Austrian Centre of Industrial Biotechnology (Austria)

Pichia pastoris is an established protein expression host mainly applied for the production of biopharmaceuticals and industrial enzymes. This methylotrophic yeast is a distinguished production system for its growth to very high cell densities, for the available strong and tightly regulated promoters, and for the options to produce gram amounts of recombinant protein per litre of culture both intracellularly and in secretory fashion. However, not every protein of interest is produced in or secreted by P. pastoris to such high titres. Frequently, protein yields are clearly lower, particularly if complex proteins are expressed that are hetero-oligomers, membrane-attached or prone to proteolytic degradation. The last few years have been particularly fruitful because of numerous activities in improving the expression of such complex proteins with a focus on either protein engineering or on engineering the protein expression host P. pastoris. This review refers to established tools in protein expression in P. pastoris and highlights novel developments in the areas of expression vector design, host strain engineering and screening for high-level expression strains. Breakthroughs in membrane protein expression are discussed alongside numerous commercial applications of P. pastoris derived proteins.

Approximately a tenth of all described fungal species can cause diseases in plants. A common feature of this process is the necessity to pass through the plant cell wall, an important barrier against pathogen attack. To this end, fungi possess a diverse array of secreted enzymes to depolymerize the main structural polysaccharide components of the plant cell wall, i.e., cellulose, hemicellulose, and pectin. Recent advances in genomic and systems-level studies have begun to unravel this diversity and have pinpointed cell wall-degrading enzyme (CWDE) families that are specifically present or enhanced in plant-pathogenic fungi. In this review, we discuss differences between the CWDE arsenal of plant-pathogenic and non-plant-pathogenic fungi, highlight the importance of individual enzyme families for pathogenesis, illustrate the secretory pathway that transports CWDEs out of the fungal cell, and report the transcriptional regulation of expression of CWDE genes in both saprophytic and phytopathogenic fungi.

More than 70 years ago, the filamentous ascomycete Trichoderma reesei was isolated on the Solomon Islands due to its ability to degrade and thrive on cellulose containing fabrics. This trait that relies on its secreted cellulases is nowadays exploited by several industries. Most prominently in biorefineries which use T. reesei enzymes to saccharify lignocellulose from renewable plant biomass in order to produce biobased fuels and chemicals. In this review we summarize important milestones of the development of T. reesei as the leading production host for biorefinery enzymes, and discuss emerging trends in strain engineering. Trichoderma reesei has very recently also been proposed as a consolidated bioprocessing organism capable of direct conversion of biopolymeric substrates to desired products. We therefore cover this topic by reviewing novel approaches in metabolic engineering of T. reesei.

Most eukaryotes develop close interactions with microorganisms that are essential for their performance and survival. Thus, eukaryotes and prokaryotes in nature can be considered as meta-organisms or holobionts. Consequently, microorganisms that colonize different plant compartments contain the plant's second genome. In this respect, many studies in the last decades have shown that plant-microbe interactions are not only crucial for better understanding plant growth and health, but also for sustainable crop production in a changing world. This mini-review acting as editorial presents retrospectives and future perspectives for plant microbiome studies as well as information gaps in this emerging research field. In addition, the contribution of this research topic to the solution of various issues is discussed.

The review compiles artificial cascades involving enzymes with a focus on the last 10 years. A cascade is defined as the combination of at least two reaction steps in a single reaction vessel without isolation of the intermediates, whereby at least one step is catalyzed by an enzyme. Additionally, cascades performed in vivo and in vitro are discussed separately, whereby in vivo cascades are defined here as cascades relying on cofactor recycling by the metabolism or on a metabolite from the living organism. The review introduces a systematic classification of the cascades according to the number of enzymes in the linear sequence and differentiates between cascades involving exclusively enzymes and combinations of enzymes with non-natural catalysts or chemical steps. Since the number of examples involving two enzymes is predominant, the two enzyme cascades are further subdivided according to the number, order, and type of redox steps. Furthermore, this classification differentiates between cascades where all reaction steps are performed simultaneously, sequentially, or in flow.

The importance of microbial root inhabitants for plant growth and health was recognized as early as 100 years ago. Recent insights reveal a close symbiotic relationship between plants and their associated microorganisms, and high structural and functional diversity within plant microbiomes. Plants provide microbial communities with specific habitats, which can be broadly categorized as the rhizosphere, phyllosphere, and endosphere. Plant-associated microbes interact with their host in essential functional contexts. They can stimulate germination and growth, help plants fend off disease, promote stress resistance, and influence plant fitness. Therefore, plants have to be considered as metaorganisms within which the associated microbes usually outnumber the cells belonging to the plant host. The structure of the plant microbiome is determined by biotic and abiotic factors but follows ecological rules. Metaorganisms are co-evolved species assemblages. The metabolism and morphology of plants and their microbiota are intensively connected with each other, and the interplay of both maintains the functioning and fitness of the holobiont. Our study of the current literature shows that analysis of plant microbiome data has brought about a paradigm shift in our understanding of the diverse structure and functioning of the plant microbiome with respect to the following: (i) the high interplay of bacteria, archaea, fungi, and protists; (ii) the high specificity even at cultivar level; (iii) the vertical transmission of core microbiomes; (iv) the extraordinary function of endophytes; and (v) several unexpected functions and metabolic interactions. The plant microbiome should be recognized as an additional factor in experimental botany and breeding strategies.

