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Physics and biology, two good partners
JID:PLREV AID:470 /DIS
[m3SC+; v 1.191; Prn:17/04/2014; 15:01] P.1 (1-2)
Available online at www.sciencedirect.com
ScienceDirect Physics of Life Reviews ••• (••••) •••–••• www.elsevier.com/locate/plrev
Comment
Physics and biology, two good partners Comment on “Physical methods for genetic transformation of fungi and yeast” by Ana Leonor Rivera, Denis Magaña-Ortíz, Miguel Gómez-Lim, Francisco Fernández, Achim M. Loske Elizabeth Ortiz-Vázquez División de Estudios de Posgrado e Investigación, Instituto Tecnológico de Mérida, Av. Tecnológico s/n Km. 4.5, CP 97118, Mérida, Yuc, Mexico Received 7 March 2014; accepted 7 March 2014
Communicated by M. Frank-Kamenetskii
Over the years, the production of heterologous proteins and other metabolites in microbial systems has become a necessity; numerous examples, such as the production of insulin, interferon, erythropoietin and other essential proteins for the treatment of several diseases, are now of vital importance. Unfortunately, the cost of protein drugs is often extremely high. A representative example would be recombinant human erythropoietin (EPO) which is used in the treatment of anemia due to kidney failure or in anticancer treatments, and costs over 2 US $ billion/kg, probably the most expensive substance in existence today [1]. The search for new production systems of heterologous proteins from rDNA is imperative. Initially, prokaryotic systems were most commonly used for these purposes. However, since prokaryotic cells are incapable of secreting proteins, and lack a posttranslational process such as glycosylation, and correct folding among others reactions, new systems are being sought [2]. Nowadays, eukaryotic microbial systems, such as yeasts and other fungi, are being used to produce these metabolites. The biotechnological production of human insulin in S. cerevisiae is considered to be the first of such successful commercial achievements and, due to its enormous medical and market value, it is now a highly important field of research [3]. Indeed, there are currently two commercial recombinant proteins produced by S. cerevisiae that have been able to successfully combat high incidence diseases: hepatitis B and human papillomavirus (HPV) [2,4,5]. Nevertheless, the proteins produced by S. cerevisiae are often hyperglycosylated, and retention of the products within the periplasmic space is frequently observed, with consequent partial degradation. The use of genera such as Pichia or Aspergillus, in which heterologous proteins are correctly glycosylated, has been implemented [4–8]. The disadvantage of high protease production by these fungi has been solved using protease-deficient strategies [7,8]. However, new eukaryotic microorganisms must be proposed in order to improve the quality and quantity of recombinant products. For this purpose, genetic transformation of filamentous fungi and yeasts is an essential tool. However, available protocols to transform fungi are inefficient, laborious, and have low reproducibility. Thus, an efficient transformation system is required. Although there are protocols based on biological systems for genetic transformation, it is important to establish new methodologies to improve transformation efficiency [9,10]. DOI of original article: http://dx.doi.org/10.1016/j.plrev.2014.01.007. http://dx.doi.org/10.1016/j.plrev.2014.03.001 1571-0645/© 2014 Elsevier B.V. All rights reserved.
