Ingegneria metabolica “smart”

Strategie di attivazione parallela

Come si ottiene un aumento di flusso?

* In lievito nello switch tra fermentazione a respirazione (DeRisi, 1997)

* Nel seme durante la mobilizzazione delle riserve lipidiche (Rylott, 2001)

* Sintesi dei lipidi durante l’embriogenesi di Arabidopsis (O’Hara, 2002)

* Altri esempi (vedi Fell)

Esaminiamo alcuni esempi di aumenti di flusso in vivo

Aumentando S

Sottraendo P

Aumentando enzima

Aumentando attività

Causano aumenti locali che faticano a propagarsi lungo la via (dampening)

Diauxic shift in yeastExploring the Metabolic and Genetic Control of Gene Expression on a Genomic Scale (DeRisi

et al., 1997)

Quali sono i geni che vengono attivati e quali vengono disattivati nella transizione da fermentazione a respirazione?

Microarray con tutti i geni di lievito ibridato con mRNA a vari tempi di crescita

Rosso = Aumento

Verde = Diminuzione

Seguiamo i trascritti nel tempo

Passando da fermentazione a respirazione cosa cambia nel

metabolismo?

PYK1

4.9 Variazione

Gene interessato

Rosso = Aumento

Verde = Diminuzione

Molti geni sono regolati in modo simile

Variazione coordinata di molti geni

E’ possibile classificare i geni in base alla regolazione: 6 classi

Lipid mobilization in Arabidopsis

germinating seeds

Schematic representation of the pathways involved in storage lipid mobilization in oilseeds: 1, ACX; 2, multifuctional protein; 3, thiolase; 4, MS; 5, ICL; 6, PEPck.

Northern analysis

(A) Stages of seedling development (B) Northern blot analysis of gene expression

from 0 to 8 days after imbibition

Rylott EL, Hooks MA, Graham IA. (2001) Co-ordinate regulation of genes involved in storage lipid mobilization in Arabidopsis thaliana. Biochem Soc Trans. 29:283-7.

Enzimi coinvolti

ACCMalonyl-CoA transacilasi

KAS III, II & IFAS - Acido grasso sintasi

Lipid synthesis during embryogenesis

FAS Components Exhibit Constant mRNA Ratios

3-oxoacyl-ACP reductase (KR)biotin carboxylase (BC)acyl-ACP thioesterase (TE) enoyl-ACP reductase (ENR)acyl-carrier protein (ACP)

O'Hara, P., et al. Plant Physiol. 2002;129:310-320

Abbondanza relativa dei trascritti

It was demonstrated recently that mRNAs encoding the four subunits of heteromeric (ACCase) acetyl-CoA carboxylase accumulate at a constant molar ratio throughout silique development in Arabidopsis. The ratios were found to be CAC1:CAC2:CAC3:(accD-A & accD-B) = 0.14:1.0:0.17:0.06 (Ke et al., 2000)

Via del triptofano in lievito

Solo la simultanea espressione di molti (tutti) i geni causa un ΔJ paragonabile al ΔEi (ΔJ ≃ CJ x ΔEi )

Evidenze sperimentaliReguloni!

La concentrazione dei metaboliti varia molto meno del flusso

* Rate limiting step concept: more misguided than even MCA initially suggested

* Agire su un solo punto è poco efficace e potrebbe essere deleterio

Il metodo universale mantiene costanti le concentrazioni dei metaboliti [Si]

evita effetti negativi dovuti all’aumento o alla riduzione di [Si]

ReferenzeReferenze ai lavori sugli aumenti naturali in vivo Vedi anche Fell ultimo cap

* DeRisi JL, Iyer VR, Brown PO. DeRisi JL, Iyer VR, Brown PO. (1997) Exploring the metabolic and genetic control of gene expression on a genomic scale. Science. 278:680-6.

* O'Hara P, Slabas AR, Fawcett T. (2002) Fatty acid and lipid biosynthetic genes are expressed at constant molar ratios but different absolute levels during embryogenesis. Plant Physiol. 129:310-20

* Rylott EL, Hooks MA, Graham IA. (2001) Co-ordinate regulation of genes involved in storage lipid mobilization in Arabidopsis thaliana. Biochem Soc Trans. 29:283-7.

* Niederberger P, Prasad R, Miozzari G, Kacser H. (1992) A strategy for increasing an in vivo flux by genetic manipulations. The tryptophan system of yeast. Biochem J. 287:473-9.

* Zhao J, Last RL.(1996) Coordinate regulation of the tryptophan biosynthetic pathway and indolic phytoalexin accumulation in Arabidopsis. Plant Cell. 8:2235-44.

* Eastmond PJ, Rawsthorne S. (2000) Ccoordinate changes in carbon partitioning and plastidial metabolism during the development of oilseed rape embryos. Plant Physiol. 122:767-74

•Universal method: Kacser and Acerenza (1993) A universal method for achieving increases in metabolite production Eur J. of Biochemistry 216:361-367

•Lütke-Eversloh T, Stephanopoulos G. (2008) Combinatorial pathway analysis for improved L-tyrosine production in Escherichia coli: identification of enzymatic bottlenecks by systematic gene overexpression. Metab Eng. 10:69-77.

Espressione di fattori di trascrizione che regolano positivamente gli enzimi della via metabolica

* Terpenoid Indole Alkaloyd (TIA)

* via dei flavonoidi cere, glucinolati...