The microbiome of plants plays a crucial role in both plant and ecosystem health. Rapid advances in multi-omics tools are dramatically increasing access to the plant microbiome and consequently to the identification of its links with diseases and to the control of those diseases. Recent insights reveal a close, often symbiotic relationship between microorganisms and plants. Microorganisms can stimulate germination and plant growth, prevent diseases, and promote stress resistance and general fitness. Plants and their associated microorganisms form a holobiont and have to be considered as co-evolved species assemblages consisting of bacterial, archaeal and diverse eukaryotic species. The beneficial interplay of the host and its microbiome is responsible for maintaining the health of the holobiont, while diseases are often correlated with microbial dysbioses. Microbial diversity was identified as a key factor in preventing diseases and can be implemented as a biomarker in plant protection strategies. Targeted and predictive biocontrol approaches are possible by developing microbiome-based solutions. Moreover, combined breeding and biocontrol strategies maintaining diversity and ecosystem health are required. The analysis of plant microbiome data has brought about a paradigm shift in our understanding of its role in health and disease and has substantial consequences for biocontrol and health issues.

In this study cutinases from Thermobifida cellulosilytica DSM44535 (Thc_Cut1 and Thc_Cut2) and Thermobifida fusca DSM44342 (Thf42_Cut1) hydrolyzing poly(ethylene terephthalate) (PET) were successfully cloned and expressed in E.coli BL21-Gold(DE3). Their ability to hydrolyze PET was compared with other enzymes hydrolyzing natural polyesters, including the PHA depolymerase (ePhaZmcl) from Pseudomonas fluorescens and two cutinases from T. fusca KW3. The three isolated Thermobifida cutinases are very similar (only a maximum of 18 amino acid differences) but yet had different kinetic parameters on soluble substrates. Their k cat and K m values on pNP–acetate were in the ranges 2.4–211.9 s –1 and 127–200 μM while on pNP–butyrate they showed k cat and K m values between 5.3 and 195.1 s –1 and between 1483 and 2133 μM. Thc_Cut1 released highest amounts of MHET and terephthalic acid from PET and bis(benzoyloxyethyl) terephthalate (3PET) with the highest concomitant increase in PET hydrophilicity as indicated by water contact angle (WCA) decreases. FTIR-ATR analysis revealed an increase in the crystallinity index A 1340 / A 1410 upon enzyme treatment and an increase of the amount of carboxylic and hydroxylic was measured using derivatization with 2-(bromomethyl)naphthalene. Modeling the covalently bound tetrahedral intermediate consisting of cutinase and 3PET indicated that the active site His-209 is in the proximity of the O of the substrate thus allowing hydrolysis. On the other hand, the models indicated that regions of Thc_Cut1 and Thc_Cut2 which differed in electrostatic and in hydrophobic surface properties were able to reach/interact with PET which may explain their different hydrolysis efficiencies.

., apolipoproteinE (ApoE). ApoE, being responsible for fat transport in the body, plays a key role in the LNP's plasma circulation time. In this work, we use small-angle neutron scattering, together with selective lipid, cholesterol, and solvent deuteration, to elucidate the structure of the LNP and the distribution of the lipid components in the absence and the presence of ApoE. While DSPC and cholesterol are found to be enriched at the surface of the LNPs in buffer, binding of ApoE induces a redistribution of the lipids at the shell and the core, which also impacts the LNP internal structure, causing release of mRNA. The rearrangement of LNP components upon ApoE incubation is discussed in terms of potential relevance to LNP endosomal escape.

Recombinant protein production is a multibillion-dollar market. The development of a new product begins with the choice of a production host. While one single perfect host for every protein does not exist, several expression systems ranging from bacterial hosts to mammalian cells have been established. Among them, yeast cell factories combine the advantages of being single cells, such as fast growth and easy genetic manipulation, as well as eukaryotic features including a secretory pathway leading to correct protein processing and post-translational modifications. In this respect, especially the engineering of yeast glycosylation to produce glycoproteins of human-like glycan structures is of great interest. Additionally, different attempts of cellular engineering as well as the design of different production processes that are leading to improved productivities are presented. With the advent of cheaper next-generation sequencing techniques, systems biotechnology approaches focusing on genome scale analyses will advance and accelerate yeast cell factories and thus recombinant protein production processes in the near future. In this review we summarize advantages and limitations of the main and most promising yeast hosts, including Saccharomyces cerevisiae, Pichia pastoris, and Hansenula polymorpha as those presently used in large scale production of heterologous proteins.

