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Characterization of Acyltransferases in Plant Secondary Metabolism: 

Characterization of Acyltransferases in Plant Secondary Metabolism Horticultural Science Laboratory of Molecular Genetics Ji-Young Lee 2005. 04. 14

Secondary Metabolites: 

Secondary Metabolites Products that are not essential for normal growth and development of plants Natural products Multiple functions throughout the plant’s life cycle

Functions and Applications of Secondary Metabolites: 

Secondary metabolites Functions and Applications of Secondary Metabolites An interesting target for plant breeding and genetic engineering

Genetic Engineering of 2’ Metabolic Pathway: 

Genetic Engineering of 2’ Metabolic Pathway Aims to either increase or decrease the quantity of a certain compound 1. Change the expression of regulatory genes => regulation of multiple biosynthesis genes 2. Control the expression of one or a few structural genes => overcoming specific rate-limiting steps in the pathway

Acyltransferases: 

Acyltransferases Acylation The process of adding an acyl group to a compound The structural and functional modification of compounds Acyltransferases A group of enzymes catalyzing the transfer of acyl groups A powerful means for engineering of secondary metabolites SAAT

Versatile Acyltransferase (VAT) Family: 

Versatile Acyltransferase (VAT) Family Protein family consisting of acyltransferases in secondary metabolism in plants and fungi Motif 1 (HXXXD):a general acid/base catalyst Motif 2:unknown function Motif 3 (DFGWG):catalytic activity Nakayama et al., Journal of Molecular Catalysis, 2003, 23, 117-132

Molecular Phylogenetic Tree of VAT: 

Molecular Phylogenetic Tree of VAT Hirokazu Suzuki et al., The Plant Journal, 2004, 38, 994-1003

Characterization of VATs: 

Characterization of VATs Benzylalcohol acyltransferae (BEAT) Deacetylvindoline 4-O-acetyltransferase (DAT) Strawberry alcohol acyltransferase (SAAT) Anthocyanin acyltransferases (AAT)

BEAT: 

BEAT Benzylacetate A type of benzoid esters A major constituent of the floral scent of Clarkia breweri Important flavorants in ripe fruit and ingredients in perfumes Benzylalcohol acyltransferase (BEAT) Catalysis the formation of the ester bezylacetate + Acetyl-CoA BEAT Benzylalcohol Benzylacetate

Isolation of BEAT Genes: 

Isolation of BEAT Genes Natalia Dudareva et al., The Plant Journal, 1998, 14, 297-304

Analysis of BEAT Activity: 

Analysis of BEAT Activity Benzylacetate standard Plant purified BEAT enzyme Spent media in which E. coli cells expressing BEAT cDNA Natalia Dudareva et al., The Plant Journal, 1998, 14, 297-304 E. coli cells expressing BEAT cDNA Spent media in which E. coli cells without BEAT cDNA

Expression of BEAT: 

Expression of BEAT Natalia Dudareva et al., The Plant Journal, 1998, 14, 297-304

DAT: 

DAT Vindoline Monoterpenoid indole alkaloids Use in cancer chemotherapy Low yields in the plant Deacetylvindoline 4-O-acetyltransferase (DAT) Catalysis of the last step in vindoline biosynthesis De Luca et al., Journal of Biological Chemistry, 1993, 15, 5504-5511

Isolation of DAT Gene: 

Isolation of DAT Gene St-Pierre et al., The Plant Journal, 1998, 14, 297-304 motif1 motif3

Chemical Modification of DAT: 

Chemical Modification of DAT St-Pierre et al., The Plant Journal, 1998, 14, 297-304 Motif1 : HXXD

Light Induction of DAT Activity: 

Light Induction of DAT Activity Enzyme activity Protein Transcripts St-Pierre et al., The Plant Journal, 1998, 14, 297-304 D : Dark L : Light

SAAT: 

SAAT Volatile esters Production form all soft fruit species during ripening Flavor components of the majority of fruits Source of energy, vitamins, minerals and antioxidants Strawberry alcohol acyltransferase (SAAT) Catalysis of transacylation from acyl-CoA to an alcohol Key enzyme in aroma biochemistry Use of the last step in volatile esters biosynthesis

Isolation of the SAAT Gene: 

Isolation of the SAAT Gene Asaph Aharoni et al., The Plant Cell, 2000, 12, 647-661

Analysis of SAAT Gene Expression: 

Analysis of SAAT Gene Expression Asaph Aharoni et al., The Plant Cell, 2000, 12, 647-661

Functional Expression of SAAT : 

Functional Expression of SAAT Standard With SAAT protein Without SAAT protein Asaph Aharoni et al., The Plant Cell, 2000, 12, 647-661

AAT in Salvia splendens: 

AAT in Salvia splendens Anthocyanin The largest subclass of plant flavonoid Attracting pollinators Protection of plants from various stresses Safety to human health Anthocyanin acyltransferases (AAT) Catalyze the acyl transfer from acyl-CoA to the sugar moiety of anthocyanin

Isolation of AAT gene: 

Isolation of AAT gene Bisdemalonylsalvianin Monodemalonylsalvianin Salvianin Malonyl-CoA CoA Ss5MaT1 Ss5MaT2 Malonyl-CoA CoA Hirokazu Suzuki et al., The Plant Journal, 2004, 38, 994-1003

Analysis of Ss5MaT gene expression: 

Analysis of Ss5MaT gene expression Hirokazu Suzuki et al., The Plant Journal, 2004, 38, 994-1003 PC : positive control NC : negative control F : flower

Applications of Acyltransferases: 

Applications of Acyltransferases Heterologous expressions Transformation SAAT gene of strawberry fruit into petunia leaves Metabolic engineering to modify flower color blue rose Modification of the functional and structural properties Only research for acyltransferase related to flavor and pigment Nakayama et al., Journal of Molecular Catalysis, 2003, 23, 117-132

Future Prospects: 

Future Prospects

References: 

References Acetyl-CoA:benzylalcohol acetyltransferase – an enzyme involved in floral scent production in Clarkia breweri., Natalia Dudareva, John C. D’Auria,Kyoung Hee Nam, Eran Pichersky, The Plant Journal, 1998, February, 297-304 The termial O-acetyltransferase involved in vindoline biosynthesis defines a new class of proteins responsible for coenzyme A-dependent acyl transfer., Venoit St-Pierre, Pierre Laflamme, Anne-Marie Alarco, Vincenzo De Luca, The plant Journal, 1998, March, 703-713 Identification of the SAAT gene involved in strawberry flavor biogenesis by use of DNA microarrays., Asaph Aharoni, Lepold C. P. Keizer, Harro J. Bouwmeester, Ann P. O’Connell., The Plant Cell, 2000, May, 647-661 Engineering secondary metabolite production in plants., R. Verpoorte and J. Memelink, Current Opinion in Biotechnology, 2000, May, 181-187 Anthocyanin acyltransferases: specificities, mechanism, phylogenetics, and applications., Toru Nakayama, Hirokazu Suzuki, Tokuzo Nishino, Journal of Molecular Catalysis B: Enzymatic, 2003, March, 117–132 Identification and characterization of a novel anthocyann malonyltransferase from scarlet sage (Salvia splendens) flowers., Hirokazu Suzuki, Shin’ya Sawade, Kazufumi Watanabe,Tokuzo Nishino, The Plant Journal, 2004, March, 994-1003

Slide27: 

Thank you for your attention!!!

Slide28: 

My experiments