Submitted Successfully!
To reward your contribution, here is a gift for you: A free trial for our video production service.
Thank you for your contribution! You can also upload a video entry or images related to this topic.
Version Summary Created by Modification Content Size Created at Operation
1 Filamentous fungi hold unlimited potential for industrial applications, from the development of meat-like products and biomaterials, to bioremediation and biofuel production. + 1990 word(s) 1990 2020-09-22 06:29:24 |
2 format correct + 5 word(s) 1995 2020-10-09 12:18:28 |

Video Upload Options

Do you have a full video?

Confirm

Are you sure to Delete?
Cite
If you have any further questions, please contact Encyclopedia Editorial Office.
Ntana, F.; Mortensen, U.H.; Sarazin, C.; Figge, R. Aspergillus. Encyclopedia. Available online: https://encyclopedia.pub/entry/2448 (accessed on 28 March 2024).
Ntana F, Mortensen UH, Sarazin C, Figge R. Aspergillus. Encyclopedia. Available at: https://encyclopedia.pub/entry/2448. Accessed March 28, 2024.
Ntana, Fani, Uffe Hasbro Mortensen, Catherine Sarazin, Rainer Figge. "Aspergillus" Encyclopedia, https://encyclopedia.pub/entry/2448 (accessed March 28, 2024).
Ntana, F., Mortensen, U.H., Sarazin, C., & Figge, R. (2020, October 08). Aspergillus. In Encyclopedia. https://encyclopedia.pub/entry/2448
Ntana, Fani, et al. "Aspergillus." Encyclopedia. Web. 08 October, 2020.
Aspergillus
Edit

Aspergilli have been widely used in the production of organic acids, enzymes, and secondary metabolites for almost a century. Today, several GRAS (generally recognized as safe) Aspergillus species hold a central role in the field of industrial biotechnology with multiple profitable applications. Since the 1990s, research has focused on the use of Aspergillus species in the development of cell factories for the production of recombinant proteins mainly due to their natively high secretion capacity. Advances in the Aspergillus-specific molecular toolkit and combination of several engineering strategies (e.g., protease-deficient strains and fusions to carrier proteins) resulted in strains able to generate high titers of recombinant fungal proteins

Aspergillus fermentation filamentous fungi genetic engineering heterologous expression recombinant protein secretion

1. Introduction

Proteins are functionally versatile biomolecules (e.g., enzymes, structural proteins, and hormones) involved in multiple biological processes in the cell. Despite their role in supporting biological systems, proteins have been extensively studied for their potential in the formulation of commercial products. They often find applicability in the production of pharmaceuticals, food, beverages, biofuels, cosmetics, detergents, etc. [1][2].

Market demand for industrially relevant proteins has guided research into exploring practices that can lead to large-scale production levels [3]. The development of recombinant DNA technology has opened up the possibility of producing recombinant proteins in heterologous expression systems that can support high production yields. In that respect, any gene can now be transferred into a production host able to generate large quantities of the corresponding protein of interest, avoiding limitations related to the conventional extraction of the protein from its native host [4]. Human insulin produced in E. coli cells was the first recombinant protein that was actually approved by the FDA for clinical use. The recombinant insulin Humulin®, originally developed by Genentech, was eventually commercialized in 1982 [3]. Since then, a plethora of other proteins with pharmaceutical and industrial applications have successfully been synthesized in heterologous expression systems and have made their way into the market [1].

Today, recombinant proteins can be synthesized using a wide range of production platforms, including bacteria, yeasts and filamentous fungi, mammalian or insect cells, and transgenic plants, to name a few. Every heterologous production system though comes with certain advantages and drawbacks (Table 1). In most cases, the structure and function of the protein of interest determines which production system is the most appropriate to be used. For example, when it comes to manufacturing therapeutic proteins of high quality, mammalian cell lines are predominantly used, as they can produce complex, human-like glycosylated proteins that are safe for patients. In fact, almost 84% of the biopharmaceutical proteins are currently produced by Chinese Hamster Ovarian (CHO) cell lines [5].

