Una visione sistematica per l

1
Una visione sistematica per lingegneria
metabolica

• Creazione di vie metaboliche

2
Ingegneria metabolica
probabilmente efficace
probab. inefficace
(1)
(2)
(3)
(4)
(5)
(7)
(8)
(6)
(9)
S
S
S
S
S
S
S
S
S
A
A
A
A
A
A
A
A
A
Y
E
B
B
B
B
B
B
B
B
B
Z
W
X
F
C
C
C
C
C
C
C
C
Q
P
P
P
P
P
Q
P
P
P
P
ATP
3
Espressione di nuovi enzimi
Ingegneria metabolica dei lipidi

• Olii ad alto contenuto di ac. Laurico (C12)
• Ac. grassi poli-insaturi (Ac. Erucico)
• Olii ad alto contenuto di acidi monoinsaturi (ac.
  oleico)
• Olii con acido ricinoleico

Manipolazioni efficaci che riguardano uno o pochi
enzimi
4

• Knutzon et al., (1999) LPAAT from coconut
  endosperm mediates the insertionof laurate at the
  sn-2 position of triacylglycerols in Lauric
  rapeseed oil and can increase total laurate
  levels. Plant Physiology 120739746.
• Katavic et al., (2000) Utility of the
  Arabidopsis FAE1 and yeast SLC1-1 genes for
  improvements in erucic acid and oil content in
  rapeseed. Biochem Soc Trans. 28935-7.
• Mietkiewska E et al., (2004) Seed-specific
  heterologous expression of a nasturtium FAE gene
  in Arabidopsis results in a dramatic increase in
  the proportion of erucic acid. Plant Physiol.
  1362665-75
• Stoutjesdijk PA et al., (2002) hpRNA-mediated
  targeting of the Arabidopsis FAD2 gene gives
  highly efficient and stable silencing. Plant
  Physiol. 1291723-31.
• Liu Q, Singh SP, Green AG. (2002) High-stearic
  and High-oleic cottonseed oils produced by
  hairpin RNA-mediated post-transcriptional gene
  silencing. Plant Physiol. 1291732-43.
• Jain et al., (2000) Enhancement of seed oil
  content by expression of glycerol-3-phosphate
  acyltransferase genes. Biochem Soc Trans.
  28958-61.
• Klaus D, Ohlrogge JB, Neuhaus HE, Dormann P.
  (2004) Increased fatty acid production in potato
  by engineering of acetyl-CoA carboxylase.
  Planta. 219389-96.
• J. Burgal, J. Shockey, C. Lu, J. Dyer, T. Larson,
  I. Graham, J. Browse, Metabolic engineering of
  hydroxy fatty acid production in plants RcDGAT2
  drives dramatic increases in ricinoleate levels
  in seed oil, Plant Biotechnol J. 6 (2008)
  819-831.
• Andrianov W et al. (2010) Tobacco as a production
  platform for biofuel overexpression of
  Arabidopsis DGAT and LEC2 genes increases
  accumulation and shifts the composition of lipids
  in green biomass. Plant Biotech. J 8277287
• Broun P, Gettner S, Somerville C. (1999) Genetic
  engineering of plant lipids. Annu Rev Nutr.
  19197-216. Review.

Maisonneuve et al. (2010) Expression of rapeseed
microsomal lysophosphatidic acid acyltransferase
isozymes enhances seed oil content in
Arabidopsis. Plant Physiol. 152670-84.
5
Importanza degli olii

• Produzione di olio vegetale mondiale ca. 120
  Mt/anno (50 Billions )
• 20 Kg/persona/anno usati in gran parte per
  lalimentazione
• Usati anche per la produzione di saponi,
  detergenti, lubrificanti, combustibili
  (biodiesel), cosmetici, fibre e vernici
• Aumenta il consumo, ma aumenta anche la
  produzione, per cui il prezzo rimane abbastanza
  costante (0.6 /Kg)
• In ordine di importanza soia, palma, colza,
  girasole (gt70 della prod.)

