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Leonardo da Vinci
Human ingenuity may makeHuman ingenuity may make
various inventions, but it will nevervarious inventions, but it will neverdevise any inventions more beautiful,devise any inventions more beautiful,nor more simple, nor more to thenor more simple, nor more to the
purpose than Nature does; because inpurpose than Nature does; because inher inventions nothing is wanting andher inventions nothing is wanting andnothing is superfluous.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?
History
1. Interest
b. Biodegradability
a. Applications
Process
a. Nutrientsb. Biochemistry
c. Extractionb. Bio ethics
Future
a. Directions
Overview
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DegradationBiodegradation Environmental
Intracellular Extracellular Thermal Hydrolytic
Depolymeraseenzymes w/ dimer
hydrolase
Excreted DPEs Conditions 0-3380C ester linkages
R--hydroxybutyricacid
acetylacetate
assimilationProduct (Iso)crotonic
acid, dimer,trimer
monomerichydroxy
acid
Griffin G. J. L.
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Factors for degradability? A ? A? A? AEOHCOOHk
tCOOH 2$
xx
Non
enzymatic
COOH groups auto catalyze
Second stage weight loss- porous- bulk ( surface in enzymatic)
Crystallinity reduces reactivity
Scission catalyzed by acids, bases(amines)
Hydroph
obicity reduces th
e catalysis rateHydroph
obicity reduces th
e catalysis rateBlocking end groups(COOH) temporary reduction in rate
Blending ( variable effects) PHBV/CABBlending ( variable effects) PHBV/CABBlending ( variable effects) PHBV/CABEnzymatic
(vivo) Low Crystallinity, low Tg enhances rate, alkylsubstituents retard
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Economics
Bacterial cost Synthetic
PolyestersPHBV$3-4/ lb projected from1994 ($8-10/lb)
Polypropylene$0.30-0.45/lb
1. Substrate ~ 10%2. Separation ~ (50 70)%
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Synthetic strategies(1)O
O
O
O O
O
Sn
OH
OH
O
O
O
O
CH3
CH3
O
O
O
O
CaprolactoneE
Stannous Octoate
Ethylene Glycol
(Catalyst)
(initiator)
D, L - lactide glycolide
rom : Taylor A. E.
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Synthetic strategies(2)
O
O
O
O
O
OH
O
O
O
O
O n
OO
O
O n
+
CaprolactoneSuccinic anhydride
1-methylimidazole
1, 2 dichloroethane
65-70 oC
Carboxylic acid termination
N2, Stannous Octate
65 oC, 3h
115 oC, 15h
Polymer
Glycidyl termination
Storey , R. F., Hickey, T. P. J. Poly Sci.,
Part A 1993, 31, 1825
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Synthetic strategies(3)
C H 2 O
C H2
O
C H2
O
C H 2 O
C H2
O
O
H
O
H
O
C H3
C H2
C
H
P C LO
O
P C LO
O
P C LO
O
P C L OO
P C L OO
P C L OO
P C L O OP C LOO
C
C
C
T r i m e th
y l p r o p a n e i n i t i a t e d P o l y - E - C a p r o l a c t o n e
X y l i t o l - i n i t i a t e d P o l y - E - C a p r o l a c t o n e
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Initiators
% conversion over time
0
20
40
60
80
100120
8 14 2032
.541
.5 57 84 105
129
187
Time (h)
%conversion
% Conversion (AEG-
Init.) (NMR)
% Conversion (AEG-
Init.) (TGA)
% Conversion (Water-
Init.) (NMR)
% Conversion (Water-
Init.) (TGA)
% Conversion (EG-
Init.) (NMR)
% Conversion (EG-
Init.) (TGA)
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Poly hydroxy alkanoates
PHAs
Propionate (H)
Butyrate (CH3)
Valerate (CH2CH3)
Caproate (C3H7)
Heptanoate (C4 H9)
Octanoate (C5 H11)
MWMWMW (50-1,000) k Da Repeat unitRepeat unitRepeat unit
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Co-polyester properties -PP
P(3HB) P(3HB-3HV) P(3HB-4HB) P(3HO-3HH) PP
Content 20% 16% 11%
Tm (C) 177 145 150 61 176
Tg (C) 2 -1 -7 -36 -10
Crystallinity (%) 70 56 45 30 60
Extension to break (%) 5 50 444 300 400
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
Tensile Strength
020
40
60
0 3 9 14 20 25
%HV
Tensile Strength(M
)
Impact Strength
0
200
400
600
0 3 9 14 20 25
%HV
Notched Izod im pact
strength ( m)
ransition emperatures
10
0
10
20
0 3 9 14 20 25
%HV
Glass Transition (oC)
Griffin G . J.L.
