Una visione sistematica per l’ingegneria metabolica

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1 Una visione sistematica per l’ingegneria metabolica
Creazione di vie metaboliche

2 Ingegneria metabolica
probabilmente efficace probab. inefficace (1) (2) (3) (4) (5) (6) (7) (8) (9) S S S S S S S S S + TF A A A A A + A A A A + Y B B B B E B + B B B B Z W C C Q C F C C C C C X P P P Q P P P P P P ATP

3 Espressione di nuovi enzimi
Q (3) P C A B S F E (4) P C A B Q S Vitamina E Vitamina A Vitamina C Folato … Manipolazioni (in)efficaci che riguardano uno o pochi enzimi

4 Kanwischer M, Porfirova S, Bergmuller E, Dormann P
Kanwischer M, Porfirova S, Bergmuller E, Dormann P. (2005) Alterations in tocopherol cyclase activity in transgenic and mutant plants of Arabidopsis affect tocopherol content, tocopherol composition, and oxidative stress. Plant Physiol. 137: Collakova E. DellaPenna D. (2003) Homogentisate phytyltransferase activity is limiting for tocopherol biosynthesis in Arabidopsis. Plant Physiol. 131: Valentin HE, Lincoln K, Moshiri F, Jensen PK, Qi Q, Venkatesh TV, Karunanandaa B, Baszis SR, Norris SR, Savidge B, Gruys KJ, Last RL. (2006) The Arabidopsis vitamin E pathway gene5-1 mutant reveals a critical role for phytol kinase in seed tocopherol biosynthesis. Plant Cell. 18: Ischebeck T, Zbierzak AM, Kanwischer M, Dormann P. (2006) A salvage pathway for phytol metabolism in Arabidopsis. J Biol Chem. 281: Van Eenennaam et al., (2003) Engineering vitamin E content: from Arabidopsis mutant to soy oil. Plant Cell. 15: Van Eenennaam et al., (2004) Elevation of seed alpha-tocopherol levels using plant-based transcription factors targeted to an endogenous locus. Metab Eng. 6:101-8. Shintani D, DellaPenna D. (1998) Elevating the vitamin E content of plants through metabolic engineering. Science 282: Rippert P, Scimemi C, Dubald M, Matringe M. (2004) Engineering plant shikimate pathway for production of tocotrienol and improving herbicide resistance. Plant Physiol. 134: Karunanandaa (2005) Metabolically engineered oilseed crops with enhanced seed tocopherol. Metabolic Engineering 7:384–400. DellaPenna D. (2005) Progress in the dissection and manipulation of vitamin E synthesis. Trends Plant Sci. 10:574-9 (review).

5 Efficacia biologica della vitE
Tocopherols Tocotrienols

6 Vit E biosynthesis TAT The combined annual consumption of tocopherols for human and animal applications was estimated at about 40,000 t in 2002, with only 10% coming from natural sources. Natural vitamin E originates almost exclusively from soybean oil, and is used predominantly for human applications Tyrosine

7 Tocopherol and tocotrienol biosynthetic pathway
A seconda che la coda isoprenica sia ridotta o meno, si avranno i tocoferoli o i tocotrienoli Copyright ©2004 American Society of Plant Biologists

8 γ-TMT in Synechocystis
Il gene per la γ-TMT (VTE4) è stato identificato per primo in batteri (il mutante corrispondente è incapace di operare la conversione da γ a α) (A) Putative tocopherol biosynthetic operon from Synechocystis (15). SLR0089 encodes γ-TMT and SLR0090 encodes p-hydroxyphenylpyruvate dioxygenase. (B) γ-TMT enzymatic reaction. γ -TMT adds a methyl group to ring carbon 5 of  γ-tocopherol. (C) HPLC profiles of tocopherols in wild-type Synechocystis PCC6803 and the γ-TMT null mutant. Total lipid extracts were isolated from each line, and tocopherols were analyzed by HPLC. Shintani D, DellaPenna D. (1998)