INTRODUCTION: The human genome-scale metabolic reconstruction details all known metabolic reactions occurring in humans, and thereby holds substantial promise for studying complex diseases and phenotypes. Capturing the whole human metabolic reconstruction is an on-going task and since the last community effort generated a consensus reconstruction, several updates have been developed. OBJECTIVES: We report a new consensus version, Recon 2.2, which integrates various alternative versions with significant additional updates. In addition to re-establishing a consensus reconstruction, further key objectives included providing more comprehensive annotation of metabolites and genes, ensuring full mass and charge balance in all reactions, and developing a model that correctly predicts ATP production on a range of carbon sources. METHODS: Recon 2.2 has been developed through a combination of manual curation and automated error checking. Specific and significant manual updates include a respecification of fatty acid metabolism, oxidative phosphorylation and a coupling of the electron transport chain to ATP synthase activity. All metabolites have definitive chemical formulae and charges specified, and these are used to ensure full mass and charge reaction balancing through an automated linear programming approach. Additionally, improved integration with transcriptomics and proteomics data has been facilitated with the updated curation of relationships between genes, proteins and reactions. RESULTS: Recon 2.2 now represents the most predictive model of human metabolism to date as demonstrated here. Extensive manual curation has increased the reconstruction size to 5324 metabolites, 7785 reactions and 1675 associated genes, which now are mapped to a single standard. The focus upon mass and charge balancing of all reactions, along with better representation of energy generation, has produced a flux model that correctly predicts ATP yield on different carbon sources. CONCLUSION: Through these updates we have achieved the most complete and best annotated consensus human metabolic reconstruction available, thereby increasing the ability of this resource to provide novel insights into normal and disease states in human. The model is freely available from the Biomodels database (http://identifiers.org/biomodels.db/MODEL1603150001).

BACKGROUND: Trichoderma is a genus of mycotrophic filamentous fungi (teleomorph Hypocrea) which possess a bright variety of biotrophic and saprotrophic lifestyles. The ability to parasitize and/or kill other fungi (mycoparasitism) is used in plant protection against soil-borne fungal diseases (biological control, or biocontrol). To investigate mechanisms of mycoparasitism, we compared the transcriptional responses of cosmopolitan opportunistic species and powerful biocontrol agents Trichoderma atroviride and T. virens with tropical ecologically restricted species T. reesei during confrontations with a plant pathogenic fungus Rhizoctonia solani. RESULTS: The three Trichoderma spp. exhibited a strikingly different transcriptomic response already before physical contact with alien hyphae. T. atroviride expressed an array of genes involved in production of secondary metabolites, GH16 ß-glucanases, various proteases and small secreted cysteine rich proteins. T. virens, on the other hand, expressed mainly the genes for biosynthesis of gliotoxin, respective precursors and also glutathione, which is necessary for gliotoxin biosynthesis. In contrast, T. reesei increased the expression of genes encoding cellulases and hemicellulases, and of the genes involved in solute transport. The majority of differentially regulated genes were orthologues present in all three species or both in T. atroviride and T. virens, indicating that the regulation of expression of these genes is different in the three Trichoderma spp. The genes expressed in all three fungi exhibited a nonrandom genomic distribution, indicating a possibility for their regulation via chromatin modification. CONCLUSION: This genome-wide expression study demonstrates that the initial Trichoderma mycotrophy has differentiated into several alternative ecological strategies ranging from parasitism to predation and saprotrophy. It provides first insights into the mechanisms of interactions between Trichoderma and other fungi that may be exploited for further development of biofungicides.

Targeted gene replacement to generate knock-outs and knock-ins is a commonly used method to study the function of unknown genes. In the methylotrophic yeast Pichia pastoris, the importance of specific gene targeting has increased since the genome sequencing projects of the most commonly used strains have been accomplished, but rapid progress in the field has been impeded by inefficient mechanisms for accurate integration. To improve gene targeting efficiency in P. pastoris, we identified and deleted the P. pastoris KU70 homologue. We observed a substantial increase in the targeting efficiency using the two commonly known and used integration loci HIS4 and ADE1, reaching over 90% targeting efficiencies with only 250-bp flanking homologous DNA. Although the ku70 deletion strain was noted to be more sensitive to UV rays than the corresponding wild-type strain, no lethality, severe growth retardation or loss of gene copy numbers could be detected during repetitive rounds of cultivation and induction of heterologous protein production. Furthermore, we demonstrated the use of the ku70 deletion strain for fast and simple screening of genes in the search of new auxotrophic markers by targeting dihydroxyacetone synthase and glycerol kinase genes. Precise knock-out strains for the well-known P. pastoris AOX1, ARG4 and HIS4 genes and a whole series of expression vectors were generated based on the wild-type platform strain, providing a broad spectrum of precise tools for both intracellular and secreted production of heterologous proteins utilizing various selection markers and integration strategies for targeted or random integration of single and multiple genes. The simplicity of targeted integration in the ku70 deletion strain will further support protein production strain generation and synthetic biology using P. pastoris strains as platform hosts.

