Unconventional microbial systems for the cost-efficient production of high-quality protein therapeutics

Biotechnology Advances 31 (2013) 140–153

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Research review paper

Unconventional microbial systems for the cost-efficient production of high-qualityprotein therapeutics

José Luis Corchero a,b,c, Brigitte Gasser d,e, David Resina f, Wesley Smith g, Ermenegilda Parrilli h,Felícitas Vázquez a,b,c,1, Ibane Abasolo a,i, Maria Giuliani h, Jussi Jäntti g, Pau Ferrer j, Markku Saloheimo g,Diethard Mattanovich d,e, Simó Schwartz Jr. a,i, Maria Luisa Tutino h, Antonio Villaverde a,b,c,⁎a CIBER en Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Spainb Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spainc Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spaind Department of Biotechnology, University of Natural Resources and Life Sciences (BOKU), Muthgasse 18, 1190 Vienna, Austriae Austrian Centre of Industrial Biotechnology (ACIB), Vienna, Austriaf Bioingenium s.l., Barcelona Science Park, Baldiri Reixac 15, 08028 Barcelona, Spaing VTT Technical Research Centre of Finland, Finlandh Dipartimento di Chimica Organica e Biochimica, Università di Napoli Federico II, Complesso Universitario M.S. Angelo, via Cinthia 4, 80126 Naples, Italyi CIBBIM-Nanomedicine, Hospital Universitari Vall d'Hebrón and Vall d'Hebrón Institut de Recerca, Universitat Autónoma de Barcelona, 08035 Barcelona, Spainj Departament d'Enginyeria Química, Escola d'Enginyeria, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain

⁎ Corresponding author at: Institut de Biotecnologia iE-mail address: [email protected] (A. Villave

1 Present address: Departament d'Enginyeria Química

0734-9750/$ – see front matter © 2012 Elsevier Inc. Allhttp://dx.doi.org/10.1016/j.biotechadv.2012.09.001

a b s t r a c t
a r t i c l e i n f o
Article history:Received 25 November 2011Revised in revised form 4 September 2012Accepted 7 September 2012Available online 15 September 2012

Keywords:Recombinant proteinBiotherapeuticsMicrobial cellsProtein foldingAggregationProtein production

Both conventional and innovative biomedical approaches require cost-effective protein drugswith high therapeuticpotency, improved bioavailability, biocompatibility, stability and pharmacokinetics. The growing longevity of thehuman population, the increasing incidence and prevalence of age-related diseases and the better comprehensionof genetic-linked disorders prompt to develop natural and engineered drugs addressed to fulfill emergingtherapeutic demands. Conventional microbial systems have been for long time exploited to producebiotherapeutics, competing with animal cells due to easier operation and lower process costs. However,both biological platforms exhibit important drawbacks (mainly associated to intracellular retention of theproduct, lack of post-translational modifications and conformational stresses), that cannot be overcomethrough further strain optimization merely due to physiological constraints. The metabolic diversityamong microorganisms offers a spectrum of unconventional hosts, that, being able to bypass some of theseweaknesses, are under progressive incorporation into production pipelines. In this review we describe themain biological traits and potentials of emerging bacterial, yeast, fungal and microalgae systems, by comparingselected leading species with well established conventional organisms with a long run in protein drugproduction.

© 2012 Elsevier Inc. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1412. Conventional protein production systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142

2.1. Escherichia coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1422.2. Saccharomyces cerevisiae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1422.3. Mammalian cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143

3. Emerging microbial systems for protein production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1443.1. Pseudoalteromonas haloplanktis improved protein folding in bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1443.2. Pichia pastoris and other non-conventional yeasts for protein humanization and enhanced secretion . . . . . . . . . . . . . . . . . . . . 1453.3. Trichoderma reesei: enhanced productivity and easy protein purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1483.4. Chlamydomonas reinhardtii, an emeging GRAS system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148

de Biomedicina, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain.rde)., Escola d'Enginyeria, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain.

rights reserved.

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141J.L. Corchero et al. / Biotechnology Advances 31 (2013) 140–153

4. Conclusions and future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149

1. Introduction

Since the production of recombinant insulin in the late 70s, theemergence of molecular biology and biotechnology has enabled thebiological fabrication of a long list of active therapeutic proteins.Today, recombinant DNA and hybridoma cell technologies are main-stream platforms to obtain most of the currently marketed proteindrugs such as monoclonal antibodies, hormones, cytokines andgrowth factors (Ferrer-Miralles et al., 2009; Walsh, 2010). Over 200protein drugs are expected to be available over the next few years totreat expanding human disorders such as diabetes, cancer, respiratory,cardiovascular and inflammation-related diseases, as well as rare dis-eases. In fact, the global market for protein drugs already exceeds US$50billion, with an average annual growth rate of almost 4%, accordingto BCC Research (Protein Drugs: Global Markets and ManufacturingTechnologies, 2008, http://www.bccresearch.com/report/protein-drugs-markets-manufacturing-bio021c.html?tab=scope&highlightKeyword=protein+drugs). Unfortunately, the costs of protein drugs are oftenextremely high. As a representative example, recombinant humanErythropoietin (EPO), which is used as treatment for anemia due tokidney failure or anticancer treatments costs over 2 US $ billion/kg,probably being the most expensive existing substance today. Enzymereplacement therapies such as for lysosomal storage diseases (LSD) rep-resent in excess of 150 K$/year per patient. Such high costs are partiallyexplained not only by the investment in product development but alsoby the expenses associated to quality analysis and control (http://www.chem.agilent.com/Library/primers/Public/5990-8561EN_LO.pdf; and(Eon-Duval et al., 2012; Medrano et al., 2012)). In addition, the imma-ture state of the current production methods raises manufacturing

Protein drug pMa

Unconventional hosts o

Midstream Downstream

Costs

Wrong or no glycosilation

Improper folding

Costs

Intracellular r

Wrong or no maturation

Wrong or no disulphide bridge formation

Upstream

Gene expression vectors and tools

Physiologic, genetic and omics data

Cold-adapted bacteria Filamentous fun

Alternative yeasts Unconventiona

Filamentous fungi Lactic acid bact

Scalability

Microalgae

Fig. 1. Main bottlenecks in the microbial production of recombinant drugs, as identified whmight be unsolvable by further process, genetic or metabolic engineering, as they still pehave been aligned with steps in the production pipeline, where unconventional hosts can insin accumulating reports (see Tables 1 and 2). While conventional systems are preferred in uppoints of view, and a plethora of tools for genetic manipulation and gene expression are avaidownstream. This is often reflected by an increase in protein folding and quality which imprthe other hand, some unconventional hosts are also appealing regarding regulatory issues

costs to a level often unaffordable from an industrial point of view,what drops pharmacologically valuable products from the marketingpipeline. With the exception of short peptides, suitable to be producedby chemical synthesis, protein pharmaceuticals have to be producedin living cells or transgenic organisms, what poses important challengesto industrially-scaled production (Kemsley, 2009). Manufacturing a re-combinant protein or an antibody might represent up to 25% of theglobal sale figures (Farid, 2007), what makes a strong pressure to findcost-effective alternatives to the current recombinant fabrication sys-tems (Chadd and Chamow, 2001; Farid et al., 2005). Among the differ-ent steps of recombinant protein production, downstream processingmight impose a load of up to 80% of the total process cost (Roqueet al., 2004; Rouf et al., 2000), a figure that can be slightly reduced byadapting the equipment, specially moving from steel bioreactors todisposable tanks (Vermasvuori, 2009). Interestingly, the major factorinfluencing the process cost is the biological platform used as cell factory(Vermasvuori, 2009).

