- Email: [email protected]
Metabolic and process engineering for enhanced production of biofuels and biochemicals
Abstracts / Journal of Biotechnology 136S (2008) S276–S289 Wang, L., Ridgway, D., Gu, T., Moo-Young, M., 2005. Bioprocessing strategies to improve heterologous protein production in filamentous fungi. Biotech. Adv. 23, 115–129. Xu, Y., Hsieh, M.Y., Narayanan, N., Anderson, W.A., Scharer, J.M., Moo-Young, M., Chou, C.P., 2005. Cytoplasmic overexpression, folding and processing of penicillin acylase precursor in E. coli. Biotechnol. Prog. 21, 1357–1365.
doi:10.1016/j.jbiotec.2008.07.591
S277
Additionally, within this presentation the design and the principles of an in situ microscope will be discussed. Here, the setup and the different functions will be described in detail and several applications will be presented in the monitoring of yeast, BHK, CHO, microcarrier cultivations and crystallisation processes. The needs of an intelligent image analysis will be discussed for different application examples.
KN-006 References Production of fine chemicals by yeast and filamentous fungi Jens Nielsen Systems Biology, Department of Chemical and Biological Engineering, Chalmers University of Technology, SE-412 96 Gothenburg, Sweden Cell factories are used extensively to produce many specific molecules used as pharmaceuticals, fine chemicals, fuels, materials and food ingredients. Through the use of directed genetic modifications of cell factories – an approach referred to as metabolic engineering – it is possible to develop novel bioprocesses that are more efficient, that are more environmentally friendly and that may produce novel compounds. Biotech processes are therefore increasingly replacing classical chemical synthesis. In this development, it is particularly interesting to develop platform cell factories that can be used for production of many different compounds. This approach has been used with great success in the field of industrial enzyme production, where, e.g. Aspergillus oryzae is used for the production of a large number of enzymes. Yeast and filamentous fungi represents very attractive cell factories for production of chemicals, as these organisms have extensive metabolic capabilities and are already implemented for industrial production of many different compounds. In this presentation technologies for development of the yeast Saccharomyces cerevisiae and the filamentous fungus Aspergillus niger as platform cell factories will be presented and it will be demonstrated how these organisms can be used for the production of a range of different chemical compounds. doi:10.1016/j.jbiotec.2008.07.592 KN-008 Non-invasive analysis systems for continuous bioprocess monitoring T. Scheper Institut für Technische Chemie, Leibniz University of Hannover, Callinstr 3, 30167 Hannover, Germany Optical sensors offer the possibility to monitor non-invasively bioprocesses on line and without any time delay. Several optical sensor systems are described in the actual sensor literature, however, only few of them are commercially available. The class of optical sensors can be subdivided in the classes of optical chemosensors (such as dissolved oxygen or pH probes), spectroscopic sensors (such as NIR, FTIR or fluorescence probes) and image analysis systems (such as in situ microscopy). The principles of the so called 2-D-spectrofluorometry will be presented for non-invasive monitoring of bioprocesses, its modelling and for down-stream process analysis. This techniques opens the possibility to monitor all fluorescent compounds within 30 s in emission and excitation scans (from 380 to 600 nm each). Thus, concentration profiles of intra- and extracellular components can be analysed and be appropriate models, and prediction of relevant bioprocess variables such as biomass and educt/product concentrations is possible.
Anton, F., Burzlaff, A., Kasper, C., Brückerhoff, T., Scheper, T., 2007. Preliminary study towards the use of in-situ microscopy for the online analysis of microcarrier cultivation. Eng. Life Sci. 7 (1), 91–96. Fritzsche, M., Barreiro, C.G., Hitzmann, B., Scheper, T., 2007. Optical pH sensing using spectral analysis. Sens. Actuators B 128, 133–137. Rudolph, G., Lindner, P., Gierse, A., Bluma, A., Martinez, G., Hitzmann, B., Scheper, T., 2008. Online monitoring of microcarrier based fibrolast cultivations with in situ microscopy. Biotechnol. Bioeng. 99 (1), 136–145. Surribas, A., Geissler, D., Gierse, A., Scheper, T., Hitzmann, B., Motesinos, J.L., Valero, F., 2006. State variables monitoring by in situ-wavelength fluorescence spectroscopy in heterologous protein production by Pichia pastoris. J. Biotechnol. 124, 412–419.
