- Email: [email protected]
Quorum sensing in filamentous fungi
S76
Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576
Kalogeris, E., Iniotaki, F., Topakas, E., Christakopoulos, P., Kekos, D., Macris, B.J., 2003. Performance of an intermittent agitation rotating drum type bioreactor for solidstate fermentation of wheat straw. Bioresour. Technol. 86, 207–213. Ninawe, S., Kuhad, R.C., 2006. Bleaching of wheat straw-rich soda pulp with xylanase from a thermoalkalophilic Streptomyces cyaneus SN32. Bioresour. Technol. 97, 2291–2295. Sigoillot, C., Camarero, S., Vidal, T., Record, E., Asther, M., Perez-Boada, M., Martınez, M.J., Sigoillot, J.C., 2005. Comparison of different fungal enzymes for bleaching high-quality paper pulps. J. Biotechnol. 115, 333–343.
doi:10.1016/j.jbiotec.2010.08.195 [I.18] Bioprocess scale-up from deepwell plates to 100 L bioreactor scale with EnBase® -Flo J. Glazyrina 1 , M. Krause 1 , S. Junne 1 , A. Neubauer 2 , A. Vasala 2 , P. Neubauer 1,2,∗ 1
Technische Universität Berlin, Germany BioSilta Oy, Finland Keywords: EnBase; Fed-batch fermentation; Recombinant protein production; Disposable bioreactor 2
EnBase® is a unique microbial cultivation platform for high cell density growth of microorganisms (Krause et al., 2010; PanulaPerälä et al., 2008). It is based on the principle of the glucose-limited fed-batch technology but applies an enzyme controlled internal delivery system for the controlled feed of glucose from a glucosecontaining polymer which allows easy scaling to any cultivation volume, from micro liter cultures to the liter scale, with the benefit that no external feeding is needed. The previous EnBase system was characterized by a 3-phase system, a storage gel containing the polymer, a gel for controlling the release to the medium, and the overlaying liquid medium. Although this set up is advantageous by providing the possibility to supply high amounts of substrate, its preparation is laborious and its application is limited to small volumes. Here we demonstrate a new liquid form of EnBase, EnBase-Flo, which consists of a simple one-phase system only (Panula-Perälä et al., 2008). Controlling the growth with Liquid-EnBase is easily applied in any scale. Cell densities above 8 g/l are obtained and the new liquid system has the same beneficial characteristics as the gelbased EnBase for recombinant proteins, a higher amount of soluble protein is produced and aggregation is low. Whilst the ultimate cell densities have been shown in the gel based format compared the liquid format is much more convenient, and cultures can be directly centrifuged to obtain the cell pellet for extraction of the target product. EnBase-Flo is especially advantageous for bioprocess scale up, which we demonstrate here by performing a scale up from microliter cultures to the 100 L scale. Preliminary investigations with a rocking-motion-type bioreactor (BIOSTAT CultiBag RM) show that the EnBase technology is also applicable in disposable bioreactors. References Krause, M., Ukkonen, K., Haataja, T., Ruottinen, M., Glumoff, T., Neubauer, A., Neubauer, P., Vasala, A., 2010. A novel fed-batch based cultivation method provides high cell-density and improves yield of soluble recombinant proteins in shaken cultures. Microb Cell Fact 9, 11. Panula-Perälä, J., Siurkus, J., Vasala, A., Wilmanowski, R., Casteleijn, M.G., Neubauer, P., 2008. Enzyme controlled glucose auto-delivery for high cell density cultivations in microplates and shake flasks. Microb. Cell Fact 7, 31.
