Plant Secondary Metabolism Engineering: Methods and Applications

340 Pages·2010·4.08 MB·English

by  Naíla Cannes do Nascimento (auth.)

M M B TM
ETHODS IN OLECULAR IOLOGY
SeriesEditor
JohnM.Walker
SchoolofLifeSciences
UniversityofHertfordshire
Hatfield,Hertfordshire,AL109AB,UK
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Plant Secondary Metabolism
Engineering
MethodsandApplications
Editedby
Arthur Germano Fett-Neto
UniversidadeFederaldoRioGrandedoSul,PortoAlegre,RS,Brazil
Editor
ArthurGermanoFett-Neto
UniversidadeFederaldo
RioGrandedoSul
CentrodeBiotecnologia
PortoAlegre-RS
Brazil
[email protected]
ISSN1064-3745 e-ISSN1940-6029
ISBN978-1-60761-722-8 e-ISBN978-1-60761-723-5
DOI10.1007/978-1-60761-723-5
SpringerNewYorkDordrechtHeidelbergLondon
LibraryofCongressControlNumber:2010923085
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Cover illustration: Fruit-bearing shoots of the Brazilian Atlantic Forest understorey tree Psychotria brachyceras
(Rubiaceae). This species accumulates an unusual monoterpene indole alkaloid, brachycerine, which is induced in
response to wounding, UV-exposure, jasmonate application, drought and osmotic stress. Its function is probably
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Preface
As sessile organisms, plants have evolved an amazing array of metabolic pathways leading
to molecules capable of responding promptly and effectively to stress situations imposed
by biotic and abiotic factors. These pathways, often recruited during biological evolu-
tion from essential primary metabolism pathways upon initial gene duplication, combine
carbon, hydrogen, oxygen, nitrogen, and sulfur into molecules that are capable of herbi-
vore deterrence, tri-trophic signaling (i.e., attracting herbivore predators and parasites),
pathogen inhibition, integrating defense responses, UV absorption, quenching of reac-
tive oxygen species, allelopathic activity, attracting pollinators, or seed dispersing animals,
heat dissipation, among other activities. Throughout the kingdom Plantae, there are tens
of thousands of molecules, frequently restricted to specific taxonomic groups, which play
this major role of plant interaction with the environment and are extremely important
for plant fitness; these are historically referred to as secondary metabolites, also known as
naturalproducts.
In spite of our detailed knowledge of only a fraction of plant secondary metabolic
pathways, particularly when considering tropical and subtropical phytodiversity, some
major patterns have emerged, revealing a complex regulation of this metabolism. Both
constitutive and inducible secondary metabolism activity can be found in different plant
species, and frequently an intermediate profile of metabolic activity between these two
strategies can be found. A strict spatial and temporal control of gene expression ensures
the correct accumulation pattern of various secondary products. Organ-specific, tissue-
specific, and cell type-specific expression of different portions of a single metabolic path-
way are not uncommon in the biosynthesis of alkaloids and terpenes, for example. The
requiredtransportofmetabolicintermediatesconstitutesanadditionallevelofregulation.
Within cells, different compartments participate in biosynthetic steps, including chloro-
plasts, cytoplasm, endoplasmic reticulum, vacuoles, nucleus, and special structures, such
as alkaloid vesicles. The induction of secondary metabolism gene expression by wound-
ing, herbivore-derived molecules, pathogen elicitors, and oxidative stress caused by heat,
drought, flooding, UV light, or temperature extremes is often mediated by integrating
signaling molecules such as salicylic acid, jasmonic acid, and their derivatives. Ontogeny
and circadian clock controlled expression are also important features of plant secondary
metabolism. At the level of gene expression, the presence of master regulatory transcrip-
tion factorsisacommon themein themetabolism ofphenolics,alkaloids, and glucosino-
lates. These regulators are considered some of the best targets for engineering secondary
metabolicpathways.
