Leonardo da Vinci

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Leonardo da Vinci
Human ingenuity may make various inventions,
but it will never devise any inventions more
beautiful, nor more simple, nor more to the
purpose than Nature does because in her
inventions nothing is wanting and nothing is
superfluous.
Jim Robbins, Second Nature, Smithsonian, July
2002, Vol. 33, No. 4, p. 78-84
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Bio-polyesters
Why (Bio) synthesis?
Overview

b. Bio ethics
History
Process
Future
1. Interest
a. Nutrients
a. Directions
a. Applications
b. Biochemistry
c. Extraction
b. Biodegradability
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Degradation
Biodegradation Biodegradation Environmental Environmental
Intracellular Extracellular Thermal Hydrolytic
Depolymerase enzymes w/ dimer hydrolase Excreted DPEs Conditions 0-3380C ester linkages
R--hydroxybutyric acid acetylacetate assimilation Product (Iso)crotonic acid, dimer, trimer monomeric hydroxy acid
Griffin G. J. L.
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Factors for degradability
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Economics
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Synthetic strategies(1)
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Synthetic strategies(2)
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Synthetic strategies(3)
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Initiators
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Poly hydroxy alkanoates PHAs

• Propionate (H)
• Butyrate (CH3)
• Valerate (CH2CH3)
• Caproate (C3H7)
• Heptanoate (C4 H9)
• Octanoate (C5 H11)

MW (50-1,000) k Da Repeat unit
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Co-polyester properties - PP
Polypropylene properties compared with some
Co-polyester content indicated is with P(3HB).
Madison LL, Huisman GW. From Doi, Y. Microbial
Polyesters VCH New York, 1990.
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PHBV properties
Griffin G. J. L.
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ISODIMORPHISM
Griffin G. J. L.
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Crystallinity
P(3HB-co-3HP), Bacterial P(3HB) Syn P(3HP)
Yi Wang, Yasuhide Inagawa, Terumi Saito,Ken-ichi
Kasuya,Yoshiharu Doi, and Yoshio Inoue.,
Enzymatic Hydrolysis of Bacterial
Poly(3-hydroxybutyrate-co-3-hydroxypropionate)s
by Poly(3-hydroxyalkanoate) Depolymerase from
Acidovorax Sp. TP4, Biomacromolecules, 3 (4), 828
-834, 2002
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Biodegradability

• Synthetic polymers w/ ester linkages
• Hydrolysis would initiate degradation
• Structure of prepolymer could propagate
• Easy to process

• Biosynthetic polymers
• Compatible chirality
• PHB content in most living organisms
• High reproducibility
• Quick yield
• Renewable

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P(3HB-3HV) in aerobic (20C) sewage sludge
0, 2, 4, 6, 8, and 10 weeks
Photograph courtesy of Dieter Jendrossek,
Georg-August-Universität, Göttingen, Germany.
Lara L. Madison and Gjalt W. Huisman
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Applications

• Chiral blocks for syn, chromatography
• Various biodegradability products
• Toner (paper recyclables)
• Insecticide packaging
• Drug release matrix in vet MEDICINE
• Piezoelectric properties in temp dep
• PHBV grades allow var phy properties
• Synthetic polymers w/ ester linkages

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History

• Maurice Lemoigne (Institut Pasteur) .25
• PHB first MENTIONED
• Baptist, Werber (W. R. Grace) 60s Lb of
  PHB produced for eval
• Imperial Chemical Industries, Ltd. 7-80s
  Pruteen developed Ae 70 biomass
• Biopol, Metabolix Inc. and Monsanto90s
  Bioidegradable bottle Wella shampoo

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Philip Ball, Consulting Editor, Nature
There is no assembly plant so delicate,
versatile and adaptive as the cell.
Jim Robbins, Second Nature, Smithsonian, July
2002, Vol. 33, No. 4, p. 78-84
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General enzymatic pathway for PHB and PHBHx
synthesis
fatty acid degradation
A three-step pathway. The three enzymes are
encoded by the genes of the phbCAB operon. A
promoter upstream of phbC transcribes the
complete operon.
Madison LL, Huisman GW.
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PHBV w/o supplements

• Generally, P(3HB-3HV) is synthesized with
  supplements of propionate, valerate, or other
  Codd fatty acids.
• Some organisms are able to form Propionyl-CoA
  through the methylmalonyl-CoA pathway, from
  succinyl-CoA in the TCA cycle.

Madison LL, Huisman GW.
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The two ketothiolases for PHBV synthesis
Steven Slater et al
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Tapping the fatty acid synthesis

• Monomers from (R)-3-hydroxyacyl-ACP intermediates
  are converted to (R)-3-hydroxyacyl-CoA through an
  acyl-ACPCoA transacylase encoded by the phaG
  gene.