CAVEAT: ci sono limiti a questa strategia?

Certo, alcuni enzimi come già molto abbondanti (es. quelli del calvin o glicolitici)

Ingegneria metabolica “in batch”

P

C

A

B

TF+

S

(6)

+

+

+Usando i fattori di trascrizione probabilmente si mantengono le “giuste proporzioni tra gli enzimi

Fig. 1. Biosynthesis of TIAs in C. roseus.

Solid arrows indicate single enzymatic conversions, whereas dashed arrows indicate multiple enzymatic conversions.

Abbreviations of enzymes:

AS, anthranilate synthase; DXS, D-1-deoxyxylulose 5-phosphate synthase; G10H, geraniol 10-hydroxylase; CPR, cytochrome P450-reductase; TDC, tryptophan decarboxylase; STR, strictosidine synthase; SGD,strictosidine b-D-glucosidase; D4H, esacetoxyvindoline 4-hydroxylase; and DAT, acetyl-CoA:4-O-deacetylvindoline 4-O-acetyltransferase.

Genes regulated by ORCA3 are underlined.

Numerosi enzimi della via sono stati identificati e clonati. Esiste un fattore di trascrizione capace di attivarli tutti insieme?

T-DNA activation tagging

Struttura del T-DNA

Punto di inserzione del T-DNA nel genoma

ORF attivata dall’inserzione

Linea cellulare selezionata con inibitori delle TDC. L’inserzione del T-DNA porta ad un aumento del flusso nella via

Molti altri geni della stessa via sono indotti nella linea cellulare

Il metabolismo secondario: Flavonoidi, Antociani e

Lignina

Genes encoding all enzymes indicated in

red are clock-controlled

Alcuni geni sembrano essere regolati in maniera molto simile dal punto di vista temporale. Può essere segno di un controllo comune mediato cioè dallo stesso fattore di trascrizione?

Myb transcription factor PAP1

I geni in rosso sono implicati nella biosintesi dei

fenilpropanoidi e sono controllati dal ritmo circadiano

Activation tagging

Il mutante pap1-D presenta una colorazione rossa (carattere dominante) e accumula antocianine (una classe di flavonoidi)

Molti geni della via dei fenilpropanoidi (e sue diramazioni: flavonoidi, antocianine) sono espressi maggiormente nel mutante.

Il mutante pap1-D presenta una maggiore attività enzimatica e più lignina.

La sovraespressione di Pap1 o Pap2 in Tabacco o Arabidopsis porta ad un’intensa pigmentazione

Come identificare i fattori implicati nella trascrizione di vie metaboliche

mutanti classici (indotti o spontanei) gene

activation tagging o sovraespressione

Coregolazione elementi comuni in cis elementi comuni in trans (?) identificazione del fattore tramite One-hybryd

Identificazione….

Attenzione: i fattori di trascrizione sono enzimi (?) e spesso agiscono in sinergia

Geni regolatori in Anthyrrinum majus

Immagini cortesia del prof. C. Martin

Diversi geni della via sono down-regulated nel mutante delila ma solo nella zona con ridotta pigmentazione

TubeLobe

Tobacco crosses: 35S:Del x 35S:Ros1Tobacco crosses: 35S:Del x 35S:Ros1

Immagini cortesia del prof. C. Martin

Piante di Arabidopsis che sovraesprimono uno solo dei due fattori non mostrano accumulo. Quando sono coespressi l’aumento di flusso è notevole.

Sinergismo!

Rosea1 + Delila can give 100-fold + activation and anthocyanin levels of up to 10 mg/g fwt. They can also increase flux through pathway branches 2.5-fold. Other regulatory combinations are not so potent

Immagini cortesia del prof. C. Martin

Fattori di trascrizione coinvolti nella regolazione del metabolismo in pianta

Broun P. (2004) Transcription factors as tools for metabolic engineering in plants. Curr Opin Plant Biol. 7:202-9.

Altri esempi:

- Cernac et al. (2006) The WRI1 gene encodes an AP2/EREBP transcription factor involved in the control of metabolism, particularly glycolysis, in the developing seeds. Plant Physiology 141:745-757. - Xie et al. (2006) Metabolic engineering of proanthocyanidins through co-expression of anthocyanidin reductase and the PAP1 MYB transcription factor. Plant J. 45:895-907. - Metabolismo degli olii in foglia: Santos Mendoza et al., (2005) FEBS Lett. 579:4666-4670. LEAFY COTYLEDON 2- Kannangara et al. (2007) The transcription factor WIN1/SHN1 regulates Cutin biosynthesis in Arabidopsis thaliana. Plant Cell. 2007 Apr;19(4):1278-94. - Aharoni et al. (2004) The SHINE clade of AP2 domain transcription factors activates wax biosynthesis, alters cuticle properties, and confers drought tolerance when overexpressed in Arabidopsis. Plant Cell. 16:2463-80.- Baud and Lepiniec (2009) Regulation of de novo fatty acid synthesis in maturing oilseeds of Arabidopsis, Plant Physiol. Biochem. 47:448–455.- Ruuska et al. (2002) Contrapuntal networks of gene expression during Arabidopsis seed filling, Plant Cell 14:1191–1206.- Shen et al. (2010) Expression of ZmLEC1 and ZmWRI1 increases seed oil production inmaize, Plant Physiol. 153:980–987.- Pouvreau et al. (2011) Duplicate maize Wrinkled1 transcription factors activate target genes involved in seed oil biosynthesis, Plant Physiol. 156:674–686.- Zhang et al. (2002) Similarity of expression patterns of knotted1 and ZmLEC1 during somatic and zygotic embryogenesis in maize (Zea mays L.), Planta 215:191–194.- Maeo et al. (2009) An AP2-type transcription factor, WRINKLED1, of Arabidopsisthaliana binds to the AW-box sequence conserved among proximal upstream regions of genes involved in fatty acid synthesis, Plant J. 60:476–487.

