Open Access

Yeast cell factory: fishing for the best one or engineering it?

Microbial Cell Factories20098:51

https://doi.org/10.1186/1475-2859-8-51

Received: 5 October 2009

Accepted: 12 October 2009

Published: 12 October 2009

Editorial

When today scientists and bioprocess engineers look at a Microbial Cell Factory for the production of a protein or a metabolite of commercial or research interest, they think at the microorganism of choice first from a (i) molecular, then from a (ii) metabolic and finally from a (iii) process point of view. Analyses and manipulations of the pathway(s) involved in the synthesis of the desired product and how this pathway(s) interacts with the overall cell functions and activities are indeed steps required to obtain high yield of the product (g of compound per g of substrate), high production (g/l) and high productivity (g/l/h). Conceptually, the whole set of biochemical reactions that take place in the microbial cell factory should be considered. This is valid for processes involving natural and/or recombinant products in wild type and/or engineered hosts.

Definitely, the whole approach was not straightforward when it was anticipated about 20 years ago by James Bailey, who proposed the development of a new discipline called Metabolic Engineering defined as "the improvement of cellular activities by manipulations of enzymatic, transport, and regulatory functions of the cell with the use of recombinant DNA technology" [1]. The development in genomic, molecular and bioinformatic tools together with high-throughput systems enormously speeded up the development of the existing as well as of new microbial cell factories.

Since 2002, Microbial Cell Factories has published more than 180 relevant manuscripts in form of Research Articles, Technical Notes, Reviews and Commentaries highlighting the role of hosting microbial cells for proteins and metabolites productions. About one fourth of all the manuscripts have the yeast Saccharomyces cerevisiae and other non-conventional yeasts as a subject. Many of these manuscripts are highly cited and, despite its young age, the Journal has become already a reference in the current yeast biotechnology literature.

A simple analysis of the manuscripts published during the last three decades clearly indicates a transition in the choice of the different yeast hosts. Indeed, starting from the early '80s, the majority of recombinant proteins produced in yeasts have been expressed using the conventional yeast S. cerevisiae. This was a direct reflection of the familiarity of molecular biologists with this yeast, combined with the deep knowledge about its genetics, biochemistry, physiology and fermentation technologies. Furthermore, S. cerevisiae is recognized by the American Food and Drug Administration (FDA) as an organism "generally regarded as safe" (GRAS). In this respect, it should be underlined that most of the recombinant pharmaceuticals so far approved for human use by the FDA and/or by the European Medicines Agency (EMEA) obtained by microbial eukaryotic cells have been obtained almost exclusively using S. cerevisiae [2]. However, it has to be said that sometimes this yeast is not the optimal host for large-scale production of heterologous proteins, especially because of its fermentation needs that require sophisticated equipment. In addition, the proteins produced by S. cerevisiae are often hyper-glycosylated and retention of the products within the periplasmic space, with a consequent partial degradation, is frequently observed. Disadvantages such as these have promoted, since the late '80s, a search for alternative hosts, trying to exploit the great biodiversity existing among the yeasts, and starting the development of expression systems using the so-called "non-conventional" yeasts.

Based on the manuscripts published by Microb Cell Fact, the most established alternative host used for the production of heterologous proteins is today the yeast Pichia pastoris, while S. cerevisiae is still the predominant host used for metabolite productions. Vaccines [3], receptors [4], industrial enzymes [5, 6] and bacterial toxins [7], are among the most prominent compounds obtained by recombinant P. pastoris cell factories. The use of different promoters, culture medium and operational strategies for P. pastoris have been reviewed [8]. The analysis of transcriptional levels of the genes involved in protein synthesis and secretion is a key factor to understand the host organism's responses to recombinant protein production, as well as their interaction with the growth conditions. The transcriptional levels of some genes related to the unfolded protein response (UPR) and central metabolism have been analysed, revealing that overexpression and secretion of a recombinant lipase seems to trigger the UPR in P. pastoris, resulting in a physiological bottleneck for the production process [5]. However, when the genome sequence was not available, strain and process development relied mainly on analogies to other well studied yeasts like S. cerevisiae [9]. Finally, the genome of P. pastoris has been published [10, 11]. A gene annotation http://bioinformatics.psb.ugent.be/webtools/bogas/ and an open access web based genome browser http://www.pichiagenome.org are now available to the scientific community. A tremendous development of this host is also confirmed by many articles over viewing the development of new fermentation [3, 4, 12, 13], scale-up [14] and modelling [15] strategies. The first example of a metabolite production in P. pastoris is related to the overexpression of the riboflavin biosynthetic pathway [16].