On the other hand,many protein drugs are often unstable during pro-duction and/or purification (Fig. 1) and tend to aggregate (Vazquez et al.,2011). Systemic administration of protein drugs is specially sensitive toaggregation (Antosova et al., 2009), which can also occur during storageor in a form of unnoticed soluble aggregates (Vazquez et al., 2011).Many side effects and undesired immunoreactions have been found tobe linked to protein instability and aggregation once in the body(Antonelli, 2008; De Groot and Scott, 2007; Fradkin et al., 2009; Richardand Prang, 2010). Then, improving protein solubility represents a contin-uous challenge in the development of protein drugs (Garcia-Fruitos et al.,2011; Martinez-Alonso et al., 2010; Rathore and Rajan, 2008; Shire,2009). Unfortunately, protein products still suffer from batch-to-batch

roduction pipelinein bottlenecks

ffering improvement

Storage/administration Regulatory

Poor activity

Aggregation

etention Side effects

Non GRAS nature

Immunogenicity

Lactic acid bacteriagi Cold-adapted bacteria

l yeasts

eria

Unconventional yeast

Filamentous fungi

Microalgae

Unconventional yeast

en using conventional hosts for protein production. At least some of these limitationsrsist despite intensive research and improvement attempts. These main bottleneckstead offer significant improvement because of specific physiological traits, as identifiedstream because they are extremely well known from genetic, metabolic and proteomiclable, alternative hosts offer different added values specially in midstream and speciallyoves product stability, solubility and performance during storage or administration. Onbecause of their GRAS character.

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142 J.L. Corchero et al. / Biotechnology Advances 31 (2013) 140–153

conformational heterogeneity, a currently unavoidable disadvantageinherent to recombinant biological production. These drawbackscomplicate the consideration of proteins as clinical drugs (http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM073497.pdf) from the regulatory point of view (Sahooet al., 2009).

Moreover, proteins with therapeutic value often undergo post-transcriptional and post-translational modifications necessary for thenatural biological function. These include potential splicing, site-specific proteolysis, proper protein folding, glycosylation and disulfidebond formation. Also, some proteins are only functional as specificoligomeric combinations, while uncontrolled multimerization results inthe formation of soluble and insoluble aggregates (Vazquez et al., 2011).Unfortunately, drug formulation cannot consistently solve conformation-al issues andprevent protein aggregation (Richard andPrang, 2010; Shire,2009). Currently, major efforts are devoted to favor proper glycosylationand folding patterns, as they are considered critical issues regarding sta-bility and efficacy of protein drugs ((Kamionka, 2011) and Fig. 1).

Noteworthy, there is an increasing need to provide the clinical settingwith protein drugs with particular biodistribution, for instance, being ca-pable of crossing the blood–brain barrier (BBB) (Brasnjevic et al., 2009;Pardridge, 2007).With the progressive aging of the population, a growingnumber of neurological diseases, such as acute and chronic brain injury,Alzheimer and Parkinson's diseases, stroke, schizophrenia and depressionhave to be now confronted. Unfortunately, a majority of the marketedprotein drugs are therefore not reaching the central nervous system.However, this issue can be approached through the production of fusionproteins capable of crossing the BBB by endothelial receptor-mediatedtranscytosis, what if successful, is expected to offer new clinical protocolsfor the treatment of neurological disorders (Brasnjevic et al., 2009). Theneed for protein drug engineering is evident in targeted drug deliv-ery. The end terminal fusion of specific ligands that permit the chi-merical drug to interact with particular cell surface receptors couldhopefully reduce drug doses and therefore, production costs andside effects (Vazquez et al., 2009). On the other hand, protein engineer-ingmight, as a side effect, favor insolubility (Baneyx andMujacic, 2004).

The ever-increasing relevance of protein drugs in the clinical scenariomakes the efficient and cost-effective production of safe and effectivepolypeptides necessary (Carter, 2011; Roskos et al., 2011; Szlachcicet al., 2011). Different host organisms might offer very different cultureand protein processing conditions, downstream opportunities and alter-native post-transcriptional–post-translational modifications. Therefore,not only upgrading the well-known production platforms (whose opti-mizationmight have reached a physiological plateau) but also the incor-poration of new biological systems is necessary to explore high-qualityprotein drug production (Fig. 1). Products with improved solubility andhigh production yields, at the same time reaching safety and stabilitystandards are particularly imperative (Carter, 2011; Roskos et al., 2011;Szlachcic et al., 2011; Tomlinson, 2004). Unlike mammalian or insectcell lines, with rather regular metabolic profiles, the vast diversity ofthemicrobial physiology offers exciting opportunities for product devel-opment (Rodriguez-Carmona and Villaverde, 2010; Vazquez andVillaverde, 2010; Villaverde, 2010). This would be possible not onlythrough the convenient engineering of conventional cell factories and as-sociated processes but especially by the identification of novel hostswithappealing properties. Here we revise the foremost emerging microbialsystems, in comparison with standard host cells, offering unusual valuesas cell factories that could contribute to reduce costs and to enhance bi-ological quality and usability of recombinant protein pharmaceuticals.

2. Conventional protein production systems

2.1. Escherichia coli

Among the 58 pharmaceutical products approved from 2006 toJune 2010, 17 are produced in E. coli (Walsh, 2010), confirming that

even with its important shortcomings E. coli is still a workhorse ofbiopharmaceutical production (Ferrer-Miralles et al., 2009). Moreover,in 2009, one of the top selling biopharmaceuticals was Lantus®, anengineered insulin produced in this bacterium. The deeply exploredgenetics, wide availability of genetic tools for gene cloning and expres-sion, accurate genomic, proteomic and metabolic profiling and the fastgrowth in inexpensive media at high cell densities make this specieshighly desirable for industrial protein production. Despite these re-markable figures and the recognized advantages of this production sys-tem, E. coli ismostly appropriate to produce structurally simple proteins(Carter, 2011).

The main constrains for the production of functional proteins inE. coli are its inability to carry out post-translational modifications,proper protein folding and efficient protein secretion (Ferrer-Miralleset al., 2009). Posttranslational modifications are often essential forcorrect protein folding and functionality. Approximately 70% of thetherapeutic proteins currently in clinical trials are glycosylated(Sethuraman and Stadheim, 2006). Until recently, it was believed thatonly eukaryotic cell lines could be used to produce N-type glycosylatedproteins. However, in 2002, Wacker et al. (2002) successfullytransferred the N-linked glycosylation system of Campylobacter jejunito E. coli, to generate a chimerical system with some room for furtherimprovement (bu-Qarn et al., 2008; Elliott et al., 2003; Pandhal andWright, 2010). On the other hand, while periplasmic expression is ingeneral successful (Anton et al., 2010; Makino et al., 2011; Mergulhaoet al., 2005), E. coli lacks generic protein secretory systems (Hannigand Makrides, 1998).

Finally, aggregation of heterologous proteins and formation of inclu-sion bodies (IBs) are extremely common events (Baneyx and Mujacic,2004). How to minimize protein deposition and how to gain solubilityhave been a matter of discussion since early recombinant DNA times(Davis et al., 1999; de Marco et al., 2007; Lee et al., 2003; Marston,1986; Sorensen andMortensen, 2005). Using weak promoters, culturingat suboptimal temperatures or coproducing folding modulators alongwith target proteins have been explored without reaching a consensusabout the choice procedures. Unveiling the side effects of chaperoneco-production (Kolaj et al., 2009; Martinez-Alonso et al., 2009b, 2010;Platas et al., 2011), the occurrence of soluble aggregates and functionallycompetent IBs (Garcia-Fruitos, 2010; Gonzalez-Montalban et al., 2007;Rodriguez-Carmona et al., 2010; Villaverde et al., 2012), and the deepcomplexity, intricacy and overlapping nature of the quality controlsystem (Martinez-Alonso et al., 2009a) have prevented a systematicapproach towards an improved solubility of the E. coli protein products.In fact, the polarity of IB formation (Rokney et al., 2009), and the closefunctional link between IBs and protein degradation, mediated bychaperone DnaK (Carrio and Villaverde, 2005; Garcia-Fruitos, 2010;Garcia-Fruitos et al., 2007; Martinez-Alonso et al., 2007, 2009b), indi-cate that IB formation is not a passive event but the result of complexcell activities, similar to those leading to aggresome formation in mam-malian cells (Kopito, 2000). Nowadays, protein aggregation still re-mains as a major, probably unaffordable drawback of the E. coli cellfactory.