Madison LL, Huisman GW.
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PhaG mediated synthesis
Silke Fiedler, Alexander Steinbüchel, and Bernd
H. A. Rehm,
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MCL PHA syn in 'thioesterase I- E. coli JMU193 on
Gluconate

1. B-ketoacyl-ACP synthase
2. B-ketoacyl-ACP reductase
3. hydroxyacyl-ACP dehydrase
4. enoyl-ACP reductase
5. 'thioesterase I
6. acyl-CoA synthase
7. acyl-CoA dehydrogenase
8. Enoyl-CoA hydratase
9. Isomerase
10. specific hydratase
11. PHA polymerase

Klinke, S. Ren, Q. Witholt, B. Kessler B
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MCL PHA syn in Transgenic Plants

• The oxidation of unsaturated fatty acids with cis
  double bonds at an even carbon are indicated by
  dashed lines.

Volker Mittendorf et al
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PHBHx Synthesis

• Aeromonas caviae
• PhaC, PhaJ
• Ralstonia eutropha
• PhbA, PhbB, BktB, (S)-3HB-CoA dehydrogenase
• Streptomyces cinnamonensis
• Crotonyl-CoA reductase

Fukui T., Abe H., and Doi Y
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Plasmid for PHBHx synthesis in R. eutrophus
The direction Of the codons is Important as
well as the promoters which are induced using
external Control agents.
Fukui T., Abe H., and Doi Y
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Gene constructs for Transgenic plants

• PTS, Peroxisomal
• targeting sequence
• PhaC1 synthase
• FatB3 thioesterase
• OCS, octopine
• synthase

Yves Poirier, Giovanni Ventre, and Daniela
Caldelari
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Amount of PHA relative to growth stage of
transgenic plants
7-day-old seedlings grown in Murashige and Skoog
media, green senesced leaves from soil-grown
plants. Average of 2 ind. measurements.
The beta-oxidation cycle is induced upon seed
germination, involved in the mobilization of
reserve lipids, so the highest amount of PHA is
expected to be synthesized at this stage. Volker
Mittendorf et al
http//www.pnas.org/cgi/content/full/95/23/13397
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(No Transcript)
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Operons

• A. Complete phbCAB
• B. Intrptd phb loci
• C. Incomp phb loci
• D. From 2 plmrsas
• E. P(3HB-3HH)
• F. msc

Madison LL, Huisman GW.
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Formation of a PHA granule

• C and Z are similar to L acting on the surface of
  the granule
• Proposed mechanism- E converts monomerCoA to
  oligomers which cleave at critical length or
  conc. E-oligomrers form compartments or PHA
  granules which coalesce to form larger bodies.

C PHA polymerase Z PHA depolymerase L lipase (
cleaves ester bonds) E soluble
enzyme TGTriglyceride
Madison LL, Huisman GW.
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S. Cerevisiae
Grown on 0.1 glucose 0.1 Oleic acid, 2
Pluronic 127 A wt, B recombinant w/ PHA
synthase, 1 um bar, arrow to PHA granules.
Poirier, Y., Erard, N., Petetot, J. M.-C. (2001).
Synthesis of Polyhydroxyalkanoate in the
Peroxisome of Saccharomyces cerevisiae by Using
Intermediates of Fatty Acid beta -Oxidation.
Appl. Environ. Microbiol. 67
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Cell medium effects (phaG)

1. PHA accumulation and composition of P. fragi
2. NH4Cl concentrations.
3. 50 ml of MM with 1.5 (wt/vol) sodium gluconate
4. At 30C for 48 h.
5. 3HA, 3-hydroxyalkanoate 3HDD1,
   3-hydroxydodecanoate 3HDD, 3-hydroxydodecanoate
   3HD, 3-hydroxydecanoate 3HO, 3-hydroxyoctanoate
   3HHx, 3-hydroxyhexanoate.

Silke Fiedler, Alexander Steinbüchel, and Bernd
H. A. Rehm Applied and Environmental
Microbiology, May 2000, Vol. 66, No. 5, p.
2117-2124.
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Promoter induction (thioesterase-I bad)

1. PHA accumulation and 'thioesterase I activity in
   E. coli
2. Arabinose as indicated.
3. 25 h after stat phase
4. GC (open circles).
5. Spectrophotometric (solid squares)
6. Control lacked the 'thioesterase I-encoding gene.
   

Klinke, S. Ren, Q. Witholt, B., Kessler B.
Appl. Environ. Microbiol. 1999, 65, 540-8.
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Promoter induction (polymerase alk)

• PHA accumulation by E. coli
• DCPK(dicyclopropylketone) conc.
• 25 h (shaded bars)
• 44 h (open bars)
• Analyzed by GC.

Klinke, S. Ren, Q. Witholt, B., Kessler B.
Appl. Environ. Microbiol. 1999, 65, 540-8.
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Enzymatic effects (PhbA BktB)

1. Comparison of PhbA and BktB in synthesis of PHBV
   in E. coli.
2. Plasmid from R. eutropha phbCB plus a
   -ketothiolase gene.
3. bktB (squares) EE245
4. phbA (triangles) EE247
5. Propionate in the medium.