Lytic polysaccharide monooxygenase (LPMO) represents a unique principle of oxidative degradation of recalcitrant insoluble polysaccharides. Used in combination with hydrolytic enzymes, LPMO appears to constitute a significant factor of the efficiency of enzymatic biomass depolymerization. LPMO activity on different cellulose substrates has been shown from the slow release of oxidized oligosaccharides into solution, but an immediate and direct demonstration of the enzyme action on the cellulose surface is lacking. Specificity of LPMO for degrading ordered crystalline and unordered amorphous cellulose material of the substrate surface is also unknown. We show by fluorescence dye adsorption analyzed with confocal laser scanning microscopy that a LPMO (from Neurospora crassa ) introduces carboxyl groups primarily in surface-exposed crystalline areas of the cellulosic substrate. Using time-resolved in situ atomic force microscopy we further demonstrate that cellulose nano-fibrils exposed on the surface are degraded into shorter and thinner insoluble fragments. Also using atomic force microscopy, we show that prior action of LPMO enables cellulases to attack otherwise highly resistant crystalline substrate areas and that it promotes an overall faster and more complete surface degradation. Overall, this study reveals key characteristics of LPMO action on the cellulose surface and suggests the effects of substrate morphology on the synergy between LPMO and hydrolytic enzymes in cellulose depolymerization. Background Lytic polysaccharide monooxygenase (LPMO) has recently been discovered to depolymerize cellulose. Results Dynamic imaging was applied to reveal the effects of LPMO and cellulase activity on solid cellulose surface. Conclusion Critical features of surface morphology for LPMO synergy with cellulases are recognized. Significance Direct insights into cellulose deconstruction by LPMO alone and in synergy with cellulases are obtained.

Several pathways modulating longevity and stress resistance converge on translation by targeting ribosomal proteins or initiation factors, but whether this involves modifications of ribosomal RNA is unclear. Here, we show that reduced levels of the conserved RNA methyltransferase NSUN5 increase the lifespan and stress resistance in yeast, worms and flies. Rcm1, the yeast homologue of NSUN5, methylates C2278 within a conserved region of 25S rRNA. Loss of Rcm1 alters the structural conformation of the ribosome in close proximity to C2278, as well as translational fidelity, and favours recruitment of a distinct subset of oxidative stress-responsive mRNAs into polysomes. Thus, rather than merely being a static molecular machine executing translation, the ribosome exhibits functional diversity by modification of just a single rRNA nucleotide, resulting in an alteration of organismal physiological behaviour, and linking rRNA-mediated translational regulation to modulation of lifespan, and differential stress response.

Besides its use for efficient production of recombinant proteins the methylotrophic yeast Pichia pastoris (syn. Komagataella spp.) has been increasingly employed as a platform to produce metabolites of varying origin. We summarize here the impressive methodological developments of the last years to model and analyze the metabolism of P. pastoris, and to engineer its genome and metabolic pathways. Efficient methods to insert, modify or delete genes via homologous recombination and CRISPR/Cas9, supported by modular cloning techniques, have been reported. An outstanding early example of metabolic engineering in P. pastoris was the humanization of protein glycosylation. More recently the cell metabolism was engineered also to enhance the productivity of heterologous proteins. The last few years have seen an increased number of metabolic pathway design and engineering in P. pastoris, mainly towards the production of complex (secondary) metabolites. In this review, we discuss the potential role of P. pastoris as a platform for metabolic engineering, its strengths, and major requirements for future developments of chassis strains based on synthetic biology principles.

Protein secretion is an essential process for living organisms. In eukaryotes, this encompasses numerous steps mediated by several hundred cellular proteins. The core functions of translocation through the endoplasmic reticulum membrane, primary glycosylation, folding and quality control, and vesicle-mediated secretion are similar from yeasts to higher eukaryotes. However, recent research has revealed significant functional differences between yeasts and mammalian cells, and even among diverse yeast species. This review provides a current overview of the canonical protein secretion pathway in the model yeast Saccharomyces cerevisiae, highlighting differences to mammalian cells as well as currently unresolved questions, and provides a genomic comparison of the S. cerevisiae pathway to seven other yeast species where secretion has been investigated due to their attraction as protein production platforms, or for their relevance as pathogens. The analysis of Candida albicans, Candida glabrata, Kluyveromyces lactis, Pichia pastoris, Hansenula polymorpha, Yarrowia lipolytica, and Schizosaccharomyces pombe reveals that many - but not all - secretion steps are more redundant in S. cerevisiae due to duplicated genes, while some processes are even absent in this model yeast. Recent research obviates that even where homologous genes are present, small differences in protein sequence and/or differences in the regulation of gene expression may lead to quite different protein secretion phenotypes.

The oxidation of alcohols to the corresponding carbonyl or carboxyl compounds represents a convenient strategy for the selective introduction of electrophilic carbon centres into carbohydrate-based starting materials. The O2-dependent oxidation of prim-alcohols by flavin-containing alcohol oxidases often yields mixtures of aldehyde and carboxylic acid, which is due to “over-oxidation” of the aldehyde hydrate intermediate. In order to directly convert alcohols into carboxylic acids, rational engineering of 5-(hydroxymethyl)furfural oxidase was performed. In an attempt to improve the binding of the aldehyde hydrate in the active site to boost aldehyde-oxidase activity, two active-site residues were exchanged for hydrogen-bond-donating and -accepting amino acids. Enhanced over-oxidation was demonstrated and Michaelis–Menten kinetics were performed to corroborate these findings.