9 Effetto sul rapporto α/γ
Sovraesprimendo il gene di Arabidopsis si ottiene una conversione quasi completa di γ in α.  aumenta il valore nutritivo Arabidopsis seed contains ≈95% γ -tocopherol; seed-specific overexpression of VTE4 resulted in the near-complete conversion of γ-tocopherol to α-tocopherol (an 80-fold increase in α-tocopherol) and a ninefold increase in vitamin E activity Shintani D, DellaPenna D. (1998)

10 Tocopherol composition and levels in leaves of 5-week-old wild-type and transgenics
Tocopherol composition and levels in leaves of 5-week-old wild-type, 35S::γ-TMT, 35S::HPT1, and double 35S:: γ-TMT/ 35S::HPT1 overexpressers. Leaf tissue (approximately 70 mg) was extracted, and individual tocopherols were separated and quantified by normal phase HPLC. Each line is represented as an average ± SD from three plants. Total tocopherol levels of 35S::HPT1 and 35S::γ-TMT/35S::HPT1 plants were significantly higher than wild-type levels (P < 0.005). The excess γ-tocopherol in double transgenics was methylated to α-tocopherol. Collakova and DellaPenna (2003)

11 Tocopherol composition and levels in seed of wild-type and transgenics
Il livello totale non cambia molto, ma cambia il rapporto tra i vari metaboliti Collakova and DellaPenna (2003)

12 Soybean oil Sovraespressione di VTE3, VTE4 o entrambi in semi di soia La sovraespressioni dei diversi geni non cambia la quantità totale ma la proporzione tra le varie forme di tocoferolo Van Eenennaam AL, et al., (2003)

13 Segregation analysis P7S-At-VTE3 P7S-At-VTE3 + P7S-At-VTE4.
Tocopherol Composition Analysis of Segregating R1 Single Soybean Seed. (A) Data for wild-type control and eight seeds of transgenic line (P7S-At-VTE3). (B) Data for wild-type control and eight seeds of transgenic line (P7S-At-VTE3 + P7S-At-VTE4). Le popolazioni derivano da piante eterozigoti in cui segregano i vari caratteri Van Eenennaam AL, et al., (2003)

14 What is really limiting Tocopherol biosynthesis?
“… the gene encoding Arabidopsis HPT, HPT1, was constitutively overexpressed in Arabidopsis. In leaves, HPT1 overexpression resulted in a 10-fold increase in HPT specific activity and a 4.4-fold increase in total tocopherol content relative to wild type. In seeds, HPT1 overexpression resulted in a 4-fold increase in HPT specific activity and a total seed tocopherol content that was 40% higher than wild type, primarily because of an increase in γ-tocopherol content. These results indicate that HPT activity is limiting in various Arabidopsis tissues…” Collakova & DellaPenna (2003) “Overexpression of VTE1 resulted in an increase in total tocopherol of at least 7-fold in leaves, and a dramatic shift from α-tocopherol to γ-tocopherol. Expression studies demonstrated that indeed VTE1 is a major limiting factor of tocopherol synthesis in leaves.” Kanwischer et al.. (2005)

15 Manipolazione del livello di VitE in foglie e semi
Impact of engineering single and multiple tocochromanol biosynthetic enzymes on total and individual tocochromanol levels and on vitamin E activity in Arabidopsis leaves (a) and seed (b) leaves: 20 pmol / mg FW seeds: 800 pmol / mg seed DellaPenna D. (2005) Solo la sovraespressione di più geni comporta aumenti significativi nel flusso Data were taken from: [15] for wild type (WT) and VTE4; from [29] for VTE2 and VTE2CVTE4; from [35] for VTE1; from [9] for barley HGGT; from [27] for TyrACHPPD and VTE2CTyrACHPPD. Because studies were from different laboratories in each case, the impact of transgenics was calculated relative to the wild-type control(s) from each study.

16 Effetto della sovra-espressione dei vari geni
Cosa succede se sovraesprimo HPPD o se aumento il supply di HPP (idrossi fenilpiruvato)?