Table 1. Comparison of the most commonly used heterologous expression systems in the field of recombinant protein production.

For the production of non-medicinal proteins, a more economical approach is usually followed, using either bacterial or fungal production hosts [1][16][10][1,6,7]. While bacteria are often suitable for smaller proteins that do not require complex post-translational modifications, production of larger and more complex proteins is usually performed in yeast, e.g., Pichia pastoris [17]. However, yeasts have the tendency to hyperglycosylate secreted proteins, and thus reduce their in vivo half-life and affect their efficacy [8]. Additional limitations including low expression levels and plasmid instability have restricted the use of some yeasts (e.g., S. cerevisiae) in the production of industrial enzymes [9]. An alternative production platform that can support low-cost synthesis of large proteins with complex modifications, but with a lesser degree of hypermannosylation during glycosylation compared to yeast is filamentous fungi. In addition, due to their saprophytic lifestyle, most filamentous fungi have already developed the ability to produce and secrete a vast amount of enzymes in order to break down and feed on organic matter [18]. Strains belonging to the genera Aspergillus, Trichoderma, and Neurospora are in fact widely used for production of recombinant proteins with industrial applications [19][20][21][22]. Several reviews have described the potentials of filamentous fungi in the production of pharmaceutical and other industrial proteins, as well as the genetic engineering approaches followed to maximize production levels [10][23][24]. In this review, we specifically focus on the use of Aspergillus species in the manufacturing of recombinant proteins. Bottlenecks in protein synthesis and secretion are discussed, while our comprehensive literature search provides a general overview of the most important genetic engineering projects and bioprocessing strategies applied over the past 30 years to improve recombinant protein yields in Aspergillus.

2. Industrial Application of Aspergilli

2.1. Traditional Uses of Aspergillus Species

The use of Aspergillus species in biotechnology begun approximately a century ago, when James Currie, a food chemist, discovered that the filamentous mold A. niger was able to produce citric acid, a food and beverage additive that was conventionally extracted from citrus fruits [25]. Since then, production of citric acid, now performed in A. niger cultures that grow on inexpensive sugar-based minimal media, has turned into a multibillion dollar business [26].

Nonetheless, industrial applications of Aspergilli are not limited to the production of citric acid. Several species have been used as prolific producers of other organic acids (e.g., itaconic), secondary metabolites, and enzymes of biotechnological significance [18]. For example, A. niger produces several enzymes used in food and feed production such as glucoamylases, proteases, and phytases [26]. A. oryzae, traditionally used in Asian cuisine, has been exploited as a cell factory for producing malate, which is used in the development of food and pharmaceutical products [27]. A. terreus has attracted interest due to its ability to produce a group of secondary metabolites called statins that are used in the production of cholesterol-lowering drugs [28]. In fact, AB Enzymes, BASF, Chr. Hansen, DuPont, and Novozymes are only a few examples of companies that have been or are still using Aspergillus species in large-scale manufacturing of commercial products such as organic acids, enzymes, proteins, and secondary metabolites [29].

2.2. The Use of Aspergillus Species in Heterologous Protein Production

Filamentous fungi are generally considered promising hosts for production of recombinant proteins, mainly due to their secretory capacity and metabolic versatility. However, only a few species appear to be able to produce competitive recombinant protein levels and even fewer have been developed into industrial production platforms. This can be attributed mainly to our incomplete knowledge of fungal physiology. For example, the mechanisms behind protein production and secretion in fungal cells are not yet fully understood for most of the species. In addition, the presence of unwanted metabolites (e.g., mycotoxins) has excluded several fungi from industrial production [29].

