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Ingegneria metabolica smart Strategie di attivazione parallela.

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Presentazione sul tema: "Ingegneria metabolica smart Strategie di attivazione parallela."— Transcript della presentazione:

1 Ingegneria metabolica smart Strategie di attivazione parallela

2 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 lembriogenesi di Arabidopsis (OHara, 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)

3 Diauxic shift in yeast 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

4 Seguiamo i trascritti nel tempo

5 Passando da fermentazione a respirazione cosa cambia nel metabolismo? PYK1 4.9 Variazione Gene interessato Rosso = Aumento Verde = Diminuzione

6 Molti geni sono regolati in modo simile

7 Variazione coordinata di molti geni E possibile classificare i geni in base alla regolazione: 6 classi

8 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.

9 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.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.

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

11 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:

12 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)

13 Via del triptofano in lievito Solo la simultanea espressione di molti (tutti) i geni causa un ΔJ paragonabile al ΔE i (ΔJ C J x ΔE i )

14 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 [S i ] evita effetti negativi dovuti allaumento o alla riduzione di [S i ]

15 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.DeRisi JL, Iyer VR, Brown PO. * 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. 129:310-20O'Hara P, Slabas AR, Fawcett T. * 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.Rylott EL, Hooks MA, Graham IA. * 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: * Zhao J, Last RL.(1996) Coordinate regulation of the tryptophan biosynthetic pathway and indolic phytoalexin accumulation in Arabidopsis. Plant Cell. 8: * Eastmond PJ, Rawsthorne S. (2000) Ccoordinate changes in carbon partitioning and plastidial metabolism during the development of oilseed rape embryos. Plant Physiol. 122: Universal method: Kacser and Acerenza (1993) A universal method for achieving increases in metabolite production Eur J. of Biochemistry 216: 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.

16 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

17 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?

18 T-DNA activation tagging Struttura del T-DNA Punto di inserzione del T-DNA nel genoma ORF attivata dallinserzione

19 Linea cellulare selezionata con inibitori delle TDC. Linserzione del T-DNA porta ad un aumento del flusso nella via Molti altri geni della stessa via sono indotti nella linea cellulare

20 Il metabolismo secondario: Flavonoidi, Antociani e Lignina Genes encoding all enzymes indicated in red are clock-controlled

21 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

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

23 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.

24 La sovraespressione di Pap1 o Pap2 in Tabacco o Arabidopsis porta ad unintensa pigmentazione

25 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

26 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 Tube Lobe

27 Tobacco 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 laumento di flusso è notevole.

28 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

29 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.

30 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: Xie et al. (2006) Metabolic engineering of proanthocyanidins through co-expression of anthocyanidin reductase and the PAP1 MYB transcription factor. Plant J. 45: Metabolismo degli olii in foglia: Santos Mendoza et al., (2005) FEBS Lett. 579: LEAFY COTYLEDON 2 - Kannangara et al. (2007) The transcription factor WIN1/SHN1 regulates Cutin biosynthesis in Arabidopsis thaliana. Plant Cell Apr;19(4): 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: Baud and Lepiniec (2009) Regulation of de novo fatty acid synthesis in maturing oilseeds of Arabidopsis, Plant Physiol. Biochem. 47:448– Ruuska et al. (2002) Contrapuntal networks of gene expression during Arabidopsis seed filling, Plant Cell 14:1191– Shen et al. (2010) Expression of ZmLEC1 and ZmWRI1 increases seed oil production in maize, Plant Physiol. 153:980– Pouvreau et al. (2011) Duplicate maize Wrinkled1 transcription factors activate target genes involved in seed oil biosynthesis, Plant Physiol. 156:674– 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– 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.

31 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):

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

33 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).

34 Lipid and fatty acid compositions, after LEC2:GR induction in leaves Fatty acid composition Lipid composition.

35 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.