doi:10.1016/j.jbiotec.2010.08.196
[I.19] Quorum sensing in filamentous fungi S. Raina, D. De Vizio, T. Keshavarz ∗ University of Westminster, United Kingdom Keywords: Filamentous fungi; Quorum sensing; Enhanced productivity Introduction: Since the discovery of quorum sensing (QS) in Vibrio fischeri and Vibrio harveyi over forty years ago (Nealson et al., 1970), extensive studies has been carried out on G- and Gram+ bacteria as well as yeasts. Different mechanisms have been proposed for this occurrence which is quite common among these species. Upon reaching a specific cell-density threshold the cells release extracellular diffusible molecules (autoinducers) or QS molecules. These molecules affect genes which in turn instigate a range of physiological and morphological responses. This process is known as QS. While homoserine lactones are studied as QS molecules in Gram- bacteria (Miller and Bassler, 2001), the dominant autoinducers in Gram+ bacteria are based on a two component signal transduction mechanism (Kleerebezem et al., 1997) employing small, post-translationally modified or unmodified peptides (Bassler, 2002). In many actinomycetes species, ␥-butyrolactones act as autoregulators and in yeasts Farnesol (Hornby et al., 2001) and Tyrosol (Narayanan and Rao, 1976) are QS molecules. In spite of extensive research on the autoinducers and their role in pathogenesis which could provide valuable information to battle infectious diseases, there is very little information on QS in filamentous fungi. This study reports for the first time the presence of QS process in filamentous fungi. The aim of this work was to investigate effect of potential QS molecules on filamentous fungi and to exploit the process for future industrial exploitation. Methods: Aspergillus sp. (A. nidulans, A. terreus) and Penicillium sp. (P. sclerotiorum strains) were used. HPLC, LC, GC, NMR were adopted to identify potential QS molecules. Results: Butyrolactone I and multicolic acid as QS molecules in A. terreus and P. sclerotiorum respectively resulted in 2.9-fold increase in the production rate of lovastatin in 5 l fermenters. Discussion: This study shows the potential for industrial exploitation of QS for enhanced productivity. Acknowledgemenst: Financial support of this work (Quorum, Proposal no. 032811, 2006-2009: Discovering Quorum Sensing in industrially useful Fungi, a novel approach at molecular level for scaling-up in white biotech., from the European Commission under FP6) is gratefully appreciated. References Hornby, J.M., Jensen, E.C., Lisec, A.D., Tasto, J.J., Jahnke, B., Shoemaker, R., Dussault, P., Nickerson, K.W., 2001. Quorum sensing in the dimorphic fungus Candida albicans is mediated by farnesol. Applied and Environmental Microbiology 67 (7), 2982–2992. Kleerebezem, M., Quadri, L.E., Kuipers, O.P., de Vos, W.M., 1997. Quorum sensing by peptide pheromones and two-component signal-transduction systems in Grampositive bacteria. Molecular Microbiology 24 (5), 895–904. Miller, M.B., Bassler, B.L., 2001. Quorum sensing in bacteria. Annual Review of Microbiology 55, 165–199. Narayanan, T.k., Rao, G.R., 1976. Production of beta-(4-hydroxyphenyl)ethanol and beta-(4-hydroxyphenyl)lactic acid by Candida species. Canadian journal of microbiology 22 (3), 349–384. Nealson, K.H., Platt, T., Hastings, J.W., 1970. Cellular Control of the Synthesis and Activity of the Bacterial Luminescent System. Journal of Bacteriology 104 (1), 313–322.
doi:10.1016/j.jbiotec.2010.08.197
Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576
Kalogeris, E., Iniotaki, F., Topakas, E., Christakopoulos, P., Kekos, D., Macris, B.J., 2003. Performance of an intermittent agitation rotating drum type bioreactor for solidstate fermentation of wheat straw. Bioresour. Technol. 86, 207–213. Ninawe, S., Kuhad, R.C., 2006. Bleaching of wheat straw-rich soda pulp with xylanase from a thermoalkalophilic Streptomyces cyaneus SN32. Bioresour. Technol. 97, 2291–2295. Sigoillot, C., Camarero, S., Vidal, T., Record, E., Asther, M., Perez-Boada, M., Martınez, M.J., Sigoillot, J.C., 2005. Comparison of different fungal enzymes for bleaching high-quality paper pulps. J. Biotechnol. 115, 333–343.
doi:10.1016/j.jbiotec.2010.08.195 [I.18] Bioprocess scale-up from deepwell plates to 100 L bioreactor scale with EnBase® -Flo J. Glazyrina 1 , M. Krause 1 , S. Junne 1 , A. Neubauer 2 , A. Vasala 2 , P. Neubauer 1,2,∗ 1
Technische Universität Berlin, Germany BioSilta Oy, Finland Keywords: EnBase; Fed-batch fermentation; Recombinant protein production; Disposable bioreactor 2
EnBase® is a unique microbial cultivation platform for high cell density growth of microorganisms (Krause et al., 2010; PanulaPerälä et al., 2008). It is based on the principle of the glucose-limited fed-batch technology but applies an enzyme controlled internal delivery system for the controlled feed of glucose from a glucosecontaining polymer which allows easy scaling to any cultivation volume, from micro liter cultures to the liter scale, with the benefit that no external feeding is needed. The previous EnBase system was characterized by a 3-phase system, a storage gel containing the polymer, a gel for controlling the release to the medium, and the overlaying liquid medium. Although this set up is advantageous by providing the possibility to supply high amounts of substrate, its preparation is laborious and its application is limited to small volumes. Here we demonstrate a new liquid form of EnBase, EnBase-Flo, which consists of a simple one-phase system only (Panula-Perälä et al., 2008). Controlling the growth with Liquid-EnBase is easily applied in any scale. Cell densities above 8 g/l are obtained and the new liquid system has the same beneficial characteristics as the gelbased EnBase for recombinant proteins, a higher amount of soluble protein is produced and aggregation is low. Whilst the ultimate cell densities have been shown in the gel based format compared the liquid format is much more convenient, and cultures can be directly centrifuged to obtain the cell pellet for extraction of the target product. EnBase-Flo is especially advantageous for bioprocess scale up, which we demonstrate here by performing a scale up from microliter cultures to the 100 L scale. Preliminary investigations with a rocking-motion-type bioreactor (BIOSTAT CultiBag RM) show that the EnBase technology is also applicable in disposable bioreactors. References Krause, M., Ukkonen, K., Haataja, T., Ruottinen, M., Glumoff, T., Neubauer, A., Neubauer, P., Vasala, A., 2010. A novel fed-batch based cultivation method provides high cell-density and improves yield of soluble recombinant proteins in shaken cultures. Microb Cell Fact 9, 11. Panula-Perälä, J., Siurkus, J., Vasala, A., Wilmanowski, R., Casteleijn, M.G., Neubauer, P., 2008. Enzyme controlled glucose auto-delivery for high cell density cultivations in microplates and shake flasks. Microb. Cell Fact 7, 31.