doi:10.1016/j.jbiotec.2008.07.593 KN-010 Metabolic and process engineering for enhanced production of biofuels and biochemicals Shang-Tian Yang Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH 43210, USA E-mail address: [email protected]. This paper will report recent progresses in metabolic and process engineering for economical production of biofuels and biochemicals from renewable biomass. Efficient microbial conversion of sugars and lignocellulosic biomass to fuels and chemicals is the core operation in biorefinery (Yang, 2006). The product yield depends on metabolic efficiency in cells as well as process conditions. Currently, many carboxylic acids, including propionic, butyric, and acetic acids, are produced mainly by petroleum-based chemical synthesis. However, the high oil price has prompted new interests in producing these carboxylic acids from biomass via fermentation. Conventional fermentation processes for propionic acid and butyric acid production are limited by low productivity and yield due to the formation of acetate as a byproduct and feedback inhibition by the main fermentation product. In order to increase the production of propionic and butyric acids, integrational mutagenesis was used to disrupt genes associated with the acetate formation pathway in P. acidipropionici and C. tyrobutyricum, respectively (Suwannakham et al., 2006; Zhu et al., 2005). With the metabolically engineered mutants, more pyruvate, the common precursor of propionic (butyric) and acetic acid in P. acidipropionici (C. tyrobutyricum), can be distributed to the production of propionic (butyric) acid, resulting in higher product yields. The potential of using these mutants to improve propionic (butyric) acid production from waste sugars and glycerol was studied in both free and immobilized cell fermentations. The mutant grew slower in batch fermentation, but produced more acids and less acetate as compared to the wild type strain. A fibrous bed bioreactor (FBB) was used to immobilize cells and further improve acid production to reach a final concentration of ∼100 g/L. The mutants adapted in the FBB showed a much higher acid tolerance. The carboxylic acids can be further converted to corresponding alcohols such as n-butanol, which is a better alternative biofuel than ethanol. The feasibility of a two-step fermentation
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Abstracts / Journal of Biotechnology 136S (2008) S276–S289
has been demonstrated with C. acetobutylicum immobilized in a fibrous bed bioreactor co-fed with glucose and butyrate. The bacterium produced butanol at an overall yield of 0.3–0.4 g/g glucose and productivity of ∼5.6 g/L h (Huang et al., 2004). The result was a doubling of butanol yield to ∼2.5 gal/bushel of corn, allowing biobutanol to compete more favorably in the fuel market. References
KN-020 Screening of novel microbial enzymes and their industrial applications Sakayu Shimizu Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan E-mail address: [email protected].
Huang, W.-C., Ramey, D.E., Yang, S.T., 2004. Continuous production of butanol by Clostridium acetobutylicum immobilized in a fibrous bed bioreactor. Appl. Biochem. Biotechnol. 113–116, 887–898. Suwannakham, S., Huang, Y., Yang, S.T., 2006. Construction and characterization of ack knock-out mutants of Propionibacterium acidipropionici for enhanced propionic acid fermentation. Biotechnol. Bioeng. 94, 383–395. Yang, S.T., 2006. Bioprocessing for Value-Added Products from Renewable Resources: New Technologies and Applications. Elsevier. Zhu, Y., Liu, X., Yang, S.T., 2005. Construction and characterization of pta gene deleted mutant of Clostridium tyrobutyricum for butyric acid fermentation. Biotechnol. Bioeng. 90, 154–166.