WIN1: wax inducer (biosintesi delle cere)

Activation of wax production in Arabidopsis plants that overexpress WIN1, an ERF-type transcription factor, and concurrent induction of wax pathway genes. Morphological phenotype of (a) a control (wt) and (b) 35S::WIN1 plants. Note the glossy appearance of 35S::WIN1-overexpressing leaves. Scanning electron microscope (SEM) images of (c) control and (d) 35S::WIN1 leaf surfaces: WIN1 overexpressors produce wax crystals, which are absent from control leaves. (Magnification: 3000x.) Stomatal cells are shown at the centre of the images. (e) Northern analysis of the expression of wax pathway genes in 35S::WIN1 and control plants: KCS1, which encodes a putative fatty acid elongase, and CER1, encoding a putative fatty acid decarbonylase, are induced in 35S::WIN1 plants.

Northern and microarray analyses of 35S::WIN1 plants indicated that several genes that are implicated in wax biosynthesis, such as ECERIFERUM1 (CER1) and 3-KETOACYL-COA SYNTHASE1 (KCS1), were upregulated in the WIN1-overexpressors

Broun P, Poindexter P, Osborne E, Jiang C-Z, Riechmann JL: WIN1, a transcriptional activator of epidermal wax accumulation in Arabidopsis. Proc Natl Acad Sci USA 2004, 101(13):4706-11

wt

35S::WIN1 35S::WIN1

b and c are representative of medium, and high levels of leaf glossiness

Total fatty acids per seed for the untransformed mutant (wri1) and wild type (WT) (a), and transgenic lines in the wri1 background (b) or the wild type background (c).

Lipid and fatty acid compositions, after LEC2:GR induction in leaves

Fatty acid composition

Lipid composition.

Transcriptional regulation of triacylglycerol biosynthesis in maturing seeds of Arabidopsis thaliana

LEAFY COTYLEDON1 (LEC1), LEC2, ABSCISIC ACID INSENSITIVE3 (ABI3), and FUSCA3 (FUS3) arenormally expressed predominantly in seeds, can induce the deposition of seed oil in vegetative tissues when ectopically activated in seedlings.

Family TF Name Summary of Role in Seed Oil Deposition

B3 domain;

AFL Clade

ABSCISIC ACID INSENSITIVE3 (ABI3),LEAFY COTYLEDON2 (LEC2),FUSCA3 (FUS3)

Master regulators of embryogenesis and seed maturation; mutation/overexpression often associated with pleiotropic effects; direct and indirect regulation of suites of genes involved in carbohydrate and lipid metabolism, including fatty acid synthesis, triacylglycerol assembly and packaging

HAP3/CBP

LEAFY COTYLEDON1 (LEC1),LEC1-LIKE (L1L)

Subunits of CCAAT binding proteins; capable of working independently of CBP; master regulators of embryogenesis and seed maturation; direct and indirect regulation of genes involved in carbohydrate and lipid metabolism

AP2 WRINKLED1 (WRI1)

Direct target of master regulators having more specific role towards seed oil biosynthesis; mutants dramatically reduced in seed oil content and wrinkled appearance; direct and indirect regulation of carbohydrate and lipid metabolism genes, particularly plastidial fatty acid synthesis

Dof GmDof4 GmDof11Transgenic expression yields higher seed oil levels; direct and indirect regulation of lipid metabolism genes; possible negative regulators of seed storage proteins

CHD3 PICKLE (PKL)Putative chromatin remodeling factor; represses master regulator genes at germination; associated with the repressive chromatin mark H3K27me3

PRC2

FERTILIZATION INDEPENDENTENDOSPERM (FIE),SWINGER (SWN),EMBRYONIC FLOWER2 (EMF2)

Components of Polycomb Repressive Complex 2 that catalyze deposition of H3K27me3; repressors of seed maturation genes in vegetative tissues

B3 domain;HSI2 Clade

HIGH-LEVEL EXPRESSION OF SUCROSE INDUCIBLEGENE2 (HSI2)/VAL1,HSI2-LIKE1 (HSIL1/VAL2),HSL2/VAL3

Act redundantly to repress AFL Clade genes and other positive regulators of seed maturation during germination and in seedlings; possible chromatin remodeling activities

AP2 APETALA2 (AP2)Negative regulator of seed size, possibly via carbohydrate metabolism in the seed coat; effects on seed oil deposition likely indirect

HD-ZIP GLABRA2 (GL2)Negative regulator of oil content; loss of seed mucilage proposed to make more C available for fatty acid synthesis

Zhong and Ye (2009) Transcriptional regulation of lignin biosynthesis. Plant Signal Behav. 4:1028-34.

How universal is the “universal method” in vivo?

