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

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

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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) (

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SLIDE 1

Production of Biofuel Precursors

University of Wisconsin at Madison

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SLIDE 2
  • One Answer:
  • Biofuels through bacterial pathways
  • Energy supply is a BIG problem:
  • Fossil fuels will not last forever
  • Straining national economy

Sean McMaster

Biochemistry & Math

(http://express.howstuffworks.com) (http://www.well-surveillance.com)

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SLIDE 3

Sean McMaster

Biochemistry & Math

  • Lignin

– Greater access to cellulose and hemi-cellulose

  • Sorbitol

– Crude biofuel from sugar and solid state catalysis

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SLIDE 4

Sean McMaster

Biochemistry & Math

Biomass Cellulose Hemicellulose Lignin

(http://www.ceres.net)

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SLIDE 5
  • Lignin peroxidase production

through E. coli

  • Increase cellulose availability
  • Enhanced production of biofuels

Lignin:: INTRO : Export : Assay : Optimization Yash Jhala

Genetics

Phanerochaete chrysosporium (www.aber.ac.uk )

Lignin

(http://www.ceres.net)

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SLIDE 6

(www.stern.de)

Lignin:: INTRO : Export : Assay : Optimization Yash Jhala

Genetics

(http://commtechlab.msu.edu) (lpmpjogja.diknas.go.id )

Lignin Peroxidase

lipD Gene White Rot Fungus Expression Develop an Assay Export Optimization Cloned into

  • E. coli
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SLIDE 7

Lignin:: INTRO : Export : Assay : Optimization Andy Braasch

Molecular Biology

f 1 O r i g i n K a n a m y c i n r e s i s t a n c e l a c I l i p D O R F X h
  • I
E c
  • R
I

1 2 3 4 5 6

pET28a-lipD Sequence pET28a-lipD Sequence 6460 bp 6460 bp

Kanamycin Resistance T7 terminator

lipD

T7-tag His-tag rbs Lac operator T7 promoter

  • Transformation into Rosetta-gami 2 (DE3) pLysS

50kDa

Induced protein

40kDa

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SLIDE 8

Lignin:: Intro : EXPORT : Assay : Optimization Ben Cox

Engineering & Math

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SLIDE 9

Synthesized export sequences: ycbK ycdO ycdB torA

  • Fuse with lipD
  • Transform into E. coli

Lignin:: Intro : EXPORT : Assay : Optimization Ben Cox

Engineering & Math

(DeLisa 2007)

A D C B

1) 100bp Ladder 2) ycdO (Sec tag) 3) ycbK (TAT tag) 4) ycdB (TAT/Sec tag) 5) torA (TAT) 1 2 3 4 5

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SLIDE 10
  • Azure B vs. Veratryl Alcohol

Lignin Peroxidase has higher specificity to Azure B Measured absorbance change

  • Oxidation of Azure B

Lignin:: Intro : Export : ASSAY : Optimization Jack Ho

BME

S N N CH3 CH3 N H H3C + S N N CH3 CH3 H2N +

Lignin peroxidase

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SLIDE 11
  • Heme group required for redox activity
  • Aminolevulinic acid (ALA) synthesis is the rate limiting step
  • Add ALA to bypass the rate limiting step
  • Add Fe2+ to accommodate more heme

Lignin:: Intro : Export : Assay : OPTIMIZATION

Peter VanderVelden

Biochemistry

Fe2+=

ALA +

H2N O O OH

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SLIDE 12
  • Lignin peroxidase
  • Bacterial expression and export
  • Increased biofuel production efficiency

Lignin:: Intro : Export : Assay : OPTIMIZATION Andy Braasch

Molecular Biology

(http://commtechlab.msu.edu) (lpmpjogja.diknas.go.id ) (www.stern.de) (http://askbobrankin.com) (http://www.ceres.net)

Organic Waste

(www.vic.gov.au)

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SLIDE 13
  • Bacterial sorbitol production