2.2. Saccharomyces cerevisiae

Since the implementation of S. cerevisiae as an expression platformfor heterologous proteins (Hitzeman et al., 1981), this yeast has beenemployed for the production of several pharmaceutical proteins,among which the most important are insulin, insulin variants andhepatitis B vaccines (Ferrer-Miralles et al., 2009) (Table 2). Combiningthe eukaryotic ability to perform post-translational modifications withthe bacterial capacity to grow at high cell densities in inexpensivemedia, S. cerevisiae (and yeasts in general) offers generally higher yieldsof recombinant proteins and better scalability than mammalian cells.Contrary to enterobacteria, yeasts are able to secrete recombinant

http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM073497.pdf




proteins to the extracellular medium, which is very appreciated indownstream processing.

S. cerevisiae, well known as a beer and bread producer, is the mostcommon yeast system used for manufacturing of therapeutic proteins(Ferrer-Miralles et al., 2009). Recombivax, a recombinant Hepatitis Bvirus (HBV) antigen was the first protein produced in this species(McAleer et al., 1984). Also, S. cerevisiaewas the first sequenced eukary-otic organism, so that its genetics and physiology arewidely known andtools for molecular biology have been very well established. Up to date,several therapeutic proteins produced in S. cerevisiae have beencommercialized, and innovative approaches for protein drug pro-duction are being explored. For instance Ardiani et al. (2010)reviewed the use of whole cell recombinant S. cerevisiae cells as ther-apeutic vaccines, where yeast cells were engineered to express viralor tumoral antigens. Such heat-inactivated yeasts have the ability tostimulate tumor or viral specific CD4+ and CD8+ T-cell responses.Also, cell wall components such as beta-1, 3-D-glucan or mannanhave strong properties as vaccine adjuvants. In 2004, Lu et al. testedthe capacity of yeast whole cells expressing mammalian mutant K-rasprotein to generate a response against tumor cells. The success at thepreclinical assays led to a phase I clinical trial with 33 patients withrasmutations. Approximately 90% of the patients showed a ras specificT-cell response.

Following the use of yeast cells as biotherapeutics, Sun et al.(2010) proposed a new advance in the use of yeast surface displayas carrier for therapeutic enzymes such as calcitonin. Today, salmoncalcitonin is used in the treatment of Paget's disease, osteoporosis,and hypercalcemia. The authors claim that orally administered yeastcells exposing salmon calcitonin on the cell wall were able to induce along term hypocalcemic affect in Wistar rats, without adverse affects(Sun et al., 2010).

On the other hand, fusion technologies are gaining a lot of attractive-ness because of their evident potential advantages over the first gener-ation therapeutic products. In general, fusion proteins are assumed toincrease the circulation half-live of the protein of interest or target tospecific receptors. In the report by Evans et al. (2010), the fusion ofalbumin to a scFv increased more than ten-fold the residence timecompared with the non-fused scFv. In the near future this new tech-nology of albumin-fusion proteins could make available a series ofnew products with a lower therapeutic dose and dose frequency.Also, non-glycosylated transferrin was fused to glucagon-like peptide1 and to exendi-4, for the treatment of diabetes (Kim et al., 2010). Toavoid yeast glycosylation-related issues, potential glycosylation siteson transferrin were removed by amino acid substitution N413E andN611E (Sadegi et al., 2006). The fusion protein GLP-1-transferrin wasproduced in S. cerevisiae and it was able to activate GLP-1 receptor.The highly stable fusion protein exhibited a half life of 2 days, comparedwith the 1 to 2 min of the native protein. Non-glycosylated transferrinwas also expressed successfully (Finnis et al., 2010). In this case,overexpression of the disulphide bond formation chaperone PDI1 in-creased approximately 10-fold the expression level of the producingstrain.

On the other hand, yeast glycoprotein expression is potentiallyenvisaged as a main source of human glycoproteins in the future(Chiba and Akeboshi, 2009; Gerngross, 2004). A huge effort is beingdone to generate a collection of new strainswith humanized sugar con-tents, starting with a S. cerevisiae mutant strain with a deletion in thealpha-1,6-mannosyltransferase OCH1 gene (Nakayama et al., 1992).Disruption of the OCH1 gene caused the loss of hyperglycosylation in se-creted proteins. Also, several studies have focused in disrupting yeastgenes involved in the extension of mannose chains (Nakanishi-Shindoet al., 1993) and in introducing new genes to catalyze the transformationof yeast glycans into human-like sugars (Chiba et al., 1998). However, acompletely humanized yeast glycan was not achieved until 2006, whenthe fully humanized synthesis of N-glycans in the methylotrophic yeastPichia pastoris was described (Hamilton et al., 2006). However, all the

commercialized therapeutic proteins produced in S. cerevisiae to dateare non-glycosylated.

2.3. Mammalian cells

Mammalian cells are a preferred choice to produce recombinantproteins due to their ability to perform proper post-translational modi-fications, often essential for therapeutic purposes (Ferrer-Miralles et al.,2009). By 2007, the production of protein drugs in mammalian expres-sion systems had reached US $20billion worldwide annually (Griffinet al., 2007), in a sector that is steadily growing (Walsh, 2010). Main is-sues of this system are limited and instable productivity (Barnes et al.,2003; Butler, 2005; Wurm, 2004). Constitutive protein production forextended cultivation times is routinely reached by integrating the re-combinant gene into the host genome (stable cell lines). In this scenario,the recombinant DNA is passed onto and maintained in all daughtercells. However, a decrease in specific productivity of protein expres-sion during prolonged culture is commonly observed due to genesilencing at the level of chromatin (epigenetic gene silencing). Reagentsthat relieve gene repression, cis-acting DNA agents or targetingtransgenes to genomic sites that favor gene expression usually driveto steady protein production (Kwaks and Otte, 2006). Cell transfectionwith the target gene along with a selectable marker gene such asdihydrofolate reductase (DHFR) or glutamine synthetase (GS), followedby the application of selective pressure (inhibitors of theDHFR or GS) re-sults in an enhancement of transgene dosage and higher productivity.These and other strategies have made the establishment of recombinantcell lines with high specific productivities (20–60 pg/cell/day for anti-body cell lines) relatively common, although the high throughputscreening for more stable clones is still a costly, time-consuming process(Wurm, 2004).

Faster and cheaper approaches for protein production are preferredfor the fast screening of the biopharmaceutical properties of proteinsets or protein isoforms. For that, recombinant DNA is often not incorpo-rated into the host cell genome and genetic selection and/or the isolationof stable transfectants is bypassed. This strategy is attracting increasinginterest because of its ability to produce large amounts of recombinantproteins within reduced times (Meissner et al., 2001; Pham et al., 2006;Wurm and Bernard, 1999). Transient gene expression (TGE) typically re-sults in protein production of milligram to gram amounts up to 10 dayspost-transfection (Baldi et al., 2007). TGE is typically performed instirred-tank bioreactors or in agitated containers including shake flasks,wave-type bioreactors, and plastic or glass bottles (Geisse et al., 2005).Volumes of up to 100 L have been proved feasible for TGE procedures(Girard et al., 2002; Tuvesson et al., 2008). However, stable gene expres-sion has usually resulted in higher specific productivities as compared toTGE (Baldi et al., 2007; Pham et al., 2006; Wurm, 2004). To make TGE amore competitive technology, considerable improvements are stillnecessary but feasible, as already shown for the therapeutic enzymeα-galactosidase A (Corchero et al., 2011).

Irrespective of the production strategy, the choice of host cells hasalso a deep impact on product properties and maximum attainableyields. Chinese hamster ovary (CHO) cells have become a standard forthe production of proteins, since they grow rapidly, offer process versa-tility, and can be cultured in adherence or in suspension-adapted culturein protein-free medium (Derouazi et al., 2004; Rosser et al., 2005).However, mouse myeloma (NS0), baby hamster kidney (BHK), humanembryonic kidney (HEK-293) and human-retina-derived (PERC6)cells have proved to be good alternatives. Volumetric yields of secretedrecombinant proteins are usually higher when using HEK-293 cells(Baldi et al., 2007). HEK-293 cells expressing the EBNA nuclear antigenhave been extensively used for small and large-scale protein expression(Durocher et al., 2002) and adapted to serum-free medium.