Globally, over 126 million tonnes of oils were
consumed in 2006/2007. Palm and soybean oils are
consumed the most at 38.9 and 37.5 million tonnes
respectively, followed by rapeseed oil (including
canola oil) at 18.9 million tonnes and sunflower
oil at 10.5 million tonnes.
6
Palm oil production
Fresh Fruit Bunches (FFB) (circa 1000 seeds/bunch)
A palm oil mill produces crude palm oil and
kernels, as primary products and biomass as
secondary product. The capacity of mills varies
between 60- 100 tons FFB/h.
http//lipidlibrary.aocs.org/processing/palmoil/in
dex.htm
http//lipidlibrary.aocs.org/index.html
7
Usi non alimentari saponi
The word 'soap' comes from the Celtic word Saipo.
Wood ash was mixed with water, then animal fat
was added. When the mixture boiled, more and more
ash was added as the water evaporated. Soap
cleaned clothes because it soaked into the fabric
and lifted up dirt, which was then carried away
in the rinse water.
http//hubpages.com/hub/Soap-Fatty-Acids-and-Plant
-Ashes--Who-Thought-of-That
8
http//chemistry.about.com/library/weekly/blsapon.
htm
Soap making
La materia prima per produrre saponi e detergenti
sono gli ACIDI GRASSI (i detergenti sono essenz.
come i saponi ma non precipitano con Ca o Mg)
http//www.vinythai.co.th/ourchemicalproducts/caus
ticsoda/mkt1soapdetergent/0,,2009-2-0,00.htm
Fuel making... methanolysis (FAME)
9
Acidi grassi
Molecole con parte polare e parte apolare
Gli acidi grassi nelle piante sono lineari con
12-22 atomi di C La maggior parte di quelli
estratti da pianta contengono linoleate,
palmitate, laurate e oleate.
C16 acido palmitico
10
Acidi grassisaturi ed insaturi
Le insaturazioni influenzano limpaccamento dei
lipidi nelle membrane e quindi la temperatura a
cui cristallizzano
11
Palmitico, stearico e oleico sono i più
abbondanti, Linsaturazione più comune è tra C7 e
C8
12
Sintesi glicerolipidi
Sintesi del Diacilglicerolofosfato (acido
fosfatidico)
13
Di e tri-acilgliceroli
Olii e grassi (animali e vegetali)
14
Destino degli acidi grassi
15
Due vie
Entrambe le vie funzionano nelle piante
16
Overview of lipid synthesis in plants
Somerville and Browse (1996) Trends Cell Biology
6148-153
17
Sintesi degli acidi grassi(cloroplasto)
18
Sintesi di Acetil-CoA e Malonil-CoA
Acetil-CoA
Oxaloacetato
Citrato
Malonil-CoA
Acetil-CoA
19
Acetyl CoA Carboxylase as RLS
Although biochemical analysis indicates that
Acetyl CoA carboxylase is a major metabolic
control point in fatty acid synthesis (ref), its
overexpression had only minor impact on seed oil
content (ref), probably due Ohlrogge J. et al,
(2000) Fatty acid synthesis from CO2 to
functional genomics Biochem Soc Trans. 28567-73.
Review
Balle! Il Malonil-CoA viene regolato a livello
del demand. Aumentare il supply non comporta un
aumento di flusso (incorporazione nei lipidi)
20
Enzimi coinvolti
ACC Malonyl-CoA transacilasi KAS III, II I FAS
- Acido grasso sintasi
21
Mutanti, mutanti, mutanti...
Grandi quantità di lipidi sono scambiate tra ER e
cloroplasto
22
La via cloroplastica solitamente produce C16-C18
I geni Fat terminano la crescita della catena di
acido grasso (staccano la catena) e determinano
quindi la sua lunghezza. Hanno attività
come acyl-ACP thioesterases (FatA and FatB
classes)
Gli acidi C16-C18 servono sintesi
triacilgliceroli Sintesi galattolipidi
Sintesi fosfolipidi Sintesi acidi grassi a
catena più lunga
23
C16-C18
Lipidi di riserva
Lipidi di membrana (via procarioti)
Lipidi di membrana (via eucarioti)
Lipidi a lunga catena
Lipidi modificati
?-ossidazione
24
Lipidi di riserva
Una volta attivati tramite CoA, gli acidi grassi
possono essere esterificati con glicerolo
3-fosfato (G-3-P) per produrre LPA, PA, DAG e TAG.
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Oil bodies