Sorbitol:: INTRO : Modeling : Cloning : Assay Joseph Yuen

BME

  • Aqueous phase reforming

HO OH OH OH OH OH H 3C C H 3

  • Sorbitol

C6H14O6

sorbitol hexane

catalyst

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SLIDE 14

Nissen 2005

  • The natural metabolic pathway
  • Upregulating sorbitol dehydrogenase production
  • Knocking out phosphofructokinase

Sorbitol:: INTRO : Modeling : Cloning : Assay Jia Luo

Biochemistry

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SLIDE 15

pfk knockout RL257 and parent strain MQ

  • btained from E. coli Genomic Stock

Center Growth curves in C & min media performed Sorbitol:: INTRO : Modeling : Cloning : Assay Jia Luo

Biochemistry

Growth curve for RL257 and MQ 1:10 subculture

0.00 0.20 0.40 0.60 0.80 1.00 1.20 2 4 6 8 10 12 time (hours) absorbance at 600nm

MQ minMedia RL257 minMedia MQ cMedia RL257 cMedia

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SLIDE 16

pfk knockout RL257 and parent strain MQ

  • btained from E. coli Genomic Stock

Center Growth curves in C & min media performed tpiA knockout obtained from the Keio collection Growth curves in C media made Sorbitol:: INTRO : Modeling : Cloning : Assay Jia Luo

Biochemistry

Growth of RL257 subculture 1:10 in C media

y = 0.2199x + 0.0207 R

2 = 0.9917

n=3

0.000 0.200 0.400 0.600 0.800 1.000 1.200 1 2 3 4 5 6

Time (hours) log (Absorbance (600nm))

Growth curve for RL257 and MQ 1:10 subculture 0.00 0.20 0.40 0.60 0.80 1.00 1.20 2 4 6 8 10 12 time (hours) absorbance at 600nm

MQ minMedia RL257 minMedia MQ cMedia RL257 cMedia

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SLIDE 17
  • Computer modeling
  • Flux distribution
  • Gene knockouts
  • Triosephosphate isomerase

triosephosphate isomerase

Mattias Gyllborg

Med Micro & Immu

Sorbitol:: Intro : MODELING : Cloning : Assay

dihydroxyacetone phosphate glyceraldehyde-3-phosphate

O OH O P O

  • O
  • O

O O P O

  • O
  • O

OH H

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SLIDE 18

Mattias Gyllborg

Med Micro & Immu

Sorbitol:: Intro : MODELING : Cloning : Assay

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SLIDE 19

Nissen 2005

  • Knocking Triosephosphate Isomerase
  • Upregulating Sorbitol Dehydrogenase production
  • Determine which cells have the highest sorbitol

yield Sorbitol:: INTRO : Modeling : Cloning : Assay Mattias Gyllborg

Med Micro & Immu

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SLIDE 20

srlD gene

Verification of srlD inserted into pBAD30 via digestion

Charlie Burns

Biochemistry

Sorbitol:: Intro : Modeling : CLONING : Assay

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SLIDE 21
  • Sorbitol Dehydrogenase Purification

Cell Cultures

(pBAD and pET Vectors)

His-Tag Nickel Column Purification Cell Lysate

  • Sorbitol Quantification

Enzyme assay allows for sorbitol quantification from cell lysate

Tanner Peelen

Biochemistry

Sorbitol:: Intro : Modeling : Cloning : ASSAY

  • Enzymatic assay

+ β-NADH

SDH

+ β-NAD

OH H O OH HO OH HO OH OH OH OH OH HO

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SLIDE 22

Tanner Peelen

Biochemistry

Sorbitol Production

  • Modeling and cell growth
  • Upregulation
  • Combining in an E.coli strain
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SLIDE 23

Tanner Peelen

Biochemistry

Outlining alternative pathways

  • Lignin breakdown
  • Sorbitol production
  • Mass production of small hydrocarbons
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SLIDE 24
  • Dr. Doug Weibel
  • Dr. Jennie Reed
  • Dr. Brian Pfleger
  • Dr. Aseem Ansari
  • Dr. Franco Cerrina
  • Dr. Ken Hammel
  • Dr. Jaehyuk Yu
  • Dr. Tom Jeffries
  • Dr. Kirk Kersten
  • Dr. Kirk Kent
  • Dr. Mike Sussman
  • Dr. Dan Cullen
  • Dr. Amber Vanden