17 VitE & metabolic shortcuts
La prefenato deidrogenasi di lievito permette la conversione di prefenato in idrossifenilpiruvato (HPP) e rappresenta quindi una scorciatoia metabolica. Il flusso a Fenilalanina è molto abbondante nelle piante (per la sintesi dei fenilpropanoidi e dei precursori della lignina) e quindi la sottrazione di una parte sotto forma di prefenato non crea complicazioni. Il lavoro sulla durrina suggerisce che il flusso a tirosina può variare di molto senza effetti negativi Rippert et al., (2004)

18 Selezione di piante resistenti a DKN
DKN è un inibitore della HPPD La compresenza di HPPD e di PDH permette un maggior grado di resistenza al DKN Rippert et al., (2004)

19 Effetto dei transgeni sull’accumulo di tocoferoli/tocotrienoli
Linea sensibile Linee più resistenti HPPD-PDH 4 tobacco plants could not reveal any expression of the PDH gene Rippert et al., (2004)

20 Sorgente inattesa per la coda di Fitolo
The phenotype of the vte5-1 mutant is consistent with the hypothesis that chlorophyll degradation-derived phytol serves as an important intermediate in seed tocopherol synthesis and forces reevaluation of the role of geranylgeranyl diphosphate reductase in tocopherol biosynthesis. Valentin et al., (2006)

21 Un mutante di Arabidopsis accumula solo 20% di tocoferolo
HPLC Analysis of Tocopherol Content in Seed Extracts from Wild-Type Arabidopsis and the Arabidopsis lt1 Mutant Wild-type trace is shown in black and lt1 in red. Tocol was used as an internal standard. Un mutante di Arabidopsis accumula solo 20% di tocoferolo Copyright ©2006 American Society of Plant Biologists Valentin, H. E., et al. Plant Cell 2006;18:

22 Tocopherol Content of Arabidopsis Seed
Il gene viene mappato e identificato (riesce a complementare il mutante). La proteina (At5g04490) possiede attività di fitolo kinasi (non mostrato) Il mutante accumula anche fitolo libero (non riesce a fosforilarlo) Valentin, H. E., et al. Plant Cell 2006;18: ; vedi anche Ischebeck et al. (2006) J Biol Chem. 281:2470-7

23 Profile of Tocopherol, Chlorophyll, and Oil Accumulation in Developing Brassica napus Seed
Il tocoferolo si accumula in concomitanza con la degradazione della clorofilla (e quindi la produzione di fitolo che da essa deriva) Valentin, H. E., et al. Plant Cell 2006;18:

24 Metabolically engineered oilseed crops with enhanced seed tocopherol
Manipolazioni su più geni permettono l’accumulo di tocoferolo e tocotrienoli in soia e Arabidopsis fino a livelli visibili ad occhio nudo Seed phenotype and HGA accumulation in transgenic Arabidopsis and soybean seed. Panels A and B show segregating Arabidopsis and soybean seed transformed with pMON69909, and pMON69943, respectively. These plasmids contained seed-specific expression constructs for Eh-TYRA, At-HPPD, and Syn-VTE2. Panels C and D show LC-MS chromatograms for HGA from Arabidopsis and soybean seed extracts harboring pMON69909, and pMON69943, respectively. Karunanandaa, B. et al. (2005)

25 Seed specific expression of At-HPPD, Eh-TYRA, At-VTE2, or Syn-VTE2 as single genes resulted in significant seed tocochromanol increases of 1.09-fold, 1.20-fold, 1.41-fold, and 1.36-fold in R2 seed populations, respectively Karunanandaa, B. et al. (2005)