doi:10.1016/j.jbiotec.2008.07.594 KN-011 Industrial biotechnology—Towards next generation biorefineries Marcel Wubbolts DSM White Biotechnology, DSM Innovation Center, Delft, The Netherlands E-mail address: [email protected]. The selectivity and catalytic proficiency of enzymes, the catalysts from Nature, form the basis of all white or industrial biotechnology processes. This holds true for fermentations, where a complex network of reactions and control elements in involved, as well as for simple biocatalytic processes that utilize a limited number of enzymes. DSM has a wealth of large scale experience with enzymes in a variety of application area’s ranging from manufacturing enantiomerically pure advanced pharmaceutical ingredients and semi-synthetic antibiotics in the pharma sector to protein modifications and bio-active peptide production in food and feed applications. In addition, break through fermentation processes requiring the coordinated interplay of many enzymes have been developed for the production of food and feed enzymes, vitamins and antibiotics. In order to fully exploit the potential of DSM’s capabilities in areas such as white biotechnology, innovation programs to explore emerging business areas new to DSM have been started. One of these programs, the emerging business area white biotechnology, focuses on the use of our technological competences such as fermentation, metabolic engineering, enzyme production and application development in area’s outside those of the current business activities. The presentation will focus on innovation using Nature’s toolbox at DSM and examples of white biotechnology applications for second generation, lignocellulose based biorefineries will be presented. doi:10.1016/j.jbiotec.2008.07.595
Over the past decade, the industrial use of microbial enzymes or enzyme systems, and so on, has developed rapidly and is gathering increasing attention, particularly their use in solving environmental problems. Here, several unique microbial enzymes or reactions recently discovered in our laboratory and now used industrially are introduced, through which I will emphasize importance of screening for potential microorganisms and mutual collaboration between academia and industries. Single cell oil production by Mortierella alpina: a filamentous fungus, M. alpina 1S-4, was obtained, through extensive screening, as an potential producer of triacylglycerol containing C20 polyunsaturated fatty acids (PUFAs) such as arachidonic acid. With this discovery as a starting point, we conducted employing methods from metabolic engineering and molecular biology for controlling cultures and breeding mutant strains. These parental and mutant strains are now used for large-scale production of a variety of PUFAs. Lactonase process for the optical resolution of racemic pantolactone: the process involves a stereospecific hydrolysis step with a novel enzyme “lactonohydrolase” as the catalyst for the resolution of dl-pantolactone (DL-PL). The enzyme from F. oxysporum, specifically hydrolyzes D-PL to d-pantoic acid (d-PA); thus DL-PL can be separated into d-PA and l-PL. The mycelia containing the enzyme were immobilized into calcium alginate gels for the practical purpose. The commercial production of d-PL has started since 1999, through which it was been shown that the present process is highly satisfactory not only economically but also environmentally (water, 49%, CO2 , 30% and BOD, 62%; comparing the former chemical resolution method). Bioreduction system for large-scale production of chiral alcohols: a novel whole cell bioreduction system, in which transformant cells co-expressing an NAD(P)H-dependent carbonyl reductase gene and that of cofactor regeneration enzyme are used as a catalyst, for asymmetric reduction of prochiral carbonyl compounds to chiral alcohols. Production of (S)-4-chloro-3-hydroxybutanoate esters by using a carbonyl reductase of Candida magnoliae is a typical successful example of this bioreduction system. Expression of other genes encoding reductase enzymes together with cofactor regenerator enzyme gene in appropriate host cells has been shown to be useful for large-scale production of various kinds of chiral alcohols. doi:10.1016/j.jbiotec.2008.07.596
doi:10.1016/j.jbiotec.2008.07.591
S277
Additionally, within this presentation the design and the principles of an in situ microscope will be discussed. Here, the setup and the different functions will be described in detail and several applications will be presented in the monitoring of yeast, BHK, CHO, microcarrier cultivations and crystallisation processes. The needs of an intelligent image analysis will be discussed for different application examples.
KN-006 References Production of fine chemicals by yeast and filamentous fungi Jens Nielsen Systems Biology, Department of Chemical and Biological Engineering, Chalmers University of Technology, SE-412 96 Gothenburg, Sweden Cell factories are used extensively to produce many specific molecules used as pharmaceuticals, fine chemicals, fuels, materials and food ingredients. Through the use of directed genetic modifications of cell factories – an approach referred to as metabolic engineering – it is possible to develop novel bioprocesses that are more efficient, that are more environmentally friendly and that may produce novel compounds. Biotech processes are therefore increasingly replacing classical chemical synthesis. In this development, it is particularly interesting to develop platform cell factories that can be used for production of many different compounds. This approach has been used with great success in the field of industrial enzyme production, where, e.g. Aspergillus oryzae is used for the production of a large number of enzymes. Yeast and filamentous fungi represents very attractive cell factories for production of chemicals, as these organisms have extensive metabolic capabilities and are already implemented for industrial production of many different compounds. In this presentation technologies for development of the yeast Saccharomyces cerevisiae and the filamentous fungus Aspergillus niger as platform cell factories will be presented and it will be demonstrated how these organisms can be used for the production of a range of different chemical compounds. doi:10.1016/j.jbiotec.2008.07.592 KN-008 Non-invasive analysis systems for continuous bioprocess monitoring T. Scheper Institut für Technische Chemie, Leibniz University of Hannover, Callinstr 3, 30167 Hannover, Germany Optical sensors offer the possibility to monitor non-invasively bioprocesses on line and without any time delay. Several optical sensor systems are described in the actual sensor literature, however, only few of them are commercially available. The class of optical sensors can be subdivided in the classes of optical chemosensors (such as dissolved oxygen or pH probes), spectroscopic sensors (such as NIR, FTIR or fluorescence probes) and image analysis systems (such as in situ microscopy). The principles of the so called 2-D-spectrofluorometry will be presented for non-invasive monitoring of bioprocesses, its modelling and for down-stream process analysis. This techniques opens the possibility to monitor all fluorescent compounds within 30 s in emission and excitation scans (from 380 to 600 nm each). Thus, concentration profiles of intra- and extracellular components can be analysed and be appropriate models, and prediction of relevant bioprocess variables such as biomass and educt/product concentrations is possible.