Traditionally, much attention has been paid to transfection efficiencyregarding the DNA:transfection reagents ratio (Durocher et al., 2002) orthe DNA dosage (Derouazi et al., 2004). However, other parameters


affecting recombinant protein production (metabolic profile or nutrientrequirements after transfection) are also under focus. In this context, asingle pulse of peptones 24 h after transfection resulted in a significantincrease in volumetric protein productivity (Pham et al., 2003, 2005).Moreover, protein production in polyethylemine (PEI)-transfected CHOcells was significantly enhanced when incubating cells between 29 °Cand 33 °C after transfection (Wulhfard et al., 2008).

As in other expression systems, recombinant protein aggregation hasbeen also observed in mammalian cells (Kopito, 2000), specially whenattempting to produce integral membrane proteins. A cell machinerytransports such protein aggregates, in amicrotubule-dependentmanner,to the centrosome, forming the organelle called aggresome (Johnstonet al., 1998; Kopito, 2000; Kopito and Sitia, 2000). The aggresome servesas a storage compartment for protein aggregates and it could be activelyinvolved in protein refolding and degradation. In fact, chaperones Hsp70and Hsp27, and components of the ubiquitin–protease system arerecruited into the aggresome (Junn et al., 2002). Also, it has been demon-strated that autophagic clearance of protein aggregates also associateswith aggresome formation (Garcia-Mata et al., 2002). Overexpressionof the cystic fibrosis transmembrane conductance regulator (CFTR) orpresenilin-1 (PS1) led to their accumulation into aggregates (Johnstonet al., 1998). The same was observed when expressing mutant forms ofsuperoxide dismutase (Johnston et al., 2000), synphilin 1 (Zaarur et al.,2008), or when expressing a chimera between green fluorescent protein(GFP) and a fragment of p115, a membrane protein (Garcia-Mata et al.,1999). Although contrary to bacterial IBs, aggresomes have not beensystematically investigated, a careful literature search indicates thattheir formation is very common in the context of mammalian cellfactories.

3. Emerging microbial systems for protein production

Apart from specific concerns related to particular production systemsor protein species, recombinant protein aggregation is a genericmatter ofconcern in conventional cell factories (Fig. 1). Particularly in E. coli andalso in S. cerevisiae and mammalian cells, many eukaryotic proteins withtherapeutic potential are exclusively found sequestered in aggresomes(Kopito, 2000) or IBs (Villaverde and Carrio, 2003). Also, heat-shock, un-folded protein responses (UPR) and other conformation-related stressesor metabolic responses are commonly triggered, compromising cell sta-bility and protein quality (Gasser et al., 2008). The increase of product sol-ubility, either by process engineering or through in vitro protein refoldingfrom aggregates (Vallejo and Rinas, 2004) is often unsuccessful. Forsoluble proteins, the occurrence of soluble aggregates has become amain concern in the context of protein quality (de Marco and Schroedel,2005; Martinez-Alonso et al., 2008). Such soluble aggregates havebeen progressively recognized because of the implementation of more

Table 1The most relevant alternative microbial systems for recombinant protein production, ident

Host platfform Model species Main features R

Cold adaptedbacteria

Pseudoalteromonashaloplanktis

Improved protein folding (

Pseudomonads Pseudomonasfluorescens

Efficient secretion (

Coryneformbacteria

Corynebacteriumglutamicum

High-level production and secretion (

Bacilli Bacillus megaterium High-level production and secretion (Lactic acidbacteria

Lactococcus lactis Secretion; GRAS (Regulatory issues) (

Filmentousfungi

Tricoderma reseei High-level production and secretion;post-translational modifications

(1

Unconventionalyeasts

Pichia pastoris Secretion; post-translational modifications (e

Moss Physcomitrellapatens

Post-translational modifications (

Algae Chlamydomonasreinhardtii

Cheap nutrients; high potential for scalability (2

refined analytical technologies for the quality control of protein drugs(Garcia-Fruitos et al., 2011). In addition, protein quality, stability and ag-gregation during storage and administration are amainmatter of concern(Frokjaer andOtzen, 2005; Hawe and Friess, 2007; Jorgensen et al., 2009).In this context, undesired aggregation, aggregation-related side effects,short half-life, limited bioavailability and inefficient pharmacokinetics im-pair the development of recombinant drugs (Shire, 2009). Therefore,there is an urgent need for the exploitation of better cell factories ableto overcome particular bottlenecks and to generically improve conforma-tional quality and productivity of proteins (listed in Fig. 1). Rather thandeveloping new strains to improve properties of the current systems(what in any case is an interesting and useful approach (Makino et al.,2011; Sorensen, 2010)), the wide diversity of the microbial metabolismprompts to select and explore new species with particular metabolicprofiles. These unconventional cell factoriesmight offer enoughbiologicalopportunities for the high quality protein production required for thera-peutic purposes. Apart from lactic acid bacteria,which have lately becomea model for the production and in vivo delivery of therapeutic proteins(Bermudez-Humaran et al., 2011; Daniel et al., 2011; Detmer andGlenting, 2006; Douillard et al., 2011; Guimaraes et al., 2009; Hu et al.,2011; Nguyen et al., 2011; Noreen et al., 2011; Peterbauer et al., 2011;Pontes et al., 2011) and whose potentials as cell factories have beenextensively revised elsewhere (Innocentin et al., 2009; Peterbaueret al., 2011; Siezen and van, 2011; Teusink et al., 2011), other microbialsystems are under continuous exploration for protein production(Table 1). The most promising among those systems (Fig. 1) are de-scribed in the following sections.

3.1. Pseudoalteromonas haloplanktis improved protein folding in bacteria

The secretory properties of Bacillus sp. have been largely explored asa prokaryotic alternative to E. coli (Pohl and Harwood, 2010; Westerset al., 2004). Also, being Generally Recognised As Safe (GRAS), lacticacid bacteria are promising cell factories (Morello et al., 2008; Noreenet al., 2011; Tremillon et al., 2010). However, in the context of intrinsicprotein quality, cold-adapted bacteria are unique emerging systemsthat deserve special attention.

Protein aggregation is mainly driven by stereospecific interactionsbetween solvent-exposed hydrophobic patches (Carrio et al., 2005;Speed et al., 1996). Some thermodynamic studies demonstrated thatthe hydrophobic effect is due to entropic drive forces and therefore it isreduced at low temperatures (Yang et al., 1992). Therefore, the produc-tion of recombinant proteins in psychrophilic bacteria, cultured at 4 °Cor below,may represent an excitingmodel to improve the conformationalquality and solubility of protein products. In this context, a few coldadapted bacterial species are under early but intense exploration as coldcell factories, among them, Pseudoalteromonas haloplanktis TAC125,

ifiying their potencial to bypass the limitations listed in Fig. 1.

eferences

Duilio et al., 2004a, 2004b; Giuliani et al., 2011)

Jin et al., 2011; Retallack et al., 2007, 2011)

Kikuchi et al., 2008, 2009)

Biedendieck et al., 2010; David et al., 2011; Stammen et al., 2010)Innocentin et al., 2009; Le et al., 2005; Miyoshi et al., 2006; Morello et al., 2008)

Collen et al., 2005; Guillemette et al., 2007; Jun et al., 2011; Nyyssonen and Keranen,995; Nyyssonen et al., 1993; Uusitalo et al., 1991)Bai et al., 2011; Gasser et al., 2006; Gurramkonda et al., 2009; Jahic et al., 2003; Porrot al., 2011; Sohn et al., 2010; Stadlmayr et al., 2010)Decker and Reski, 2007, 2008)

Franklin and Mayfield, 2005; Gong et al., 2011; Potvin and Zhang, 2010; Specht et al.,010)


being a representative example. P. haloplanktis TAC125 is a Gram-negative bacterium isolated from an Antarctic coastal seawater sample(Birolo et al., 2000) and it can be classified as a Eurypsychrophile (i.e. abacterium growing in a wide range of low temperatures; (Atlas andBartha, 1993)), which duplicates in the range between 0 °C and 30 °C(Tutino et al., 2001). However, the bacterium still grows at fast speedeven at lower temperatures and, when provided with sufficient nutri-ents and aeration, it reaches very high cell density (up to A600=20) at0 °C. This growth performance makes it one of the fastest growingpsychrophiles so far characterized and an attractive host as cell factoryfor proteins.

