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Metabolic engineering
- 1. METABOLIC ENGINEERING…… Metabolic engineeringis generally defined as the redirection of one or more enzymatic reactions to produce new compounds in an organism, improve the production of existing compounds, or mediate the degradation of compounds. Dr. Soumitra Paul, MPP Lab, C.U.
- 2. • The useof term “engineering” implies that there is some precise understanding of the system, that is being modified • Rate-limiting steps must be known • A typical metabolic-engineering approach focuses on a particular metabolic intermediate or product such as starch, vitamin E, carotenoids, amino acids etc. Metabolic engineering……….. Dr. Soumitra Paul, MPP Lab, C.U.
- 3. • The mostsuccessful metabolic-engineering approaches are those that have introduced new pathways into plants e.g. production of provitamin A in rice • It’s a matter of great importance to perform a metabolite profiling of transgenic plants (even in case of failure of experiment) in order to diagnose the problem • Unexpected results can happen e.g. dwarf tomato plants instead carotenoid rich plants • Commercial projects must characterize their plants in order to release plant into a market Dr. Soumitra Paul, MPP Lab, C.U.
- 4. BACKGROUND……. • Over thepast few years, significant advances have been made in metabolic engineering through the application of genomics and proteomics technologies to elucidate and characterize metabolic pathways in a holistic manner, rather than on a step by step basis. • Further work has been carried out on the modeling of metabolic pathways on a genomic scale, which has shown that metabolic pathways are controlled at multiple levels and any form of perturbation can have wide ranging effects at the whole system level. • Hence, the attention has now shifted towards more complex and sophisticated strategies in which several steps in a given pathway are modified simultaneously to achieve optimal flux. Dr. Soumitra Paul, MPP Lab, C.U.
- 5. GOALS OF PLANTMETABOLIC ENGINEERING ….. There are three basic goals of metabolic engineering in plants: 1. The production of more of a specific desired compound. 2. The production of less of a specific unwanted compound. 3. The production of novel compound. PLANT METABOLIC ENGINEERING DESIRED COMPOUND UNWANTED COMPOUND NOVEL COMPOUND INCREASE DECREASE IMPORT Dr. Soumitra Paul, MPP Lab, C.U.
- 6. Several ways tometabolically engineer an organism • Block a metabolic flux (re-channel) • Channel a metabolic flux into new cell compartments • Induce a metabolic flux (can lead to unexpected results) • Introduce a new metabolic pathway into organism (the most successful way) Dr. Soumitra Paul, MPP Lab, C.U.
- 7. Approaches to PlantMetabolic Engineering Dr. Soumitra Paul, MPP Lab, C.U.
- 8. Incorporation of newpathway in plants 1. Engineering for VLC-PUFAs in Arabidopsis: Incorporation of Δ9-elongase, Δ8-desturase and Δ5-desaturase genes from bacteria, with seed specific promoter. Recently, reconstruction through altering DHA metabolism by ɷ-3 desaturase from Phytopthora infestans into Brassica juncea. 2. Engineering for folate metabolism, Overexpression of ADCS (aminodeoxychorismate synthase) and GCHI (GTP cyclohydrolaseI) gene increases PABA and pteridine molecules, ultimately rises folate levels in tomato 3. Enhancing ascorbate bioosynthesis Overexpression of rat L-gluconolactone oxidase in tobacco, myo-inositol oxidase in Arabidopsis, supressing malate dehydrogenase in mitocondria in tomato Dr. Soumitra Paul, MPP Lab, C.U.
- 9. ENGINEERING METABOLIC PATHWAYS….. Carbohydrate Metabolism Aminoacid Metabolism Polyamine Metabolism Lipid Metabolism Alkaloids Terpenoids Flavonoids Volatiles Dr. Soumitra Paul, MPP Lab, C.U.
- 10. CARBOHYDRATE METABOLISM….. Starch: •Starch is a storage carbohydrate that accumulates transiently in leaves and stably in seeds, tubers and roots. • It is a staple source of dietary carbohydrate for animals and also has a large number of industrial uses. • Metabolic engineering has been used in an attempt to increase starch yields and to modify the properties of starch by changing the relative proportions of its two components – amylose and amylopectin. Dr. Soumitra Paul, MPP Lab, C.U.
- 11. ENGINEERING THE STARCHBIOSYNTHETIC PATHWAY…… • The allosteric enzyme ADP-glucose pyrophosphorylase (AGP) plays a key role in regulating starch biosynthesis in cereal seeds and is likely the most important determinant of seed sink strength. • Plant AGPs are heterotetrameric, consisting of two large and two small subunits. • Both wheat (Smidansky et al 2002) and rice (Smidansky et al 2003) have been transformed with a modified form of maize (Zea mays L.) Shrunken2 gene (Sh2r6hs), which encodes an altered AGP large subunit. • This altered large subunit gives rise to a maize AGP heterotetramer with decreased sensitivity to its negative allosteric effector, orthophosphate, and more stable interactions between large and small subunits. Dr. Soumitra Paul, MPP Lab, C.U.
