Metabolic engineering of clostridium cellulolyticum for advanced biofuel production

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Engineering Conferences International ECI Digital Archives BioEnergy IV: Innovations in Biomass Conversion for Heat, Power, Fuels and Chemicals Proceedings Spring 6-13-2013 Metabolic engineering of

1 Engineering Conferences International ECI Digital Archives BioEnergy IV: Innovations in Biomass Conversion for Heat, Power, Fuels and Chemicals Proceedings Spring Metabolic engineering of clostridium cellulolyticum for advanced biofuel production Andrea Liedtke Fraunhofer IME Stefan Jennewein IME Follow this and additional works at: Part of the Chemical Engineering Commons Recommended Citation Andrea Liedtke and Stefan Jennewein, "Metabolic engineering of clostridium cellulolyticum for advanced biofuel production" in "BioEnergy IV: Innovations in Biomass Conversion for Heat, Power, Fuels and Chemicals", Manuel Garcia-Perez,Washington State University, USA Dietrich Meier, Thünen Institute of Wood Research, Germany Raffaella Ocone, Heriot-Watt University, United Kingdom Paul de Wild, Biomass & Energy Efficiency, ECN, The Netherlands Eds, ECI Symposium Series, (2013). This Conference Proceeding is brought to you for free and open access by the Proceedings at ECI Digital Archives. It has been accepted for inclusion in BioEnergy IV: Innovations in Biomass Conversion for Heat, Power, Fuels and Chemicals by an authorized administrator of ECI Digital Archives. For more information, please contact

2 Federal Ministry of Education and Research Fraunhofer Institute for Molecular Biology and Applied Ecology BioEnergie 2021 Metabolic Engineering of Clostridium cellulolyticum for advanced biofuel production Andrea Liedtke & Dr. Stefan Jennewein Bioenergy IV, Otranto

3 Fraunhofer Institute for Molecular Biology and Applied Ecology Industrial Biotechnology Research areas Metabolic Engineering Biosynthetic Pathway Elucidation Anaerobic Biotechnology 2

4 Biobutanol a biofuel Set of problems Demand for diverse and sustainable supplies of transport fuels is increasing (Dürre, 2011) CO/CO2 emissions have to be decreased (Dürre, 2011) Ignition of interest in production of liquid biofuels, especially from non-foodstock competing biomass (Dürre, 2011) Biobutanol - a solution Superior to ethanol as a fuel additive (Dürre, 2007) billion pound produced petrochemically per year (Lee et al. 2008) Butanol from cellulosic biomass poses a solution (Jang et al. 2012) Dürre P, 2011.Fermentative production of butanol-the academic perspective. Current Opinion in Biotechnology. 22: Dürre P, Biobutanol: an attractive biofuel. Biotechnology Journal. 2: Jang SY, Joungmin L, Malaviya A, Seung DY, Cho JH and Lee SY, Butanol production from renewable biomass: Rediscovery of metabolic pathways and metabolic engineering. Biotechnol. J. 7, Lee SY, Park JH, Jang SH, Nielsen LK, Kim J and Jung KS, Fermentative butanol production by Clostridia. Biotechnology and Bioengineering. 101,2:

5 Cellulosic waste biomass to biofuel Advantages Cellulosic biomass: low cost, largely available, near-zero greenhouse gas emissions (Carere et al. 1998) Cellulolysis- Biological Cellulose to Ethanol approach Microbial fermentation process Pre-requirement: physical or chemical pretreatment of lignocellulose Followed by chemical/enzymatic hydrolysis of cellulose (e.g.: alkaline peroxide, cellulase enzymes-however quite cost intesive) (Zhu et al. 2009) Hydrolysis without external enzymes possible by some microorganisms possessing cellulosome Carere CR, Sparling R, Cicek N and Levin DB, Third generation biofuels via direct cellulose fermentation. Int.J. Mol. Sci 9, Zhu JY, Pan XY, Wang GS, Gleisner R, Sulfite pretreatment (SPORL) for robust enzymatic saccharification of spruce and red pine. Bioresour technol. 100 (8):