doi:10.1016/j.jbiotec.2010.08.196
[I.19] Quorum sensing in filamentous fungi S. Raina, D. De Vizio, T. Keshavarz ∗ University of Westminster, United Kingdom Keywords: Filamentous fungi; Quorum sensing; Enhanced productivity Introduction: Since the discovery of quorum sensing (QS) in Vibrio fischeri and Vibrio harveyi over forty years ago (Nealson et al., 1970), extensive studies has been carried out on G- and Gram+ bacteria as well as yeasts. Different mechanisms have been proposed for this occurrence which is quite common among these species. Upon reaching a specific cell-density threshold the cells release extracellular diffusible molecules (autoinducers) or QS molecules. These molecules affect genes which in turn instigate a range of physiological and morphological responses. This process is known as QS. While homoserine lactones are studied as QS molecules in Gram- bacteria (Miller and Bassler, 2001), the dominant autoinducers in Gram+ bacteria are based on a two component signal transduction mechanism (Kleerebezem et al., 1997) employing small, post-translationally modified or unmodified peptides (Bassler, 2002). In many actinomycetes species, ␥-butyrolactones act as autoregulators and in yeasts Farnesol (Hornby et al., 2001) and Tyrosol (Narayanan and Rao, 1976) are QS molecules. In spite of extensive research on the autoinducers and their role in pathogenesis which could provide valuable information to battle infectious diseases, there is very little information on QS in filamentous fungi. This study reports for the first time the presence of QS process in filamentous fungi. The aim of this work was to investigate effect of potential QS molecules on filamentous fungi and to exploit the process for future industrial exploitation. Methods: Aspergillus sp. (A. nidulans, A. terreus) and Penicillium sp. (P. sclerotiorum strains) were used. HPLC, LC, GC, NMR were adopted to identify potential QS molecules. Results: Butyrolactone I and multicolic acid as QS molecules in A. terreus and P. sclerotiorum respectively resulted in 2.9-fold increase in the production rate of lovastatin in 5 l fermenters. Discussion: This study shows the potential for industrial exploitation of QS for enhanced productivity. Acknowledgemenst: Financial support of this work (Quorum, Proposal no. 032811, 2006-2009: Discovering Quorum Sensing in industrially useful Fungi, a novel approach at molecular level for scaling-up in white biotech., from the European Commission under FP6) is gratefully appreciated. References Hornby, J.M., Jensen, E.C., Lisec, A.D., Tasto, J.J., Jahnke, B., Shoemaker, R., Dussault, P., Nickerson, K.W., 2001. Quorum sensing in the dimorphic fungus Candida albicans is mediated by farnesol. Applied and Environmental Microbiology 67 (7), 2982–2992. Kleerebezem, M., Quadri, L.E., Kuipers, O.P., de Vos, W.M., 1997. Quorum sensing by peptide pheromones and two-component signal-transduction systems in Grampositive bacteria. Molecular Microbiology 24 (5), 895–904. Miller, M.B., Bassler, B.L., 2001. Quorum sensing in bacteria. Annual Review of Microbiology 55, 165–199. Narayanan, T.k., Rao, G.R., 1976. Production of beta-(4-hydroxyphenyl)ethanol and beta-(4-hydroxyphenyl)lactic acid by Candida species. Canadian journal of microbiology 22 (3), 349–384. Nealson, K.H., Platt, T., Hastings, J.W., 1970. Cellular Control of the Synthesis and Activity of the Bacterial Luminescent System. Journal of Bacteriology 104 (1), 313–322.
doi:10.1016/j.jbiotec.2010.08.197