P. haloplanktis TAC125 was the first Antarctic Gram-negativebacterium from which the genome was fully sequenced and carefullyannotated (Medigue et al., 2005). Genomic and metabolic features ofthis species, accounting for its remarkable versatility and fast growthcompared with other bacteria from aqueous environments, were dis-covered by combining genome sequencing and further in silico and invivo analyses. Among several relevant traits, it is worth mentioningthat the bacterium is remarkably well adapted to protection against re-active oxygen species (ROS) under cold condition, as experimentallydemonstrated by the relevant production of classical oxidative stressprotecting enzymes (such as the superoxide dismutase SodB, thethioredoxin reductase TrxB, the thioredoxin-dependent peroxide re-ductase AhpCB, and the catalase) (Wilmes et al., 2010) and the develop-ment of a novel anti-ROS and anti-RNS strategy using a 2-on-2haemoglobin (Parrilli et al., 2010). Also, the in silico proteome composi-tion revealed a specific bias that provides a way to resist to proteinaging features involving asparagine cyclization and deamidation (aside reaction which has to be put to a minimum during the productionof some proteins (Weintraub and Manson, 2004). Finally, the bacterialgenome is characterized by a quite high number of rRNA and tRNAgenes (106, sometimes organized in long runs of repeated sequences),which may account for its relevant capacity for translation in the cold.The host versatility was recently widened by the development ofan efficient genetic scheme, allowing the construction of genometargeted insertion/deletion mutants and permitting to create geneticallyengineered strains with improved features regarding protein production(Parrilli et al., 2006, 2010).

P. haloplanktis TAC125 was also the first Antarctic bacterium inwhich an efficient gene-expression technology was set up, by the prop-er combination of selected genetic elements (Duilio et al., 2004a; Tutinoet al., 2001) into a modified E. coli cloning vector (Tutino et al., 2002).Several generations of cold-adapted gene-expression vectors allowthe production of recombinant proteins either by constitutive (Duilioet al., 2004a) or inducible systems (Papa et al., 2007), and to addressthe product towards any cell compartment or to the extra-cellular me-dium (Parrilli et al., 2008). Secreted products have generally simplifiedand cheaper downstream processing due to the low contaminationfrom host proteins. Additionally, extra-cytoplasmic translocation canprovide a method to guarantee the N-terminal authenticity of theexpressed polypeptide, because it often involves the cleavage of a signalsequence (Mergulhao et al., 2005). This processing removes theunwanted initial methionine, whose presence can reduce the biologicalactivity and stability of the product (Liao et al., 2004) or in the case oftherapeutic proteins, elicit an immunogenic response.

Beneficial effects in using this cold-adapted protein productionplatform compared to the conventional mesophilic E. coli have beenreported during the production of antibody fragments (Dragosits etal., 2011; Giuliani et al., 2011) (Table 2) and in the production ofsome “difficult proteins” such as the two case-study proteins, namelythe human nerve growth factor (h-NGF, (Vigentini et al., 2006)) andalpha-glucosidase from S. cerevisiae (Papa et al., 2007). The matureh-NGF production imposes several issues to the cell factory, as its cor-rect folding requires the formation of three non consecutive disulphidebonds (the so-called cysteine-knot fold) and homodimerization. Whilewhen produced in E. coli, this protein accumulates into IBs (Rattenholl

et al., 2001), the production of mature h-NGF in P. haloplanktisTAC125 results in fully soluble and periplasmically translocated protein,accumulating in almost fully dimeric form (Vigentini et al., 2006).Alpha-glucosidase from S. cerevisiae (Papa et al., 2007) is a prototypeof eukaryotic proteins which results largely insoluble when expressedin E. coli. Its recombinant production in P. haloplanktis TAC125 gives arecombinant enzyme totally soluble and highly active, with an en-hancement of both the product folding and quality (Papa et al., 2007).

The fact that insoluble protein aggregates have never been foundin recombinant P. haloplanktis TAC125 (even when high productionyields, namely several hundred milligram protein per liter of culture,were reached (Papa et al., 2007) suggests that its cellular physico-chemical conditions and/or folding processes are quite differentfrom those observed in mesophilic bacteria. A recent study (Pietteet al., 2010) seems to support this latter hypothesis. By applying a differ-ential proteomics approach, a peculiar expression of protein chaperonesof P. haloplanktis TAC125 at its environmental growth temperature(0–4 °C) was described. Indeed, in this condition, the bacteriumstrongly overproduces the trigger factor whereas it represses theproduction of most other HSP chaperones (i.e. DnaK, GroEl, sHSP,Hsp90 and Dsb) to almost undetectable levels. Since the classical cellularfunction of these downregulated chaperones is to assist co- or post-translationally protein folding and prevent or relieve misfolding, itcan be concluded that nascent polypeptide folding largely relies onribosome-bound trigger factor. This chaperone interacts with virtuallyall nascent polypeptides and can be regarded as the primary foldingfactor for the growth of P. haloplanktis TAC125.

The next challenge towards the industrial application of P. haloplanktisTAC125 as non-conventional systems for the recombinant protein pro-duction is the development of an efficient fermentation scheme toup-scale the recombinant protein production in automatic bioreactors.Recently, a synthetic medium for P. haloplanktis TAC125 was developedand used for chemostat cultivation (Giuliani et al., 2011). Moreover, itwas demonstrated that a P. haloplanktis TAC125 fed-batch fermentationstrategy could be also established, which is feasible at a laboratory scaleor for industrial purposes (Wilmes et al., 2010).

The efficiency of cold-adapted expression systems was alsodemonstrated by the production of other difficult proteins andbiopharmaceuticals such as antibody fragments (Dragosits et al.,2011; Giuliani et al., 2011) (Table 2).

3.2. Pichia pastoris and other non-conventional yeasts for protein humaniza-tion and enhanced secretion

Since the implementation of S. cerevisiae as an expression platformfor heterologous proteins (Hitzeman et al., 1981), this yeast has beenemployed for the production of several pharmaceutical proteins(Ferrer-Miralles et al., 2009) (Table 2). However, a number of alterna-tive yeast expression systems have been developed since then(reviewed in (Boer et al., 2007)). Due to their later availability, theseproduction platforms lag behind in their entry to the biopharmaceuticalmarket. Nevertheless, several products from P. pastoris like humanserum albumin, insulin, interferon-alpha, and hepatitis B vaccine aremarketed in India and/or Japan (Shekhar, 2008). In 2009, the FDA ap-proved the recombinant kallikrein inhibitor ecallantide (Kalbitor,Dyax, US), a synthetic peptide produced with P. pastoris for the treat-ment of hereditary angioedema (HAE) and for the prevention of bloodloss in cardiothoracic surgery. Other therapeutic products produced inP. pastoris are in the clinical pipeline or in development (Meyer et al.,2008). Clinical trials have so far involved a humanized monoclonal an-tibody (ALD518, directed against human interleukin IL-6, which is al-ready clinically validated for the treatment of rheumatoid arthritis,and currently in clinical phase II for treatment of cancer by AlderBiopharmaceuticals, US), a malaria vaccine candidate (Anders et al.,2010), and a fusion protein consisting of an anti-carcinoembrionic anti-gen (CEA) single chain fragment scFv and the enzyme carboxypeptidase

Table 2Mammalian or difficult-to-express proteins produced in P. haloplanktis, P. pastoris, T. reesei and C. reinhardtii.