26 VTE4, VTE3 HPPD, TYRA, VTE2 VTE4, VTE3 HPPD, TYRA, VTE2 When single genes were expressed in crops, only seed specific expression of Eh-TYRA in canola resulted in a significant increase of the average seed tocochromanol content of all events, with up to two-fold WT tocochromanol levels for the best performing event. However, when combinations of two or more tocochromanol genes were expressed, seed tocochromanol levels increased significantly in Arabidopsis, and canola for all gene combinations Correlation between α-tocopherol and total tocochromanol levels in F2 segregating soybean seed obtained through crossing homozygous soybean lines transformed with pMON69943, and pMON Plasmids pMON69943 and pMON67227 harbored the P7Sα‘-CTP2-At-HPPD-E9 3′, P7Sα‘-CTP1-Eh-TYRA-E9 3′, PArc−5-CTP1-Syn-VTE2-Arc 3′, and P7sα’-At-VTE4-E9 3′, P7sα’-At-VTE3-E9 3′ expression cassettes, respectively Total vitamin E content in F2 seed combining the high total tocochromanol phenotype with the high a-tocopherol and high a-tocotrienol phenotype, was calculated to be up to 11-fold higher than in WT-soybean seed. Giocando sui geni e sulla loro espressione si può ottenere l’accumulo di quantità e proporzioni diverse dei vari composti. Con l’attivazione parallela, migliora l’omeostasi dei metaboliti e la germinabilità. Karunanandaa, B. et al. (2005)

27 Folate L’assunzione di folato riduce i casi di spina bifida (40-80%)
Google immagini: neural tube defects NTD (attenzione: immagini forti!) Alcune micotossine (fumonisine) riducono l’assunzione di folato Nei paesi come il Messico e Guatemala dove c’è forte consumo di mais contaminato con fumonisine c’è maggiore incidenza di NTD Diventa interessante la biofortificazione con folato Ulteriori informazioni a:

28 Monoglutamyl tetrahydrofolate
5 10 Monoglutamyl tetrahydrofolate 4-amino-4-deoxychorismate (ADC)

29 Overexpression of GTP cyclohydrolase-1 from E. coli in Arabidopsis
(A) Levels of pterins in T2 and T3 transgenic lines and nontransgenic Arabidopsis. (B) Values for transgenic T3 line 5-21 (value 0.02) and nontransgenic plants (solid bars) were compared with values from the literature (29) for other plant sources. The levels of neopterin observed in transgenic plants were up to 1,100-fold higher than the corresponding levels in nontransgenic plants. The quantities of pterins present in transgenic lines were highly correlated with the amount of EcGCH detected. Hossain et al. (2004) Proc Natl Acad Sci USA 101:5158–5163

30 Total folate levels of T3 transgenic lines and nontransgenic (NT) Arabidopsis
Folato e pterine Plot of folate values as a function of total pterins in transgenic and nontransgenic lines Comparison of folate levels for transgenic and nontransgenic Arabidopsis plants (solid bars) and values reported in the literature Hossain et al. (2004) Proc Natl Acad Sci USA 101:5158–5163

31 Folato e pterine: overexpressing GCHI in ripening tomato Fruit
Changes in total pteridine content of control (V2) and GCHI+ (M9) fruit during ripening. Data are means of three replicates and SE. MG, mature green stage de la Garza R. D. et.al. PNAS 2004;101:

32 Analysis of folates in GCHI+ and vector-control fruits by HPLC with electrochemical detection
A) Total folate contents of red-ripe fruit of 12 independent GCHI+ and 10 independent control transformants. (B) Analysis of the one-carbon substituents of folates from GCHI+ and control fruits. (C) Polyglutamyl tail length of 5-methyl-THF from control and GCHI+ fruit. de la Garza R. D. et.al. PNAS 2004;101:

33 At-ADCS overexpression
Total PABA accumulation in red ripe fruit from 25 independent AtADCS+ primary transformants AtADCS overexpression increases PABA content in tomato fruit. (A) Levels of AtADCS mRNA relative to actin in ripening tomato fruit, determined by real-time PCR (red and red ripe stages are 3 and 7 days after breaker stage, respectively). Data are means and SE of 2(–      CT) values calculated from three PCR replicates for the AtADCS and actin genes. Transgenic fruits were sampled from T1 AtADCS+ plants. (B) Total PABA accumulation in red ripe fruit from 25 independent AtADCS+ primary transformants (T0) analyzed by HPLC with fluorescence detection. Control value is the mean of five independent vector-alone transformants. Values for each AtADCS+ line are from single fruits. WT, wild type; fw, fresh weight. Hossain et al. (2004) Proc Natl Acad Sci USA 101:5158–5163

