Ingegneria metabolica “smart” Strategie di attivazione parallela

Come si ottiene un aumento di flusso? Aumentando S Sottraendo P Aumentando enzima Aumentando attività Causano aumenti locali che faticano a propagarsi lungo la via (dampening) Esaminiamo alcuni esempi di aumenti di flusso in vivo * 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)

Diauxic shift in yeast Rosso = Aumento Verde = Diminuzione Exploring 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 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. (A) Stages of seedling development (B) Northern blot analysis of gene expression from 0 to 8 days after imbibition

Enzimi coinvolti ACC Malonyl-CoA transacilasi KAS III, II & I FAS - Acido grasso sintasi

Lipid synthesis during embryogenesis 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 FAS Components Exhibit Constant mRNA Ratios

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 Provate a suggerire motivi per la discrepanza tra Δei e ΔJ Solo la simultanea espressione di molti (tutti) i geni causa un ΔJ paragonabile al ΔEi (ΔJ ≃ CJ x ΔEi )

Evidenze sperimentali Reguloni! 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]

Referenze Referenze 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.

Ingegneria metabolica “in batch” Espressione di fattori di trascrizione che regolano positivamente gli enzimi della via metabolica P C A B TF + S (6) * Terpenoid Indole Alkaloyd (TIA) * via dei flavonoidi cere, glucinolati... Usando i fattori di trascrizione probabilmente si mantengono le “giuste proporzioni tra gli enzimi CAVEAT: ci sono limiti a questa strategia? Certo, alcuni enzimi come già molto abbondanti (es. quelli del calvin o glicolitici)

Fig. 1. Biosynthesis of TIAs in C. roseus. Solid arrows indicate single enzymatic conversions, whereas dashed arrows indicate multiple enzymatic conversions. Numerosi enzimi della via sono stati identificati e clonati. Esiste un fattore di trascrizione capace di attivarli tutti insieme? 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.

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 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

Myb transcription factor PAP1 I geni in rosso sono implicati nella biosintesi dei fenilpropanoidi e sono controllati dal ritmo circadiano 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?

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 Lobe Tube

Tobacco crosses: 35S:Del x 35S:Ros1 Piante di Arabidopsis che sovraesprimono uno solo dei due fattori non mostrano accumulo. Quando sono coespressi l’aumento di flusso è notevole. Immagini cortesia del prof. C. Martin

Immagini cortesia del prof. C. Martin 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 Sinergismo! 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 in maize, 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 Arabidopsis thaliana 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) 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 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

wt b and c are representative of medium, and high levels of leaf glossiness 35S::WIN1 35S::WIN1

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).

Fatty acid composition Lipid and fatty acid compositions, after LEC2:GR induction in leaves Santos Mendoza (2005) LEAFY COTYLEDON 2 activation is sufficient to trigger the accumulation of oil and seed specific mRNAs in Arabidopsis leaves. FEBS Letters 579:4666–4670 Lipid composition.

Transcriptional regulation of triacylglycerol biosynthesis in maturing seeds of Arabidopsis thaliana Model for the transcriptional regulation of triacylglycerol biosynthesis in maturing seeds of Arabidopsis thaliana. Factors that induce or repress lipogenesis are presented in open boxes. Target genes encoding actors of the fatty acid and triacylglycerol biosynthetic networks are presented below the transcriptional regulators, together with the cis-regulatory elements recognized by these regulators. For the sake of clarity, crosstalk between activators and repressors of the maturation program have been omitted. ASIL1, ARABIDOPSIS 6B-INTERACTING PROTEIN 1-LIKE1; AP2-EREBP, APETALA2-ethylene responsive element-binding protein; bZIP, basic leucine zipper; CBF, CAAT box-binding factor; ER, endoplasmic reticulum; FA, fatty acid; OB, oil body; FUS3, FUSCA3; LEC1,2, LEAFY COTYLEDON1,2; L1L, LEAFY COTYLEDON1-LIKE; TAG, triacylglycerol; Trihelix DNA BP, trihelix DNA binding protein; VAL1, 2, 3, VP1/ABSCISIC ACID INSENSITIVE3-LIKE1, 2, 3; WRI1, WRINKLED1. 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.

