasts are particularly amenable to studying the mechanistic basis of high-yielding recombinant protein production experiments using synthetic biology tools. In the postgenome era, significant advances in the yeast toolbox of genetic elements have been made toward synthetic biology approaches from classical genetic engineering. Genome-scale metabolic models based on whole-genome sequence information are available not only for S. cerevisiae (Osterlund et al., 2012), but also for other nonconventional yeasts such as P. pastoris and Y. lipolytica (De Schutter et al., 2009; Sherman et al., 2009). Moreover, genome-scale engineering of S. pombe by large-scale gene deletion was shown to be applicable for the construction of host strains designed for enhanced production of recombinant proteins (Sasaki et al., 2013). A genome-scale model consisting of core components in the secretory machinery and reactions for the protein secretory machinery was recently constructed using a bottom-up approach in S. cerevisiae (Feizi et al., 2013). This model offers knowledge of the protein secretory machinery in other eukaryotic organisms as well as in yeast. More importantly, it could be utilized to estimate the metabolic and energy demands on the secretory machinery and thereby finally assess targets of metabolic engineering for improving protein secretion. Importantly, new platform strains and molecular cloning tools as well as in vivo glycoengineering to produce humanized glycoforms have made yeast a more practical host for therapeutic recombinant protein production. The synthetic biology approach of targeting glycosylation engineering should facilitate implementation of the humanized glycosylation pathway as well as assembly of synthetic glycosylation processes to attach several kinds of artificial oligosaccharide chains. The creation of glycoproteins with tailor-made glycoforms and differing pharmacodynamics using glycoengineered yeast strains will surely pave the way for the production of new glycoprotein pharmaceuticals with better properties than natural variants. Therefore, it is predicted that synthetic biology approach would further expand the use of various yeasts as a major workhorse for production of next-generation biopharmaceutical proteins. In the respect of industrial applications, high cell-density fermentation of yeast is one of the important strategies to increase the ultimate yield of recombinant proteins. However, the move from low-density cultivation at shake flasks to high-density cultivation in bioreactors requires considerable re-optimization of several culturing conditions, such as pH, aeration, and carbon source feed rate (Cereghino et al., 2002). Moreover, high cell-density cultivation of yeast not only produces more contaminating proteins but also causes an accumulation of lipid-like substances in high-salt medium. In the case of the production of recombinant human interleukin-6 (rhIL-6) in P. pastoris, PEG-8000 precipitation was shown to be an effective approach to eliminate unnecessary contaminating proteins, thus facilitating the downstream purification process (Li et al., 2011). On the other hand, it has been reported that specific productivity of antibodies produced by a glycoengineered P. pastoris in high cell-density cultivation was improved through oxygen-limited cultivation. Moreover, the oxygen-limited process was successfully scaled to 1200 L-manufacturing pilot scale, which showed not only increased antibody concentration (up to 1.9 g L−1) but also improved N-glycosylation pattern and reduced antibody fragmentation (Berdichevsky et al., 2011). Thus, it is apparent that intensive efforts are needed to develop physiologically rational and suitable cultivation process with protein-specific adjustments to upgrade yield and quality of recombinant proteins. As systems and synthetic biology approaches would provide more comprehensive information on host physiology, along with more rationally designed host engineering suitable for large-scale cultivation, yeast cell factories will become increasingly more efficient and economic platforms for the production of recombinant therapeutic proteins, which are equipped with robust and commercially viable processes. This work was supported by a grant from the Korea Ministry of Education and Science Technology (Global Frontier Program for the Intelligent Synthetic Biology No. NRF-2013M3A6A8073554) and by a grant from the Korean Ministry of Rural Development Administration (Next-Generation BioGreen 21 Program No. ADFC2013-PJ009586). H. Kim was a recipient of a 2011 Chung-Ang University Excellent Student Scholarship. AUTHORS' CONTRIBUTION K.H. and Y.S.J. contributed equally to this work. Conflict of interest statement. None declared. 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TI - Yeast synthetic biology for the production of recombinant therapeutic proteins
JF - FEMS Yeast Research
DO - 10.1111/1567-1364.12195
DA - 2015-02-01
UR - https://www.deepdyve.com/lp/oxford-university-press/yeast-synthetic-biology-for-the-production-of-recombinant-therapeutic-h080GLRJOk
SP - 1
EP - 16
VL - 15
IS - 1
DP - DeepDyve
ER -