During the past years, great efforts have been dedicated to the development of yeasts fermenting xylose efficiently. Also in this respect, the Pichia genus has been an important source of hydrolytic enzymes, especially the ones involved in the xylose fermentation. The NAD(P)H-dependent Pichia stipitis xylose reductase (PsXR) is one of the key enzymes for xylose fermentation, and has been cloned into the commonly used ethanol-producing yeast S. cerevisiae. In order to eliminate the redox imbalance due to the preference of this enzyme for NADPH, successful strategies have been implemented to alter the coenzyme specificity [17]. Other non-conventional yeasts (Hansenula polymorpha, Arxula adeninivorans [1822], Candida boidinii [22], Pichia methanolica [22], Xanthophyllomyces dendrorhous [23] and Kluyveromyces lactis [24, 25] covered a less relevant space on Microb Cell Fact.

Going back to the prominent role covered by S. cerevisiae, a clear transition in the application fields has been observed. Indeed, the majority of the articles published in these last years by Microb Cell Fact are about the production of metabolites instead of heterologous proteins. The backer's yeast has long been utilized as a very efficient biocatalyst, thanks to its enzymatic capabilities, native or manipulated. Remarkably, it can be easily engineered to become able to produce small, huge and complex molecules, like a variety of flavonoids [26, 27], isoprenoids and polyunsatured fatty acids [27], biofuels [28], fine chemicals and API (active pharmaceutical ingredient) [29], alkaloids [30] and organic acids [31]. In very recent years, many efforts have been devoted to widen the substrate range for S. cerevisiae. In particular, specific attention has been dedicate to make S. cerevisiae able to ferment xylose and/or arabinose [3236] or to improve its growth on sucrose [37]. S. cerevisiae has been also the center of attention as a model for basic and applied virus research, including the analysis of the function of individual proteins from pathogenic viruses, the elucidation of key processes in viral replication and the use of this yeast in antiviral drug development and vaccine production [38]. In this respect, S. cerevisiae metabolome served as a tool to prioritize host targets [39]. Metabolite profiling has been also used for the characterization of the metabolic shift between oxidative and fermentative growth [40] as well as for the development of new technologies leading to yeast artificial chromosomes employed for random assembly of biosynthetic pathways [26]. A couple of articles have also been published about the modulation of the ribosomal subunit ratio [41] and regulation of gene expression [42] to maximize recombinant protein yield in S. cerevisiae, an enduring bottleneck in the post-genomic sciences.

Finally, an interesting comparative review of different microbial cell factories for recombinant protein production has considerably helped to describe the extremely rich landscape of in-vivo protein folding processes [43]. The effects of the composition of growth media in relation to strain development for industrial application and for heterologous protein and for bulk bio-commodity production have also been reviewed [44].

In the end, taking into consideration everything said so far, Microb Cell Fact articles show that manipulation of yeast cell factories for improving host properties and productions can be obtained by changing the external and/or the internal environment of the cell. Modulations of cell performances changing the external environment have long been and are still practiced by choosing selective operating conditions during the processes. Traditionally, internal changes have been and are still achieved by random mutagenesis, selection and, more directly, by means of rDNA approaches.

Based on my experience, I (DP) do believe that the best yeast cell factory could be obtained by fishing for the wild type or recombinant yeast cell factory having the best fitness for that specific production/process. I do also believe that it will be obtained by a deep analysis and following direct engineering of the whole set of biochemical reactions involved in the biosynthesis. Judging from the articles published by Microb Cell Fact, it seems clear that the first approach is more successful for the production of metabolites, while the second is for the production of proteins. At the same, we are both aware that selection remains the most powerful tool to catch the desired cell factory.