• I triacilgliceroli (TAG) si accumulano negli Oil
  bodies che si originano a livello dellER
• Esistono vari tipi di olii
• Saturi C16-C18 a sn-1 e 3 (es. cacao)
• Saturi C8-C14 in tutte le posizioni (es. cocco)
• Insaturi (oliva, colza)

Canola production worth 1.5-2 B per year to
Western Canada 1 increase in oil content worth
10-20 million
27
I lipid bodies sono circondati da oleosina
28
FAE acido grasso elongasi
Lipidi a lunga catena
Acidi grassi a lunga catena (Very Long Chain
Fatty Acids -VLCFA) Altre modificazioni possibili
sono Epossidazione Ossidrilazione
Ciclizzazione
La sovraespressione di FAE porta alla formazione
di una maggior proporzione di VLCFA
29
Gli acidi grassi vengono allungati oltre C18 nel
reticolo endoplasmatico
30
Acidi grassi inusuali
Lipidi modificati
Diverse di queste strutture sono usate come
precursori nella chimica di sintesi (nylon)
Plant factories per la produzione di
detergenti, plastica, lubrificanti, fibre
Esempio 1-Octene is a high-demand feedstock with
a global consumption of over half a million tons
per year that is primarily used as a comonomer in
the expanding production of linear low density
polyethylene. È ottenuto a partire da acidi
grassi ?-7 come ac. palmitoleico o cis-vaccenico.
Plants synthesize gt200 different FA structures
with attractive functional properties
31
Erucic acid
U6.50/kg (for 1,000kgs)
Erucamide, unsaturated long chain carboxylic acid
amide (221 n-9), is used as a slip agent ,
anti-fogging or lubricant for plastic films
(polyolefin) which can be used in food packing
material. It is used as a dispersant in printing
and dying. It is used in paper and textile
industry for water-proof as well as corrosion
inhibitor in oil wells. It is used for the 
synthesis of organic chemicals and surfactants
used in detergent, ore floating agent, fabric
softener, anti-static agent, germicide,
insecticide, emulsifier, anti-caking agent,
lubricant and water treatment agent.
La variabilità genetica nelle varie specie è già
stata abbondantemente sfruttata per creare
varietà con profili diversi di acidi grassi...
32
erucic acid
Canola (CANadian Oil Low Acid) varietà di colza
(rapeseed) creata per incrocio e selezione per
ridurre il contenuto di ac. Erucico (che era
ritenuto tossico)
Analogamente per migliorare le qualità alimentari
dellolio di lino
ftp//ftp.fao.org/es/esn/food/bio-10t.pdf
33
...e dellolio di girasole
...e di soia
34
Manipolazioni del metabolismo
Large-scale new industrial uses of engineered
plant oils are on the horizon but will require a
better understanding of factors that limit the
accumulation of unusual fatty acid structures in
seeds. Thelen Ohlrogge
(2002)
Major goals Increase content of healthy
fatty acids and reduce unhealthy fatty
acids. Improve oil stability to expand
applications and reduce the need for
hydrogenation. Expand the repertoire of fatty
acids available at low cost and high volume
through exploitation of genetic diversity and
enzyme engineering. Increase oil content to
reduce production costs.
Più recentemente si è sfruttata anche
lingegneria genetica in combinazione con il
breeding
35
Reazioni plastidiali di sintesi e modificazione
degli acidi grassi esplorate nei transgeni(il
numero accanto alle frecce corrisponde al numero
in tabella nella colonna evidenziata in verde)
Thelen and Ohlrogge (2002)
36
Incorporazione degli acidi grassi nei
trigliceridi
Reazioni plastidiali di sintesi e modificazione
degli acidi grassi esplorate nei transgeni
Thelen and Ohlrogge (2002)
37
Selected Examples of Fatty Acid Engineering in
Transgenic Plants
38
Modificazione del profilo di acidi grassi in soia
The first trait-modified crop introduced by
Monsanto, by Pioneer Hi-bred a division of DuPont
and by Asoyia is low linolenic soybean. Achieved
through conventional breeding methods using
marker assisted selection, these seeds have
reduced the linolenic acid level to under 3
(Monsanto and DuPont) and 1 (Asoyia), reducing
oxidation potential. Oil from these seeds
primarily is used for frying and spray coating on
snack foods and crackers. Use of this oil
improves the stability against oxidation and
extends product shelf life and fry life compared
to liquid soybean oil. Not having to hydrogenate
the oil, trans fat levels in food are
significantly reduced while maintaining or
slightly reducing saturated fat levels.
Wilkes RS (2008) Low linolenic soybeans and
beyond. Lipid Technology 20277-279 http//onlinel
ibrary.wiley.com/doi/10.1002/lite.200800072/pdf
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TREUSTM brand High Oleic Soybean TREUS Low
Linolenic Soybean Oil
Low linolenic soybean oil produced from Pioneer
brand soybean varieties, and previously marketed
as NUTRIUM Low Linolenic Soybean Oil, will now be
marketed as TREUS Low Linolenic Soybean Oil.
Acres planted with Pioneer brand low linolenic
soybeans grew from about 35,000 in 2005, 200,000
in 2006, 500,000 in 2007, 1.8 million acres in
2008.
VISTIVE is a new range of winter oilseed rape
with a high oleic, low linolenic fatty acid oil
profile (HOLL) (conventional)
http//www.vistive.eu/about/default.asp
41
Soia ad alto acido oleico
42
Because these soybeans differ from commodity
soybeans in their fatty acid content, an identity
preservation system has been established in the
USA beginning at the farm and includes grain
elevators, processors, and oil refiners to assure
that customers receive identity preserved oil
made from low linolenic acid soybeans.
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Major vegetable oils currently or soon-to-be
available to food processors and other
manufacturers
http//www.mccormickcompany.net/pioneer/cropinsigh
ts/70.pdf
45
palmitoleic 161?9 and cis-vaccenic 181?11
Nguyen et al., (2010) Metabolic engineering of
seeds can achieve levels of ?-7 fatty acids
comparable with the highest levels found in
natural plant sources. Plant Physiol 1541897-904
A plant oil containing high (70) content of ?-7
FA would represent a new and sustainable
feedstock for 1-octene production. Heterologous
expression of the milkweed desaturase in
Arabidopsis failed to produce detectable ?-7 FA,
and when the Doxantha desaturase was expressed in
Brassica napus, it resulted in the accumulation
of only approx. 9 ?-7 FA
46
Biochemical evidence has con?rmed that the fab1
lesion is in KASII. Expression of Com25 in fab1
increased the accumulation of 161?9 and 181?11
to approx. 23 and approx. 16 respectively,
yielding a total of approximately 39 ?-7 FA.
increased ?-7 FA to as much as 71
47
Niente è più insensato di una risposta a una
domanda che non si pone (R. Niehbur)
Primo es. produzione di ac. Laurico
Introdotta una FAT (Fatty acid -ACP Thioesterase)
BTE in colza.
Thelen and Ohlrogge (2002)
48
Rapeseed BTE (Bay Thio Esterase) plants

