Una visione sistematica per
l’ingegneria metabolica
Creazione di vie metaboliche
Ingegneria metabolica
probabilmente efficace
probab. inefficace
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
S
S
S
S
S
S
S
S
S
+A
A
A
A
+
A
A
A
A
A
TF
+
B
B
B
B E
B
+
B
B
B
B
Y
Z
W
C
C Q
C F
C
P
P
P Q
P
ATP
P
C
C
C
C
P
P
P
P
X
Espressione di nuovi enzimi
(3)
(4)
S
S
A
A
B
B E
C Q
C F
P
P Q
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
 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
120:739746.
 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. 28:935-7.
 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. 136:2665-75
 Stoutjesdijk PA et al., (2002) hpRNA-mediated targeting of the Arabidopsis FAD2 gene gives highly efficient and
stable silencing. Plant Physiol. 129:1723-31.
 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. 129:1732-43.
 Jain et al., (2000) Enhancement of seed oil content by expression of glycerol-3-phosphate acyltransferase genes.
Biochem Soc Trans. 28:958-61.
 Klaus D, Ohlrogge JB, Neuhaus HE, Dormann P. (2004) Increased fatty acid production in potato by engineering of
acetyl-CoA carboxylase. Planta. 219:389-96.
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 8:277–287
Broun P, Gettner S, Somerville C. (1999) Genetic engineering of plant lipids. Annu Rev Nutr. 19:197-216. Review.
Maisonneuve et al. (2010) Expression of rapeseed microsomal lysophosphatidic acid
acyltransferase isozymes enhances seed oil content in Arabidopsis. Plant Physiol. 152:670-84.
Importanza degli olii
 Produzione di olio vegetale mondiale: ca.
120 Mt/anno (50 Billions $)
 20 Kg/persona/anno; usati in gran parte per
l’alimentazione
 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 (>70% 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.
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 60100 tons FFB/h.
http://lipidlibrary.aocs.org/processing/palmoil/index.htm
http://lipidlibrary.aocs.org/index.html
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
http://chemistry.about.com/librar
y/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++)
Detergent making
http://www.vinythai.co.th/ourchemicalproducts/causticsoda/
mkt1soapdetergent/0,,2009-2-0,00.htm
Fuel making... methanolysis (FAME)
Molecole con parte
polare e parte apolare
Acidi grassi
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
Acidi grassi:
saturi ed insaturi
Le insaturazioni influenzano l’impaccamento dei lipidi nelle
membrane e quindi la temperatura a cui “cristallizzano”
Palmitico, stearico e oleico sono i più abbondanti, L’insaturazione più comune è tra C7 e C8*
Sintesi glicerolipidi
sn-1
sn-2
I carboni del glicerolo3P sono
identificati con questi simboli
sn-3
Sintesi del Diacilglicerolofosfato (acido fosfatidico)
Di e tri-acilgliceroli
Olii e grassi
(animali e
vegetali)
Destino degli acidi grassi
AT
Sintesi di lipidi
nel cloroplasto
FAT
Esporto al RE
Due vie
Entrambe le vie funzionano nelle piante
Overview of lipid synthesis in plants
Via eucariotica
Marcatore della via procariotica
Via procariotica
Lipidi plastidiali derivati dalla
via eucariota
Somerville and Browse (1996) Trends Cell Biology 6:148-153
Sintesi degli
acidi grassi
(cloroplasto)
Sintesi di Acetil-CoA e Malonil-CoA
Acetil-CoA
Citrato
Acetil-CoA
Oxaloacetato
Malonil-CoA
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. 28:567-73. Review
Balle! Il Malonil-CoA viene regolato a livello del demand.
Aumentare il supply non comporta un aumento di flusso
(incorporazione nei lipidi)
Enzimi coinvolti
ACC
Malonyl-CoA transacilasi
KAS III, II & I
FAS - Acido grasso sintasi
Mutanti,
mutanti,
mutanti...
Grandi quantità di lipidi
sono scambiate tra ER
e cloroplasto
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
Lipidi di riserva
C16-C18
Lipidi di membrana
(via procarioti)
Lipidi di membrana
(via eucarioti)
Lipidi a lunga catena
-ossidazione
Lipidi modificati
Lipidi di riserva
Una volta attivati tramite CoA,
gli acidi grassi possono essere
esterificati con glicerolo 3fosfato (G-3-P) per produrre
LPA, PA, DAG e TAG.