- 12. • Both transgenicwheat and rice lines showed increase in seed weight and total plant biomass. Results indicate increased availability and utilization of resources in response to enhanced seed sink strength. STARCH B I O S Y N T H E T I C PATHWAY Dr. Soumitra Paul, MPP Lab, C.U.
- 13. • Amylose contentin wheat has been markedly increased with RNAi approach, by suppressing simultaneously the expression of SBEIIa and SBEIIb, which are the isoforms of the starch branching enzyme (Regina et al 2006). Modifying Relative proportions of Amylose and Amylopectin Scanning electron microscopy identified gross changes in granule size and structure as shown in the figure above. Compared with the untransformed control, starch granules from endosperms with reduced SBEIIa and SBEIIb expression displayed significant morphological alterations. Scanning electron micrographs of isolated starch granules. NB1 (nontransformed control wheat) (a), 087 (hp-SBEIIa wheat) (b), and 008 (hp-SBEIIb wheat) (c). Dr. Soumitra Paul, MPP Lab, C.U.
- 14. AMINO ACIDMETABOLISM: • Amino acid synthesis pathways have been targets for metabolic engineering predominantly to increase the abundance of essential amino acids- Lysine, threonine, methionine and tryptophan. • Lysine biosynthesis in plants is regulated primarily by a lysine-mediated feedback inhibition of dihydrodipicolinate synthase (DHPS). • Efforts have been made to improve lysine production in plants by metabolic engineering, by utilizing bacterial DHPS enzymes that are much less sensitive to lysine inhibition than their plant counterparts. • Transgenic plants expressing the bacterial DHPS in the plastid, overproduced lysine by upto more than 100-fold compared to control nontransformed plants (Galili 2002). Dr. Soumitra Paul, MPP Lab, C.U.
- 15. The Aspartate familybiosynthetic pathway of the essential amino acid. Abbreviations: AK,aspartate kinase; DHPS,dihydrodipicolinate synthase; HSD,homoserine dehydrogenase; HSK,homoserine kinase; TS,threonine synthase; CGS,cystathionine c-synthase; CBL,cystathionine b-lyase; MS; methionine synthase; SAMS; S-adenosylmethionine synthetase. Dr. Soumitra Paul, MPP Lab, C.U.
- 16. POLYAMINE METABOLISM: •Polyamines are small aliphatic amines that are derived from the amino acids ornithine and arginine by decarboxylation. • Polyamines have several important roles in plant physiology and development. The three major polyamines found in plants are putrescine, spermidine and spermine. • Polyamine accumulation specifically in the rice grains was possible by expressing ODC under the control of the wheat seed-specific, low molecular weight glutenin promoter, and such lines could be very useful to improve the nutritional properties of rice grains without affecting the growth or development of vegetative parts of plants. • The primary targets in the polyamine biosynthesis pathway are the ornithine decarboxylase (ODC) and arginine decarboxylase (ADC). Putrescine Spermidine Spermine Dr. Soumitra Paul, MPP Lab, C.U.
- 17. Polyamine biosynthesis in plants Another key enzymein the polyamine biosynthesis, SAMDC, has also been targeted. The Datura stramonium SAMDC gene was expressed in rice and the expected increase in SAMDC activity increased the levels of spermidine and spermine accumulated in seeds (Thu-Hang et al., 2002). Further, yeast SAMDC has also been expressed in tomato, increasing its nutritional value (Mehta et al., 2002). Dr. Soumitra Paul, MPP Lab, C.U.
- 18. LIPID METABOLISM: •The modification of lipid metabolism to change the quantity and quality of fatty acids in plants has important applications in the food industry. • Plants represent a significant renewable source of fatty acids because many species accumulate them in the form of triacylglycerol as major storage components in seeds. • Recently, Liu et al 2002, utilized a hairpin RNA (hpRNA) mediated RNAi method to downregulate two key fatty acid desaturase genes encoding stearoyl-acyl-carrier protein 9-desaturase and oleoyl-phosphatidylcholine 6-desaturase. Downregulation of these two genes in cotton resulted in nutritionally improved high stearic and high oleic cotton seed oils which are essential fatty acids for better health of human heart. Dr. Soumitra Paul, MPP Lab, C.U.