Aspergillus is a genus that has been studied extensively due to its value as a model organism in fungal research (A. nidulans) and its industrial importance in citric acid and enzyme production (A. niger, A. oryzae) [26]. Several molecular tools (e.g., synthetic promoters and terminators, selection markers, RNA interference-RNAi, and CRISPR-Clusters of Regularly Interspaced Short Palindromic Repeats-associated technologies), suitable for Aspergillus species, have also been developed, facilitating efficient and targeted manipulation of their genomes [30][31]. CRISPR/Cas, for example, a system developed to create site-specific double strand DNA breaks, has been successfully applied in editing the genome of A. niger [32][33][34][35], A. nidulans [35], A. oryzae [36], A. fumigatus [37], and other aspergilli [35]. With a relatively well-understood physiology (growth and development, gene expression, and secretion machinery) and several molecular tools available, the GRAS A. niger has already been used in industrial production of recombinant proteins, such as calf chymosin [38], human lactoferrin [39], and the plant-derived sweetener neoculin [40]. Nevertheless, heterologous protein production in Aspergillus species is not always efficient, leading to low production titers. In such cases, strategies that are usually applied to improve titers involve genetic engineering of the production strains and establishing the appropriate fermentation conditions.

3. Genetic Engineering Approaches for Aspergillus Strain Improvement

Due to their capacity to secrete large quantities of proteins into the culture medium, Aspergillus species, and especially A. niger, are considered promising candidates for the development of large-scale heterologous protein production platforms. However, production yields for heterologous proteins are usually much lower compared to the ones detected for the native proteins. Failure to achieve the desired protein amounts in Aspergillus cultures can be attributed to limitations related to transcription, translation, and the post-translation processing and modifications during protein production. Additionally, bottlenecks in the fungal secretion machinery and the problem of extracellular degradation by fungal proteases further hinder the efficient production of foreign proteins in Aspergillus species [41].

4. Fermentation Conditions for Improved Heterologous Production in Aspergillus

Development of most heterologous expression platforms begins with strain improvement, which hopefully results in obtaining strains able to produce large quantities of a specific protein. Once strain improvement is complete, the fermentation process for production of the desirable protein in large-scale has to be established [10][42]. Designing and setting up fungal fermentations is a complex process that has to be repeated every time a newly engineered strain is used or a new protein is to be produced. This process requires several optimization steps, starting from finding the optimal growth medium and fermentation parameters (temperature, pH, and oxygenation) to choosing the appropriate type of fermentation and the fungal morphology that favors high production yields of the specific protein [43][44][45].

5. Conclusions and Future Perspectives

Filamentous fungi hold unlimited potential for industrial applications, from the development of meat-like products and biomaterials, to bioremediation and biofuel production. One of their best qualities, largely exploited by the industry, is their innate capacity for the secretion of enzymes, which facilitate downstream processing and product recovery. Moreover, their ability to produce complex proteins with post-translational modifications and the fact that they can be cultivated on inexpensive media makes them a promising alternative for production of eukaryotic proteins. Despite their undeniable potential though, filamentous fungi have not yet been exploited to the fullest in the industrial production of recombinant proteins.

Advances in the molecular toolkit available for genetic manipulation of several Aspergillus species opened up the path for developing them into production systems for recombinant proteins. Nevertheless, due to a number of factors described in the review, aspergilli have not yet met the expected production levels. Many studies that focused on engineering different steps of protein synthesis and secretion, or generating protease-deficient strains, have resulted in a significant increase of protein yields. Additionally, optimization of the fungal fermentation process has further improved protein production. However, there are aspects of the fungal physiology that limit protein production and remain unclear. Continuous data input from “omics” studies sheds light on the complex fungal mechanisms related to protein quality control and secretion stress, as well as their impact on protein productivity. The knowledge generated from these studies combined with advances in the field of synthetic biology will soon place Aspergillus, and possibly other filamentous fungi, in the race for the most efficient recombinant protein production system. Its potential as a large-scale production platform not only for recombinant proteins, but also for organic acids, bioactive compounds, enzymes, and peptides, as well as new perspectives related to the use of Aspergillus in waste treatment and bioremediation processes, prove that this fungus can provide sustainable solutions for multiple and diverse markets and industries.