Summary of Role in Seed Oil Deposition 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 GmDof11 Transgenic 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 INDEPENDENT ENDOSPERM (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 INDUCIBLE GENE2 (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 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 http://lipidlibrary.aocs.org/plantbio/transfactors/index.htm

Zhong and Ye (2009) Transcriptional regulation of lignin biosynthesis 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 How universal is the “universal method” in vivo?

 mRNA is not equal to protein  flux changes over long times If a mRNA level changes, what happens to other ones in the same metabolic pathway? PSY (Phytoene Synthase) PDS (Phytoene Desaturase)  mRNA is not equal to protein  flux changes over long times Two-gene scatterplot Use data from many different tissues, mutants, conditions… Pearson correlation coefficient 39

A square matrix PSY (Phytoene Synthase) 1.00 0.12 0.01 0.03 0.04 0.20 At3g21500 At4g15560 At5g11380 At5g62790 At2g02500 At2g26930 At1g63970 At5g60600 At4g34350 1.00 0.12 0.01 0.03 0.04 0.20 0.25 0.19 0.35 0.75 0.66 0.65 0.73 0.31 0.40 0.21 0.18 0.15 0.69 0.70 0.78 0.68 0.77 0.72 0.67 0.56 0.80 0.60 0.88 0.74 PSY (Phytoene Synthase) 40

From numbers to colours Gene 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 41

The Red Square… Gene ABCDEFGHIJKLMNOPQRS Group 1 Group 1 & 3 are coregulated Group 3 Coregulated genes close in the list will appear as a red square Apply the correlation analysis to the entire “metabolic genome” (enzymes, transporters….)

Isoprenoid biosynthesis two indipendent pathways in plants: A cytosolic B plastidial 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.

Plastidial pathway: Carotenoids Phytyl Plastoquinone Phylloquinone Tocopherol Mono-terpenes Phytochrome Gibberellic acid Abscissic acid. Figure from Lange and Ghassemian (2003)

1000 2000 2750 500 genes 1414 45

500 genes 46

◄ GGPP synthases: 10 isoforms Plastidial IPP Cytosolyc IPP (meval.) 100 genes ◄ GGPP synthases: 10 isoforms Carotenoid Chlorophyll 47

GA GGPP Prenyl group Phytyl PP Chlorophyll GGPP synthase GGPP Prenyl group Phytyl PP Chlorophyll (At3g20160) At3g29430 At3g32040 At4g36810 (At4g38460) At3g29430 and At3g32040 provide GGPP for… Which GGPP synthase isoform works in the carotenoid pathway? 48

Migliori correlatori tra tutti i geni di Arabisopsis At3g29430 At3g29430 1.0000 geranylgeranyl pyrophosphate synthase, putative At3g29410 0.8313 terpene synthase/cyclase family protein At4g33720 0.7440 pathogenesis-related protein, putative At5g15180 0.7352 peroxidase, putative At1g53940 0.7341 GDSL-motif lipase/hydrolase family protein At2g24400 0.7081 auxin-responsive protein, putative / small auxin up RNA (SAUR_D) At5g59680 0.7022 leucine-rich repeat protein kinase, putative At5g24410 0.6942 glucosamine/galactosamine-6-phosphate isomerase-related At1g73780 0.6873 protease inhibitor/seed storage/lipid transfer protein At3g47210 0.6867 expressed protein At3g59370 0.6865 expressed protein At1g33900 0.6857 avirulence-responsive protein, putative At5g03570 0.6855 iron-responsive transporter-related At3g32040 0.6847 geranylgeranyl pyrophosphate synthase, putative At1g21210 0.6831 wall-associated kinase 4 At5g37450 0.6827 leucine-rich repeat transmembrane protein kinase, putative At1g11540 0.6810 expressed protein At3g49860 0.6770 ADP-ribosylation factor, putative At2g31085 0.6739 Clavata3 / ESR-Related-6 (CLE6) At1g49030 0.6734 expressed protein At1g66020 0.6725 terpene synthase/cyclase family protein At3g05950 0.6709 germin-like protein, putative At5g15725 0.6668 expressed protein At3g01190 0.6644 peroxidase 27 (PER27) (P27) (PRXR7) At4g31875 0.6620 expressed protein At2g38600 0.6569 acid phosphatase class B family protein At3g46400 0.6532 leucine-rich repeat protein kinase, putative Migliori correlatori tra tutti i geni di Arabisopsis (R value in linear plots) At3g29430 is possibly involved in terpene synthesis 49