According to Metabolic Control Analysis, the parallel activation (multisite modulation) of enzymes within a biochemical pathway is the optimal strategy for changing fluxes retains metabolite and control homeostasis

If a mRNA level changes, what happens to other ones in the same metabolic pathway?

PSY (Phytoene Synthase)

PD

S (

Ph

ytoe

ne

Des

atu

rase

)

Two-gene scatterplot

Pearson correlation coefficient

mRNA is not equal to protein flux changes over long times

Use data from many different tissues, mutants, conditions…

A square matrix

At3g21500 At4g15560 At5g11380 At5g62790 At2g02500 At2g26930 At1g63970 At5g60600 At4g34350

At3g21500 1.00 0.12 0.01 0.03 0.04 0.20 0.25 0.19 0.19

At4g15560 0.12 1.00 0.35 0.75 0.66 0.65 0.66 0.73 0.73

At5g11380 0.01 0.35 1.00 0.35 0.31 0.40 0.21 0.18 0.15

At5g62790 0.03 0.75 0.35 1.00 0.69 0.70 0.78 0.78 0.68

At2g02500 0.04 0.66 0.31 0.69 1.00 0.77 0.72 0.67 0.56

At2g26930 0.20 0.65 0.40 0.70 0.77 1.00 0.80 0.67 0.60

At1g63970 0.25 0.66 0.21 0.78 0.72 0.80 1.00 0.88 0.74

At5g60600 0.19 0.73 0.18 0.78 0.67 0.67 0.88 1.00 0.77

At4g34350 0.19 0.73 0.15 0.68 0.56 0.60 0.74 0.77 1.00

PSY (Phytoene Synthase)

PS

Y (

Ph

ytoe

ne

Syn

thas

e)

From numbers to coloursGene A Gene B

Gene A

Essentially the same strategy published recently by Toufighi K, et al. (2005) Plant J. 43:153-63

The Botany Array Resource: e-Northerns, Expression Angling, and promoter analyses.

Gene B

Gene ABCDEFGHIJKLMNOPQRS

Coregulated genes close in the list will appear as a red square

Group 1

Group 3Group 1 & 3

are coregulated

The Red Square…

Apply the correlation analysis to the entire “metabolic genome” (enzymes, transporters….)

Isoprenoid biosynthesis

two indipendent pathways in plants:

BA

Lange and Ghassemian (2003) Genome organization in Arabidopsis thaliana: a survey for genes involved in isoprenoid and chlorophyll metabolism. Plant Mol Biol. 51:925-48.

A cytosolicB plastidial

B

Plastidial pathway:

Carotenoids Phytyl Plastoquinone Phylloquinone Tocopherol Mono-terpenes Phytochrome Gibberellic acid Abscissic acid.

Figure from Lange and Ghassemian (2003)

500

genes

1000 2000 27501414

500 genes

100 genes

◄ GGPP synthases: 10 isoforms

Plastidial IPP

Cytosolyc IPP (meval.)

Carotenoid

Chlorophyll

Which GGPP synthase isoform works in the carotenoid pathway?

GGPPPhytyl PP

Chlorophyll

Prenyl group

GGPP synthase

At3g29430 and At3g32040 provide GGPP for…

At4g36810(At4g38460)

(At3g20160) At3g29430 At3g32040

GA

At3g29430

At3g29430 1.0000 geranylgeranyl pyrophosphate synthase, putativeAt3g29410 0.8313 terpene synthase/cyclase family proteinAt4g33720 0.7440 pathogenesis-related protein, putativeAt5g15180 0.7352 peroxidase, putativeAt1g53940 0.7341 GDSL-motif lipase/hydrolase family proteinAt2g24400 0.7081 auxin-responsive protein, putative / small auxin up RNA (SAUR_D)At5g59680 0.7022 leucine-rich repeat protein kinase, putativeAt5g24410 0.6942 glucosamine/galactosamine-6-phosphate isomerase-relatedAt1g73780 0.6873 protease inhibitor/seed storage/lipid transfer proteinAt3g47210 0.6867 expressed proteinAt3g59370 0.6865 expressed proteinAt1g33900 0.6857 avirulence-responsive protein, putativeAt5g03570 0.6855 iron-responsive transporter-relatedAt3g32040 0.6847 geranylgeranyl pyrophosphate synthase, putativeAt1g21210 0.6831 wall-associated kinase 4At5g37450 0.6827 leucine-rich repeat transmembrane protein kinase, putativeAt1g11540 0.6810 expressed proteinAt3g49860 0.6770 ADP-ribosylation factor, putativeAt2g31085 0.6739 Clavata3 / ESR-Related-6 (CLE6)At1g49030 0.6734 expressed proteinAt1g66020 0.6725 terpene synthase/cyclase family proteinAt3g05950 0.6709 germin-like protein, putativeAt5g15725 0.6668 expressed proteinAt3g01190 0.6644 peroxidase 27 (PER27) (P27) (PRXR7)At4g31875 0.6620 expressed proteinAt2g38600 0.6569 acid phosphatase class B family proteinAt3g46400 0.6532 leucine-rich repeat protein kinase, putative

At3g29430 is possibly involved in terpene synthesis

Migliori correlatori tra tutti i geni di Arabisopsis

(R value in linear plots)