Enzymatic cascade reactions experience tremendous attention by cutting short conventional step-by-step synthesis in a highly efficient and elegant fashion. Focusing on ω-transaminases, this review provides an overview of different biocatalytic strategies to afford a variety of (chiral) amines employing diverse cascade systems: Cascades to shift the reaction equilibrium as well as cascades for the amination of alcohols and nonactivated C–H bonds are discussed. Cascades enable the deracemization of rac -amines, other ones involve biocatalyzed C–C bond formation and C–C bond hydrolysis. Finally, the potential of spontaneous ring closure reactions initiated by ω-transaminases is illustrated.

•Glycosylated full length antibodies are currently produced in mammalian cells.•Antibody fragments can be produced in microbial organisms.•Strain engineering allows production of full length antibodies in microbials.•Microbials provide several advantages over mammalian cells. Monoclonal antibodies (mAbs) and antibody fragments represent the most important biopharmaceutical products today. Because full length antibodies are glycosylated, mammalian cells, which allow human-like N-glycosylation, are currently used for their production. However, mammalian cells have several drawbacks when it comes to bioprocessing and scale-up, resulting in long processing times and elevated costs. By contrast, antibody fragments, that are not glycosylated but still exhibit antigen binding properties, can be produced in microbial organisms, which are easy to manipulate and cultivate. In this review, we summarize recent advances in the expression systems, strain engineering, and production processes for the three main microbials used in antibody and antibody fragment production, namely Saccharomyces cerevisiae, Pichia pastoris, and Escherichia coli. Monoclonal antibodies (mAbs) and antibody fragments represent the most important biopharmaceutical products today. Because full length antibodies are glycosylated, mammalian cells, which allow human-like N-glycosylation, are currently used for their production. However, mammalian cells have several drawbacks when it comes to bioprocessing and scale-up, resulting in long processing times and elevated costs. By contrast, antibody fragments, that are not glycosylated but still exhibit antigen binding properties, can be produced in microbial organisms, which are easy to manipulate and cultivate. In this review, we summarize recent advances in the expression systems, strain engineering, and production processes for the three main microbials used in antibody and antibody fragment production, namely Saccharomyces cerevisiae, Pichia pastoris, and Escherichia coli. IntroductionOver the past three decades, the biopharmaceutical market has become a significant component of the global pharmaceutical market accounting for around 40% of its sales. The use of organisms as biopharmaceutical production factories offers several advantages over chemical synthesis. Microorganisms can produce high molecular weight compounds such as proteins [1Lee J.Y. Bang D. Challenges in the chemical synthesis of average sized proteins: sequential vs. convergent ligation of multiple peptide fragments.Biopolymers. 2010; 94: 441-447Crossref PubMed Scopus (21) Google Scholar] and carry out highly enantio- and regio-selective reactions by their native enzymatic machinery – these reactions are hard to achieve by chemical synthesis. The use of microorganisms also enables repeated implementation of immobilized enzymes or cells resulting in the reduction of the overall production costs [2Bolivar J.M. et al.Shine a light on immobilized enzymes: real-time sensing in solid supported biocatalysts.Trends Biotechnol. 2013; 31: 194-203Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar]. Finally, processes employing microorganisms do not generate organic and inorganic pollutants, such as mercury and toluene [3Chelliapan S. Sallis P.J. Removal of organic compound from pharmaceutical wastewater using advanced oxidation processes.J. Sci. Ind. Res. 2013; 72: 248-254Google Scholar].The biopharmaceutical market originated in the late 1970s with the establishment of recombinant DNA techniques. The industrial interest materialized almost immediately and in 1982 the US Food and Drug Administration (FDA) approved the commercialization of humulin, the human insulin analog, recombinantly produced in the bacterium E. coli [4Walsh G. New biopharmaceuticals: a review of new biologic drug approvals over the years, featuring highlights from 2010 and 2011.Process Development Forum. BioPharm International, 2012http://www.processdevelopmentforum.com/articles/new-biopharmaceuticals-a-review-of-new-biologic-drug-approvals-over-the-years-featuring-highlights-from-2010-and-2011/Google Scholar]. For a while the FDA only allowed the transformation of bacteria and the expression of small, non-glycosylated proteins, like insulin, due to concern about introducing new toxicities such as contaminating bacterial substances, which raise immunogenic reactions in patients. However, with the development of selectable resistance markers, like antibiotic resistance markers, and the possibility of production in eukaryotic organisms, the FDA began showing increasing flexibility towards biotechnological innovation, leading to a continually increasing number of approved new biological entities (NBEs). In 2012, the biopharmaceutical market turnover was estimated at around 100–120 billion US dollars per year [5Butler M. Meneses-Acosta A. Recent advances in technology supporting biopharmaceutical production from mammalian cells.Appl. Microbiol. Biotechnol. 2012; 96: 885-894Crossref PubMed Scopus (124) Google Scholar], with more than 200 biopharmaceutical proteins already on the market [6Berlec A. Strukelj B. Current state and recent advances in biopharmaceutical production in Escherichia coli, yeasts and mammalian cells.J. Ind. Microbiol. Biotechnol. 