- 19. ENGINEERING SECONDARY METABOLIC PATHWAYS…… Alkaloids: •Alkaloids are the largest group of secondary metabolite synthesized by plants and the pathways, enzymes and regulatory genes involved in their synthesis have been extensively studied. • A very interesting example of metabolic engineering of alkaloid is replacement of morphine with non-narcotic alkaloid reticuline in opium poppy (Papaver somniferum). • In 2004, Allen et al, reported gene silencing in transgenic opium poppy using RNAi. They interfered multiple steps of complex biochemical pathway with an hpRNA construct designed to silence all members of the multigene Codeine reductase (COR) family. Dr. Soumitra Paul, MPP Lab, C.U.
- 20. • After thegene silencing through RNAi , the transgenic plants accumulated (S)- reticuline, a precursor non-narcotic alkaloid which occurs seven enzymatic steps upstream of codeinone in the pathway. The (S)- reticuline accumulation in transgenic opium poppy occurred at the expense of morphine, codeine, opium and thebaine. Dr.SoumitraPaul,MPPLab,C.U.
- 21. TERPENOIDS • The terpenoids,sometimes called isoprenoids, are a large and diverse class of naturally- occurring organic chemicals, derived from five-carbon isoprene units assembled and modified in thousands of ways. • Despite their diversity, all terpenoids are synthesized from the common precursors dimethylallyl pyrophosphate and isopentyl pyrophosphate. This occurs through two distinct pathways – the mevalonate independent pathway and the mevalonate pathway, both of which have been targets for metabolic engineering. • Metabolic engineering of biosynthetic pathway for production of β-carotene in rice and reduction of toxic gossypol in cotton have been done successfully. Dr. Soumitra Paul, MPP Lab, C.U.
- 22. Science, 2005 Changing Subcellularlocalization of an Enzyme Dr. Soumitra Paul, MPP Lab, C.U.
- 23. • In 2006,Sunilkumar et al. reported successful disruption of gossypol biosynthesis in cottonseed tissue by interfering with the expression of the -cadinene synthase gene during seed development. The gossypol content was reduced only in the seeds of cotton, without affecting its levels in any other part of the plant. Reduction of Gossypol in Cotton • Cotton seeds have high quality protein content which makes it a nutrient rich resource for food but it cannot be utilized due to the presence of toxic gossypol within the seed tissue. • Gossypol is a cardio and hepatotoxic terpenoid, which makes it unsafe for human and monogastric animal consumption. Hence any means, which could yield gossypol-free cottonseed, would significantly contribute to human nutrition and health. Dr. Soumitra Paul, MPP Lab, C.U.
- 24. Structures and proposedbiosynthetic pathway of gossypol and other terpenoids in cotton plants. δ-cadinene synthase enzyme was targeted in the seed through RNAi to interfere with gossypol biosynthesis Dr. Soumitra Paul, MPP Lab, C.U.
- 25. Engineering the Carotenoid BiosyntheticPathway • Much attention has been paid to the carotenoid metabolic pathway, which produces pigments with roles in light harvesting and photoreception, and which form vital components of human and animal diets. • The principal example of carotenoid metabolic engineering in plants is of course the synthesis of β-carotene in rice endosperm (Golden rice), which normally accumulates GGPP (geranyl geranyl pyrophosphate) but lacks the subsequent enzymes in the pathway. • The four enzyme activities missing in the β-carotene synthesis pathway in cereal grains are phytoene synthase, phytoene desaturase, ζ-carotene desaturase and lycopene β-cyclase. Dr. Soumitra Paul, MPP Lab, C.U.
- 26. Engineering the ProvitaminA)b-Carotene) Biosynthetic Pathway into (Carotenoid-Free) Rice Endosperm Xudong Ye, Salim Al-Babili, Andreas Kloti, Jing Zhang, Paola Lucca, Peter Beyer, Ingo Potrykus * Vitamin A deficiency causes symptoms ranging from night blindness to those of total blindness * In Southeast Asia, it is estimated that a quarter of a million children go blind each year because of this nutritional deficiency * It is estimated that 125 million children worldwide are deficient in vitamin A * Oral delivery of vitamin A is problematic mainly due to the lack of infrastructure Dr. Soumitra Paul, MPP Lab, C.U.
- 27. • No ricecultivars produce provitamin A in the endosperm therefore recombinant technologies rather than conventional breeding are required • Immature rice endosperm is capable of synthesizing the early intermediate geranylgeranyl diphosphate, which can be used to produce the uncolored carotene phytoene by expressing the enzyme phytoene synthase (psy) in rice endosperm Continue…. Dr. Soumitra Paul, MPP Lab, C.U.
- 28. • The synthesisof b-carotene requires the complementation with three additional plant enzymes : phytoene desaturase (pds) and z-carotene desaturase (zds), each catalyzing the introduction of two double bonds, and lycopene b-cyclase, encoded by the lcy gene. • To reduce the transformation effort, a bacterial carotene desaturase ,(crt1) capable of introducing all four double bonds required, was used • Transit peptide was attached to crt1 • A transit peptide exists in plant psy Dr. Soumitra Paul, MPP Lab, C.U.