Calvin cycleAt4g26520 fructose-bisphosphate aldolase, cytoplasmicAt4g26530 fructose-bisphosphate aldolase, putativeAt4g38970 fructose-bisphosphate aldolase, putativeAt2g21330 fructose-bisphosphate aldolase, putativeAt5g56630 phosphofructokinase family proteinAt5g47810 phosphofructokinase family proteinAt4g32840 phosphofructokinase family proteinAt2g22480 phosphofructokinase family proteinAt4g26390 pyruvate kinase, putativeAt3g55440 triosephosphate isomerase, cytosolic, putativeAt2g29560 enolase, putativeAt1g07110 fructose-6-phosphate 2-kinase / fructose-2,6-bisphosphatase (F2KP)At1g13440 glyceraldehyde 3-phosphate dehydrogenase, cytosolic, putativeAt1g42970 glyceraldehyde-3-phosphate dehydrogenase B, chloroplast (GAPB)At3g26650 glyceraldehyde 3-phosphate dehydrogenase A, chloroplast (GAPA)At3g04120 glyceraldehyde-3-phosphate dehydrogenase, cytosolic (GAPC)At3g12780 phosphoglycerate kinase, putativeAt1g58150 hypothetical proteinAt1g56190 phosphoglycerate kinase, putativeAt1g22170 phosphoglycerate/bisphosphoglycerate mutase family proteinAt1g78040 pollen Ole e 1 allergen and extensin family proteinAt3g08590 2,3-biphosphoglycerate-independent phosphoglycerate mutaseAt5g04120 phosphoglycerate/bisphosphoglycerate mutase family proteinAt3g22960 pyruvate kinase, putativeAt5g52920 pyruvate kinase, putativeAt2g21170 triosephosphate isomerase, chloroplast, putativeAt5g61410 ribulose-phosphate 3-epimerase, chloroplast, putative /At1g71100 ribose 5-phosphate isomerase-relatedAt3g04790 ribose 5-phosphate isomerase-relatedAt2g45290 transketolase, putativeAt3g60750 transketolase, putativeAt1g32060 phosphoribulokinase (PRK) / phosphopentokinaseAt1g43670 fructose-1,6-bisphosphatase, putativeAt3g54050 fructose-1,6-bisphosphatase, putativeAt3g55800 sedoheptulose-1,7-bisphosphatase, chloroplastAt5g35790 glucose-6-phosphate 1-dehydrogenase / G6PD (APG1)At1g09420 glucose-6-phosphate 1-dehydrogenase, putative / G6PD, putativeAt5g24420 glucosamine/galactosamine-6-phosphate isomerase-relatedAt5g24410 glucosamine/galactosamine-6-phosphate isomerase-relatedAt3g49360 glucosamine/galactosamine-6-phosphate isomerase family proteinAt1g13700 glucosamine/galactosamine-6-phosphate isomerase family proteinAt5g44520 ribose 5-phosphate isomerase-relatedAt2g01290 expressed proteinAt5g39320 UDP-glucose 6-dehydrogenase, putativeAt5g64290 oxoglutarate/malate translocator, putativeAt5g35630 glutamine synthetase (GS2)At4g37930 glycine hydroxymethyltransferaseAt1g23310 glutamate:glyoxylate aminotransferase 1 (GGT1)At3g19710 branched-chain amino acid aminotransferase, putativeAt1g32450 proton-dependent oligopeptide transport (POT) family protein

3.5 in log scale >3000

Reducing glucosinolates in Arabidopsis

Glucosinolates are sulphur rich compounds from brassicas

Some beneficial, other toxic (quantity!)Upon wounding are converted into toxic

productsTwo branchesMutants isolated

Aliphatic GSL

Indolic GSL

Short chain

Long chain

Beekwilder et al., (2008) PLoS 3:e2068.

Glucosinolate pathway

Phase 2 - core structure synthesis

Amino Acid

S-AlkylThioidroximate

GSTs

Aci-Nitro compoundCYP83s

Thioidroximate

C-S Lyase

Desulfo-glucosinolate

UGTs

GlucosinolateST5s

AldoximeCYP79s

Cytoplasm

Step 1: Oxidation Step 2: Oxidation

Step 5: GlucosylationStep 6: Sulfatation

Step 3: Conjugation

Step 4: C-S Clevage

Glucosinolates: sulfur-rich secondary metabolites

Amino acid

Transamination

Oxydativedecarboxylation

Isomerization

Condensation

Chloroplast

Phase 1 - side chain elongation

Oxo-acid

2-alkyl-malic acid

3-alkyl-malic acid

Export

Oxo-acid

Amino acid (n+1)C

Several rounds of chain elongation are possible

Kroymann et al., Plant Physiology (2001) 127:1077–1088,

Phase 3 - Side Chain Modification

Various oxidations on the side chain

Cytoplasm

compartimentation -transport

SAT 52 – Serine O-acetyltrasferaseCysteine SynthaseGlycosil hydrolase family 1 proteinABC Transporter

At3g49680At3g19710At5g23010At3g58990At4g13430At2g43100At4g13770At3g03190At1g78370At1g74090At1g18590At2g46650At1g65860At1g62560At4g12030At5g61420At1g21440At4g03060At4g03050

At5g05260At4g39950At2g22330At4g31500At1g74100At2g30870At1g27130At2g30860At4g30530At5g05730At5g17990At3g54640At2g04400At4g39980At5g56760At3g59760At2g04400At1g59870At2g20610At1g24100At4g39940At2g14750