Concluding, the ideal solution would be a laboratory created biological system capable of replication and evolution, fed only by simple carbon and energy sources. The future is already under way: the chemical synthesis, assembly, and cloning of a bacterial genome in S. cerevisiae has been already described [45, 46].

Authors’ Affiliations

(1)
Department of Biotechnology and Biosciences, University of Milano Bicocca

References

  1. Bailey JE: Toward a science of metabolic engineering. Science. 1991, 252: 1668- 10.1126/science.2047876.View ArticleGoogle Scholar
  2. Ferrer-Miralles N, Domingo-Espín J, Corchero JL, Vázquez E, Villaverde A: Microbial factories for recombinant pharmaceuticals. Microbial Cell Factories. 2009, 8: 17- 10.1186/1475-2859-8-17.View ArticleGoogle Scholar
  3. Gurramkonda C, Adnan A, Gäbel T, Lünsdorf H, Ross A, Nemani SK, Swaminathan S, Khanna N, Rinas U: Simple high-cell density fed-batch technique for high-level recombinant protein production with Pichia pastoris: Application to intracellular production of Hepatitis B surface antigen. Microbial Cell Factories. 2009, 8: 13- 10.1186/1475-2859-8-13.View ArticleGoogle Scholar
  4. Singh S, Gras A, Fiez-Vandal C, Ruprecht J, Rana R, Martinez M, Strange PG, Wagner R, Byrne B: Large-scale functional expression of WT and truncated human adenosine A2A receptor in Pichia pastoris bioreactor cultures. Microbial Cell Factories. 2008, 7: 28- 10.1186/1475-2859-7-28.View ArticleGoogle Scholar
  5. Resina D, Bollók M, Khatri NK, Valero F, Neubauer P, Ferrer P: Transcriptional response of P. pastoris in fed-batch cultivations to Rhizopus oryzae lipase production reveals UPR induction. Microbial Cell Factories. 2007, 6: 21- 10.1186/1475-2859-6-21.View ArticleGoogle Scholar
  6. Colao MC, Lupino S, Garzillo AM, Buonocore V, Ruzzi M: Heterologous expression of lcc1 gene from Trametes trogii in Pichia pastoris and characterization of the recombinant enzyme. Microbial Cell Factories. 2006, 5: 31- 10.1186/1475-2859-5-31.View ArticleGoogle Scholar
  7. Gurkan C, Ellar DJ: Recombinant production of bacterial toxins and their derivatives in the methylotrophic yeast Pichia pastoris. Microbial Cell Factories. 2005, 4: 33- 10.1186/1475-2859-4-33.View ArticleGoogle Scholar
  8. Cos O, Ramón R, Montesinos JL, Valero F: Operational strategies, monitoring and control of heterologous protein production in the methylotrophic yeast Pichia pastoris under different promoters: A review. Microbial Cell Factories. 2006, 5: 17- 10.1186/1475-2859-5-17.View ArticleGoogle Scholar
  9. Sauer M, Branduardi P, Gasser B, Valli M, Maurer M, Porro D, Mattanovich D: Differential gene expression in recombinant Pichia pastoris analysed by heterologous DNA microarray hybridisation. Microbial Cell Factories. 2004, 3: 17- 10.1186/1475-2859-3-17.View ArticleGoogle Scholar
  10. De Schutter K, Lin YC, Tiels P, Van Hecke A, Glinka S, Weber-Lehmann J, Rouzé P, Peer Van de Y, Callewaert N: Genome sequence of the recombinant protein production host Pichia pastoris. Nat Biotechnol. 2009, 6: 561-10.1038/nbt.1544. 10.1038/nbt.1544.View ArticleGoogle Scholar
  11. Mattanovich D, Graf A, Stadlmann J, Dragosits M, Redl A, Maurer M, Kleinheinz M, Sauer M, Altmann F, Gasser B: Genome, secretome and glucose transport highlight unique features of the protein production host Pichia pastoris. Microbial Cell Factories. 2009, 8: 29- 10.1186/1475-2859-8-29.View ArticleGoogle Scholar