As can be easily realized, effective engineering of plant secondary metabolism path-
ways is not a trivial task, mostly due to the various levels of regulation and the often
observed close dependence of subcellular, tissue, and organ differentiation for effective
expression of metabolic steps. Nonetheless, examples of successful engineering of plants,
plant cell cultures, and heterologous expression of plant secondary metabolism genes in
microbes are available. The purpose of this book is to provide a source of detailed prac-
tical information on some of the most important methods employed in the engineering
v
vi Preface
of plant secondary metabolism pathways and in the acquisition of essential knowledge in
performing this activity. Some examples in which the application of these methods has
allowed significant advances in the major goal of tailoring plants or microorganisms to
becomefactoriesofrelevantplantsecondarymetabolitesarehighlighted.Someemerging
strategies for metabolic engineering, still incipiently used in secondary metabolism, are
also described, pointing to future directions in the field. The book is intended to be a
resourceful reference tool for researchers (molecular biologists, plant physiologists, bio-
chemists, chemical engineers, agronomists) engaged in the challenging task of modifying
some of the most intricate products of plant evolution. Hopefully, it will assist scientists’
efforts in the goal of sustainably supplying, in a fast changing planet, the ever growing
needs of humankind for natural chemicals, such as pharmaceuticals, nutraceuticals, agro-
chemicals,foodandchemicaladditives,biofuels,andbiomass.
ArthurGermanoFett-Neto
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
1. PlantSecondaryMetabolismandChallengesinModifyingItsOperation:
AnOverview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
NaílaCannesdoNascimentoandArthurGermanoFett-Neto
2. SuppressionSubtractiveHybridizationasaTooltoIdentifyAnthocyanin
Metabolism-RelatedGenesinAppleSkin . . . . . . . . . . . . . . . . . . . . . 15
YusukeBanandTakayaMoriguchi
3. Identification of Regulatory Protein Genes Involved in Alkaloid
BiosynthesisUsingaTransientRNAiSystem . . . . . . . . . . . . . . . . . . . 33
YasuyukiYamada,NobuhikoKato,YasuhisaKokabu,QingyunLuo,
JosephGogoDubouzetandFumihikoSato
4. Site-Directed Mutagenesis and Saturation Mutagenesis
for the Functional Study of Transcription Factors Involved
inPlantSecondaryMetaboliteBiosynthesis . . . . . . . . . . . . . . . . . . . . 47
SitakantaPattanaik, JoshuaR.Werkman, QueKong,
andLingYuan
5. Isolation of Proteins Binding to Promoter Elements of Alkaloid
Metabolism-RelatedGenesUsingYeastOne-Hybrid . . . . . . . . . . . . . . . 59
DéboraVomEndtandGiancarloPasquali
6. Modulation of Carotenoid Accumulation in Transgenic Potato
byInducingChromoplastFormationwithEnhancedSinkStrength . . . . . . . 77
JoyceVanEck,XiangjunZhou,ShanLu,andLiLi
7. Over-expressionofRate-LimitingEnzymestoImproveAlkaloidProductivity . . 95
TomoyaTakemura,Yit-laiChow,TakehikoTodokoro,TakuyaOkamoto,
andFumihikoSato
8. Microbial Expression of Alkaloid Biosynthetic Enzymes
forCharacterizationofTheirProperties . . . . . . . . . . . . . . . . . . . . . . 111
HiromichiMinami,NobuhiroIkezawa,andFumihikoSato
9. Producing a Recombinant Flavin-Containing Monooxygenase
from Coffea arabica in Escherichia coli for Screening of Potential
NaturalSubstrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
IgorCesarinoandPauloMazzafera
10. Efficient Production of Active Form Recombinant Cassava
HydroxynitrileLyaseUsingEscherichiacoliinLow-TemperatureCulture . . . . 133
HisashiSemba,EitaIchige,TadayukiImanaka,HaruyukiAtomi,
andHidekiAoyagi
vii
viii Contents
11. Introduction of the Early Pathway to Taxol Biosynthesis
in Yeast by Means of Biosynthetic Gene Cluster Construction
UsingSOE-PCRandHomologousRecombination . . . . . . . . . . . . . . . 145