Oil bodies
I triacilgliceroli (TAG) si
accumulano negli Oil bodies
che si originano a livello
dell’ER
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 $
I “lipid bodies” sono
circondati da oleosina
Lipidi a lunga catena
FAE – acido grasso elongasi
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
Gli acidi grassi vengono allungati
oltre C18 nel reticolo endoplasmatico
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 ω-7 come ac.
palmitoleico o cis-vaccenico.
Plants synthesize >200 different FA structures with attractive functional properties
Erucic acid
U$6.50/kg (for 1,000kgs)
Erucamide, unsaturated long chain carboxylic acid amide (22:1 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...
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 dell’olio di lino
ftp://ftp.fao.org/es/esn/food/bio-10t.pdf
...e dell’olio di girasole
...e di soia
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 l’ingegneria genetica in combinazione con il breeding
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)
Incorporazione degli acidi grassi nei trigliceridi
Reazioni plastidiali di
sintesi e modificazione
degli acidi grassi
esplorate nei transgeni
Thelen and Ohlrogge (2002)
Selected Examples of Fatty Acid Engineering in Transgenic Plants
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 20:277-279
http://onlinelibrary.wiley.com/doi/10.1002/lite.200800072/pdf
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
Soia ad alto acido oleico
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.
Major vegetable oils currently or soon-tobe available to food processors and other
manufacturers
http://www.mccormickcompany.net/pioneer/cropinsights/70.pdf
palmitoleic 16:1Δ and cis-vaccenic 18:1Δ
9
11
Nguyen et al., (2010) Metabolic engineering of seeds can achieve levels of ω-7 fatty acids
comparable with the highest levels found in natural plant sources. Plant Physiol 154:1897-904
A plant oil containing high (70%) content of
ω-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 ω-7 FA, and when the Doxantha
desaturase was expressed in Brassica napus, it
resulted in the accumulation of only approx.
9% ω-7 FA
Biochemical evidence has confirmed that the fab1
lesion is in KASII. Expression of Com25 in fab1
increased the accumulation of 16:1Δ9 and 18:1Δ11
to approx. 23% and approx. 16% respectively,
yielding a total of approximately 39% ω-7 FA.
… increased ω-7 FA to as much as 71%
Primo es.: produzione di ac. Laurico
“Niente è più insensato di una risposta a una domanda che non si pone”
(R. Niehbur)
Introdotta una FAT
(Fatty acid -ACP
Thioesterase) BTE
in colza.
Thelen and Ohlrogge (2002)
Rapeseed BTE (Bay Thio Esterase) plants
 L’olio 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
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 12:0-LPA and various 14C-labeled acyl-CoAs. PA, Phosphatitic acid.
Knutzon, D. S., et al. Plant Physiol. 1999;120:739-746
Correlation of 12:0-LPAAT activity
with 12:0 accumulation at sn-2
LPAAT activity using 12:0-CoA and 12:0-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 12:0 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. 1999;120:739-746
Relationship between total
laurate content and trilaurin
Nelle piante con sola BTE, trilaurina
era <3% 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 dell’atteso sulla base della [12:0]
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. 1999;120:739-746
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. 1999;120:739-746
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. 1999;120:739-746
Correlation of oil levels with BTE and CLP.
L’eccesso
viene
degradato
Nelle piante CLP- esiste un limite (60%) nell’accumulo 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
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 phospholipid:diacylglycerol
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).
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 2–4% 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, 1435–1444.
Bibliografia
Mittendorf et al., PNAS (1998)
Somerville and Browse (1996) Trends Cell Biology 6:148-153
Ohlrogge J. et al, (2000) Fatty acid synthesis: from CO2 to functional genomics Biochem Soc Trans.
28:567-73. 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 122:389-401
Thelen JJ, Ohlrogge JB. (2002) Metabolic engineering of fatty acid biosynthesis in plants. Metab
Eng., 4:12-21. 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. 120:739-746
Klaus D, Ohlrogge JB, Neuhaus HE, Dormann P. (2004) Increased fatty acid production in potato by
engineering of acetyl-CoA carboxylase. Planta 219:389-96.