JID:PLREV AID:470 /DIS
2
[m3SC+; v 1.191; Prn:17/04/2014; 15:01] P.2 (1-2)
E. Ortiz-Vázquez / Physics of Life Reviews ••• (••••) •••–•••
Physical methods, such as electroporation, biolistics, agitation with glass beads, vacuum infiltration and shock waves, have contributed significantly towards improving the capacities and have enabled the design of genetically manipulated strains of different fungi which are very well discussed by Rivera et al. [9]. All of them are relatively simple; however some methods such as biolistic could be rather expensive. Shock waves have been reported as a novel and highly efficient method for genetic transformation of fungi, but with the disadvantage of the relatively high cost of the shock wave source and the fact that expertise in shock wave physics is required [10]. The enormous and diverse list of microorganisms that have been transformed using physical methods can give us an idea of the potential of these techniques. Even though some physical genetic transformation methods are only applied in a few fungal species [9]. Filamentous fungi and unconventional yeasts offer enormous potential for efficient and large scale production of recombinant gene products. Moreover, protein secretion provides a platform for the eukaryotic style post-translational modification of proteins. Fungi are cheap to cultivate and down-stream processing is made easy with no need to break cells open for product recovery [4,8]. References [1] Corchero JL, Gasser B, Resina B, Smith W, Parrilli E, Vázquez F, et al. Unconventional microbial systems for the cost-efficient production of high-quality protein therapeutics. Biotechnol Adv 2013;31:140–53. [2] Porro D, Sauer M, Branduardi P, Mattanovich D. Recombinant protein production in yeasts. Mol Biotechnol 2005;31:245–59. [3] KazemiSeresht A, Palmqvist EA, Olsson L. The impact of phosphate scarcity on pharmaceutical protein production in S. cerevisiae: linking transcriptomic insights to phenotypic responses. Microb Cell Fact 2011;10:104. [4] Adrio JL, Demain AL. Fungal biotechnology. Int Microbiol 2003;6:191–9. [5] Deshpande N, Wilkins MR, Packer N, Nevalainen H. Protein glycosilation pathways in filamentous fungi. Glycobiology 2008;18:626–37. [6] Macauley-Patrick S, Fazenda ML, McNeil B, Harvey LM. Heterologous protein production using the Pichia pastoris expression system. Yeast 2005;22:249–70. [7] Ward OP. Production of recombinant proteins by filamentous fungi. Biotechnol Adv 2012;30:1119–39. [8] Nevalainen H, Peterson R. Making recombinant proteins in filamentous fungi-are we expecting too much? Front Microbiol 2014;5:75. [9] Rivera AL, Magaña-Ortíz D, Gómez-Lim M, Fernández F, Loske AM. Physical methods for genetic transformation of fungi and yeast. Phys Life Rev 2014. http://dx.doi.org/10.1016/j.plrev.2014.01.007 [in this issue]. [10] Magaña-Ortíz D, Coconi-Linares N, Ortiz-Vazquez E, Fernández F, Loske AM, Gómez-Lim MA. A novel and highly efficient method for genetic transformation of fungi employing shock waves. Fungal Genet Biol 2013;56:9–16.
[m3SC+; v 1.191; Prn:17/04/2014; 15:01] P.1 (1-2)
Available online at www.sciencedirect.com
ScienceDirect Physics of Life Reviews ••• (••••) •••–••• www.elsevier.com/locate/plrev
Comment
Physics and biology, two good partners Comment on “Physical methods for genetic transformation of fungi and yeast” by Ana Leonor Rivera, Denis Magaña-Ortíz, Miguel Gómez-Lim, Francisco Fernández, Achim M. Loske Elizabeth Ortiz-Vázquez División de Estudios de Posgrado e Investigación, Instituto Tecnológico de Mérida, Av. Tecnológico s/n Km. 4.5, CP 97118, Mérida, Yuc, Mexico Received 7 March 2014; accepted 7 March 2014
Communicated by M. Frank-Kamenetskii
Over the years, the production of heterologous proteins and other metabolites in microbial systems has become a necessity; numerous examples, such as the production of insulin, interferon, erythropoietin and other essential proteins for the treatment of several diseases, are now of vital importance. Unfortunately, the cost of protein drugs is often extremely high. A representative example would be recombinant human erythropoietin (EPO) which is used in the treatment of anemia due to kidney failure or in anticancer treatments, and costs over 2 US $ billion/kg, probably the most expensive substance in existence today [1]. The search for new production systems of heterologous proteins from rDNA is imperative. Initially, prokaryotic systems were most commonly used for these purposes. However, since prokaryotic cells are incapable of secreting proteins, and lack a posttranslational process such as glycosylation, and correct folding among others reactions, new systems are being sought [2]. Nowadays, eukaryotic microbial systems, such as yeasts and other fungi, are being used to produce these metabolites. The biotechnological production of human insulin in S. cerevisiae is considered to be the first of such successful commercial achievements and, due to its enormous medical and market value, it is now a highly important field of research [3]. Indeed, there are currently two commercial recombinant proteins produced by S. cerevisiae that have been able to successfully combat high incidence diseases: hepatitis B and human papillomavirus (HPV) [2,4,5]. Nevertheless, the proteins produced by S. cerevisiae are often hyperglycosylated, and retention of the products within the periplasmic space is frequently observed, with consequent partial degradation. The use of genera such as Pichia or Aspergillus, in which heterologous proteins are correctly glycosylated, has been implemented [4–8]. The disadvantage of high protease production by these fungi has been solved using protease-deficient strategies [7,8]. However, new eukaryotic microorganisms must be proposed in order to improve the quality and quantity of recombinant products. For this purpose, genetic transformation of filamentous fungi and yeasts is an essential tool. However, available protocols to transform fungi are inefficient, laborious, and have low reproducibility. Thus, an efficient transformation system is required. Although there are protocols based on biological systems for genetic transformation, it is important to establish new methodologies to improve transformation efficiency [9,10]. DOI of original article: http://dx.doi.org/10.1016/j.plrev.2014.01.007. http://dx.doi.org/10.1016/j.plrev.2014.03.001 1571-0645/© 2014 Elsevier B.V. All rights reserved.