34 Accumulation of folate, PABA, and pteridines in G+ x A+ (GCHI+/AtADCS+) late red ripe tomatoes. Values for controls (nontransgenic segregants) and for single G+ and A+ transgenics are shown for comparison. Nei transgeni (ADCS e GCH) l’omeostasi dei metaboliti è andata a pallino Nessuno dei due enzimi ha un alto CJ (non sono limitanti nel senso classico del termine) Manipolarli entrambi permette di ottenere un aumento significativo (10X) Doppi transgenici (GCHI+/AtADCS+)

35 Storozhenko et al. (2007) Folate fortification of rice by metabolic engineering
NATURE BIOTECHNOLOGY 25:

36 Figure 2 Analysis of PABA, pterins and folate content in seeds of transgenic rice plants. (a) Total PABA and folate levels in seeds of A-lines. (b) Total pterins and folate levels in G-lines. (c) Total PABA, pteridines and folate levels in GA-lines. (d) Representation of the main folate species in seeds of GA lines as compared to WT seeds calculated as percentages of total folate for each line. Values are averages of five GA transgenic lines or three independent measurements of WT seeds. Error bars indicate s.d. H4PteGlu, tetrahydrofolate; 5-CH3-H4PteGlu, 5-methyltetrahydrofolate; PteGlu, folic acid; 5-CHO-H4PteGlu, 5-formyltetrahydrofolate; 10-CHO-PteGlu, 10-formylfolic acid; 5,10-CH¼H4PteGlu, 5,10-methenyltetrahydrofolate. (e) Relative abundances of polyglutamated and monoglutamated folates in seeds of transgenic GA lines and WT. (f) Changes in total folate levels in seeds of GA lines upon cooking. Except for d, values are means of two independent seed samples, error bars indicate s.d. Samples from seeds of homozygous T2 and T3 plants are underlined with regular and bold lines, respectively. V, control transformed with the ‘empty’ vector.

37 Waller et al. (2010) reported that the expression “of three downstream pathway genes […] increased by up to 7.8-, 2.8-, and 1.7-fold, apparently in response to the build-up of specific folate pathway metabolites”. Thus the substantial accumulation of folate reported in some publications might be due to a partial parallel activation stimulated by feedforward mechanisms rather than high flux control coefficients of the enzymes targeted for manipulation.

38 Hanson and Gregory III (2011) Folate Biosynthesis, Turnover, and Transport in Plants. Annu. Rev. Plant Biol. 62:

39 Trasportatori Fig. 1. Compartmentation of folate and SAM metabolism. DHP, dihydropteroate; E4P, erythrose-4-phosphate; HC, homocysteine; HMDPH, hydromethyldihydropterin; Met, methionine; pABA, para-aminobenzoate; PEP, phosphoenolpyruvate; SAHC, S-adenosylhomocysteine; SAM, S-adenosylmethionine; THF, tetrahydrofolate. Vacuole I trasportatori non sono in genere considerati, ma sono parte integrante della via metabolica!! Weber et al. (2007) FEBS Letters 581:2215–2222

40 Pompa ATP-dipendente (ABC)
(5) P C A B S ATP L’ingegnerizzazione di trasportatori potrebbe stimolare l’accumulo MOLTO più efficacemente Raichaudhuri (2009) J. BIOL. CHEM. 284:

41 I mutanti KO hanno una maggiore sensibilita a Metotrexate

42 Transgenic multivitamin corn through biofortification of endosperm with three vitamins representing three distinct metabolic pathways The transgenic kernels contained 169-fold the normal amount of β-carotene, 6-fold the normal amount of ascorbate, and double the normal amount of folate. Naqvi et al., (2009) PNAS 106: 7762–7767.