  12. Surribas A, Resina D, Ferrer P, Valero F: Rivoflavin may interfere with on-line monitoring of secreted green fluorescence protein fusion proteins in Pichia pastoris. Microbial Cell Factories. 2007, 6: 15- 10.1186/1475-2859-6-15.View ArticleGoogle Scholar
  13. Jahic M, Wallberg F, Bollok M, Garcia P, Enfors SO: Temperature limited fed-batch technique for control of proteolysis in Pichia pastoris bioreactor cultures. Microbial Cell Factories. 2003, 2: 6- 10.1186/1475-2859-2-6.View ArticleGoogle Scholar
  14. Holmes WJ, Darby RAJ, Wilks MDB, Smith R, Bill RM: Developing a scalable model of recombinant protein yield from Pichia pastoris: the influence of culture conditions, biomass and induction regime. Microbial Cell Factories. 2009, 8: 35- 10.1186/1475-2859-8-35.View ArticleGoogle Scholar
  15. Maurer M, Kühleitner M, Gasser B, Mattanovich D: Versatile modeling and optimization of fed batch processes for the production of secreted heterologous proteins with Pichia pastoris. Microbial Cell Factories. 2006, 5: 37- 10.1186/1475-2859-5-37.View ArticleGoogle Scholar
  16. Marx H, Mattanovich D, Sauer M: Overexpression of the riboflavin biosynthetic pathway in Pichia pastoris. Microbial Cell Factories. 2008, 7: 23- 10.1186/1475-2859-7-23.View ArticleGoogle Scholar
  17. Liang L, Zhang J, Lin Z: Altering coenzyme specificity of Pichia stipitis xylose reductase by the semi-rational approach CASTing. Microbial Cell Factories. 2007, 6: 36- 10.1186/1475-2859-6-36.View ArticleGoogle Scholar
  18. Kensy F, Zang E, Faulhammer C, Tan RK, Büchs J: Validation of a high-throughput fermentation system based on online monitoring of biomass and fluorescence in continuously shaken microtiter plates. Microbial Cell Factories. 2009, 8: 31- 10.1186/1475-2859-8-31.View ArticleGoogle Scholar
  19. Stöckmann C, Scheidle M, Dittrich B, Merckelbach A, Hehmann G, Melmer G, Klee D, Büchs J, Kang HA, Gellissen G: Process development in Hansenula polymorpha and Arxula adeninivorans, a re-assessment. Microbial Cell Factories. 2009, 8: 22- 10.1186/1475-2859-8-22.View ArticleGoogle Scholar
  20. Steinborn G, Böer E, Scholz A, Tag K, Kunze G, Gellissen G: Application of a wide-range yeast vector (CoMed™) system to recombinant protein production in dimorphic Arxula adeninivorans, methylotrophic Hansenula polymorpha and other yeasts. Microbial Cell Factories. 2006, 5: 33- 10.1186/1475-2859-5-33.View ArticleGoogle Scholar
  21. Dmytruk OV, Dmytruk KV, Abbas CA, Voronovsky AY, Sibirny AA: Engineering of xylose reductase and overexpression of xylitol dehydrogenase and xylulokinase improves xylose alcoholic fermentation in the thermotolerant yeast Hansenula polymorpha. Microbial Cell Factories. 2008, 7: 21- 10.1186/1475-2859-7-21.View ArticleGoogle Scholar
  22. Hartner FS, Glieder A: Regulation of methanol utilisation pathway genes in yeasts. Microbial Cell Factories. 2006, 5: 39- 10.1186/1475-2859-5-39.View ArticleGoogle Scholar
  23. Martín JF, Gudiña E, Barredo JL: Conversion of β-carotene into astaxanthin: Two separate enzymes or a bifunctional hydroxylase-ketolase protein?. Microbial Cell Factories. 2008, 7: 3- 10.1186/1475-2859-7-3.View ArticleGoogle Scholar