• Lolio di colza di piante wt contiene solo il 7
  di ac. Grassi saturi (C16 e C18)
• Piante che esprimono una BTE (Bay Thio-Esterase)
  riescono ad accumulare fino al 60 di ac.laurico,
  ma non oltre
• Il limite è probabilmente da attribuirsi alla
  LPAAT endogena di colza che non riesce ad
  incorporare laurato in sn-2
• Trasformare colza con una LPAAT da cocco che
  riesce a incorporare laurato in sn-2

49
LPAAT substrate specificity of CLP-expressing
canola seeds
A pool of mid-maturation seeds from a control
plant (white bars) and from a transgenic plant,
pCGN5511-LP004-5 (black bars), were assayed for
LPAAT substrate specificity using 120-LPA and
various 14C-labeled acyl-CoAs. PA, Phosphatitic
acid.
Knutzon, D. S., et al. Plant Physiol.
1999120739-746
50
Correlation of 120-LPAAT activity with 120
accumulation at sn-2
LPAAT activity using 120-CoA and 120-LPA was
determined in membrane fractions derived from
developing untransformed, control canola seeds,
as well as from independent CLP LP004
transformants. In addition, the proportion of
120 at sn-2 was measured in 20-seed pools of
mature F1 seeds derived from crosses of the same
set of CLP plants with a homozygous
BTE-containing line, DH22. The figure correlates
these two determinations. ?, Control ,
individual CLP  BTE F1 seed lots. PA,
Phosphatitic acid.
Each primary transformant was crossed with DH22,
a homozygous BTE line that contains 51 mol
laurate. Since the BTE parent was homozygous,
each resulting F1 seed should have a complement
of BTE alleles.
Knutzon, D. S., et al. Plant Physiol.
1999120739-746
51
Relationship between total laurate content and
trilaurin
Nelle piante con sola BTE, trilaurina era lt3
anche con il laurato al 47
, Canola lines transformed with BTE alone ?,
seeds from F2, cross CLP X BTE plants -,
theoretical, assuming laurate at random
F1 plants from crosses of several LPAAT
transformants with the 50 mol laurate
containing BTE homozygous line DH22 were grown
and CLP-homozygous F2 lines selected in the next
generation.
Le piante con entrambi i transgeni accumulano più
trilaurina dellatteso sulla base della 120
Seed oil was extracted from transformed canola
plants and the total laurate content of the oil
and proportion of trilaurin compared with other
TAGs in the oil were determined. Each symbol
reflects a sample derived from a seed pool from
one dihaploid plant as described. , Canola lines
transformed with BTE alone ?, seeds from F2
plants resulting from crosses of several
different CLP transformants with the homozygous
BTE plant. The line was calculated by assuming
that laurate was positioned randomly at all three
positions of the triglyceride.
Knutzon, D. S., et al. Plant Physiol.
1999120739-746
52
Laurate proportion at sn-2 is dependent on total
laurate levels and coconut LPAAT
?, CLP-positive plant , CLP-negative plant
The primary CLP transformants were crossed with
the homozygous BTE line. A F1 plant harboring CLP
and BTE alleles was grown. Independently
segregating F2 microspores derived from this
plant were made diploid and grown into
(homozygous) dihaploid plants as described. The
presence or absence of the coconut LPAAT gene in
the individual dihaploid plants was determined
via PCR of leaf tissue. All plants were selfed,
and oil was extracted from the resulting seeds.
The sn-2 analysis of seed oil was executed using
R. arrhizus lipase. Each symbol represents a seed
pool derived from one dihaploid plant. ?,
CLP-positive plant , CLP-negative plant. For
this analysis, we selected the top 12 laurate
producers of the generated dihaploids, as well as
randomly chosen plants throughout the laurate
range.
Knutzon, D. S., et al. Plant Physiol.
1999120739-746
53
Coconut LPAAT can boost laurate levels
Le piante CLP- non riescono ad accumulare oltre
il 60 di laurato.
I transgeni con la sola BTE si fermano al 60 di