Anton, F., Burzlaff, A., Kasper, C., Brückerhoff, T., Scheper, T., 2007. Preliminary study towards the use of in-situ microscopy for the online analysis of microcarrier cultivation. Eng. Life Sci. 7 (1), 91–96. Fritzsche, M., Barreiro, C.G., Hitzmann, B., Scheper, T., 2007. Optical pH sensing using spectral analysis. Sens. Actuators B 128, 133–137. Rudolph, G., Lindner, P., Gierse, A., Bluma, A., Martinez, G., Hitzmann, B., Scheper, T., 2008. Online monitoring of microcarrier based fibrolast cultivations with in situ microscopy. Biotechnol. Bioeng. 99 (1), 136–145. Surribas, A., Geissler, D., Gierse, A., Scheper, T., Hitzmann, B., Motesinos, J.L., Valero, F., 2006. State variables monitoring by in situ-wavelength fluorescence spectroscopy in heterologous protein production by Pichia pastoris. J. Biotechnol. 124, 412–419.
doi:10.1016/j.jbiotec.2008.07.593 KN-010 Metabolic and process engineering for enhanced production of biofuels and biochemicals Shang-Tian Yang Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH 43210, USA E-mail address: [email protected]. This paper will report recent progresses in metabolic and process engineering for economical production of biofuels and biochemicals from renewable biomass. Efficient microbial conversion of sugars and lignocellulosic biomass to fuels and chemicals is the core operation in biorefinery (Yang, 2006). The product yield depends on metabolic efficiency in cells as well as process conditions. Currently, many carboxylic acids, including propionic, butyric, and acetic acids, are produced mainly by petroleum-based chemical synthesis. However, the high oil price has prompted new interests in producing these carboxylic acids from biomass via fermentation. Conventional fermentation processes for propionic acid and butyric acid production are limited by low productivity and yield due to the formation of acetate as a byproduct and feedback inhibition by the main fermentation product. In order to increase the production of propionic and butyric acids, integrational mutagenesis was used to disrupt genes associated with the acetate formation pathway in P. acidipropionici and C. tyrobutyricum, respectively (Suwannakham et al., 2006; Zhu et al., 2005). With the metabolically engineered mutants, more pyruvate, the common precursor of propionic (butyric) and acetic acid in P. acidipropionici (C. tyrobutyricum), can be distributed to the production of propionic (butyric) acid, resulting in higher product yields. The potential of using these mutants to improve propionic (butyric) acid production from waste sugars and glycerol was studied in both free and immobilized cell fermentations. The mutant grew slower in batch fermentation, but produced more acids and less acetate as compared to the wild type strain. A fibrous bed bioreactor (FBB) was used to immobilize cells and further improve acid production to reach a final concentration of ∼100 g/L. The mutants adapted in the FBB showed a much higher acid tolerance. The carboxylic acids can be further converted to corresponding alcohols such as n-butanol, which is a better alternative biofuel than ethanol. The feasibility of a two-step fermentation
S278
Abstracts / Journal of Biotechnology 136S (2008) S276–S289
has been demonstrated with C. acetobutylicum immobilized in a fibrous bed bioreactor co-fed with glucose and butyrate. The bacterium produced butanol at an overall yield of 0.3–0.4 g/g glucose and productivity of ∼5.6 g/L h (Huang et al., 2004). The result was a doubling of butanol yield to ∼2.5 gal/bushel of corn, allowing biobutanol to compete more favorably in the fuel market. References
KN-020 Screening of novel microbial enzymes and their industrial applications Sakayu Shimizu Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan E-mail address: [email protected].