Cytochrome b5Flavin-contaning monooxygenaseFlavin-contaning monooxygenaseBile acid Sodium symporterMYB 28Mutase family proteinAOP2 - DioxygenaseAOP3 -Dioxygenase

CYP79A2

CYP83B1

ST5a

SUR1

AKN2

AKN1

UGT74B1

F17I23

CYP79B2

CYP79B3

ASA1

TSA1

ATGSTF10

CYP83A1

AOP3

AOP2

ATGSTU13

IGPS

DHS1

OASC

BCAT4

B5 #1

MAM1

F17J16

ST5b

MYB28

F16J13

MFL8

ATGSTF11

ST5c

T3P18

ATGSTU20

F28J8

T9E8

TRP 1

BCAT3

F12P19

ATGSTF9

CYP79A2CYP79B2CYP79B3CYP83B1ST5a - SulfotransferaseGlutathione S-TransferaseGlutathione S-TransferaseGlutathione S-Transferase

GLUCOSINOLATE FROMTRYPTOPHAN AND PHENYLALANINE

Anthranilate synthaseASA1 -Anthranilate synthase α subunitTSA1 - Trp synthase, alpha subunitTRP1- P-ribosyl-anthranilate synthaseIGPS Indole-3-glycerol p synthaseDHS1 – DAHP synthetase 1

TRYPTOPHAN BIOSYNTHESIS

SUR1 - C-S LyaseUGT74B1 – S-Glucosil TrasferaseAKN2 – Adenylylsulfate kinase 2AKN1 – Adenylylsulafte kinase 1

SHARED GENES(PAPS BIOSYNTHESIS,C-S LYASE AND GLUCOSYL

TRANSFERASE)

BCAT3BCAT4MAM 1 – 2 isopropylmalate synthase 3Aconitase C-terminal domainAconitase family proteinAconitase C-terminal domain

Branched-chain aminoacid aminotransferase

HOMOMETHIONINE BIOSYNTHESIS

CYP83A1Glutathione-S TransferaseGlutathione-S TransferaseST5b – SulfotransferaseST5c – Sulfotransferase

GLUCOSINOLATE FROM HOMOMETIONINE

SAT52

PEN3PEN2

Phase II – GLS from Trp and Phe

TRP Biosynthesis

Phase II Shared genes(PAPS Biosynthesis, C-S

Lyase, Glucosyl Transferase)

Phase I - Homomet Biosynthesis

Phase II – GLS from Homomet

Phase III, transport and regulation – GLS from

HOMOMET

Aromatic branch

Aliphatic branch

CYP83A1

BCAT4

B5 #1

MAM1

F17J16

ST5b

MYB28

F16J13

MFL8

ATGSTF11

ST5c

SUR1

T3P18

ATGSTU20

F28J8

T9E8

METHIONINE SIDE-CHAINELONGATION

Phase I and II enzymes are co-regulating

Monooxygenase “GLUCOSINOLATE FROM FENIL.-OMOMET.”

Glutathione S-transferase

Glutathione S-transferase

C-S Lyase “GLUCOSIN. FROM PHENILAL-TRYPT-HOMOMET.”

Sulfotransferase “GLUCOSINOLATE FROM HOMOMET.”

Sulfotransferase “GLUCOSINOLATE FROM HOMOMET.”

Aminotransferase“HOMOMET.–LEUCINE BIOSYNTHESIS”

2-isopropylmalate Synthase “HOMOMET BIOSYNTHESIS”

Aconitase C-terminal domain “LEUC.-HOMOMET.BIOSYNTHESIS”

Aconitase C-terminal domain“HOMOMET. BIOSYNTHESIS”

Aconitate hydratase

Sodium symporter family protein

Transcription factor

Cytochrome b5

Flavin conteining monooxygenase family protein

Mutase family protein

At4g13770At3g03190At1g78370At2g20610At1g18590At1g74090At3g19710At5g23010At3g58990At2g43100At4g13430At4g12030At5g61420At2g46650At1g62560At1g21440

GLUCOSINOLATE BIOSYNTHESIS

Phase II - GLS biosynthesis (Met derived)

Phase I - GLS biosynthesis (Met derived) SIDE-CHAIN

ELONGATION

Candidate genes for transport, regulation... (MET derived GLS)

O

NH2

SCH3 OH

Methionine

O

O

SCH3 OH

2 oxo 4 methylthio butanoic acid

OH OS

CH3

OHO

OH

2-(2'methytio)ethyl malic acid

OH

O

SCH3 OH

O

OH3-(2'methytio)ethyl malic acid

O

O

SCH3 OH

2 oxo 5 methylthio pentanoic acid

SCH3 NH2

OOH

Homomethionine

Aminotransferase Methylthioalkylmalate synthase

Aconitase

AcetylCoA CoA

At5g23100At3g19710

At3g58890

At2g43100

-Chetoglutarato An amino acid

NAD+NADH

CO2

-Chetoglutarato An amino acid

MAM1BCAT4

BCAT3

Myb28 (At5g61420)

ATG

SALK_136312

PROM EX3

LBa1 LB51

134 214 484344

TGA

ATG

BRC_H161Lb

16231

Effect of knocking out Myb28?