PiaDahmandStefanJennewein
12. BiocatalyticSynthesisofTritium(3H)-LabelledTaxa-4(5),11(12)-diene,
thePathwayCommittingPrecursoroftheTaxoidDiterpenoids . . . . . . . . . 165
HansSchmeerandStefanJennewein
13. USER Cloning and USER Fusion: The Ideal Cloning Techniques
forSmallandBigLaboratories . . . . . . . . . . . . . . . . . . . . . . . . . . 185
HussamH.Nour-Eldin,FernandoGeu-Flores,
andBarbaraA.Halkier
14. Enrichment of Carotenoids in Flaxseed by Introducing
aBacterialPhytoeneSynthaseGene . . . . . . . . . . . . . . . . . . . . . . . . 201
MasakiFujisawaandNorihikoMisawa
15. Metabolic Engineering by Plastid Transformation
asaStrategytoModulateIsoprenoidYieldinPlants . . . . . . . . . . . . . . . 213
TomohisaHasunuma,AkihikoKondo,andChikahiroMiyake
16. Engineering High Yields of Secondary Metabolites in Rubia Cell
CulturesThroughTransformationwithRolGenes . . . . . . . . . . . . . . . . 229
VictorP.Bulgakov,YuriN.Shkryl,andGalinaN.Veremeichik
17. Flow Cytometric Methods to Investigate Culture Heterogeneities
forPlantMetabolicEngineering . . . . . . . . . . . . . . . . . . . . . . . . . 243
VishalGaurav,MartinE.Kolewe,andSusanC.Roberts
18. Phenylpropanoid Biosynthesis in Leaves and Glandular Trichomes
ofBasil(OcimumbasilicumL.) . . . . . . . . . . . . . . . . . . . . . . . . . . 263
CíceroDeschampsandJamesE.Simon
19. Fusion with Fluorescent Proteins for Subcellular Localization
ofEnzymesInvolvedinPlantAlkaloidBiosynthesis . . . . . . . . . . . . . . . . 275
PatríciaDuarte,JohanMemelink,andMarianaSottomayor
20. ImmunohistochemicalLocalisationofaPutativeFlavonoidTransporter
inGrapeBerries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291
ElisaPetrussa,EnricoBraidot,MarcoZancani,CarloPeresson,
AlbertoBertolini,SoniaPatui,ValentinoCasolo,SabinaPassamonti,
FrancescoMacrì,andAngeloVianello
21. ElectrogenicBromosulfaleinTransportinIsolatedMembraneVesicles:
Implementation in Both Animal and Plant Preparations for the Study
ofFlavonoidTransporters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307
SabinaPassamonti,FedericaTramer,ElisaPetrussa,EnricoBraidot,
andAngeloVianello
SubjectIndex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337
Contributors
HIDEKI AOYAGI •GraduateSchoolofLifeandEnvironmentalSciences,Universityof
Tsukuba,Ibaraki,Japan
HARUYUKI ATOMI •DepartmentofSyntheticChemistryandBiologicalChemistry,
GraduateSchoolofEngineering,KyotoUniversity,Kyoto,Japan
YUSUKE BAN •GraduateSchoolofLifeandEnvironmentalSciences,Universityof
Tsukuba,Tsukuba,Ibaraki,Japan
ALBERTO BERTOLINI •DepartmentofPlantBiologyandProtection,SectionofPlant
Biology,UdineUniversity,Udine,Italy
ENRICO BRAIDOT •DepartmentofPlantBiologyandProtection,SectionofPlant
Biology,UdineUniversity,Udine,Italy
VICTOR P. BULGAKOV •BioengineeringGroup,InstituteofBiologyandSoilScience,
FarEastBranchofRussianAcademyofSciences,Vladivostok,Russia
VALENTINO CASOLO •DepartmentofPlantBiologyandProtection,SectionofPlant
Biology,UdineUniversity,Udine,Italy
IGOR CESARINO •DepartmentofPlantBiology,InstituteofBiology,StateUniversityof
Campinas,Campinas,SP,Brazil
YIT-LAI CHOW •DivisionofIntegratedLifeScience,GraduateSchoolofBiostudies,
KyotoUniversity,Kyoto,Japan
PIA DAHM •InstituteofOrganicChemistryandBiochemistry,TechnicalUniversityof
Darmstadt,Darmstadt,Germany
CÍCERO DESCHAMPS •DepartmentofAgronomy,FederalUniversityofParana,
Curitiba,PR,Brazil
PATRÍCIA DUARTE •IBMC-InstituteofCellularandMolecularBiology,Porto
University,Porto,Portugal
JOSEPH GOGO •BiotransformationTeam,ScionResearch,Rotorua,NewZealand
JOYCE VAN ECK BOYCE •ThompsonInstituteforPlantResearch,CornellUniversity,
Ithaca,NY,USA
DÉBORA VOM ENDT •StateUniversityofRioGrandedoSul,NovoHamburgo,RS,
Brazil
ARTHUR GERMANO FETT-NETO •DepartmentofBotanyandPlantPhysiology
Laboratory,CenterforBiotechnology,FederalUniversityofRioGrandedoSul,Porto
Alegre,RS,Brazil
ix