6 Consolidated bioprocessing using Clostridium cellulolyticum Biomass Pretreatment Product Clostridium cellulolyticum Fermentation Butanol Source: US Department of Energy 5

Clostridia Gram +, anaerobic bacteria Metabolically diverse Carbon sources: sugars, organic acids, alcohols, syngas Natural products: ethanol, acetate,

7 Clostridia Gram +, anaerobic bacteria Metabolically diverse Carbon sources: sugars, organic acids, alcohols, syngas Natural products: ethanol, acetate, acetone, butyrate, 1,3-propanediol, hexanol, butanol, Natural butanol producer C. acetobutylicum Dürre 2005, Handbook on Clostridia CRC Press Taylor and Francis Group 6

Clostridium cellulolyticum - a cellulosome holding microbe strictly anaerobic Extracellular multienzymatic complex cellulosome (Desvaux, 2005) Introduction of biosynthetic pathway into C.

8 Clostridium cellulolyticum - a cellulosome holding microbe strictly anaerobic Extracellular multienzymatic complex cellulosome (Desvaux, 2005) Introduction of biosynthetic pathway into C. cellulolyticum Advantages for use in biofuel production Cellulases of cellulosome no additional enzymes required Anaerobic fermentation possible in Clostridia - more energy efficient With genetically engineered C. cellulolyticum it is possible to integrate cellulosic hydrolysis as well as solvent production in one consolidated process 7 Desvaux M, Clostridium cellulolyticum: model organism of mesophilic cellulolytic clostridia. FEMS Microbiology reviews 29:

Introduction and expression of C. acetobutylicum butanol production pathway in C. cellulolyticum Cellulose C. cellulolyticum s cellulosome Glucose Glycolysis Native butanol synthesis pathway in C.

9 Introduction and expression of C. acetobutylicum butanol production pathway in C. cellulolyticum Cellulose C. cellulolyticum s cellulosome Glucose Glycolysis Native butanol synthesis pathway in C. acetobutylicum Acetyl-CoA Heterologous butanol synthesis in C. cellulolyticum Thl Thl (atob) Acetoacetyl-CoA hbd hbd 3-Hydroxybutyryl-CoA crt Crotonyl-CoA Bcd-etfAB crt bcd TerTd Butyryl-CoA Butyraldehyde Butanol adhe adhe adhe adhe 8

10 Introduction and expression of C. acetobutylicum butanol production pathway in C. cellulolyticum 9

11 Introduction and expression of C. acetobutylicum butanol production pathway in C. cellulolyticum Heterologous gene cluster psos shuttle vector Vector transformation via electroporation 10

12 Initial peptide analysis in E.coli using a Targeted Proteomics approach 11

13 Initial peptide analysis in E.coli using a Targeted Proteomics approach 12

14 CoA-Ester analysis in wild type and heterologous C. cellulolyticum 13

15 Outlook - Take home message Succesfull expression of butanol syntheis genes in E.coli Successfull introduction of butanol synthesis vectors in C. cellulolyticum Butyryl-CoA as an intermediate could be verified in Clostridium cellulolyticum Optimisation of the synthesis pathway towards butanol Genomic integration of the synthesis pathway into C. cellulolyticum avoidance of issues regarding plasmid instabillity 14

16 Acknowledgements Prof. Dr. Fischer Dr. Stefan Jennewein Bernhard Güntner Sebastian de Vries Nicole Schnass Daniel Volke Benedikt Engels Dr. Andrea Veit Dr. Gabriele Philipps Christian Janke Jens Lennartz Federal Ministry of Education and Research Thank you very much for your attention! Questions? 15

External superheating in biomass power plants with pebble bed regenerators

Engineering Conferences International ECI Digital Archives BioEnergy IV: Innovations in Biomass Conversion for Heat, Power, Fuels and Chemicals Proceedings Spring 6-11-2013 External superheating in biomass