Recombinant Protein Origin Observations Yield Company or reference

P. haloplanktisα-Glucosidase Saccharomyces

cerevisiaeActive 27 mg/L (Papa et al., 2007)

Fab (anti-idiotypic antibodyAb2/3 H6 Fab)

Murin 4 mg/L (Giuliani et al., 2011)(Dragosits et al., 2011)

NGF nerve growth factor Human Mature hNG produced at 4 °C 33 mg/L (Vigentini et al., 2006)

P. pastorisscFv Human or

murineSeveral different scFvsCo-expression of chaperones, fed batch

Several mg/L>4000 mg/L

(Gasser and Mattanovich, 2007)(Damasceno et al., 2007; Pla et al., 2006)

Fab Human ormurine

Several different Fabs Several mg/L up to g/L (Gasser and Mattanovich, 2007)

IgG human AOX, GAP small scaleAOX glyco-engineered, optimized fed batchconditions

0–200 mg/L1900 mg/L

(Barnard et al., 2010; Gasser andMattanovich, 2007)(Berdichevsky et al., 2011)GlycoFi (Merck & Co)

EPO human Glycosylation Man17(GlcNAc)2, fed batchGlycoengineered with terminal sialic acid

130 mg/LNo titer given

(Celik et al., 2009)(Hamilton et al., 2006)

tPA Human Active (1,650 U/mL), shake flask 10 mg/L (Majidzadeh et al., 2010)GM-CSF Human Glycoengineered, fed batch 760 mg/L (Jacobs et al., 2009)Hirudin Medical leech Deletion of protease KEX1, fed batch

Addition of ascorbic acid, fed batch2400 mg/L intact2900 mg/L intact of 5 g/L total

(Ni et al., 2008)(Xiao et al., 2006)

VLPs HbsAg Up to 7 g/L, 30–40% soluble forVLP assembly

(Lunsdorf et al., 2011)

T. reeseiChymosin Bovine CBH1 signal sequence

CBH1 carrier fusionActive

40 mg/L150 mg/L

(Harkki et al., 1989)(Uusitalo et al., 1991)

Fab Murine CBH1 signal sequenceCBH1 carrier fusion

1 mg/L150 mg/L

(Nyyssonen and Keranen, 1995)

TPA Human Active 20 mg/mL Uusitalo J, VTT, Technical Resear Centre,unpublished data

Herceptin IgG Human 90% Kex2 cleavage of CBH1 carrier 3000 mg/L (Baldwin, 2006)

C. reinhardtiiHuman Ab Human Human IgG1 directed against anthrax

protective antigen 83 (PA83)100 mg/g of dry algal biomass (Tran et al., 2009)

Human Ab Human MAb against herpes simplex virus (HSV)glycoprotein D

na (Mayfield et al., 2003)

Vascular endothelial growthfactor (VEGF)

Human Without its signal peptide. 2% of total soluble protein (Rasala et al., 2010)

High mobility group proteinB1 (HMGB1)

Human 2.5% of total soluble protein (Rasala et al., 2010)

Tumor necrosis factor-relatedapop-tosis-inducing ligand(TRAIL)

Human 0.43%–0.67% of total solubleprotein

(Zongqi Yang et al., 2006)

Glutamic acid decarboxylase65 (hGAD65)

Human Key autoantigen in type 1 diabetes 0.25–0.3% of total solubleprotein

(Wang et al., 2008)

na: Data not available. scFv: single chain variable fragments; Fab: antigen binding fragment; IgG: immunoglobulin G; tPA: tissue plasminogen activator; GM-CSF: granulocytemacrophagecolony-stimulating factor, EPO: erythropoietin.


G2 for antibody-directed enzyme pro-drug therapy against breastcancer (MFECP1, Cancer Research Technology, UK, (Tolner et al.,2007)), among others. A similar range of biopharmaceuticals producedby another methylothropic yeast, Hansenula polymorpha, is also on themarket, including the hepatitis B vaccine Hepavax-Gene (Crucell, NL),insulin and interferon alpha-2 (Rhein-Biotech, DE).

In the last years, the published evidence of using alternativeyeasts for protein production exceeds that of S. cerevisiae. Also, inthe very few systematic comparisons between different yeast pro-duction systems (Dragosits et al., 2011; Mack et al., 2009; Mulleret al., 1998), clear advantages of the non conventional species overS. cerevisiae, specially for the production of secreted proteins havebeen found. The comparative production of the anticoagulant peptidehirudin in different expression systems (Demain and Vaishnav, 2009)demonstrated the significantly higher productivities achieved withP. pastoris and H. polymorpha. Since strong promoters are alreadyavailable for non conventional yeasts (Mattanovich et al., 2012),and gene copy number amplification (Klabunde et al., 2002; Marxet al., 2009) provides a tool to optimize total transcription rate in

these species, the folding and secretion pathway are the major sourceof advantages of alternative yeasts.

In this regard, evidence shows that the secretion pathway ofS. cerevisiae differsmore fromhigher eukaryotes than that of P. pastorisand other non conventional yeasts. Organelle structure and proliferationare different (Papanikou and Glick, 2009), and the regulation pattern ofUPR, the major regulon controlling folding limitations, shows significantdifferences between these two yeast species (Graf et al., 2008). A com-parison of the response of P. pastoris and S. cerevisiae to oxygen limitationrevealed a significant difference in heterologous protein secretion, beinghigher in P. pastoris and further increasing upon oxygen limitation, whilethe lower level in S. cerevisiae remained constant (Baumann et al.,2011b). Furthermore, ergosterol synthesis geneswere strongly regulatedin P. pastoris contrary to S. cerevisiae, which prompts to speculate thatlimited ergosterol synthesis upon oxygen limitation may play a role inthe superior secretion (Baumann et al., 2011a).

Protein folding and secretion are complex processes involvingnumerous cellular assisting proteins. Successful cell engineering byoverexpression or deletion of such assisting proteins has been recently


achieved (Idiris et al., 2010), specially for the production of secreted an-tibody fragments (Gasser and Mattanovich, 2007; Gasser et al., 2006).By comparison of engineered variants of recombinant proteins, it hasbeen demonstrated that secretion levels of closely related proteins cor-relate with their thermodynamic stability (Whyteside et al., 2011).

One of the most important features of yeast expression systems istheir ability to perform eukaryotic post-translational protein modifica-tions, such as N- and O-glycosylation, disulfide bond formation, and olig-omerization. The majority of therapeutic proteins display one or morepost-translational modifications (PTMs), and these PTMs are often es-sential for the functionality of these proteins. Glycosylation representsthe most complex and the most widespread PTM, being associatedwith 40% of all approved products (Walsh, 2010). However, proteinglycosylation in yeast (and other fungi) is different from that inmammalian cells. For instance, yeasts modify their glycoproteinswith heterologous high-mannose N-glycans, whereas in humans,N-glycans are mainly of the complex or hybrid type. This difference isoften detrimental to a therapeutic protein's biochemical and/or func-tional properties.

This problem can be circumvented by engineering the glycosylationpathways to produce homogeneous and human-like glycan struc-tures (De Pourcq et al., 2010). Most progress has been achieved inreengineering the N-glycosylation pathway in several yeast systems(S. cerevisiae, P. pastoris, Yarrowia lipolytica, etc.). Basically, the endoge-nous N-glycosylation pathway is abolished and a humanized pathwayis introduced. Nevertheless, only P. pastoris has been successfullyglyco-engineered to provide a set of strains producing homogeneousglycoproteins with different, well defined human glycoforms. Fromthe commercial point of view, an important part of this engineeringeffort has culminated with the development of engineered P. pastorisstrains capable of producing proteins uniformly glycosylated (byGlycofi, subsidiary of Merck & Co, US), sialic acid-capped products(Hamilton et al., 2006), and the Glycoswitch technology developedby VIB-Ghent (Jacobs et al., 2009), commercialized through ResearchCorporation Technologies, US.

Engineering of the O-glycosylation pathway is not as advanced asN-glycosylation engineering. Although some strategies based on partialsuppression of O-glycosylation by addition of specific inhibitors of thispathway (e.g. rhodanine-3-acetic acid) have been successfully test-ed (e.g. in the methylotrophic yeast Ogataea minuta producing IgG(Kuroda et al., 2008)), such strategies seem difficult or even unfeasiblefor industrial scale-up. Since O-mannosylation is required for thesurvival of yeast cells, complete suppression of the endogenous pathwayis a challenging, still remaining goal (De Pourcq et al., 2010). In spite ofthis limitation, some engineering approaches have shown the possibilityto introduce mucin-type O-glycosylation in yeast (Chigira et al., 2008) orto reduce the level of mannose type O-glycosylation (Evans et al., 2009).