  24. Rodríguez P, Leiro RF, Trillo MC, Cerdán ME, Siso MIG, Becerra M: Secretion and properties of a hybrid Kluyveromyces lactis-Aspergillus niger β-galactosidase. Microbial Cell Factories. 2006, 5: 41- 10.1186/1475-2859-5-41.View ArticleGoogle Scholar
  25. Gonzalez-Siso MI, Garcia-Leiro A, Tarrio N, Cerdan ME: Sugar metabolism, redox balance and oxidative stress response in the respiratory yeast Kluyveromyces lactis. Microbial Cell Factories. 2009, 8: 46- 10.1186/1475-2859-8-46.View ArticleGoogle Scholar
  26. Neasby M, Nielsen SV S, Nielsen CA F, Green T, Tange TO, Simon E, Knechtle P, Hansson A, Schwab MS, Titiz O, Folly C, Archila RE, Maver M, Sint Fiet SVAN, Boussemghoune T, Janes M, Kumar ASS, Sonkar SP, Mitra PP, Kumar Benjamin VA, Korrapati N, Suman I, Hansen EH, Thybo T, Goldsmith N, Santana Sorensen A: Yeast artificial chromosomes employed for random assembly of biosynthetic pathways and production of diverse compounds in Saccharomyces cerevisiae. Microbial Cell Factories. 2009, 8: 45- 10.1186/1475-2859-8-45.View ArticleGoogle Scholar
  27. Chemler JA, Yan Y, Koffas MAG: Biosynthesis of isoprenoids, polyunsaturated fatty acids and flavonoids in Saccharomyces cerevisiae. Microbial Cell Factories. 2006, 5: 20- 10.1186/1475-2859-5-20.View ArticleGoogle Scholar
  28. Steen EJ, Chan R, Prasad N, Myers S, Petzold CJ, Redding A, Ouellet M, Keasling JD: Metabolic engineering of Saccharomyces cerevisiae for the production of n-butanol. Microbial Cell Factories. 2008, 7: 36- 10.1186/1475-2859-7-36.View ArticleGoogle Scholar
  29. Pscheidt B, Glieder A: Yeast cell factories for fine chemical and API production. Microbial Cell Factories. 2008, 7: 25- 10.1186/1475-2859-7-25.View ArticleGoogle Scholar
  30. Cardillo AB, Talou JR, Giulietti AM: Expression of Brugmansia candida Hyoscyamine 6beta-Hydroxylase gene in Saccharomyces cerevisiae and its potential use as biocatalyst. Microbial Cell Factories. 2008, 7: 17- 10.1186/1475-2859-7-17.View ArticleGoogle Scholar
  31. Branduardi P, Sauer M, De Gioia L, Zampella G, Valli M, Mattanovich D, Porro D: Lactate production yield from engineered yeasts is dependent from the host background, the lactate dehydrogenase source and the lactate export. Microbial Cell Factories. 2006, 5: 4- 10.1186/1475-2859-5-4.View ArticleGoogle Scholar
  32. Bettiga M, Bengtsson O, Hahn-Hägerdal B, Gorwa-Grauslund MF: Arabinose and xylose fermentation by recombinant Saccharomyces cerevisiae expressing a fungal pentose utilization pathway. Microbial Cell Factories. 2009, 8: 40- 10.1186/1475-2859-8-40.View ArticleGoogle Scholar
  33. Salusjärvi L, Kankainen M, Soliymani R, Pitkänen JP, Penttilä M, Ruohonen L: Regulation of xylose metabolism in recombinant Saccharomyces cerevisiae. Microbial Cell Factories. 2008, 7: 18- 10.1186/1475-2859-7-18.View ArticleGoogle Scholar
  34. Petschacher B, Nidetzky B: Altering the coenzyme preference of xylose reductase to favor utilization of NADH enhances ethanol yield from xylose in a metabolically engineered strain of Saccharomyces cerevisiae. Microbial Cell Factories. 2008, 7: 9-View ArticleGoogle Scholar
  35. Karhumaa K, Sanchez RG, Hahn-Hägerdal B, Gorwa-Grauslund MF: Comparison of the xylose reductase-xylitol dehydrogenase and the xylose isomerase pathways for xylose fermentation by recombinant Saccharomyces cerevisiae. Microbial Cell Factories. 2007, 6: 5- 10.1186/1475-2859-6-5.View ArticleGoogle Scholar