References

  1. Puetz, J.; Wurm, F.M. Recombinant proteins for industrial versus pharmaceutical purposes: A review of process and pricing. Processes 2019, 7, 476.
  2. Choi, J.-M.; Han, S.-S.; Kim, H.-S. Industrial applications of enzyme biocatalysis: Current status and future aspects. Biotechnol. Adv. 2015, 33, 1443–1454.
  3. Baeshen, N.A.; Baeshen, M.N.; Sheikh, A.; Bora, R.S.; Ahmed, M.M.M.; Ramadan, H.A.I.; Saini, K.S.; Redwan, E.M. Cell factories for insulin production. Microb. Cell Fact. 2014, 13, 141.
  4. Khan, S.; Ullah, M.W.; Siddique, R.; Nabi, G.; Manan, S.; Yousaf, M.; Hou, H. Role of recombinant DNA technology to improve life. Int. J. Genomics 2016, 2016, 1–14.
  5. Walsh, G. Biopharmaceutical benchmarks 2018. Nat. Biotechnol. 2018, 36, 1136–1145.
  6. Gupta, S.K.; Shukla, P. Advanced technologies for improved expression of recombinant proteins in bacteria: Perspectives and applications. Crit. Rev. Biotechnol. 2016, 36, 1089–1098.
  7. Baeshen, M.N.; Al-Hejin, A.M.; Bora, R.S.; Ahmed, M.M.M.; Ramadan, H.A.I.; Saini, K.S.; Baeshen, N.A.; Redwan, E.M. Production of biopharmaceuticals in E. coli: Current scenario and future perspectives. J. Microbiol. Biotechnol. 2015, 25, 953–962.
  8. Baghban, R.; Farajnia, S.; Rajabibazl, M.; Ghasemi, Y.; Mafi, A.; Hoseinpoor, R.; Rahbarnia, L.; Aria, M. Yeast expression systems: Overview and recent advances. Mol. Biotechnol. 2019, 61, 365–384.
  9. Xie, Y.; Han, X.; Miao, Y. An effective recombinant protein expression and purification system in Saccharomyces cerevisiae. Curr. Protoc. Mol. Biol. 2018, 123, e62.
  10. Nevalainen, H.; Peterson, R. Making recombinant proteins in filamentous fungi—Are we expecting too much? Front. Microbiol. 2014, 5, 1–10.
  11. Contreras-Gómez, A.; Sánchez-Mirón, A.; García-Camacho, F.; Molina-Grima, E.; Chisti, Y. Protein production using the baculovirus-insect cell expression system. Biotechnol. Prog. 2014, 30, 1–18.
  12. Hunter, M.; Yuan, P.; Vavilala, D.; Fox, M. Optimization of protein expression in mammalian cells. Curr. Protoc. Protein Sci. 2019, 95, e77.
  13. Houdebine, L.-M. Production of pharmaceutical proteins by transgenic animals. Comp. Immunol. Microbiol. Infect. Dis. 2009, 32, 107–121.
  14. Łojewska, E.; Kowalczyk, T.; Olejniczak, S.; Sakowicz, T. Extraction and purification methods in downstream processing of plant-based recombinant proteins. Protein Expr. Purif. 2016, 120, 110–117.
  15. Yao, J.; Weng, Y.; Dickey, A.; Wang, K. Plants as factories for human pharmaceuticals: Applications and challenges. Int. J. Mol. Sci. 2015, 16, 28549–28565.
  16. Ferreira, R.d.G.; Azzoni, A.R.; Freitas, S. Techno-economic analysis of the industrial production of a low-cost enzyme using E. coli: The case of recombinant β-glucosidase. Biotechnol. Biofuels 2018, 11, 81.
  17. Demain, A.L.; Vaishnav, P. Production of recombinant proteins by microbes and higher organisms. Biotechnol. Adv. 2009, 27, 297–306.
  18. Meyer, V.; Basenko, E.Y.; Benz, J.P.; Braus, G.H.; Caddick, M.X.; Csukai, M.; de Vries, R.P.; Endy, D.; Frisvad, J.C.; Gunde-Cimerman, N.; et al. Growing a circular economy with fungal biotechnology: A white paper. Fungal Biol. Biotechnol. 2020, 7, 5.