43 phytoene synthase (psy1) cDNA from Zea mays
crtI gene (encoding carotene desaturase) from Pantoea ananatis (formerly Erwinia uredovora) rice dehydroascorbate reductase (dhar) cDNA, E. coli folE gene encoding GTP cyclohydrolase (GCH1)

44 Bibliography folate de la Garza et al. (2007) PNAS USA 104:4218–4222.
Hossain et al., (2004) PNAS USA 101: Valentin et al., (2005) Plant Cell 18:212–224 Storozhenko et al., (2007) Folate fortification of rice by metabolic engineering Nat. Biotech. 25: Sahr et al., (2006) Folate synthesis in plants: purification, kinetic properties, and inhibition of aminodeoxychorismate synthase. Biochem J. 396: Weber et al. (2007) Making the connections – The crucial role of metabolite transporters at the interface between chloroplast and cytosol. FEBS Letters 581:2215–2222.

45 Vitamin C Agius (2003) Engineering increased vitamin C levels in plants by overexpression of a D-galacturonic acid reductase. Nat Biotechnol. 21: Linster & Clarke (2008) L-Ascorbate biosynthesis in higher plants: the role of VTC2. Trends Plant Sci. 13: Linster et al., (2007) Arabidopsis VTC2 encodes a GDP-L-galactose phosphorylase, the last unknown enzyme in the Smirnoff-Wheeler pathway to ascorbic acid in plants. J Biol Chem. 282: Espandi VitC con nuovi papers, vedi review (Rossi & Morandini)

46 Planta (2012) 235:553–564 Three genes, representing diverse steps putatively involved in plant AsA biosynthesis pathways, were cloned and independently expressed in Solanum lycopersicum (tomato) under the control of the CaMV 35S promoter. Yeast-derived GDPmannose pyrophosphorylase (GMPase) and arabinono-1,4- lactone oxidase (ALO), as well as myo-inositol oxygenase 2 (MIOX2) from Arabidopsis thaliana

47 The ‘‘Smirnoff–Wheeler’’ pathway is considered the principal route for de novo synthesis of AsA
and involves the conversion of D-mannose into AsA via a series of L-galactose containing intermediates Cronje et al. (2012)

48 RT-PCR products showing transcription of the arabinono-1,4-lactone oxidase (ALO) gene in leaves (top) and green fruit (bottom) of transformed plants using TIP41 as a constitutively expressed control gene

49 GDP-mannose pyrophosphorylase (GMPase)
Cronje et al. (2012)

50 Increase in GMPase activity was concomitant with increased ascorbate levels in all tissues measured (Table 2). Ascorbate content in leaves was increased up to 66% compared with 50 and 35% in green and red fruit, respectively. Most transgenic ALO lines displayed increased ascorbate levels (P<0.05) in leaf tissue, typically between 21 and 54% (Table 3). Levels in green fruit were increased up to 25% (P<0.1), while red fruit contained levels invariant from the wild type. In leaf material, increased MIOX activity was associated with up to 30% reduction in ascorbate content (Table 4). Conversely, transgenic green fruit with increased MIOX activity displayed up to 35% increased ascorbate levels (P<0.1). Cronje et al. (2012)

51 Cronje et al. (2012)

52 Table 2. AsA content and relative content and AsA biosynthetic gene transcription level in single gene transgenic lines. Transcription levels of the six genes and AsA content were checked in p1304 control lines and assigned the value of 1. No significant differences were observed between WT and p1304 in gene transcription levels. The results were checked in at least three independent lines of each transformant when the T4 samples were 5 weeks old. Means ± SE, n ≥ 3. Zhou et al. (2012) Engineering ascorbic acid biosynthetic pathway in Arabidopsis leaves by single and double gene transformation. Biol. Plant. 56: Table 3. Relative gene expression and AsA accumulation in GGT-GLDH and GGT-GPP transgenic lines. Gene transcription in WT control was assigned the value of 1. Means ± SE, n = 3.

53 Vitamin A Vedere ppt sulla vitA
Naqvi et al., (2009) Transgenic multivitamin corn through biofortification of endosperm with three vitamins representing three distinct metabolic pathways. PNAS 106: 7762–7767

54 Morandini (2013) Plant Biotechnology Journal