36 FamilyTF NameSummary 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/C BP 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 AP2WRINKLED1 (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 DofGmDof4 GmDof11 Transgenic expression yields higher seed oil levels; direct and indirect regulation of lipid metabolism genes; possible negative regulators of seed storage proteins CHD3PICKLE (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 AP2APETALA2 (AP2) Negative regulator of seed size, possibly via carbohydrate metabolism in the seed coat; effects on seed oil deposition likely indirect HD-ZIPGLABRA2 (GL2) Negative regulator of oil content; loss of seed mucilage proposed to make more C available for fatty acid synthesis

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

38 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

39 If a mRNA level changes, what happens to other ones in the same metabolic pathway? PSY (Phytoene Synthase) PDS (Phytoene Desaturase) 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…

40 A square matrix At3g21500At4g15560At5g11380At5g62790At2g02500At2g26930At1g63970At5g60600At4g34350 At3g At4g At5g At5g At2g At2g At1g At5g At4g PSY (Phytoene Synthase)

41 From numbers to colours Gene AGene B Gene A Essentially the same strategy published recently by Toufighi K, et al. (2005) Plant J. 43: The Botany Array Resource: e-Northerns, Expression Angling, and promoter analyses. Gene B

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

43 Isoprenoid biosynthesis two indipendent pathways in plants: B A Lange and Ghassemian (2003) Genome organization in Arabidopsis thaliana: a survey for genes involved in isoprenoid and chlorophyll metabolism. Plant Mol Biol. 51: A cytosolic B plastidial B

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

45 500 genes500 genes

46 500 genes500 genes

47 100 genes100 genes GGPP synthases: 10 isoforms Plastidial IPP Cytosolyc IPP (meval.) Carotenoid Chlorophyll

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

49 At3g29430 At3g geranylgeranyl pyrophosphate synthase, putative At3g terpene synthase/cyclase family protein At4g pathogenesis-related protein, putative At5g peroxidase, putative At1g GDSL-motif lipase/hydrolase family protein At2g auxin-responsive protein, putative / small auxin up RNA (SAUR_D) At5g leucine-rich repeat protein kinase, putative At5g glucosamine/galactosamine-6-phosphate isomerase-related At1g protease inhibitor/seed storage/lipid transfer protein At3g expressed protein At3g expressed protein At1g avirulence-responsive protein, putative At5g iron-responsive transporter-related At3g geranylgeranyl pyrophosphate synthase, putative At1g wall-associated kinase 4 At5g leucine-rich repeat transmembrane protein kinase, putative At1g expressed protein At3g ADP-ribosylation factor, putative At2g Clavata3 / ESR-Related-6 (CLE6) At1g expressed protein At1g terpene synthase/cyclase family protein At3g germin-like protein, putative At5g expressed protein At3g peroxidase 27 (PER27) (P27) (PRXR7) At4g expressed protein At2g acid phosphatase class B family protein At3g 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)

50 Calvin cycle At4g26520 fructose-bisphosphate aldolase, cytoplasmic At4g26530 fructose-bisphosphate aldolase, putative At4g38970 fructose-bisphosphate aldolase, putative At2g21330 fructose-bisphosphate aldolase, putative At5g56630 phosphofructokinase family protein At5g47810 phosphofructokinase family protein At4g32840 phosphofructokinase family protein At2g22480 phosphofructokinase family protein At4g26390 pyruvate kinase, putative At3g55440 triosephosphate isomerase, cytosolic, putative At2g29560 enolase, putative At1g07110 fructose-6-phosphate 2-kinase / fructose-2,6-bisphosphatase (F2KP) At1g13440 glyceraldehyde 3-phosphate dehydrogenase, cytosolic, putative At1g42970 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, putative At1g58150 hypothetical protein At1g56190 phosphoglycerate kinase, putative At1g22170 phosphoglycerate/bisphosphoglycerate mutase family protein At1g78040 pollen Ole e 1 allergen and extensin family protein At3g ,3-biphosphoglycerate-independent phosphoglycerate mutase At5g04120 phosphoglycerate/bisphosphoglycerate mutase family protein At3g22960 pyruvate kinase, putative At5g52920 pyruvate kinase, putative At2g21170 triosephosphate isomerase, chloroplast, putative At5g61410 ribulose-phosphate 3-epimerase, chloroplast, putative / At1g71100 ribose 5-phosphate isomerase-related At3g04790 ribose 5-phosphate isomerase-related At2g45290 transketolase, putative 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 At5g24410 glucosamine/galactosamine-6-phosphate isomerase-related At3g49360 glucosamine/galactosamine-6-phosphate isomerase family protein At1g13700 glucosamine/galactosamine-6-phosphate isomerase family protein At5g44520 ribose 5-phosphate isomerase-related At2g01290 expressed protein At5g39320 UDP-glucose 6-dehydrogenase, putative At5g64290 oxoglutarate/malate translocator, putative At5g35630 glutamine synthetase (GS2) At4g37930 glycine hydroxymethyltransferase At1g23310 glutamate:glyoxylate aminotransferase 1 (GGT1) At3g19710 branched-chain amino acid aminotransferase, putative At1g32450 proton-dependent oligopeptide transport (POT) family protein 3.5 in log scale >3000