Calvin cycle 3.5 in log scale  >3000 50 At4g26520 fructose-bisphosphate aldolase, cytoplasmic At4g26530 fructose-bisphosphate aldolase, putative At4g38970 fructose-bisphosphate aldolase, putative At5g56630 phosphofructokinase family protein At2g21330 fructose-bisphosphate aldolase, putative At5g47810 phosphofructokinase family protein At4g32840 phosphofructokinase family protein At2g22480 phosphofructokinase family protein At3g55440 triosephosphate isomerase, cytosolic, putative At4g26390 pyruvate kinase, putative At2g29560 enolase, putative At1g07110 fructose-6-phosphate 2-kinase / fructose-2,6-bisphosphatase (F2KP) At1g13440 glyceraldehyde 3-phosphate dehydrogenase, cytosolic, putative At3g26650 glyceraldehyde 3-phosphate dehydrogenase A, chloroplast (GAPA) At1g42970 glyceraldehyde-3-phosphate dehydrogenase B, chloroplast (GAPB) At3g04120 glyceraldehyde-3-phosphate dehydrogenase, cytosolic (GAPC) At3g12780 phosphoglycerate kinase, putative At1g58150 hypothetical protein At1g22170 phosphoglycerate/bisphosphoglycerate mutase family protein At1g56190 phosphoglycerate kinase, putative At1g78040 pollen Ole e 1 allergen and extensin family protein At3g08590 2,3-biphosphoglycerate-independent phosphoglycerate mutase At5g04120 phosphoglycerate/bisphosphoglycerate mutase family protein At5g52920 pyruvate kinase, putative At3g22960 pyruvate kinase, putative At2g21170 triosephosphate isomerase, chloroplast, putative At5g61410 ribulose-phosphate 3-epimerase, chloroplast, putative / At1g71100 ribose 5-phosphate isomerase-related At2g45290 transketolase, putative At3g04790 ribose 5-phosphate isomerase-related At3g60750 transketolase, putative At1g32060 phosphoribulokinase (PRK) / phosphopentokinase At1g43670 fructose-1,6-bisphosphatase, putative At3g54050 fructose-1,6-bisphosphatase, putative At3g55800 sedoheptulose-1,7-bisphosphatase, chloroplast At5g35790 glucose-6-phosphate 1-dehydrogenase / G6PD (APG1) At1g09420 glucose-6-phosphate 1-dehydrogenase, putative / G6PD, putative At5g24420 glucosamine/galactosamine-6-phosphate isomerase-related At3g49360 glucosamine/galactosamine-6-phosphate isomerase family protein At5g24410 glucosamine/galactosamine-6-phosphate isomerase-related At1g13700 glucosamine/galactosamine-6-phosphate isomerase family protein At5g44520 ribose 5-phosphate isomerase-related At2g01290 expressed protein At5g64290 oxoglutarate/malate translocator, putative At5g39320 UDP-glucose 6-dehydrogenase, putative At5g35630 glutamine synthetase (GS2) At4g37930 glycine hydroxymethyltransferase At1g23310 glutamate:glyoxylate aminotransferase 1 (GGT1) At1g32450 proton-dependent oligopeptide transport (POT) family protein At3g19710 branched-chain amino acid aminotransferase, putative 3.5 in log scale  >3000 50

Reducing glucosinolates in Arabidopsis Glucosinolates are sulphur rich compounds from brassicas Some beneficial, other toxic (quantity!) Upon wounding are converted into toxic products Two branches Mutants isolated

Beekwilder et al., (2008) PLoS 3:e2068. Short chain Aliphatic GSL Long chain Indolic GSL Beekwilder et al., (2008) PLoS 3:e2068.