laurato (? non riescono ad andare oltre)
Each symbol represents a seed-pool analysis of an
individual dihaploid plant. All dihaploid plants
resulting from crosses described in Figure 4 were
sorted via PCR into a CLP-containing (CLP ) and
a CLP-free (CLP  ) populations. Plants of both
populations were grouped into 1 laurate
intervals.
Knutzon, D. S., et al. Plant Physiol.
1999120739-746
54
Correlation of oil levels with BTE and CLP.
Leccesso viene degradato
Nelle piante CLP- esiste un limite (60)
nellaccumulo di laurato, limite che viene
superato nelle piante CLP (che esprimono LPAAT)
The total seed oil mass as a percentage of dry
weight of the dihaploid plants described in
Figure 4 were determined by NMR. The data are
shown separately for CLP-free (CLP-) and
CLP-containing (CLP) populations, with the oil
percentage plotted against total laurate levels
55
Successful increase in lipid content by
increasing demand (Bouvier-Nave et al., 2000
Maisonneuve et al., 2010 Oakes et al., 2011
Petrie et al., 2012 Taylor et al., 2001, 2009b
Zheng et al., 2008) especially mediated by
augmenting the diacylglycerol transacylase (DGAT)
activity, the major TAG biosynthetic enzyme (for
more examples, see Table 1). When a change in
composition without substantial increase in
overall content is the desired target, this is
achieved by expressing the required biosynthetic
enzymes (be they thioesterases, desaturases,
elongases or hydroxylases) as well as the
acyltransferases or other activities that are
able to incorporate the specific fatty acids into
lipids/TAG (Burgal et al., 2008 Hoffmann et al.,
2008 Li et al., 2010 Mietkiewska et al., 2004
Nguyen et al., 2010 Ruiz-Lopez et al., 2012a
Sayanova et al., 2012 Taylor et al., 2009a
Truksa et al., 2006 Wilkes, 2008 Wu et al.,
2005 additional examples in Ruiz-Lopez et al.,
2012b).
traditional Kennedy pathway (green)
The increased preference for hydroxyacids by a
DGAT or a phospholipiddiacylglycerol
acyltransferase from Ricinus stimulated the
incorporation of ricinoleic acid into TAG from
17 to respectively 30 and 25 of total seed
lipid (Burgal et al., 2008 Kim et al., 2011),
and similar situations were reported for lauric
(Knutzon et al., 1999) and vernolic acid (Li et
al., 2010). When the supply of fatty acids is not
matched by an increase in demand, a futile cycle
of fatty acid degradation via b-oxidation and
sucrose re-synthesis is triggered (Moire et al.,
2004 Poirier et al., 1999 Voelker et al., 1996).
56
The parallel activation of many genes, especially
using transcriptional regulators coordinating
genes involved in TAG synthesis, achieved
remarkable flux increases, even in vegetative
tissues (Andrianov et al., 2010 Gao et al.,
2009 Naqvi et al., 2010 Pouvreau et al., 2011
Sanjaya et al., 2011 Shen et al., 2010 Slocombe
et al., 2009 reviewed in Baud and Lepiniec,
2010 Lu et al., 2011 Weselake et al., 2009).
Both positive and negative regulators of oil
content have been identified (LEC1 and LEC2, WRI,
PKL and ASIL1) and exploited to this purpose
An often overlooked factor limiting lipid
accumulation in Arabidopsis seeds is oxygen,
because hemoglobin overexpression boosts lipid
accumulation by 40 in absolute values per seed
and as percentage of seed dry weight (Vigeolas et
al., 2011). The haemoglobin maintained a higher
ATP/ADP ratio even under low (4) external
oxygen. However strange it may appear, seeds of
various species experience an internal O2
concentration in the 24 range (v/v) (see
references in Vigeolas et al., 2011)
Vigeolas, H., Huehn, D. and Geigenberger, P.
(2011) Nonsymbiotic hemoglobin-2 leads to an
elevated energy state and to a combined increase
in polyunsaturated fatty acids and total oil
content when overexpressed in developing seeds of
transgenic Arabidopsis plants. Plant Physiol.
155, 14351444.
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Bibliografia