In addition to the development of yeast systems with the ability togenerate products with therapeutically acceptable (and homogeneous)glycoprofiles (Liu et al., 2011), there is a notable trend towards theengineering of the glycocomponent of glycosylated biopharmaceuticalsto modify or enhance therapeutic attributes (Walsh, 2010). For instance,enzyme replacement therapies for LSD using recombinant enzymes pro-duced by mammalian cell lines and glycan receptors as delivery targets(including mannose receptors and cation-independent mannose-6-Preceptors) have been clinically applied to several LSDs, includingGaucherdisease, Fabry disease, and others (Tsuji et al., 2011). In this case, theterminal mannose residues of the glycans are essential for targetingthe enzyme to the mannose-6-P (M6P) receptors in the cell typeswhere the enzyme substrate accumulates. Interestingly, recent develop-ments in yeast glycoengineering show the potential of these systems.For this purpose, yeast strains bearing the OCH1 disruption (that is, elim-inating the alpha-1,6-mannosyltransferase initiating the synthesis of thehypermannosylated glycan structures in yeast), and co-overexpressingone of the genes involved in mannosylphosphorylation of N-glycans(e.g. MNN4, MNN6 and PNO1) have been constructed in O. minuta

(Akeboshi et al., 2009) and Y. lipolytica (Callewaert et al., 2009). Notably,Tsuji et al. (2011) have recently shown the therapeutic effect in mice of arecombinant human lysosomal β-hexosaminidase A (HexA) with termi-nal M6P residues on their N-glycans produced in the above mentionedglycoengineered O. minuta strain.

Importantly, cultivation conditions seem to have an important im-pact on protein glycosylation. For instance, Berdichevsky et al. (2011)have recently demonstrated that O2-limitation impacts on N-glycancomposition and galactosylation in glycoengineered P. pastoris strainsexpressing monoclonal antibodies. Medium composition strongly de-termines the degree of phosphorylation of the N-glycan population(Montesino et al., 1999). Hence, careful control of cultivation processconditions of glycoengineered yeast strains is necessary regarding identi-ty, authenticity, glycan homogeneity, etc. of the products. Moreover,significant variability exists in P. pastoris in terms of fermentation perfor-mance between genotypically similar clones with respect to cell fitness,secreted protein titer, and glycan homogeneity, so comprehensivescreening and selection of engineered strains are required for industrialscale-up (Barnard et al., 2010).

While a lot of emphasis has been put on engineering yeasts forcorrect human-like glycosylation, other post-translational modifica-tions have lagged behind. Moreover, for the production of complexmultimeric biopharmaceuticals correct interactions between subunitsare requested. In particular, the assembly of LC and HC to full lengthIgG or Fab has turned out to be very inefficient. The few attempts toimprove complex formation focused so far on the acceleration of disul-fide formation by overexpression of Pdi1, improved folding capacity ofthe cell by overexpression of chaperones (Gasser et al., 2006), or onthe reduction of proteolytic activity of the host by deletion of vacuolarand/or secreted proteases (Kuroda et al., 2007). Additionally, the sup-pression of O-mannosylation has been found to be beneficial for IgG as-sembly and secretion (Kuroda et al., 2008), leading to the assumptionthat non-native protein modifications hamper multimer assembly andtarget the protein to the degradation pathway instead. As the assemblyof IgG is based on the replacement of BiP bound to the HC by LC throughhydrophobic interactions prior to or simultaneouslywith disulfide bondformation between the chains, hydrophilization of the regions byO-glycosylation to prevent aggregation in the ER may be counterpro-ductive in this case.

Contrary to bacterial expression systems, the generation of a nativeN-terminus is usually not a critical issue with yeasts. Most biopharma-ceutical proteins are targeted for secretion using the S. cerevisiaealpha-mating factor signal peptide, which is cleaved off during secretorytransport in the Golgi by Kex2 protease and Ste13 amino-dipeptidase.Overexpression of these two processing enzymes has improved secre-tion andprotein quality in some cases such as interferon and interleukins(Degelmann et al., 2002), where incorrect N-terminal processing occurs.Interferons are also prone to degradation. This can be circumventedby efficient design of media, as shown for Y. lipolytica (Gasmi et al.,2011) or cultivation conditions as done for P. pastoris (Sinha et al.,2005), both leading to the inhibition of proteolytic activity. Alteringthe cultivation conditions also reduces the potential aggregation ofinterferon-alpha (Hao et al., 2007).

Another field of application is the production of recombinantvaccines and immunoadjuvants, with virus-like particles (VLPs) aspromising vaccine candidates. Yeast derived Hepatitis B surface antigen(HBsAg), was the first recombinant vaccine approved by the FDA in1986. It needs to assemble into VLPs to be immunogenic, and is currentlyproduced intracellularly using S. cerevisiae, H. polymorpha or P. pastoris.Recently, it was reported that the actual VLP assembly takes place duringdownstream processing, whereas intracellular HBsAg is assembled intomulti-layered lamellar structures within an extended ER. This indicatesthat the fast HBsAg synthesis overloads VLP assembly and thereby blockssecretion (Lunsdorf et al., 2011). Also HBV core protein (HBc) is a poten-tial component of novelHBVvaccines, and is also interesting as a packagerin medical nanotechnology, as it spontaneously assembles into VLPs.


Interestingly, P. pastoris derived HBc is phosphorylated like the nativevirus protein, but the proteins produced in E. coli or S. cerevisiae are not(Freivalds et al., 2011). Dengue VLPs have been also produced and secret-ed in P. pastoris (Liu et al., 2010). In general, yeasts seem to be excellentexpression platforms for the often highly glycosylated proteins of viruses,which are prone to aggregation and lowsolubility in bacteria (Chuck et al.,2009).

3.3. Trichoderma reesei: enhanced productivity and easy proteinpurification

T. reesei is an efficient secretory organism for which productionyields of industrially applicable native enzymes in excess of 100 g/Lhave been commonly reported (Cherry and Fidantsef, 2003). This fil-amentous fungus is a soil based microbe able to utilize cellulose ascarbon source, allowing for both low cost fermentation media andalso a strong induction of recombinant gene expression when usingthe cellobiohydrolase Ι (cbh1) promoter (Enari, 1983). Therapeuticprotein production in T. reesei is an emerging but promising field(Table 2), particularly considering that themajor N-glycan form synthe-sized by T. reesei (N-acetyl-D-glucosamine, GlcNAc2 Man5) is a suitableprecursor for mammalian glycosylation (Salovuori et al., 1987; Stalset al., 2004). Thus, the possibilities for humanization of the T. reeseiglycosylation pathway might be higher than in yeast systems.Human N-acetyleglucosaminyltransferase I has already been expressedin T. reesei to transfer a GLcNAc residue to the GlcNac2Man5 fungalglycans (Maras et al., 1999). In terms of potential for pharmaceuticalprotein production, T. reesei is not only well established in large scalefermentation but is also already approved as a GRAS organism for foodapplications, thereby presenting a platform for progression towardsregulatory approval of the system for therapeutic uses of producedproteins.

T. reesei is known to be an efficient producer of highly active enzymes,anticipating a potential for the production of therapeutic proteins. Todate, the production of highly stable antibodies at g/L yields has beenthe major area of success (Table 2). On the other hand, the endogenousproteases naturally secreted by this fungus represent a challenge for thestability of any heterologous protein. Initial strategies demonstrated byboth Chymosin (Harkki et al., 1989) and Fab (Nyyssonen et al., 1993) pro-duction were based on the addition of the CBH1 signal sequence up-stream of the target protein (Uusitalo et al., 1991), directing the proteininto the secretory pathway. This approach resulted in relatively low pro-tein yields (40 and 1 mg/mL respectively). The addition of CBH1 as a se-cretion carrier protein, replacing its cellulose binding domain by apassenger recombinant protein can significantly increase yields up to150-fold. Yield enhancement can be accounted not only by stabilizationofmRNA (Nyyssonen and Keranen, 1995), but probably also by improvedfolding and subcellular trafficking through the secretory machinery. Theidea of using a secretion carrier to boost production has been developedfurther by the introduction of optimized Kex2 cleavage sites for the pro-teolytic separation of the carrier and product during secretion throughthe Golgi complex (Baldwin, 2006).