Melting Point (oC )
050
100
150
200
0 3 9 14 20 25
%HV
Meltingpoint
Melting Point (oC )
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ISODIMORPHISM
Melting Point
7
7
7
%HV
M P
P M P
Gr ff G J L
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Crystallinity
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 fromAcidovoraxSp. TP4, Biomacromolecules, 3 (4), 828-834, 2002
P(3HB-co-3HP), Bacterial P(3HB) & Syn P(3HP)
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Biodegradability
Synthetic polymers w/ester linkages
Hydrolysis wouldinitiate degradation
Structure of
prepolymer couldpropagate
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 courtesyof Dieter Jendrossek,Georg-August-Universitt, Gttingen,
Germany.
Lara L. Madison and Gjalt W. Huisman
Degradation in soil
0
20
40
60
80
1 2 3 5 7 10 13 17 18Time in wee ks
%
Carbonlost
% labelled
carbon
lost
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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) .2525
PHB first MENTIONEDPHB first MENTIONED
Baptist,Werber(W. R. Grace) 60s60sLb of PHB produced for evalLb of PHB produced for eval
Imperial Chemical Industries, Ltd. 77--80s80s
Pruteen developed Ae 70% biomassPruteen developed Ae 70% biomass Biopol, Metabolix Inc. and Monsanto90s90s
Bioidegradable bottle & Wella shampooBioidegradable bottle & Wella shampoo
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Philip Ball, Consulting Editor, Nature
There is no assemblyThere is no assembly
plant so delicate,plant so delicate,versatile and adaptive asversatile and adaptive as
the cell.the cell.
Jim Robbins, Second Nature, Smithsonian, July 2002, Vol. 33, No. 4, p. 78-84
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General enzymatic pathway forPHB and
PHBHx synthesis
Madison LL, Huisman GW.
fatty acid degradation
Athree-step pathway. The three
enzymes are encoded by the genesofthe phbCAB operon. A promoter
upstream ofphbCtranscribes the
complete operon.
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PHBV w/o supplements
Generally, P(3HB-3HV)is synthesized withsupplements ofpropionate, valerate, orother Codd fatty acids.
Some organisms areable to form Propionyl-CoA through themethylmalonyl-CoApathway, from succinyl-CoA in the TCA cycle.
Madison LL, Huisman GW.
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The two ketothiolases forPHBV synthesis
Steven Slateret al
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Tapping the fatty acid synthesis
Monomers from (R)-3-hydroxyacyl-ACP
intermediates areconverted to (R)-3-hydroxyacyl-CoAthrough an acyl-
ACP:CoAtransacylaseencoded by the
phaG gene.
Madison LL, Huisman GW.
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PhaG mediated synthesis
Silke Fiedler, Alexander Steinbch
el, and Bernd H. A. Reh
m,
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MCL PHA syn in 'thioesterase
I- E. coli JMU193 onGluconate1. B-ketoacyl-ACP synthase
2. B-ketoacyl-ACP reductase
3. hydroxyacyl-ACP
dehydrase4. enoyl-ACP reductase
5. 'thioesterase I
6. acyl-CoA synthase
7. acyl-CoA dehydrogenase
8. Enoyl-CoA hydratase9. 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 withcis doublebonds at an evencarbon are indicatedby dashed lines.
VolkerMittendorf et al
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PHBHx Synthesis
Fukui T., Abe H., and Doi Y
Aeromonas caviae
PhaC, PhaJ
Ralstonia eutropha
PhbA, PhbB, BktB, (S)-3HB-CoAdehydrogenase
Streptomycescinnamonensis
Crotonyl-CoA reductase
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Plasmid forPHBHx synthesis in R. eutrophus
Fukui T., Abe H., and Doi Y
The direction
Of the codons isImportant as
well as the
promoters
which are
inducedusing external
Control agents..