2013; 40: 257-274Crossref PubMed Scopus (139) Google Scholar], and is expected to reach 170 billion US dollars in 2014. This exceptionally high market turnover is largely derived from the marketing of mAbs and antibody fragments that currently represent the fastest growing class of approved biopharmaceutical products. In fact, production of full length mAbs (Figure 1) is the most important biopharmaceutical venture to date, with several therapeutic products reaching blockbuster status (e.g., Avastin, Herceptin, Remicade, Rituxan, Humira, and Erbitux).More recently, interest has grown in the production of antibody fragments that can be used not only in therapeutic applications but also in immunodetection, purification, and bioseparation applications [7de Marco A. Biotechnological applications of recombinant single-domain antibody fragments.Microb. Cell Fact. 2011; 10: 44Crossref PubMed Scopus (136) Google Scholar]. Antibody fragments still exhibit antigen binding properties and can be produced in microbials, which are easy to manipulate and cultivate. In this review, we summarize recent advances in the expression system, strain engineering, and production process for the three main microbials for antibody fragment production, namely S. cerevisiae, P. pastoris, and E. coli, and highlight ongoing research that may allow full length antibody production in these organisms in the future.mAbs and antibody fragments: an overviewA full length mAb consists of the constant Fc (crystallizable fragment) domain and an antigen binding domain, comprising the Fv (variable fragment) and the Fab region (antibody binding fragment; Figure 1). Native full length mAbs are glycosylated during their synthesis. Although the glycosylated Fc domain does not directly interact with antigens, it stabilizes the antibody and is important for antibody-dependent, cell-mediated cytotoxicity. Moreover, glycosylation strongly impacts the clearance rate of the recombinant mAb from the body, and incompatible glycoforms can cause severe immunogenic effects in patients. Thus, much current work is focused on optimizing and controlling glycosylation events in mammalian cells [8Li F. et al.Cell culture processes for monoclonal antibody production.MAbs. 2010; 2: 466-479Crossref PubMed Scopus (461) Google Scholar], which at this time are the most often used cell type for the production of mAbs (Box 1).Box 1Production of mAbs in mammalian cells: advantages and drawbacksMammalian cells are used most often for production of mAbs due to their ability to perform post-translational modifications (PTM), especially human-like N-glycosylation. Their use simplifies subsequent medical applications by eliminating the risk of an immunogenic response in patients due to incompatible N-glycans on the protein. Chinese Hamster Ovary (CHO) cell lines are used most frequently to generate full length mAbs with human-like Fc N-glycosylation and production titers of around 10 g/l [8Li F. et al.Cell culture processes for monoclonal antibody production.MAbs. 2010; 2: 466-479Crossref PubMed Scopus (461) Google Scholar]. However, the use of mammalian cells for heterologous expression several drawbacks such as and risk of and for the of et of cell culture in mammalian in In 2010; PubMed Scopus Google Scholar], the of is still to cell highly J.Y. et of of recombinant Chinese cells in with Microbiol. 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In work is ongoing to the glycosylation events in P. and allow production of full length mAbs in this (Box of Pichia allows mAb can be used for the production of antibody fragments and mAbs (e.g., D. et of recombinant Fab fragment from Pichia by PubMed Google For the glycosylation is not only for and biological but also for and in P. the but S. The of N-glycosylation in Microbiol. PubMed Scopus Google et of a new of in of in Pichia and PubMed Scopus Google Scholar]. The of on P. is of this high in J.M. et advances in the expression of in Pichia PubMed Scopus Google Scholar]. Thus, the of the N-glycosylation in P. has an important The for an was out et of to glycosylation in the Pichia Sci. PubMed Scopus Google Scholar], and an and an et to heterologous glycosylation enzymes to the or the of Pichia 2011; PubMed Scopus Google Scholar]. The human and an resulting in the of the human et of human in PubMed Scopus Google Scholar]. In was a et human monoclonal antibodies from 2010; PubMed Scopus Google Scholar], an in the of from was and a human was to the of Saccharomyces for et of for engineering in Pichia Full Text Full Text PDF PubMed Scopus Google Scholar]. the of a strain the human and in which the an of the was out et of the the of P. PubMed Scopus Google Scholar], leading to the of of a of the S. domain and the of and human allowed the production of allowed production of N-glycans using the technology et N-glycosylation in Pichia using PubMed Scopus Google Scholar], are used to of the native glycosylation was by of its and the of the N-glycans of leading to human et of to produce PubMed Scopus Google Scholar]. the of high D. et of in Pichia 2011; PubMed Scopus Google Scholar] and of to et of of therapeutic produced in Pichia Microbiol. Biotechnol. 