  19. Havlik, D.; Brandt, U.; Bohle, K.; Fleißner, A. Establishment of Neurospora crassa as a host for heterologous protein production using a human antibody fragment as a model product. Microb. Cell Fact. 2017, 16, 128.
  20. Landowski, C.P.; Mustalahti, E.; Wahl, R.; Croute, L.; Sivasiddarthan, D.; Westerholm-Parvinen, A.; Sommer, B.; Ostermeier, C.; Helk, B.; Saarinen, J.; et al. Enabling low cost biopharmaceuticals: High level interferon alpha-2b production in Trichoderma reesei. Microb. Cell Fact. 2016, 15, 104.
  21. Magaña-Ortíz, D.; Fernández, F.; Loske, A.M.; Gómez-Lim, M.A. Extracellular expression in Aspergillus niger of an antibody fused to Leishmania sp. antigens. Curr. Microbiol. 2018, 75, 40–48.
  22. Arnau, J.; Yaver, D.; Hjort, C.M. Strategies and challenges for the development of industrial enzymes using fungal cell factories. In Grand Challenges in Fungal Biotechnology; Springer: Cham, Switzerland, 2020; pp. 179–210. ISBN 9783030295417.
  23. Sun, X.; Su, X. Harnessing the knowledge of protein secretion for enhanced protein production in filamentous fungi. World J. Microbiol. Biotechnol. 2019, 35, 54.
  24. Meyer, V. Genetic engineering of filamentous fungi—Progress, obstacles and future trends. Biotechnol. Adv. 2008, 26, 177–185.
  25. Currie, J.N. The citric acid fermentation of Aspergillus niger. J. Biol. Chem. 1917, 31, 15–37.
  26. Cairns, T.C.; Nai, C.; Meyer, V. How a fungus shapes biotechnology: 100 years of Aspergillus niger research. Fungal Biol. Biotechnol. 2018, 5, 13.
  27. Dai, Z.; Zhou, H.; Zhang, S.; Gu, H.; Yang, Q.; Zhang, W.; Dong, W.; Ma, J.; Fang, Y.; Jiang, M.; et al. Current advance in biological production of malic acid using wild type and metabolic engineered strains. Bioresour. Technol. 2018, 258, 345–353.
  28. Barrios-González, J.; Miranda, R.U. Biotechnological production and applications of statins. Appl. Microbiol. Biotechnol. 2010, 85, 869–883.
  29. Meyer, V.; Andersen, M.R.; Brakhage, A.A.; Braus, G.H.; Caddick, M.X.; Cairns, T.C.; de Vries, R.P.; Haarmann, T.; Hansen, K.; Hertz-Fowler, C.; et al. Current challenges of research on filamentous fungi in relation to human welfare and a sustainable bio-economy: A white paper. Fungal Biol. Biotechnol. 2016, 3, 6.
  30. Martins-Santana, L.; Nora, L.C.; Sanches-Medeiros, A.; Lovate, G.L.; Cassiano, M.H.A.; Silva-Rocha, R. Systems and synthetic biology approaches to engineer fungi for fine chemical production. Front. Bioeng. Biotechnol. 2018, 6, 117.
  31. Meyer, V.; Wu, B.; Ram, A.F.J. Aspergillus as a multi-purpose cell factory: Current status and perspectives. Biotechnol. Lett. 2011, 33, 469–476.
  32. Leynaud-Kieffer, L.M.C.; Curran, S.C.; Kim, I.; Magnuson, J.K.; Gladden, J.M.; Baker, S.E.; Simmons, B.A. A new approach to Cas9-based genome editing in Aspergillus niger that is precise, efficient and selectable. PLoS ONE 2019, 14, e0210243.