  36. Karhumaa K, Wiedemann B, Hahn-Hägerdal B, Boles E, Gorwa-Grauslund MF: Co-utilization of L-arabinose and D-xylose by laboratory and industrial Saccharomyces cerevisiae strains. Microbial Cell Factories. 2006, 5: 18- 10.1186/1475-2859-5-18.View ArticleGoogle Scholar
  37. Badotti F, Dário MG, Alves SL, Cordioli MLA, Miletti LC, de Araujo PS, Stambuk BU: Switching the mode of sucrose utilization by Saccharomyces cerevisiae. Microbial Cell Factories. 2008, 7: 4- 10.1186/1475-2859-7-4.View ArticleGoogle Scholar
  38. Galao RP, Scheller N, Alves-Rodrigues I, Breinig T, Meyerhans A, Díez J: Saccharomyces cerevisiae: a versatile eukaryotic system in virology. Microbial Cell Factories. 2007, 6: 32- 10.1186/1475-2859-6-32.View ArticleGoogle Scholar
  39. Schneider K, Krömer JO, Wittmann C, Alves-Rodrigues I, Meyerhans A, Diez J, Heinzle E: Metabolite profiling studies in Saccharomyces cerevisiae: an assisting tool to prioritize host targets for antiviral drug screening. Microbial Cell Factories. 2009, 8: 12- 10.1186/1475-2859-8-12.View ArticleGoogle Scholar
  40. Frick O, Wittmann C: Characterization of the metabolic shift between oxidative and fermentative growth in Saccharomyces cerevisiae by comparative 13C flux analysis. Microbial Cell Factories. 2005, 4: 30- 10.1186/1475-2859-4-30.View ArticleGoogle Scholar
  41. Bonander N, Darby RAJ, Grgic L, Bora N, Wen J, Brogna S, Poyner DR, O'Neill MAA, Bill RM: Altering the ribosomal subunit ratio in yeast maximizes recombinant protein yield. Microbial Cell Factories. 2009, 8: 10- 10.1186/1475-2859-8-10.View ArticleGoogle Scholar
  42. Quintero MJ, Maya D, Arévalo-Rodríguez M, Cebolla , Chávez S: An improved system for estradiol-dependent regulation of gene expression in yeast. Microbial Cell Factories. 2007, 6: 10- 10.1186/1475-2859-6-10.View ArticleGoogle Scholar
  43. Gasser B, Saloheimo M, Rinas U, Dragosits M, Rodríguez-Carmona E, Baumann K, Giuliani M, Parrilli E, Branduardi P, Lang C, Porro D, Ferrer P, Tutino ML, Mattanovich D, Villaverde A: Protein folding and conformational stress in microbial cells producing recombinant proteins: a host comparative overview. Microbial Cell Factories. 2008, 7: 11- 10.1186/1475-2859-7-11.View ArticleGoogle Scholar
  44. Hahn-Hägerdal B, Karhumaa K, Larsson CU, Gorwa-Grauslund M, Görgens J, van Zyl WH: Role of cultivation media in the development of yeast strains for large scale industrial use. Microbial Cell Factories. 2005, 4: 31- 10.1186/1475-2859-4-31.View ArticleGoogle Scholar
  45. Gibson DG, Benders GA, Axelrod KC, Zaveri J, Algire MA, Moodie M, Montague MG, Venter JC, Smith HO, Hutchison CA: One-step assembly in yeast of 25 overlapping DNA fragments to form a complete synthetic Mycoplasma genitalium genome. Proc Natl Acad Sci USA. 2008, 105: 20404- 10.1073/pnas.0811011106.View ArticleGoogle Scholar
  46. Lartigue C, Vashee S, Algire MA, Chuang RY, Benders GA, Ma L, Noskov VN, Denisova EA, Gibson DG, Assad-Garcia N, Alperovich N, Thomas DW, Merryman C, Hutchison CA, Smith HO, Venter JC, Glass JI: Creating Bacterial Strains from Genomes That Have Been Cloned and Engineered in Yeast. Science. 2009, 325: 1693-6. 10.1126/science.1173759.View ArticleGoogle Scholar

Copyright

© Porro and Branduardi; licensee BioMed Central Ltd. 2009

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Advertisement