Production of Biofuel Precursors University of Wisconsin at Madison - PowerPoint PPT Presentation

Production of Biofuel Precursors University of Wisconsin at Madison

• Energy supply is a BIG problem : Fossil fuels will not last forever • Straining national economy • (http://express.howstuffworks.com) ( http://www.well-surveillance.com ) •One Answer : • Biofuels through bacterial pathways Sean McMaster Biochemistry & Math

• Lignin • Sorbitol – Greater access to cellulose – Crude biofuel from sugar and hemi-cellulose and solid state catalysis Sean McMaster Biochemistry & Math

Cellulose Lignin ( http://www.ceres.net ) Biomass Hemicellulose Sean McMaster Biochemistry & Math

Lignin peroxidase production • through E. coli Increase cellulose availability • Enhanced production of biofuels • Lignin Phanerochaete chrysosporium (http://www.ceres.net) (www.aber.ac.uk ) Lignin :: INTRO : Export : Assay : Optimization Yash Jhala Genetics

Expression Export White Rot Cloned into lipD Gene Fungus E. coli Develop an Assay Optimization (http://commtechlab.msu.edu) (www.stern.de) (lpmpjogja.diknas.go.id ) Lignin Peroxidase Lignin :: INTRO : Export : Assay : Optimization Yash Jhala Genetics

T7 terminator lipD X h o I Kanamycin 0 Resistance 0 0 6 n i g i O r 1 f T7-tag His-tag l i p D O e R c F n a t s i s e r 1 n 0 c i 50kDa y 0 0 m a n a K E c o R rbs 0 I 0 0 5 Lac operator pET28a-lipD Sequence pET28a-lipD Sequence 6460 bp 6460 bp T7 promoter 40kDa 0 0 0 2 c I 4 a 0 l 0 Induced protein 0 0 0 3 0 • Transformation into Rosetta-gami 2 (DE3) pLysS Lignin :: INTRO : Export : Assay : Optimization Andy Braasch Molecular Biology

Lignin :: Intro : EXPORT : Assay : Optimization Ben Cox Engineering & Math

Synthesized export sequences: ycdO ycbK • Fuse with lipD ycdB torA (DeLisa 2007) • Transform into E. coli A C B 1 2 3 4 5 D 1) 100bp Ladder 2) ycdO (Sec tag) 3) ycbK (TAT tag) 4) ycdB (TAT/Sec tag) 5) torA (TAT) Lignin :: Intro : EXPORT : Assay : Optimization Ben Cox Engineering & Math

• Azure B vs. Veratryl Alcohol Lignin Peroxidase has higher specificity to Azure B Measured absorbance change • Oxidation of Azure B N H 3 C CH 3 N S N + H CH 3 Lignin peroxidase N CH 3 H 2 N S N + CH 3 Lignin :: Intro : Export : ASSAY : Optimization Jack Ho BME

Heme group required for redox activity • Aminolevulinic acid (ALA) synthesis is the rate limiting step • Add ALA to bypass the rate limiting step • Add Fe 2+ to accommodate more heme • ALA + Fe 2+ = OH H 2 N O O Lignin :: Intro : Export : Assay : OPTIMIZATION Peter VanderVelden Biochemistry

(www.stern.de) (lpmpjogja.diknas.go.id ) (http://commtechlab.msu.edu) Organic Waste (http://www.ceres.net) (http://askbobrankin.com) (www.vic.gov.au) • Lignin peroxidase • Bacterial expression and export • Increased biofuel production efficiency Lignin :: Intro : Export : Assay : OPTIMIZATION Andy Braasch Molecular Biology

• Sorbitol C 6 H 14 O 6 • Aqueous phase reforming sorbitol hexane OH OH catalyst HO H 3 C OH C H 3 OH OH • Bacterial sorbitol production Sorbitol :: INTRO : Modeling : Cloning : Assay Joseph Yuen BME

• The natural metabolic pathway • Knocking out phosphofructokinase • Upregulating sorbitol dehydrogenase production Nissen 2005 Sorbitol :: INTRO : Modeling : Cloning : Assay Jia Luo Biochemistry

� pfk knockout RL257 and parent strain MQ obtained from E. coli Genomic Stock Center � Growth curves in C & min media performed Growth curve for RL257 and MQ 1:10 subculture MQ minMedia 1.20 absorbance at 600nm RL257 1.00 minMedia 0.80 0.60 MQ cMedia 0.40 0.20 RL257 0.00 cMedia 0 2 4 6 8 10 12 time (hours) Sorbitol :: INTRO : Modeling : Cloning : Assay Jia Luo Biochemistry

Growth curve for RL257 and MQ 1:10 � pfk knockout RL257 and parent strain MQ subculture MQ minMedia 1.20 absorbance at 600nm RL257 1.00 obtained from E. coli Genomic Stock minMedia 0.80 MQ 0.60 cMedia 0.40 Center 0.20 RL257 0.00 cMedia 0 2 4 6 8 10 12 � Growth curves in C & min media time (hours) performed Growth of RL257 subculture 1:10 in C media 1.200 log (Absorbance 1.000 (600nm)) 0.800 0.600 y = 0.2199x + 0.0207 0.400 2 = 0.9917 R 0.200 n=3 0.000 0 1 2 3 4 5 6 Time (hours) � tpiA knockout obtained from the Keio collection � Growth curves in C media made Sorbitol :: INTRO : Modeling : Cloning : Assay Jia Luo Biochemistry

• Computer modeling • Flux distribution • Gene knockouts • Triosephosphate isomerase O O P P - O - O O O triosephosphate - O - O isomerase O OH dihydroxyacetone glyceraldehyde-3-phosphate OH H O phosphate Sorbitol :: Intro : MODELING : Cloning : Assay Mattias Gyllborg Med Micro & Immu

Sorbitol :: Intro : MODELING : Cloning : Assay Mattias Gyllborg Med Micro & Immu

• Upregulating Sorbitol Dehydrogenase production • Knocking Triosephosphate Isomerase • Determine which cells have the highest sorbitol yield Nissen 2005 Sorbitol :: INTRO : Modeling : Cloning : Assay Mattias Gyllborg Med Micro & Immu

� srlD gene Verification of srlD inserted into pBAD30 via digestion Sorbitol :: Intro : Modeling : CLONING : Assay Charlie Burns Biochemistry

• Enzymatic assay HO SDH OH OH OH O + β -NADH + β -NAD HO OH HO H OH OH OH OH • Sorbitol Dehydrogenase Purification Cell Cultures His-Tag Nickel Column Purification (pBAD and pET Vectors) Cell Lysate • Sorbitol Quantification Enzyme assay allows for sorbitol quantification from cell lysate Sorbitol :: Intro : Modeling : Cloning : ASSAY Tanner Peelen Biochemistry

Sorbitol Production • Modeling and cell growth • Upregulation • Combining in an E.coli strain Tanner Peelen Biochemistry

Outlining alternative pathways • Lignin breakdown • Sorbitol production • Mass production of small hydrocarbons Tanner Peelen Biochemistry

Dr. Doug Weibel Dr. Mike Sussman Dr. Ken Hammel Basudeb Bhattacharyya Dr. Jennie Reed Dr. Dan Cullen Dr. Jaehyuk Yu Matt Copeland Dr. Brian Pfleger Dr. Amber Vanden Dr. Tom Jeffries Jenna Eun Dr. Aseem Ansari Wymelenberg Dr. Kirk Kersten Hannah Tuson Dr. Franco Cerrina Dr. John Ralph Dr. Kirk Kent Shane Flickinger

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