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Gene constructs for Transgenic
plants
Yves Poirier, Giovanni Ventre, and Daniela Caldelari
PTS,Peroxisomaltargeting
sequencePhaC1
synthaseFatB3
thioesteraseOCS, octopine
synthase
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Amount of PHA relative to
growth stage of transgenic plants7-day-old seedlings grown
in Murashige and Skoog
media, green & senesced
leaves from soil-grown
plants. Average of2 ind.
measurements.
http://www.pnas.org/cgi/content/full/95/23/13397
The beta-oxidation cycle is induced upon seedgermination, involved in the mobilization of reservelipids, so the highest amount of PHA is expected to besynthesized at this stage. VolkerMittendorf et al
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Some Organisms and their copolyesters or enzymes and genesSome Organisms and their copolyesters or enzymes and genes
Rhodosporiillu
m rubrum
C3- C6
(of specialinterest)
Pseudomonas
oleo
PHA
depolymerasePHA synthase
phaC2
Rhodocyclusgelatinosus
PHA synthase phaC1
Rhodococcusruber
Alcaligeneseutrophus
PHA synthase
CoA reductase
phbC-A-B
Pseudomonad C4- C12 Streptomycescinnamonensis
Crotonyl-CoA
reductase
ccrSc
AeromonasCaviae
P(3HB-CO-3HHx)
even C-
alkanoates or
plant oils
PHA synthase
enoyl-CoA
hydratase
phaC-JAc
Escherichiacoli
P(3HB-CO-4HB)
glucose or
glutamate
PHA gluconate
Ralstonia
eutropha
3HB, 3HA glucose
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A. CompletephbCAB
B. Intrptd phblociC. Incomp phbloci
D. From 2plmrsasE. P(3HB-3HH)F. msc
Operons
Madison LL, Huisman GW.
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Formation of a PHA granule
C and Z are similar to Lacting on the surface ofthe granule
Proposed mechanism:- Econverts monomerCoA tooligomers which cleave atcritical length or conc. E-oligomrers formcompartments orPHAgranules which coalesce toform larger bodies.
C= PHA polymerase
Z= PHA depolymerase
L= lipase ( cleaves ester bonds)
E= soluble enzyme
TG=Triglyceride Madison LL, Huisman GW.
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S. Cerevisiae
Poirier, Y., Erard, N., Petetot, J.M.-C. (2001). Synthesis of Polyhydroxyalkanoate in the Peroxisome of Saccharomyces cerevisiae by UsingIntermediates ofFatty Acid beta -Oxidation.Appl. Environ. Microbiol. 67
Grown on 0.1% glucose 0.1% Oleic acid, 2% Pluronic 127Awt, B recombinant w/ PHA synthase, 1 um bar, arrow to PHAgranules.
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Cell medium effects (phaG)
1. PHA accumulation andcomposition ofP. fragi
2. NH4Cl concentrations.
3. 50 ml of MM with 1.5%(wt/vol) sodiumgluconate
4. At 30C for 48 h.
5. 3HA, 3-hydroxyalkanoate;3HDD:1, 3-hydroxydodecanoate;3HDD, 3-hydroxydodecanoate;3HD, 3-hydroxydecanoate; 3HO,3-hydroxyoctanoate; 3HHx, 3-hydroxyhexanoate. Silke Fiedler, Alexander Steinbchel, and Bernd H.Silke Fiedler, Alexander Steinbchel, and Bernd H. A. RehmA. RehmApplied and Environmental Microbiology,Applied and Environmental Microbiology, May 2000, Vol. 66, No. 5, p. 2117May 2000, Vol. 66, No. 5, p. 2117--2124.2124.
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Promoter induction (thioesterase-I [bad])
1. PHA accumulation and
'thioesterase I activity inE. coli
2. % Arabinose as indicated.
3. 25 h after stat phase
4. GC (open circles).
5. Spectrophotometric (solidsquares)
6. Control lacked the'thioesterase I-encodinggene.
Klinke, S.; Ren, Q.; Witholt, B., Kessler B. ;Appl. Environ. Microbiol. 1999, 65, 540-8.