  33. Sarkari, P.; Marx, H.; Blumhoff, M.L.; Mattanovich, D.; Sauer, M.; Steiger, M.G. An efficient tool for metabolic pathway construction and gene integration for Aspergillus niger. Bioresour. Technol. 2017, 245, 1327–1333.
  34. Cairns, T.C.; Feurstein, C.; Zheng, X.; Zhang, L.H.; Zheng, P.; Sun, J.; Meyer, V. Functional exploration of co-expression networks identifies a nexus for modulating protein and citric acid titres in Aspergillus niger submerged culture. Fungal Biol. Biotechnol. 2019, 6, 18.
  35. Nødvig, C.S.; Nielsen, J.B.; Kogle, M.E.; Mortensen, U.H. A CRISPR-Cas9 system for genetic engineering of filamentous fungi. PLoS ONE 2015, 10, e0133085.
  36. Katayama, T.; Tanaka, Y.; Okabe, T.; Nakamura, H.; Fujii, W.; Kitamoto, K.; Maruyama, J.I. Development of a genome editing technique using the CRISPR/Cas9 system in the industrial filamentous fungus Aspergillus oryzae. Biotechnol. Lett. 2016, 38, 637–642.
  37. Fuller, K.K.; Chen, S.; Loros, J.J.; Dunlap, J.C. Development of the CRISPR/Cas9 system for targeted gene disruption in Aspergillus fumigatus. Eukaryot. Cell 2015, 14, 1073–1080.
  38. Dunn-Coleman, N.S.; Bloebaum, P.; Berka, R.M.; Bodie, E.; Robinson, N.; Armstrong, G.; Ward, M.; Przetak, M.; Carter, G.L.; LaCost, R.; et al. Commercial levels of chymosin production by Aspergillus. Bio/Technology 1991, 9, 976–981.
  39. Ward, P.P.; Lo, J.-Y.; Duke, M.; May, G.S.; Headon, D.R.; Conneely, O.M. Production of biologically active recombinant human lactoferrin in Aspergillus Oryzae. Nat. Biotechnol. 1992, 10, 784–789.
  40. Nakajima, K.I.; Asakura, T.; Maruyama, J.I.; Morita, Y.; Oike, H.; Shimizu-Ibuka, A.; Misaka, T.; Sorimachi, H.; Arai, S.; Kitamoto, K.; et al. Extracellular production of neoculin, a sweet-tasting heterodimeric protein with taste-modifying activity, by Aspergillus oryzae. Appl. Environ. Microbiol. 2006, 72, 3716–3723.
  41. Van Den Hondel, C.A.M.J.J.; Punt, P.J.; Van Gorcom, R.F.M. Heterologous Gene Expression in Filamentous Fungi; Academic Press, Inc.: Cambridge, MA, USA, 1991.
  42. Sisniega, H.; Río, J.-L.; Amaya, M.-J.; Faus, I. Strategies for large-scale production of recombinant proteins in filamentous fungi. In Microbial Processes and Products; Humana Press: Totowa, NJ, USA, 2005; Volume 18, pp. 225–237.
  43. El-Enshasy, H.A. Filamentous fungal cultures—Process characteristics, products, and applications. In Bioprocessing for Value-Added Products from Renewable Resources; Elsevier: Amsterdam, The Netherlands, 2007; pp. 225–261.
  44. Workman, M.; Andersen, M.R.; Thykaer, J. Integrated approaches for assessment of cellular performance in industrially relevant filamentous fungi. Ind. Biotechnol. 2013, 9, 337–344.
  45. Wang, L.; Ridgway, D.; Gu, T.; Moo-Young, M. Bioprocessing strategies to improve heterologous protein production in filamentous fungal fermentations. Biotechnol. Adv. 2005, 23, 115–129.
More
Information
Contributors MDPI registered users' name will be linked to their SciProfiles pages. To register with us, please refer to https://encyclopedia.pub/register : , , ,
View Times: 962
Revisions: 2 times (View History)
Update Date: 09 Oct 2020
1000/1000