51 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

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

53 Glucosinolate pathway Phase 2 - core structure synthesis Amino Acid S-Alkyl Thioidroximate GSTs Aci-Nitro compound CYP83s Thioidroximate 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

54 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 Oxo-acid Amino acid (n+1)C Several rounds of chain elongation are possible

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

56 Phase 3 - Side Chain Modification Various oxidations on the side chain Cytoplasm compartimentation -transport

57 SAT 52 – Serine O-acetyltrasferase Cysteine Synthase Glycosil hydrolase family 1 protein ABC Transporter At3g49680 At3g19710 At5g23010 At3g58990 At4g13430 At2g43100 At4g13770 At3g03190 At1g78370 At1g74090 At1g18590 At2g46650 At1g65860 At1g62560 At4g12030 At5g61420 At1g21440 At4g03060 At4g03050 At5g05260 At4g39950 At2g22330 At4g31500 At1g74100 At2g30870 At1g27130 At2g30860 At4g30530 At5g05730 At5g17990 At3g54640 At2g04400 At4g39980 At5g56760 At3g59760 At2g04400 At1g59870 At2g20610 At1g24100 At4g39940 At2g14750 Cytochrome b5 Flavin-contaning monooxygenase Bile acid Sodium symporter MYB 28 Mutase family protein AOP2 - Dioxygenase AOP3 -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 CYP79A2 CYP79B2 CYP79B3 CYP83B1 ST5a - Sulfotransferase Glutathione S-Transferase GLUCOSINOLATE FROM TRYPTOPHAN AND PHENYLALANINE 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 TRYPTOPHAN BIOSYNTHESIS SUR1 - C-S Lyase UGT74B1 – S-Glucosil Trasferase AKN2 – Adenylylsulfate kinase 2 AKN1 – Adenylylsulafte kinase 1 SHARED GENES (PAPS BIOSYNTHESIS,C-S LYASE AND GLUCOSYL TRANSFERASE) BCAT3 BCAT4 MAM 1 – 2 isopropylmalate synthase 3 Aconitase C-terminal domain Aconitase family protein Aconitase C-terminal domain Branched-chain amino acid aminotransferase HOMOMETHIONINE BIOSYNTHESIS CYP83A1 Glutathione-S Transferase ST5b – Sulfotransferase ST5c – Sulfotransferase GLUCOSINOLATE FROM HOMOMETIONINE SAT52 PEN3 PEN2 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