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Promoter induction (polymerase [alk])
1. PHA accumulationby E. coli
2. DCPK(dicyclopropylketone) conc.
3. 25 h (shaded bars)
4. 44h
(open bars)5. 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 ofPhbA and
BktB in synthesis of
PHBV inE. coli.
2. Plasmidfrom R. eutropha
phb Bplus 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 inR. eutropha
2. wt (open squares)
3. phaR strains (solid)4. 72 h.
5. Average value for2cultures
6. PhaP regulates PHBsyn, PhaR regulatesPhaP 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
Madison LL, Huisman GW.,
E coli is proposed totransport Ca+ out and
DNA
in. The Ca+
(green) is liganded tocarbonyl oxygen andthe polyphosphate
molecule within thehelix, transport isfacilitated byenzymatic action on
the molecule.
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Transgenic
Aeromonas.caviae
Alcaligeneseutrophus
96% yield
P. putida
T. pfennigii P. putida 3HB-co-3HHx-co-3HOctanoate
Alcaligenes
eutrophus
E coli ketothiolase
50%-80%
Cosmid
pVK102
Plants P(3HB-co-3HV)
PCR PHA synthase
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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/ Fattyacid synthase
Microbial mats
Results---- Unsuccessful
(0.34% fiber weight)
0.025to 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 g of PHA/gof dry mat
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Mats NESpain & Mass.
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Concentrated pasteConcentrated pasteConcentrated paste
cellcell lysislysis
purification (purification (depolymerizationdepolymerizationdepolymerization))
centrifugationcentrifugation
Extraction(Phy, Chem, Biochem)(Phy, Chem, Biochem)
Griffin G. J. L.
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solvent extraction
PhysicalPhysical
Pretreatment with methanol or acetone toincrease permeability, removes lipids and
denatures proteinso chloroform
o methylene chloride
o di and tri chloroethane
o propylene carbonate Purification expensive , high volumes of
solvents, crystalline precipitates with nonsolvents methanol, diethyl ether orhexane.
Griffin G. J. L.
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Sodium hypochlorite digestion
ChemicalChemical
Degrades and dissolves cell wall leavingpolymer granules intact
Depolymerization due to alkalinity reduced bytreating with phenyl acetic acid and freezedrying before procedure. surfactants
Difficult to remove sodium hypochlorite.Careful control of pH and digestion timeimproved purity (95%)
Griffin G. J. L.
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Enzymatic digestion
BiochemicalBiochemicalBiochemical
Lysozymes,& deoxy ribonuclease treatmentto solubilize peptidoglycans and proteins.
Weakened cell walls ruptured ultrasonically.90% PHA and some peptidoglycans andproteins. Closest to vivo state.
May be further purified with solvents. MWmay be controlled by heat treatment andspray drying.
Griffin G. J. L.
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Some Stats and Concerns
E coli O157:H7.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 milliongoatsJennifer 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
Ge et ca y E eere Foo ro s
0
20
40
60
80
100
1996 1997 1998 1999 2000 2001
Year
o
s
of
acres
o abean
orn
Jennifer Ackerman, F d How afe? How altered?, National Geographic, May 2002, Vol. 201, No. 5, p2-50.
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Pros and Cons
Higher Yields- Tocombat world hunger.
FewerPesticides- Lessspraying soilconservation withherbicide resistant plants.
BetterNutrition-
Enhanced foods rich innutrients.
Docile farm animals
Gene Flow-mutantshard to manage
Toxin build up- BtCrops add it to soil.
Allergens-Foods
may contain chemicalby 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.
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Preventive measure
Bt Cropresistantinsects
moatrefuge
insects
Susceptible offspring
Presumably by growingregular crops near the
Bt crops , the cross-breeding of insects willdelay development ofresistant strains.
Bt = Bacillus thuringiensis(source of genes capable of producing insecticide)
Bt Corn toxic to monarch caterpillarsJennifer 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
Nutrients Inexpensive and abundant substrate
Organism Bio Stress, balance between vital cell activities and mfgSide effects of long term use,
genetic and/ or other changes in cell chemistry
Regulation triggers inducing enzyme production & releasechemically induced promoters to turn genes on and off.
Process Natural excretion or secretion desired, keeping organism viableMonitoring desired, possibly with controllable viral infections,
Purification ease of extraction, storage in specific areas, natural excretion
Bio Ethics Sanctity of life and other issues