JID:PLREV AID:470 /DIS
2
[m3SC+; v 1.191; Prn:17/04/2014; 15:01] P.2 (1-2)
E. Ortiz-Vázquez / Physics of Life Reviews ••• (••••) •••–•••
Physical methods, such as electroporation, biolistics, agitation with glass beads, vacuum infiltration and shock waves, have contributed significantly towards improving the capacities and have enabled the design of genetically manipulated strains of different fungi which are very well discussed by Rivera et al. [9]. All of them are relatively simple; however some methods such as biolistic could be rather expensive. Shock waves have been reported as a novel and highly efficient method for genetic transformation of fungi, but with the disadvantage of the relatively high cost of the shock wave source and the fact that expertise in shock wave physics is required [10]. The enormous and diverse list of microorganisms that have been transformed using physical methods can give us an idea of the potential of these techniques. Even though some physical genetic transformation methods are only applied in a few fungal species [9]. Filamentous fungi and unconventional yeasts offer enormous potential for efficient and large scale production of recombinant gene products. Moreover, protein secretion provides a platform for the eukaryotic style post-translational modification of proteins. Fungi are cheap to cultivate and down-stream processing is made easy with no need to break cells open for product recovery [4,8]. References [1] Corchero JL, Gasser B, Resina B, Smith W, Parrilli E, Vázquez F, et al. Unconventional microbial systems for the cost-efficient production of high-quality protein therapeutics. Biotechnol Adv 2013;31:140–53. [2] Porro D, Sauer M, Branduardi P, Mattanovich D. Recombinant protein production in yeasts. Mol Biotechnol 2005;31:245–59. [3] KazemiSeresht A, Palmqvist EA, Olsson L. The impact of phosphate scarcity on pharmaceutical protein production in S. cerevisiae: linking transcriptomic insights to phenotypic responses. Microb Cell Fact 2011;10:104. [4] Adrio JL, Demain AL. Fungal biotechnology. Int Microbiol 2003;6:191–9. [5] Deshpande N, Wilkins MR, Packer N, Nevalainen H. Protein glycosilation pathways in filamentous fungi. Glycobiology 2008;18:626–37. [6] Macauley-Patrick S, Fazenda ML, McNeil B, Harvey LM. Heterologous protein production using the Pichia pastoris expression system. Yeast 2005;22:249–70. [7] Ward OP. Production of recombinant proteins by filamentous fungi. Biotechnol Adv 2012;30:1119–39. [8] Nevalainen H, Peterson R. Making recombinant proteins in filamentous fungi-are we expecting too much? Front Microbiol 2014;5:75. [9] Rivera AL, Magaña-Ortíz D, Gómez-Lim M, Fernández F, Loske AM. Physical methods for genetic transformation of fungi and yeast. Phys Life Rev 2014. http://dx.doi.org/10.1016/j.plrev.2014.01.007 [in this issue]. [10] Magaña-Ortíz D, Coconi-Linares N, Ortiz-Vazquez E, Fernández F, Loske AM, Gómez-Lim MA. A novel and highly efficient method for genetic transformation of fungi employing shock waves. Fungal Genet Biol 2013;56:9–16.