2012; PubMed Scopus Google Scholar]. it to use P. for the production of full length mAbs (e.g., et of a Pichia process for antibody 2011; PubMed Scopus Google to S. cerevisiae, P. over to high cell for g/l cell weight M. et of and of Pichia a Scopus Google Scholar], on the risk of The production processes in P. are most as The possibility of are due to on the and leading to and production, is a significant of yeasts over mammalian cells and has already for the production of with P. S. et of antibody production in Pichia using in and PubMed Scopus Google Scholar]. In a recent a for in This enables the of for this D. et for a Pichia to process 2012; PubMed Scopus Google P. is a for the production of antibody In fact, recombinant therapeutic antibody fragments are already on the which is a recombinant domain antibody fragment used for and a recombinant domain antibody fragment used for recent and ongoing advances in P. is of increasing interest for the production of glycosylated full length mAbs (Box bacterium E. to on the ability to reach high cell and easy expression are used for the production of recombinant The bacterium E. coli was the microbial for the production of recombinant and still for 40% of the biopharmaceutical compounds produced today. the of in several therapeutic proteins, such as antibody fragments the have produced in this G. Biotechnol. 2010; PubMed Scopus Google expression in bacterial is by a like the the E. coli or the and a binding high et the production in Escherichia coli with a 2011; PubMed Scopus Google Scholar]. Although can be by these cause a severe for E. coli, cell and cell Thus, new expression systems, on of the heterologous have G. et Escherichia coli expression 2010; Google Scholar]. In to the with the a using a E. coli strain was also et for therapeutic applications of antibiotic resistance the Biotechnol. 2010; PubMed Scopus Google production in E. coli allows high it is often with (e.g., F. et of the and production of in Escherichia Biotechnol. 2013; PubMed Scopus Google This E. coli from expression of and the that be in the of the This can be by the of et of and on production of Fv antibody in Escherichia 2011; PubMed Scopus Google Scholar] or by the of the to the by to a peptide at the et and cell in bacteria and Sci. 2010; PubMed Scopus Google Scholar]. the has already for antibody fragments et of antibody fragment the Escherichia coli by with the in the 2013; PubMed Scopus Google Scholar]. However, is recombinant production in also a production as already for proteins et production of recombinant in Escherichia coli and its recent Ind. Microbiol. Biotechnol. 2012; PubMed Scopus Google to the high rate of E. coli, high cell are currently used for the production of antibody fragments of antibody fragment Escherichia coli in and of Biotechnol. 2013; Google Scholar]. processes with E. coli are in as processes and the production of the an to a engineering have to or at the native et engineering for in 2012; PubMed Scopus Google Scholar] and engineering the et of an Escherichia coli strain in the for culture at high and cell an to PubMed Scopus Google Scholar]. recent is to the the and of of strain The of the strain rate and rate for allows the of which are processing that real-time on a process are for that P. et for Sci. 2013; 96: Scopus Google Scholar]. can be by of process during M. of on recombinant production in Escherichia 2011; PubMed Scopus Google Scholar]. The of enables that the by P. et on the of from in 2013; PubMed Scopus Google antibody fragments, which are not glycosylated, can be produced in E. coli and the are already in (e.g., G. Biotechnol. 2010; PubMed Scopus Google production of full length mAbs in E. coli was recently, the mAbs not glycosylated et production of recombinant in Escherichia coli and its recent Ind. Microbiol. Biotechnol. 2012; PubMed Scopus Google Scholar]. The of the N-glycosylation in and the possibility of introducing it E. coli et of and in Escherichia Microbiol. 2011; PubMed Scopus Google Scholar] may the for the production of the glycosylated Fc domain et glycosylation of antibody fragments in Escherichia 2011; PubMed Scopus Google Scholar] and the expression of full length mAbs in E. coli. For this pharmaceutical are and in E. coli to their production and length mAbs as as antibody fragments represent the most important and class of today. to the for mAbs are still produced in mammalian cells, which several drawbacks to bioprocessing and By contrast, antibody fragments, which are not glycosylated but antigen binding properties, can also be produced in microbial Recent advances in the production of full length mAbs and antibody fragments with mammalian cells and microbials are in advances in the production of full length mAbs and antibody fragments with and expression engineering and of M. et of for therapeutic cell Biotechnol. 2010; PubMed Scopus Google et using 2010; Google et in mammalian cell development for recombinant 2013; Scopus Google E. et of a cell engineering to effects in cells.J. Biotechnol. 2010; PubMed Scopus Google expression of several of of of et of for of industrial of Saccharomyces 2013; PubMed Scopus Google et of a bacterial in Saccharomyces Full Text Full Text PDF PubMed Scopus Google M. et of in Pichia by of PubMed Scopus Google A. et from by of its Microbiol. Biotechnol. 2010; PubMed Scopus Google et of the in Pichia allows expression of and human PubMed Scopus Google et response heterologous in Saccharomyces Microbiol. Biotechnol. 2013; PubMed Scopus Google expression of G. et Escherichia coli expression 2010; Google et for therapeutic applications of antibiotic resistance the Biotechnol. 2010; PubMed Scopus Google et of and on production of Fv antibody in Escherichia 2011; PubMed Scopus Google and cell et in mammalian cell development for recombinant 2013; Scopus Google of and et of for of industrial of Saccharomyces 2013; PubMed Scopus Google M. et of antibody Fab fragment in Pichia its of and Microbiol. Biotechnol. 2012; PubMed Scopus Google an of the of the to the et of antibody fragment the Escherichia coli by with the in the 2013; PubMed Scopus Google of on biological et of heterologous in Saccharomyces 2012; PubMed Scopus Google et of on recombinant expression in Pichia 2013; PubMed Scopus Google M. et of antibody Fab fragment in Pichia its of and Microbiol. Biotechnol. 