58 CYP83A1 BCAT4 B5 #1 MAM1 F17J16 ST5b MYB28 F16J13 MFL8 ATGSTF11 ST5c SUR1 T3P18 ATGSTU20 F28J8 T9E8 METHIONINE SIDE-CHAIN ELONGATION Phase I and II enzymes are co-regulating Monooxygenase GLUCOSINOLATE FROM FENIL.-OMOMET. Glutathione S-transferase C-S Lyase GLUCOSIN. FROM PHENILAL-TRYPT-HOMOMET. Sulfotransferase GLUCOSINOLATE FROM HOMOMET. AminotransferaseHOMOMET.–LEUCINE BIOSYNTHESIS 2-isopropylmalate Synthase HOMOMET BIOSYNTHESIS Aconitase C-terminal domain LEUC.-HOMOMET.BIOSYNTHESIS Aconitase C-terminal domainHOMOMET. BIOSYNTHESIS Aconitate hydratase Sodium symporter family protein Transcription factor Cytochrome b5 Flavin conteining monooxygenase family protein Mutase family protein At4g13770 At3g03190 At1g78370 At2g20610 At1g18590 At1g74090 At3g19710 At5g23010 At3g58990 At2g43100 At4g13430 At4g12030 At5g61420 At2g46650 At1g62560 At1g21440 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)

59 MAM1 BCAT4 BCAT3

60 Myb28 (At5g61420) ATG SALK_ PROM EX3 LBa1LB TGA ATG BRC_H161Lb

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

62 Wt and Myb28-KO metabolome wt ko Methylsulfinyloctyl Methylsulfinylheptyl GSL unknown

63 myb28, myb29 and myb28myb29

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

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

66 Insect feeding

67 Effect of the double KO

68 Too late!

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

70 Open issues 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…

71 At5g57800 At5g578001CER1 protein, putative (WAX2) At5g expressed protein At2g beta-ketoacyl-CoA synthase family (FIDDLEHEAD) (FDH) At3g protease inhibitor/seed storage/lipid transfer protein (LTP) family protein At1g ABC transporter family protein At1g very-long-chain fatty acid condensing enzyme (CUT1) At4g mannitol dehydrogenase, putative At2g ABC transporter family protein At5g proton-dependent oligopeptide transport (POT) family protein At4g multidrug resistance P-glycoprotein, putative At5g expressed protein At1g CER1 protein (another?) At1g ABC transporter family protein At2g GDSL-motif lipase/hydrolase family protein One vs. all analysis for At5g57800CER1 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

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

73 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 dellATP 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].

74 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, (art. del 1973). [7] Kacser e Acerenza, Eur. J. Biochem. (1993) 216: [8] Hofmeyr & Cornish-Bowden (2000) Regulating the cellular economy of supply and demand. FEBS Lett. 476: [9] Koebmann et al. (2002) The glycolytic flux in Escherichia coli is controlled by the demand for ATP. J. Bacteriol. 184: [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: [12] Morandini (2009) Rethinking metabolic control. Plant Science 176: [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– [14] Morandini (2010) Inactivation of allergens and toxins. N Biotechnol. 27: [15] Tattersall DB et al., (2001) Resistance to an herbivore through engineered cyanogenic glucoside synthesis. Science 293: [16] Nielsen et al., (2008) Metabolon formation in dhurrin biosynthesis. Phytochemistry 69:88-98.

75 [17] DellaPenna D. (2005) Progress in the dissection and manipulation of vitamin E synthesis. Trends Plant Sci 10: [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: [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: [21] Thelen JJ, Ohlrogge JB. (2002) Metabolic engineering of fatty acid biosynthesis in plants. Metab Eng. 4: [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: [23] Wu (2007) Doubled sugar content in sugarcane plants modified to produce a sucrose isomer. Pl. Biotech. J. 5: [24] Basnayake S. (2012) Field performance of transgenic sugarcane expressing isomaltulose synthase. Plant Biotechnology Journal 10: [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: In rosso sono evidenziati quelli da leggere con attenzione ai fini dellesame. Ulteriori riferimenti bibliografici si trovano nei singoli file di powerpoint delle lezioni. Chiunque desiderasse gli articoli originali basta me li chieda.

76 Cosa è naturale? Luomo fa parte della natura? Da cosa viene la specialità delluomo? Su cosa si fonda? Gli esseri umani e la tecnologia sono una cosa sola?

77 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

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