Huang, W.-C., Ramey, D.E., Yang, S.T., 2004. Continuous production of butanol by Clostridium acetobutylicum immobilized in a fibrous bed bioreactor. Appl. Biochem. Biotechnol. 113–116, 887–898. Suwannakham, S., Huang, Y., Yang, S.T., 2006. Construction and characterization of ack knock-out mutants of Propionibacterium acidipropionici for enhanced propionic acid fermentation. Biotechnol. Bioeng. 94, 383–395. Yang, S.T., 2006. Bioprocessing for Value-Added Products from Renewable Resources: New Technologies and Applications. Elsevier. Zhu, Y., Liu, X., Yang, S.T., 2005. Construction and characterization of pta gene deleted mutant of Clostridium tyrobutyricum for butyric acid fermentation. Biotechnol. Bioeng. 90, 154–166.
doi:10.1016/j.jbiotec.2008.07.594 KN-011 Industrial biotechnology—Towards next generation biorefineries Marcel Wubbolts DSM White Biotechnology, DSM Innovation Center, Delft, The Netherlands E-mail address: [email protected]. The selectivity and catalytic proficiency of enzymes, the catalysts from Nature, form the basis of all white or industrial biotechnology processes. This holds true for fermentations, where a complex network of reactions and control elements in involved, as well as for simple biocatalytic processes that utilize a limited number of enzymes. DSM has a wealth of large scale experience with enzymes in a variety of application area’s ranging from manufacturing enantiomerically pure advanced pharmaceutical ingredients and semi-synthetic antibiotics in the pharma sector to protein modifications and bio-active peptide production in food and feed applications. In addition, break through fermentation processes requiring the coordinated interplay of many enzymes have been developed for the production of food and feed enzymes, vitamins and antibiotics. In order to fully exploit the potential of DSM’s capabilities in areas such as white biotechnology, innovation programs to explore emerging business areas new to DSM have been started. One of these programs, the emerging business area white biotechnology, focuses on the use of our technological competences such as fermentation, metabolic engineering, enzyme production and application development in area’s outside those of the current business activities. The presentation will focus on innovation using Nature’s toolbox at DSM and examples of white biotechnology applications for second generation, lignocellulose based biorefineries will be presented. doi:10.1016/j.jbiotec.2008.07.595
Over the past decade, the industrial use of microbial enzymes or enzyme systems, and so on, has developed rapidly and is gathering increasing attention, particularly their use in solving environmental problems. Here, several unique microbial enzymes or reactions recently discovered in our laboratory and now used industrially are introduced, through which I will emphasize importance of screening for potential microorganisms and mutual collaboration between academia and industries. Single cell oil production by Mortierella alpina: a filamentous fungus, M. alpina 1S-4, was obtained, through extensive screening, as an potential producer of triacylglycerol containing C20 polyunsaturated fatty acids (PUFAs) such as arachidonic acid. With this discovery as a starting point, we conducted employing methods from metabolic engineering and molecular biology for controlling cultures and breeding mutant strains. These parental and mutant strains are now used for large-scale production of a variety of PUFAs. Lactonase process for the optical resolution of racemic pantolactone: the process involves a stereospecific hydrolysis step with a novel enzyme “lactonohydrolase” as the catalyst for the resolution of dl-pantolactone (DL-PL). The enzyme from F. oxysporum, specifically hydrolyzes D-PL to d-pantoic acid (d-PA); thus DL-PL can be separated into d-PA and l-PL. The mycelia containing the enzyme were immobilized into calcium alginate gels for the practical purpose. The commercial production of d-PL has started since 1999, through which it was been shown that the present process is highly satisfactory not only economically but also environmentally (water, 49%, CO2 , 30% and BOD, 62%; comparing the former chemical resolution method). Bioreduction system for large-scale production of chiral alcohols: a novel whole cell bioreduction system, in which transformant cells co-expressing an NAD(P)H-dependent carbonyl reductase gene and that of cofactor regeneration enzyme are used as a catalyst, for asymmetric reduction of prochiral carbonyl compounds to chiral alcohols. Production of (S)-4-chloro-3-hydroxybutanoate esters by using a carbonyl reductase of Candida magnoliae is a typical successful example of this bioreduction system. Expression of other genes encoding reductase enzymes together with cofactor regenerator enzyme gene in appropriate host cells has been shown to be useful for large-scale production of various kinds of chiral alcohols. doi:10.1016/j.jbiotec.2008.07.596