Glucosinolate pathway Phase 2 - core structure synthesis Amino Acid S-Alkyl Thioidroximate GSTs Aci-Nitro compound CYP83s C-S Lyase Desulfo- glucosinolate UGTs Glucosinolate ST5s Aldoxime CYP79s Cytoplasm Step 1: Oxidation Step 2: Oxidation Step 5: Glucosylation Step 6: Sulfatation Step 3: Conjugation Step 4: C-S Clevage

Glucosinolates: sulfur-rich secondary metabolites Amino acid Transamination Oxydative decarboxylation Isomerization Condensation Chloroplast Phase 1 - side chain elongation Oxo-acid 2-alkyl-malic acid 3-alkyl-malic acid Export 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

Aromatic branch Aliphatic branch Phase II Shared genes Phase II – TRYPTOPHAN BIOSYNTHESIS SHARED GENES (PAPS BIOSYNTHESIS,C-S LYASE AND GLUCOSYL TRANSFERASE) HOMOMETHIONINE BIOSYNTHESIS GLUCOSINOLATE FROM TRYPTOPHAN AND PHENYLALANINE GLUCOSINOLATE FROM HOMOMETIONINE At5g05260 At4g39950 At2g22330 At4g31500 At1g74100 At2g30870 At1g27130 At2g30860 At4g30530 At5g05730 At5g17990 At3g54640 At2g04400 At4g39980 At5g56760 At3g59760 At1g59870 At2g20610 At1g24100 At4g39940 At2g14750 CYP79A2 CYP79A2 CYP79B2 CYP79B3 CYP83B1 ST5a - Sulfotransferase Glutathione S-Transferase CYP79B2 Aromatic branch CYP79B3 Phase II – GLS from Trp and Phe CYP83B1 ST5a ATGSTF10 ATGSTU13 ATGSTF9 F17I23 Anthranilate synthase ASA1 -Anthranilate synthase α subunit TSA1 - Trp synthase, alpha subunit TRP1- P-ribosyl-anthranilate synthase IGPS Indole-3-glycerol p synthase DHS1 – DAHP synthetase 1 ASA1 TSA1 TRP Biosynthesis TRP 1 IGPS DHS1 SAT52 SAT 52 – Serine O-acetyltrasferase Cysteine Synthase Glycosil hydrolase family 1 protein ABC Transporter OASC PEN2 PEN3 Phase II Shared genes (PAPS Biosynthesis, C-S Lyase, Glucosyl Transferase) SUR1 SUR1 - C-S Lyase UGT74B1 – S-Glucosil Trasferase AKN2 – Adenylylsulfate kinase 2 AKN1 – Adenylylsulafte kinase 1 UGT74B1 AKN2 AKN1 At3g49680 At3g19710 At5g23010 At3g58990 At4g13430 At2g43100 At4g13770 At3g03190 At1g78370 At1g74090 At1g18590 At2g46650 At1g65860 At1g62560 At4g12030 At5g61420 At1g21440 At4g03060 At4g03050 BCAT3 Aliphatic branch BCAT3 BCAT4 MAM 1 – 2 isopropylmalate synthase 3 Aconitase C-terminal domain Aconitase family protein Branched-chain amino acid aminotransferase BCAT4 MAM1 Phase I - Homomet Biosynthesis F17J16 T9E8 MFL8 CYP83A1 CYP83A1 Glutathione-S Transferase ST5b – Sulfotransferase ST5c – Sulfotransferase ATGSTF11 Phase II – GLS from Homomet ATGSTU20 ST5b ST5c B5 #1 Cytochrome b5 Flavin-contaning monooxygenase Bile acid Sodium symporter MYB 28 Mutase family protein AOP2 - Dioxygenase AOP3 -Dioxygenase F12P19 Phase III, transport and regulation – GLS from HOMOMET T3P18 F16J13 MYB28 F28J8 AOP2 AOP3