As in conventional production systems, unfolded protein inducedstress might represent a major obstacle in heterologous protein pro-duction in T. reesei. Incomplete folding events in the ER (Saloheimoet al., 1999), post-translation modification and secretion (Collenet al., 2005) of foreign proteins have been shown to trigger the UPR(Saloheimo et al., 2003). Down regulation of target gene expressionoccurs when a UPR response triggers regulatory binding events onthe CBH1 promoter, reducing transcription of genes encoding themajor secreted proteins (Pakula et al., 2003). The regulatory rangeof the secretion stress responses has been investigated in T. reesei,finding mechanisms such as nucleosome gene induction (Arvaset al., 2006) to be involved. Elucidation of the stress pathways is clearlya critical factor to unlock the full potential of T. reesei, and the manipu-lation of these pathways by the co-production of specific chaperones

and foldases and by blocking stress routes may be among the futurestrategies to improve recombinant protein expression.

In this context, an intriguing approach is the exploitation of intra-cellular protein body forming peptides such as ZERA (Torrent et al.,2009) or endogenous hydrophobin (Mustalahti et al., 2011) as fusionpartners. Such systems allow the accumulation of fusion proteins insu-latedwithin a protein body structure. Their purification can be achievedby taking advantage of the highly hydrophobic properties of the fusion,by mechanical gravity separation or by two-phase extraction, with theneed of additional downstream purification.

Development of improved T. reesei strains for the production oftherapeutic proteins should concentrate onboth overcoming thebottle-necks of expression and purification and on refining the molecularmechanisms involved in determining protein folding and reaching anappropriate tertiary structure, in order to yield molecules with high ef-ficacy and immunogenic compatibility with humans.

3.4. Chlamydomonas reinhardtii, an emeging GRAS system

The term “microalgae” refers to a diverse photosynthetic group ofprokaryotic (cyanobacteria) and eukaryotic organisms. Historically,microalgae have been used in several applications, ranging from en-hancing the nutritional value of animal feed to as producers of highlyvaluable molecules, like polyunsaturated fatty acid oils or human nu-tritional supplements and pigments (Apt and Behrens, 1999; Hempelet al., 2011; Adarme-Vega et al., 2012; Spolaore et al., 2006; Yu et al.,2011).

The feasibility of microalgae to be genetically modified opens a waynot only to enhance productivity of their traditional products but alsoto produce chemicals and proteins with industrial and pharmaceuticalapplications. Microalgae have recently attracted the attention of re-searchers as an alternative to currently used protein production systems(Gong et al., 2011; Specht et al., 2010; Walker et al., 2005). Microalgaeoffer the benefits of plants (sharing the basic photosynthetic mecha-nism), coupled with high productivities associated with microbial pro-duction. Being most microalgae photoautotrophs, they require onlylight, water and basic nutrients for their culture. Some species can alsobe grown as heterotrophs in fermenters without light as energy source,thus requiring a supply of sugars for energy and as a carbon source. Atthe same time, and due to their microscopic size, microalgae can begrown in large scale liquid cultures (either in controlled, closed bioreac-tors or in open ponds). The potential for large-scale culture (microalgaehave the ability to be grown on scales ranging from a few mL to500,000 L in a cost-effective manner) makes microalgae a desirabletarget for their exploitation as cell factories for the synthesis ofhigh-value therapeutic proteins. Regarding economic issues, andaccording to a recombinant antibody production study, the cost ofproduction per gram of functional antibody was US $150 and $0.05in mammalian and plant expression systems, respectively, and only$0.002 in microalgae, (Mayfield et al., 2003).

Concerning recombinant protein production, transgenic algae canbe generated quickly, requiring only a few weeks between the produc-tion of transformants and their scale up to production volumes. Sincethere is no gene flow by means of pollen or other vehicles of gene es-cape, transgenic microalgae are environmentally safe (Janssen et al.,2003). Green algae fall, in addition, into the GRAS category.

Despite the increasing examples of successful transformation ofmany microalgal species, strains of Chlamydomonas, Chlorella, Volvox,Haematococcus and Dunaliella remain the most widely explored(Griesbeck et al., 2006; Raja et al., 2008; Rosenberg et al., 2008). Currentwork is mainly performed with C. reinhardtii, as it is the best character-ized microalgal species for which stable genetic transformation at bothchloroplast (Boynton et al., 1988) and nuclear (Debuchy et al., 1989;Fernandez et al., 1989) levels were first reported. Codon-optimized re-porter genes have been expressed in the C. reinhardtii chloroplast(Franklin and Mayfield, 2005; Mayfield and Schultz, 2004) to examine


a variety of promoter and translational elements (Barnes et al., 2005).Using this strategy, GFP accumulation up to 0.5% of total soluble proteinwas achieved in transgenic chloroplasts (Barnes et al., 2003; Franklin etal., 2002).

Although recombinant protein production is notably hindered by lowexpression levels, the continuing development of genetic engineeringtools for microalgae has allowed the expression of fully functional anti-bodies (Franklin and Mayfield, 2005; Tran et al., 2009), therapeutics(Boehm, 2007; Weathers et al., 2010), and bactericides (Li and Tsai,2009) (Table 2), making this system highly promising for furtherdevelopment.

4. Conclusions and future prospects

Thirty years of recombinant DNA technologies have produced lessprotein drugs for human therapy than initially envisaged, since thebiological production of recombinant proteins causes a set of meta-bolic stresses that ultimately affect the yield and the quality of theproducts. Such events not only raise the production costs to oftenunaffordable levels but they also impair the biological effect of thedrugs once administered, due to conformational defects and aggrega-tion. Genetic and metabolic engineering of conventional recombinantprotein platforms have shown a potential to improve folding, stability,productivity and post-translational modifications of proteins intendedas biopharmaceuticals. However, physiological limitations of the cur-rent protein production platforms do not offer much more room forthe improvement of product quality and yield, and often, they do notpermit to overcome regulatory constraints. This fact, together with theurgent need for improved drugs in innovative medicines stronglyprompts to explore alternative hostswith unusual properties. The phys-iological diversity of the microbial world offers opportunities for theidentification and selection of organisms as unconventional hosts forprotein production, suitable for regulatory demands. Among the grow-ing spectrum of such emerging hosts, some of them, display appealingproperties for Biopharma. They include convenient glycosylationpatterns, cold-adapted fast growth, GRAS nature and high productiv-ity (Fig. 1). The alacrity in adapting unusual microbes as protein drugproducers combined with the awareness of regulatory issues isexpected to provide new tool-boxes for the progressive incorpora-tion into the marked of drugs that had been previously discardedfor further development because of their poor quality or productivityissues.

Acknowledgments

The authors appreciate the financial support to recombinant proteinproduction in microbial systems through ERANET-IB08-007 and theirlinked national projects (EUI2008-03610 and TEKES 40333/08). Thisworkhas also been supported by the FederalMinistry of Economy, Familyand Youth (BMWFJ), the Federal Ministry of Traffic, Innovation andTechnology (bmvit), the Styrian Business Promotion Agency SFG, theStandortagentur Tirol and ZIT-Technology Agency of the City of Viennathrough the COMET-Funding Program managed by the Austrian Re-search Promotion Agency FFG. We also appreciate the support fromAntartide 2010 to MLT and EP, from MINECO (IT2009-0021) to AVand LT, from AGAUR (2009SGR-108 to AV and 2009SGR-281 andXarxa de Referència en Biotecnologia, XRB, to PF). AV and SS are alsosupported by The Biomedical Research Networking Center in Bioengi-neering, Biomaterials and Nanomedicine (CIBER-BBN, Spain), an initia-tive funded by theVI National R&D&i Plan 2008–2011, Iniciativa Ingenio2010, Consolider Program, CIBER Actions and financed by the Institutode Salud Carlos III with assistance from the European Regional Develop-ment Fund. AV has been granted with an ICREA ACADEMIA award(from ICREA, Catalonia, Spain).

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Unconventional microbial systems for the cost-efficient production of high-quality protein therapeutics

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