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- 30. High iron/zinc biofortifiedRice Low Phytate Rice Golden Rice b-carotene + Vit E rice Hyperfortified α linolenic acid Rice (ALA) for PUFA Insulin promoting rice Improved protein-potato (Ama1) Carotenoids enriched potato Canola with b-carotene Vitamin C food crop High iron rice Vitamin E + b-carotene maize Biofortified Food Crops Major GM Rice for nutritional improvement Needs to focus Folate, Lysine, Vitamin E enrichment and more………. Dr. Soumitra Paul, MPP Lab, C.U.
- 31. Biosynthetic pathway ofTocopherols & Tocotrienols Dr. Soumitra Paul, MPP Lab, C.U.
- 32. Vitamin E- Maize HGGT catalyzes an analogous reaction to HPT, only it is highly specific for GGDP whereas HPT uses PDP as its prenyl substitute. Results from the expression of barley HGGT in transgenic plants suggest that this enzyme has strong substrate specificity for geranylgeranyl diphosphate, rather than phytyl diphosphate. Expression of HGGT enzyme in tobacco calli and Arabidopsis leaves resulted in accumulation of Vitamin E antioxidants in the form of tocotrienols ,principally as γ- Tocotrienols, and generated little or no change in the content of Tocopherols (Cahoon et al, 2003) Barley HGGT gene was over-expressed in maize seeds, leading to a 20-fold increase in tocotrienol level, which translated to an eight- fold increase in total tocols (tocopherols and tocotrienols) (Cahoon et al, 2003).Dr. Soumitra Paul, MPP Lab, C.U.
- 33. Genes involved in carotenoid biosynthesis Cloned/ transferred Crop species Remarks Reference Y1 clonedMaize Importance of such regulatory gene in rice is conceptualized Buckner et al. 1990 crtI (Phytene desaturase) cloned/ transformed Erwinia uredovora/ Tobacco/ Herbicide resistance; Increased Misawa et al 1990, 1993 crtE cloned Erwinia herbicola coding for GGPP synthase Math et al 1992 A gene cluster cloned Erwinia herbicola For complete carotenoid pathway To et al 1994 psy transformed Tomato Resulted in dwarfism redirecting the metabolites from gibberellin pathway Fray et al 1995 lcy cloned Daffodil Lycopene to beta-carotene Al-Babili et al 1996 psy cloned/ transformed Daffodil/ Rice Accumulation of phytoene in rice endosperm Scheldz et al 1996; Burkardt et al 1997 crtB (phytoene synthase) transformed Brassica Overexpression led to increase in carotenoids and other metabolites Shewmaker et al 1999 Selected historical developments in carotenoid metabolism in relation to plant metabolic engineeringDr.SoumitraPaul,MPPLab,C.U.
- 34. Co-transformationLBA4404/pZPsC + LBA4404/pZLcyH Anther culture Hemizygous T309GoldenRice (Ye et al. 2000) Dihaploid homozygous T309 Golden Rice (Baisakh et al. 2001b) IR64 1st Backcrossing F1IR64 x IR64BC1F1 x x 2nd Backcrossing BC2F1 Marker-free Selfing BC2F2 Marker-free PCR analysis Molecular analysis Phenotyping Molecular analysis Selection of hph negative transgenic progenies PCR screening and Southern confirmation IR64 NILs Marker-free Phenotyping HPLC BC1F1 progenies Marker-free Flow chart for the Development of Marker-free Near-isogenic golden Rice lines of IR64 Dr. Soumitra Paul, MPP Lab, C.U.
- 35. Development of highiron riceDr.SoumitraPaul,MPPLab,C.U.
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- 37. ANTISENSE TECHNOLOGY It isa loss of function technique where the genes are knocked out for investigation. DNA promoter coding sequence messenger RNA protein ANTISENSE promoter coding sequence NO protein Dr. Soumitra Paul, MPP Lab, C.U.
- 38. DOWNREGULATION OF PHYTICACID BIOSYNTHESIS….. The biosynthesis of phytic acid is a complex pathway controlled by various enzymes. Of which myo inositol 3 phosphate synthase (MIPS) and inositol phosphate kinase (IPK1) plays the crucial role (Suzuki et al. 2007). Hence, suppression of any of these enzymes by RNA interference (RNAi) may prove to be effective in reducing phytic acid levels in rice. Proposed InsP synthetic pathways in plants. Left: the PLC- independent pathway. Right: the PLC-dependent pathway (Suzuki et al 2007). Dr. Soumitra Paul, MPP Lab, C.U.