MYB 28

0

2

4

6

8

10

12

14

16

C 2 6 9 C 2 6 9

Leaf Root

RG

E

MYB 29

-0.5

0.0

0.5

1.0

1.5

2.0

C 2 6 9 C 2 6 9

Leaf Root

RG

E

MAM-L

-50

0

50

100

150

200

250

300

350

400

C 2 6 9 C 2 6 9

Leaf Root

RG

E

CYP83A1

-0.2

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

C 2 6 9 C 2 6 9

Leaf Root

RG

E

Aconitase

-0.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

C 2 6 9 C 2 6 9

Leaf Root

RG

E

RT-PCR on 2 controls and 2 KOs

Wt and Myb28-KO metabolome

Time5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00

%

-2

98

5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00

%

-2

98

5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00

%

-2

98

5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00

%

-2

98

M09108 1: TOF MS ES- BPI

3.42e426.481394

339.0445

20.171062

447.0345

18.98999

739.1770

4.38230

436.0256

2.90152

436.038217.53;922;420.0454

5.31279

565.046010.88572

323.1347

32.861729

477.0618

27.461445

505.1335

37.261962

487.1212

39.662088

223.0983

46.992474

333.1882

M09107 1: TOF MS ES- BPI

3.43e420.061055

492.0634

4.42232

436.01792.74144

422.0223

18.96998

739.179414.40757

478.0881

5.24275

565.045510.88572

323.1338

26.451392

339.0452

54.25;2856;791.4733

51.522712

476.1041

32.861729

477.0614

27.441444

505.1348

42.602242

478.085637.261962

487.1232

46.972473

333.1925

M09106 1: TOF MS ES- BPI

3.42e426.411390

339.0430

20.17;1062;447.02924.38230

436.020516.79883

385.1132

5.29278

565.043610.91574

323.1335

54.22;2854;791.475332.801727

477.0533

27.441444

389.1235

37.261962

487.1258

51.502711

1046.5104

39.612085

223.0984

46.972473

333.1924

M09105 1: TOF MS ES- BPI

3.43e420.121059

492.0636

4.40231

436.01692.78145

422.0237

18.98999

739.173214.40757

478.0864

5.27277

565.047110.91574

323.1330

26.401389

339.0451

54.34;2861;791.4747

51.602717

492.0991

32.861729

477.0608

27.461445

389.1248

42.622243

478.087537.261962

487.1224

51.142692

552.234846.992474

333.1934wt

ko

Methylsulfinyloctyl

Methylsulfinylheptyl

GSL unknown

myb28, myb29 and myb28myb29

Mutating Myb28 and Myb29

Beekwilder et al., (2008) PLoS 3:e2068.

Regulators

Reducing glucosinolate content...

...stimulates pest growth and damage!

Beekwilder et al., (2008) PLoS 3:e2068

Insect feeding

Effect of the double KO

Too late!

What is the distribution of all the R values in the matrix?

2828 genes

La spalla di valori alti e positivi di R all’interno dei geni metabolici è la testimonianza che esiste molta coregolazione

Open issuesExplore enzyme subsets

Pathway identification

Clustering of enzymes

Shared cis-elements / regulators

Suggest substrate for enzymes / trasporters

LimitationsOther levels of regulation

Co-regulation does not mean necessarily…

At5g57800At5g57800 1 CER1 protein, putative (WAX2)At5g20270 0.8287 expressed proteinAt2g26250 0.8044 beta-ketoacyl-CoA synthase family (FIDDLEHEAD) (FDH)At3g43720 0.7894 protease inhibitor/seed storage/lipid transfer protein (LTP) family proteinAt1g17840 0.7892 ABC transporter family proteinAt1g68530 0.7864 very-long-chain fatty acid condensing enzyme (CUT1)At4g39330 0.7792 mannitol dehydrogenase, putativeAt2g26910 0.7755 ABC transporter family proteinAt5g13400 0.7735 proton-dependent oligopeptide transport (POT) family proteinAt4g25960 0.7679 multidrug resistance P-glycoprotein, putativeAt5g14410 0.766 expressed proteinAt1g02205 0.7563 CER1 protein (another?)At1g51500 0.7379 ABC transporter family proteinAt2g04570 0.7234 GDSL-motif lipase/hydrolase family protein

One vs. all analysis for At5g57800 CER1 protein, putative (WAX2) (Log)

CUT1 (very-long-chain fatty acid condensing enzyme, At1g68530) shows good correlation with At1g51500 (R=0.815), an ABC transporter protein

Transporters

Pighin et al., Science (2004) 306:622-625

WT

cer5

cer5

Cer5 (At1g51500)

Wax analyses of Arabidopsis stem surface (cuticle) or epidermal peel extracts (total epidermis).

ProgrammaRipasso di cinetica enzimatica e approccio classico al controllo dei flussi [1,6]. Fondamenti di Analisi del Controllo Metabolico (MCA): proprietà locali e sistemiche, elasticità e coefficienti di controllo del flusso e delle concentrazione [1,6,7]. Trattazione dei sistemi Supply-Demand in generale [8] e dell’ATP in particolare [9]. Rate limiting steps e ingegneria metabolica [10, 11 e 12].