Steven Slater, Kathryn L. Houmiel, Minhtien Tran,
Timothy A. Mitsky, Nancy B. Taylor, Stephen R.
Padgette, and Kenneth J. Gruys Steven S
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Phasin effects (PhaR PhaP)

1. PhaP in R. eutropha
2. wt (open squares)
3. phaR strains (solid)
4. 72 h.
5. Average value for 2 cultures
6. PhaP regulates PHB syn, PhaR regulates PhaP syn.

Gregory M. York,1 JoAnne Stubbe,1,2 and Anthony
J. Sinskey1
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Ca Pump in P(3HB) helix imbedded in a membrane
E coli is proposed to transport Ca out and DNA
in. The Ca (green) is liganded to carbonyl
oxygen and the polyphosphate molecule within the
helix, transport is facilitated by enzymatic
action on the molecule.
Madison LL, Huisman GW.,
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Transgenic
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Transgenic w/ modifications

• Plants ---
• Zea Mays Corn
• G hirsutum (cotton).
• Alfalfa
• Arabidopsis thaliana w/ Synthase modification 
• Yeast ---
• S. Cervisiae  w/ Oleic acid
• Insect cells w/ Fatty acid synthase
• Microbial mats

• Results----
• Unsuccessful
• (0.34 fiber weight)
• 0.025 to 1.8 g kg-1 dry weight
• 0.2 to 4.0 g kg-1 dry weight
• 0.5 of cell dry weight
• 250 to 500 µg of PHA/g of dry mat

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Mats NE Spain Mass.
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Extraction (Phy, Chem, Biochem)
cell lysis
centrifugation
purification (depolymerization)
Concentrated paste
Griffin G. J. L.
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solvent extraction Physical

• Pretreatment with methanol or acetone to increase
  permeability, removes lipids and denatures
  proteins
• chloroform
• methylene chloride
• di and tri chloroethane
• propylene carbonate
• Purification expensive , high volumes of
  solvents, crystalline precipitates with non
  solvents methanol, diethyl ether or hexane.

Griffin G. J. L.
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Sodium hypochlorite digestion Chemical

• Degrades and dissolves cell wall leaving polymer
  granules intact
• Depolymerization due to alkalinity reduced by
  treating with phenyl acetic acid and freeze
  drying before procedure. surfactants
• Difficult to remove sodium hypochlorite. Careful
  control of pH and digestion time improved purity
  (95)

Griffin G. J. L.
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Enzymatic digestion Biochemical

• Lysozymes, deoxy ribonuclease treatment to
  solubilize peptidoglycans and proteins.
• Weakened cell walls ruptured ultrasonically. 90
  PHA and some peptidoglycans and proteins. Closest
  to vivo state.
• May be further purified with solvents. MW may be
  controlled by heat treatment and spray drying.

Griffin G. J. L.
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Some Stats and Concerns

• E coli O157H7.virulent strain(60)
• rBGH..(14 days)
• Processed foods(60)
• Salmonella DT104(resists 5 antibiot)
• Bacteria in a colon 200 x all humans
• US..76mill...325k.5k
• 20 milliongoats

Jennifer Ackerman, Food How safe? How altered?,
National Geographic, May 2002, Vol. 201, No. 5,
p2-50. Jim Robbins, Second Nature, Smithsonian,
July 2002, Vol. 33, No. 4, p78-84.
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How Altered? 50-fold over 6 yrs
Jennifer Ackerman, Food How safe? How altered?,
National Geographic, May 2002, Vol. 201, No. 5,
p2-50.
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Pros and Cons

• Higher Yields- To combat world hunger.
• Fewer Pesticides- Less spraying soil
  conservation with herbicide resistant plants.
• Better Nutrition-Enhanced foods rich in
  nutrients.
• Docile farm animals

• Gene Flow-mutants hard to manage
• Toxin build up- Bt Crops add it to soil.
• Allergens-Foods may contain chemical by products
  of alteration.

Jennifer Ackerman, Food How safe? How altered?,
National Geographic, May 2002, Vol. 201, No. 5,
p2-50. Jim Robbins, Second Nature, Smithsonian,
July 2002, Vol. 33, No. 4, p78-84.
51
Preventive measure
Presumably by growing regular crops near the Bt
crops , the cross- breeding of insects will
delay development of resistant strains.
Bt Crop resistant insects
moat refuge insects
Susceptible offspring
 
Bt Bacillus thuringiensis (source of genes
capable of producing insecticide) Bt Corn toxic
to monarch caterpillars Jennifer Ackerman, Food
How safe? How altered?, National Geographic, May
2002, Vol. 201, No. 5, p2-50.
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Biomimicry

• Larvae mandiblesChain saw
• Cocklebur.Velcro
• Snake skin . Entropy
• GeckoAdhesive(re)
• SpiderDragline(tendons)
• GiraffeLubricant

Jim Robbins, Second Nature, Smithsonian, July
2002, Vol. 33, No. 4, p. 78-84
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Proposed directions