Wymelenberg

  • Dr. John Ralph

Basudeb Bhattacharyya Matt Copeland Jenna Eun Hannah Tuson Shane Flickinger

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SLIDE 25

References

  • Guzman L., Belin D., CarsonM., and Beckwith L. (1995). Tight regulation, modulation and high-level expression by vectors containing the arabinose

pBAD promoter. Journal of Bacteriology. 177(14): 4121-4130.

  • Lorenzo Nissen, Gaspar Pérez-Martínez, María J. Yebra (2005) Sorbitol synthesis by an engineered Lactobacillus casei strain expressing a sorbitol-6-phosphate

dehydrogenase gene within the lactose operon FEMS Microbiology Letters 249 (1) , 177–183 doi:10.1016/j.femsle.2005.06.010

  • Roe, A. J., C. O'Byrne, D. McLaggan, and I. R. Booth. 2002. Inhibition of Escherichia coli growth by acetic acid: a problem with methionine biosynthesis and

homocysteine toxicity. Microbiology 148:2215-2222.

  • Ladero, V., Ramos, A., Wiersma, A., Goffin, P., Schanck, A., Kleerebezem, M., et al. (2007) High-level production of the low-calorie sugar sorbitol by

Lactobacillus plantarum through metabolic engineering. Appl Environ Microbiol 73: 1864–1872.

  • Metzger J (2006) Production of liquid hydrocarbons from biomass. Angew. Chem. Int. Ed. 2006, 45, 696-698.
  • Lovingshimera, M. R., Siegeleb, D., & Reinharta, G. D. (2006). Construction of an inducible, pfkA and pfkB deficient strain of Escherichia coli for the

expression and purification of phosphofructokinase from bacterial sources. Protein Expression and Purification , 46 (2), 475-482.

  • Karacaoğlan, V., & Özer, I. (2005). Steady-state kinetic properties of sorbitol dehydrogenase from chicken liver. Comparative Biochemistry and Physiology , 140,

309-312.

  • Archibald, F. S. A new assay for lignin-type peroxidases employing the dye azure B. Applied and Environmental Microbiology, 58(9), 3110-3116.
  • The International Energy Agency. 26 Aug. 2008. Biofuels for Transport: An International Perspective. April 2004.

http://www.iea.org/textbase/nppdf/free/2004/biofuels2004.pdf

  • Doyle, Wendy A., Andrew T. Smith. “Expression of lignin peroxidase H8 in Escherichia coli: folding and activation of the recombinant enzyme with Ca2+ and

haem.” Biochemistry Journal 315 (1996):15-19.

  • Hammel, Kenneth E., Dan Cullen. “Role of fungal peroxidases in biological ligninolysis.” Current Opinion in Plant Biology 11 (2008): 349-355.
  • “Cellulose.” Encyclopedia Britannica. 2008. Encyclopedia Britannica Online. 28 Oct. 2008.
  • DeLisa, Matthew P., Danelle Tullman, and George Georgiou. “Folding quality in export of proteins by the bacterial twin arginine translocation pathway.”

Proceedings of the National Academy of Sciences 100 (2003):6155-6120.

  • Tullman-Ereck, Danielle, Matthew P. DeLisa, Yasuaki Kawarasaki, Pooya Iranpour, Brian Ribnicky, Tracy Palmer, and George Georgiou. “Export Pathway

Selectivity of E. coli Twin Arginine Translocation Signal Peptides.” Journal of Biological Chemistry 282 (2007): 8309-83116.

  • Brenda: the comprehensive enzyme information system. 2008.2. Technical University of Braunschweig Department of Bioinformatics and Biochemistry. 2

July 2008. <http://www.brenda-enzymes.info/>