Phase II - GLS biosynthesis (Met derived) GLUCOSINOLATE BIOSYNTHESIS Phase II - GLS biosynthesis (Met derived) METHIONINE SIDE-CHAIN ELONGATION At4g13770 At3g03190 At1g78370 At2g20610 At1g18590 At1g74090 At3g19710 At5g23010 At3g58990 At2g43100 At4g13430 At4g12030 At5g61420 At2g46650 At1g62560 At1g21440 Monooxygenase “GLUCOSINOLATE FROM FENIL.-OMOMET.” Glutathione S-transferase C-S Lyase “GLUCOSIN. FROM PHENILAL-TRYPT-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 CYP83A1 ATGSTF11 ATGSTU20 SUR1 ST5c ST5b Phase I and II enzymes are co-regulating BCAT4 Phase I - GLS biosynthesis (Met derived) SIDE-CHAIN ELONGATION MAM1 F17J16 MFL8 T9E8 F16J13 MYB28 Candidate genes for transport, regulation... (MET derived GLS) B5 #1 T3P18 F28J8

BCAT4 MAM1 BCAT3

Myb28 (At5g61420) 1623 1 134 214 344 484 ATG TGA LBa1 LB51 SALK_136312 BRC_H161Lb PROM ATG EX3 1 134 214 344 484 1623 ATG TGA

Effect of knocking out Myb28? RT-PCR on 2 controls and 2 KOs

Wt and Myb28-KO metabolome Methylsulfinyloctyl Methylsulfinylheptyl GSL unknown wt

myb28, myb29 and myb28myb29

Beekwilder et al., (2008) PLoS 3:e2068. Mutating Myb28 and Myb29 Regulators Beekwilder et al., (2008) PLoS 3:e2068.

Reducing glucosinolate content... ...stimulates pest growth and damage! Beekwilder et al., (2008) PLoS 3:e2068

Insect feeding

Effect of the double KO

Too late!

2828 genes What is the distribution of all the R values in the matrix? La spalla di valori alti e positivi di R all’interno dei geni metabolici è la testimonianza che esiste molta coregolazione

Open issues Limitations Other levels of regulation Explore enzyme subsets Pathway identification Clustering of enzymes Shared cis-elements / regulators Suggest substrate for enzymes / trasporters Limitations Other levels of regulation Co-regulation does not mean necessarily… 70

One vs. all analysis for At5g57800 CER1 protein, putative (WAX2) (Log) At5g20270 0.8287 expressed protein At2g26250 0.8044 beta-ketoacyl-CoA synthase family (FIDDLEHEAD) (FDH) At3g43720 0.7894 protease inhibitor/seed storage/lipid transfer protein (LTP) family protein At1g17840 0.7892 ABC transporter family protein At1g68530 0.7864 very-long-chain fatty acid condensing enzyme (CUT1) At4g39330 0.7792 mannitol dehydrogenase, putative At2g26910 0.7755 ABC transporter family protein At5g13400 0.7735 proton-dependent oligopeptide transport (POT) family protein At4g25960 0.7679 multidrug resistance P-glycoprotein, putative At5g14410 0.766 expressed protein At1g02205 0.7563 CER1 protein (another?) At1g51500 0.7379 ABC transporter family protein At2g04570 0.7234 GDSL-motif lipase/hydrolase family protein CUT1 (very-long-chain fatty acid condensing enzyme, At1g68530) shows good correlation with At1g51500 (R=0.815), an ABC transporter protein

Transporters Cer5 (At1g51500) WT cer5 Cer5 (At1g51500) Wax analyses of Arabidopsis stem surface (cuticle) or epidermal peel extracts (total epidermis). cer5 Pighin et al., Science (2004) 306:622-625

Programma Ripasso 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].

Generali (MCA e metabolismo): 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?

Pontificia Università Lateranense, Sabato, 21 ottobre 2006 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 XVI Pontificia 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