• Mittendorf et al., PNAS (1998)
• Somerville and Browse (1996) Trends Cell Biology
  6148-153
• Ohlrogge J. et al, (2000) Fatty acid synthesis
  from CO2 to functional genomics Biochem Soc
  Trans. 28567-73. Review
• http//www.canr.msu.edu/lgc/ database A CATALOG
  OF GENES FOR PLANT GLYCEROLIPID BIOSYNTHESIS
  (Paper TOWARDS A FUNCTIONAL CATALOG OF THE PLANT
  GENOME A SURVEY OF GENES FOR LIPID BIOSYNTHESIS,
  Plant Physiology 122389-401
• Thelen JJ, Ohlrogge JB. (2002) Metabolic
  engineering of fatty acid biosynthesis in plants.
  Metab Eng., 412-21. Review.
• Knutzon, D. S., et al. (1999) Lysophosphatidic
  acid acyltransferase from coconut endosperm
  mediates the insertion of laurate at the sn-2
  position of triacylglycerols in lauric rapeseed
  oil and can increase total laurate levels. Plant
  Physiol. 120739-746
• Klaus D, Ohlrogge JB, Neuhaus HE, Dormann P.
  (2004) Increased fatty acid production in potato
  by engineering of acetyl-CoA carboxylase. Planta
  219389-96.