2012; PubMed Scopus Google to the et of antibody fragment the Escherichia coli by with the in the 2013; PubMed Scopus Google et of cell culture in mammalian in In 2010; PubMed Scopus Google S. of three cell culture 2011; et cell in Saccharomyces recombinant the use of a strain as a microbial cell Cell Fact. 2010; PubMed Scopus Google et to a for recombinant Pichia Cell Fact. 2011; 10: PubMed Scopus Google the native the et engineering for in 2012; PubMed Scopus Google et of an Escherichia coli strain in the for culture at high and cell an to PubMed Scopus Google in a new in current are towards optimizing the production of mAbs and antibody fragments in microbial organisms, mammalian cells in several such as the of and high cell processes on and Although mAbs are still most frequently produced in mammalian cells, ongoing with yeasts (Box and E. coli et of and in Escherichia Microbiol. 2011; PubMed Scopus Google et glycosylation of antibody fragments in Escherichia 2011; PubMed Scopus Google Scholar] are the for the production of glycosylated full length mAbs in microbial IntroductionOver the past three decades, the biopharmaceutical market has become a significant component of the global pharmaceutical market accounting for around 40% of its sales. The use of organisms as biopharmaceutical production factories offers several advantages over chemical synthesis. Microorganisms can produce high molecular weight compounds such as proteins [1Lee J.Y. Bang D. Challenges in the chemical synthesis of average sized proteins: sequential vs. convergent ligation of multiple peptide fragments.Biopolymers. 2010; 94: 441-447Crossref PubMed Scopus (21) Google Scholar] and carry out highly enantio- and regio-selective reactions by their native enzymatic machinery – these reactions are hard to achieve by chemical synthesis. The use of microorganisms also enables repeated implementation of immobilized enzymes or cells resulting in the reduction of the overall production costs [2Bolivar J.M. et al.Shine a light on immobilized enzymes: real-time sensing in solid supported biocatalysts.Trends Biotechnol. 2013; 31: 194-203Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar]. Finally, processes employing microorganisms do not generate organic and inorganic pollutants, such as mercury and toluene [3Chelliapan S. Sallis P.J. Removal of organic compound from pharmaceutical wastewater using advanced oxidation processes.J. Sci. Ind. Res. 2013; 72: 248-254Google Scholar].The biopharmaceutical market originated in the late 1970s with the establishment of recombinant DNA techniques. The industrial interest materialized almost immediately and in 1982 the US Food and Drug Administration (FDA) approved the commercialization of humulin, the human insulin analog, recombinantly produced in the bacterium E. coli [4Walsh G. New biopharmaceuticals: a review of new biologic drug approvals over the years, featuring highlights from 2010 and 2011.Process Development Forum. BioPharm International, 2012http://www.processdevelopmentforum.com/articles/new-biopharmaceuticals-a-review-of-new-biologic-drug-approvals-over-the-years-featuring-highlights-from-2010-and-2011/Google Scholar]. For a while the FDA only allowed the transformation of bacteria and the expression of small, non-glycosylated proteins, like insulin, due to concern about introducing new toxicities such as contaminating bacterial substances, which raise immunogenic reactions in patients. However, with the development of selectable resistance markers, like antibiotic resistance markers, and the possibility of production in eukaryotic organisms, the FDA began showing increasing flexibility towards biotechnological innovation, leading to a continually increasing number of approved new biological entities (NBEs). In 2012, the biopharmaceutical market turnover was estimated at around 100–120 billion US dollars per year [5Butler M. Meneses-Acosta A. Recent advances in technology supporting biopharmaceutical production from mammalian cells.Appl. Microbiol. Biotechnol. 2012; 96: 885-894Crossref PubMed Scopus (124) Google Scholar], with more than 200 biopharmaceutical proteins already on the market [6Berlec A. Strukelj B. Current state and recent advances in biopharmaceutical production in Escherichia coli, yeasts and mammalian cells.J. Ind. Microbiol. Biotechnol. 2013; 40: 257-274Crossref PubMed Scopus (139) Google Scholar], and is expected to reach 170 billion US dollars in 2014. This exceptionally high market turnover is largely derived from the marketing of mAbs and antibody fragments that currently represent the fastest growing class of approved biopharmaceutical products. In fact, production of full length mAbs (Figure 1) is the most important biopharmaceutical venture to date, with several therapeutic products reaching blockbuster status (e.g., Avastin, Herceptin, Remicade, Rituxan, Humira, and Erbitux).More recently, interest has grown in the production of antibody fragments that can be used not only in therapeutic applications but also in immunodetection, purification, and bioseparation applications [7de Marco A. Biotechnological applications of recombinant single-domain antibody fragments.Microb. Cell Fact. 2011; 10: 44Crossref PubMed Scopus (136) Google Scholar]. Antibody fragments still exhibit antigen binding properties and can be produced in microbials, which are easy to manipulate and cultivate. In this review, we summarize recent advances in the expression system, strain engineering, and production process for the three main microbials for antibody fragment production, namely S. cerevisiae, P. pastoris, and E. coli, and highlight ongoing research that may allow full length antibody production in these organisms in the