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Ingegneria metabolica “smart”

Ingegneria metabolica “smart”. Strategie di attivazione parallela. Causano aumenti locali che faticano a propagarsi lungo la via (dampening). Come si ottiene un aumento di flusso?. Aumentando S Sottraendo P Aumentando enzima Aumentando attività.

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Ingegneria metabolica “smart”

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  1. Ingegneria metabolica “smart” Strategie di attivazione parallela

  2. Causano aumenti locali che faticano a propagarsi lungo la via (dampening) Come si ottiene un aumento di flusso? • Aumentando S • Sottraendo P • Aumentando enzima • Aumentando attività 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)

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

  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 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

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

  11. 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

  12. Abbondanza relativa dei trascritti It was demonstrated recently that mRNAs encoding the four subunitsof heteromeric (ACCase) acetyl-CoA carboxylase accumulate at a constant molar ratio throughoutsilique development in Arabidopsis. The ratios were found to beCAC1:CAC2:CAC3:(accD-A & accD-B) = 0.14:1.0:0.17:0.06 (Ke etal., 2000)

  13. Via del triptofano in lievito Solo la simultanea espressione di molti (tutti) i geni causa un ΔJ paragonabile al ΔEi (ΔJ ≃ CJx ΔEi )

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

  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. • * 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.

  16. Ingegneria metabolica “in batch” + + (6) S + + TF A B C P Espressione di fattori di trascrizione che regolano positivamente gli enzimi della via metabolica * 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)

  17. Fig. 1. Biosynthesis of TIAs in C. roseus. Solidarrows indicate single enzymatic conversions,whereas dashed arrows indicate multiple enzymaticconversions. 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-phosphatesynthase; G10H, geraniol 10-hydroxylase; CPR,cytochrome P450-reductase; TDC, tryptophandecarboxylase; STR, strictosidine synthase; SGD,strictosidine b-D-glucosidase; D4H, esacetoxyvindoline4-hydroxylase; and DAT, acetyl-CoA:4-O-deacetylvindoline 4-O-acetyltransferase. Genes regulated by ORCA3 are underlined.

  18. T-DNA activation tagging Struttura del T-DNA Punto di inserzione del T-DNA nel genoma ORF attivata dall’inserzione

  19. 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

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

  21. 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?

  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 un’intensa 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 Lobe Tube Immagini cortesia del prof. C. Martin Diversi geni della via sono down-regulated nel mutante delila ma solo nella zona con ridotta pigmentazione

  27. 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

  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 141:745-757. - 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.

  31. 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

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

  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. Fatty acid composition Lipid and fatty acid compositions, after LEC2:GR induction in leaves 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. Zhong and Ye (2009) Transcriptional regulation of lignin biosynthesis. Plant Signal Behav. 4:1028-34.

  37. 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

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

  39. A square matrix PSY (Phytoene Synthase) PSY (Phytoene Synthase)

  40. From numbers to colours Gene A Gene B Gene A Gene B 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.

  41. The Red Square… Group 1 & 3 are coregulated Gene ABCDEFGHIJKLMNOPQRS Group 1 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….)

  42. Isoprenoid biosynthesistwo indipendent pathways in plants: B A B A cytosolicB 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.

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

  44. 1000 2000 2750 1414 500 genes

  45. 500 genes

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

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

  48. Migliori correlatori tra tutti i geni di Arabisopsis (R value in linear plots) 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 At3g29430 is possibly involved in terpene synthesis

  49. 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 At3g08590 2,3-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

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