Tipi di ingegneria metabolica: a- Inattivazione di enzimi e allergeni (via del gossipolo [13], ODAP e glucosidi cianogenici) e review generale [14]); b- Creazione di vie metaboliche ex novo o potenziamento di vie endogene già presenti (Glucosidi cianogenici [15,16], Vitamina E [17, 18], Folato [19], laurato [20, 21]); c- Aumento del demand (aumento del contenuto in aa, aumento del contenuto in zucchero) [22-24]; e- Amido in patata: strategie diverse [25]; f- Utilizzo dei fattori di trascrizione (Terpenoid Indole Alkaloyd, Flavonoidi, cuticola, glucosinolati...) [10,11,26].

Bibliografia (ref 2-4 sono testi generali sul metabolismo delle piante e la sua manipolazione)

Generali (MCA e metabolismo):[1] Fell, Understanding the control of Metabolism Portland Press (1997) (in Biblioteca biologica)[2] Dennis/Turpin Plant Metabolism (1998) Longman; nuova edizione.[3] Lea/Leegood Plant Biochemistry and Molecular Biology (1993) Wiley & sons.[4] Foyer e Quick (Eds) A molecular approach to primary metabolism in higher plants; Taylor and Francis (1997)

Articoli originali[6] Kacser, Burns, & Fell, The control of flux (1995) Biochem. Soc. Trans. 23, 341-366 (art. del 1973).[7] Kacser e Acerenza, Eur. J. Biochem. (1993) 216:361-367[8] Hofmeyr & Cornish-Bowden (2000) Regulating the cellular economy of supply and demand. FEBS Lett. 476:47-51.[9] Koebmann et al. (2002) The glycolytic flux in Escherichia coli is controlled by the demand for ATP. J. Bacteriol. 184:3909-16[10] Morandini & Salamini (2003) Plant biotechnology and Breeding, allied for years to come Trends Pl. Sci. 8:70-5.[11] Morandini, Salamini & Gantet, (2005) Engineering of Plant Metabolism for Drug and Food. Curr. Med. Chem. – Immun., Endoc. & Metab. Agents 5:103-112[12] Morandini (2009) Rethinking metabolic control. Plant Science 176:441-451[13] Sunilkumar et al., (2005) Engineering cottonseed for use in human nutrition by tissue-specific reduction of toxic gossypol. P.N.A.S. 103:18054–18059.[14] Morandini (2010) Inactivation of allergens and toxins. N Biotechnol. 27:482-93.[15] Tattersall DB et al., (2001) Resistance to an herbivore through engineered cyanogenic glucoside synthesis. Science 293:1826-8. [16] Nielsen et al., (2008) Metabolon formation in dhurrin biosynthesis. Phytochemistry 69:88-98.

[17] DellaPenna D. (2005) Progress in the dissection and manipulation of vitamin E synthesis. Trends Plant Sci 10:574-9.[18] Valentin (2006) The Arabidopsis vitamin E pathway gene5-1 mutant reveals a critical role for phytol kinase in seed tocopherol biosynthesis. Plant Cell. 18:212-24.[19] Hossain et al. (2004) Enhancement of folates in plants through metabolic engineering. Proc Natl Acad Sci USA 101:5158–5163.[20] 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.[21] Thelen JJ, Ohlrogge JB. (2002) Metabolic engineering of fatty acid biosynthesis in plants. Metab Eng. 4:12-21.[22] Chong et al. (2007) Growth and metabolism in sugarcane are altered by the creation of a new hexose-phosphate sink. Plant Biotechnol J. 5:240-53.[23] Wu (2007) Doubled sugar content in sugarcane plants modified to produce a sucrose isomer. Pl. Biotech. J. 5:109-17.[24] Basnayake S. (2012) Field performance of transgenic sugarcane expressing isomaltulose synthase. Plant Biotechnology Journal 10:217-225[25] Geigenberger et al., (2004) Metabolic control analysis and regulation of the conversion of sucrose to starch in growing potato tubers. Plant, Cell and Environment 27:655–673.[26] Broun P. (2004) Transcription factors as tools for metabolic engineering in plants. Curr Opin Plant Biol. 7:202-9.

In rosso sono evidenziati quelli da leggere con attenzione ai fini dell’esame.Ulteriori riferimenti bibliografici si trovano nei singoli file di powerpoint delle lezioni. Chiunque desiderasse gli articoli originali basta me li chieda.

Cosa è naturale?

L’uomo fa parte della natura?

Da cosa viene la specialità dell’uomo? Su cosa si fonda?

Gli esseri umani e la tecnologia sono una cosa sola?

Un compito...

Il docente universitario ha il compito non solo di indagare la verità e di suscitarne perenne stupore, ma anche di promuoverne la conoscenza in ogni sfaccettatura e di difenderla da interpretazioni riduttive e distorte. Porre al centro il tema della verità non è un atto meramente speculativo, ristretto a una piccola cerchia di pensatori; al contrario, è una questione vitale per dare profonda identità alla vita personale e suscitare la responsabilità nelle relazioni sociali.

Di fatto, se si lascia cadere la domanda sulla verità e la concreta possibilità per ogni persona di poterla raggiungere, la vita finisce per essere ridotta ad un ventaglio di ipotesi, prive di riferimenti certi.

BENEDETTO XVIPontificia Università Lateranense, Sabato, 21 ottobre 2006

http://www.vatican.va/holy_father/benedict_xvi/speeches/2006/october/documents/hf_ben-xvi_spe_20061021_lateranense_it.html