Preface	5
	Contents	7
	Contributors	9
	Part I: Biotechnological Production of Selected Natural Products	12
	1: Vanilla: The Most Popular Flavour	13
	1.1 Introduction	13
	1.2 Vanilla Orchids, Vanilla Flowers and the Pod	14
	1.3 When the Green Becomes Black – Vanilla Pod Curing	17
	1.4 Vanillin Is the Key Flavour Compound of the Complex Vanilla Extract	18
	1.5 How Does the Vanilla Plant Form Vanillin?	20
	1.6 Flavour Synthesis by Brewing – Bioengineering of Vanillin Biosynthesis	21
	1.7 Biotechnology-Based Production of Vanillin from Eugenol, Iso-Eugenol, Ferulic Acid and Glucose	24
	1.8 Future Perspectives and Final Remarks	29
	References	30
	2: Rosmarinic Acid and Related Metabolites	35
	2.1 Occurrence and Structures of Rosmarinic Acid and Related Metabolites	36
	2.2 Biosynthetic Pathway of Rosmarinic Acid	42
	2.3 Production of RA in Untransformed Aseptic In Vitro Cultures	44
	2.3.1 Species from the Family Lamiaceae	44
	2.3.2 Species of the Family Boraginaceae	50
	2.3.3 Non-vascular Plant Species	52
	2.4 Production of Rosmarinic Acid in Hairy Roots	52
	2.4.1 Hairy Roots of Lamiaceae Species	52
	2.4.2 Hairy Roots of Boraginaceae Species	57
	2.5 Production of Rosmarinic Acid and Related Caffeic Acid Esters in Microorganisms	58
	2.6 In Vitro Formation of Non-natural Hydroxycinnamic Acid Esters and Amides by “Rosmarinic Acid Synthase”	60
	2.7 Conclusion and Outlook	61
	References	61
	3: Bioproduction of Resveratrol	71
	3.1 Introduction	71
	3.2 Biosynthesis of Resveratrol and Its Analogs	72
	3.3 Bioproduction of Resveratrol in Microorganisms	73
	3.3.1 Yeast	74
	3.3.2 E. coli	78
	3.3.3 Other Bacteria	79
	3.4 Bioproduction of Resveratrol Analogs and Derivatives	80
	3.5 Strategies in Metabolic Engineering of Resveratrol Production	81
	3.5.1 Pathway Engineering to Increase Precursor Supply	82
	3.5.2 Protein Engineering	83
	3.6 Conclusion and Perspective	84
	References	85
	4: Anthocyanin Production in Engineered Microorganisms	90
	4.1 Anthocyanins and Their Industrial Applications	91
	4.1.1 Pharmaceutical Applications	91
	4.1.2 Food Colorants	92
	4.1.3 Cosmetic Industry	92
	4.1.4 Other Fields	93
	4.2 Plant-Based Anthocyanin Production	93
	4.2.1 Extraction from Plants	94
	4.2.2 Anthocyanin Production from Suspension Cell Culture	94
	4.3 Anthocyanin Production in Microorganisms	96
	4.3.1 Engineering of Pathway Enzymes	97
	4.3.2 Supply of Cofactors and Cosubstrates	99
	4.3.3 Engineering Anthocyanin Secretion	100
	4.3.4 Optimization of the Production Process	100
	4.4 Conclusions and Future Perspectives	101
	References	102
	5: Microbial Synthesis of Plant Alkaloids	107
	5.1 Introduction	109
	5.2 Reconstitution of MIA Biosynthetic Pathways	114
	5.2.1 Precursor Pathways	115
	5.2.2 Strictosidine Formation	116
	5.2.3 Downstream Derivatization	117
	5.3 Reconstitution of BIA Biosynthetic Pathways	118
	5.3.1 Upstream BIA Pathways	118
	5.3.2 Microbial Production of Morphinan Alkaloids	121
	5.3.3 Microbial Production of Protoberberine, Benzophenanthridine, and Phthalideisoquinoline Alkaloids	122
	5.3.3.1 Synthesis of Dihydrosanguinarine and Sanguinarine in Yeast	122
	5.3.3.2 Noscapine Synthesis in Yeast	123
	5.4 Microbial Engineering Challenges	124
	5.4.1 Functional Expression of Cytochromes P450 (CYPs)	124
	5.4.2 Taming Enzyme Promiscuity	125
	5.4.3 Limiting Efflux of Intermediates	127
	5.5 Optimization Strategies	127
	5.5.1 Genetic and Pathway Engineering Techniques	127
	5.5.1.1 Building Combinatorial Enzyme Libraries	128
	5.5.1.2 Pathway Assembly	128
	5.5.1.3 Tuning Gene Expression	129
	5.5.2 Cultivation Methods	130
	5.6 Conclusions and Future Directions	131
	References	132
	6: Caffeine	139
	6.1 Introduction	139
	6.2 Distribution of Caffeine in Plants	139
	6.3 Biosynthesis of Caffeine from Xanthosine	141
	6.4 The Caffeine Synthase Gene Family in Plants	145
	6.5 Biotechnological Production of Caffeine by Genetic Engineering	148
	References	148
	7: Taxol® Biosynthesis and Production: From Forests to Fermenters	152
	7.1 Introduction	152
	7.2 The Biosynthesis of Taxol	155
	7.3 Taxol Production by Endophytes	161
	7.4 Taxol Production by Plant Cell Cultures	165
	7.5 Microbial Biotechnology for the Production of Taxol	176
	7.6 Concluding Remarks	179
	References	180
	Part II: Technologies for Metabolic, Enzyme and Process Engineering	193
	8: Commercial-Scale Tissue Culture for the Production of Plant Natural Products: Successes, Failures and Outlook	194
	8.1 Introduction	194
	8.2 Commercial Products from Plant Tissue Culture	197
	8.3 Considerations for Commercial Targets	201
	8.4 Future Opportunities	217
	References	219
	9: Tailoring Natural Products with Glycosyltransferases	224
	9.1 Introduction	225
	9.2 The Significance of Glycosylation in Plants	227
	9.2.1 Glycosylation Increases Solubility	227
	9.2.2 Glycosylation Increases Stability	227
	9.2.3 Glycosylation Controls Sequestration/Compartmentalization	228
	9.2.4 Glycosylation Affects Bioactivity and -Availability	229
	9.2.5 Glycosylation Reduces Toxicity	230
	9.2.6 Glycosylation Affects Perception	231
	9.3 Glucoside/Glucose Ester Synthesis	231
	9.4 Family 1 Plant Glycosyltransferases	233
	9.5 Substrates of Family 1 Plant Glycosyltransferases	234
	9.5.1 Secondary Metabolites	234
	9.5.1.1 Phenylpropanoids	235
	9.5.1.2 Flavonoids, Anthocyanins	236
	9.5.1.3 Dihydrochalcones, Acylphloroglucinol, Stilbenes, Curcumin	236
	9.5.1.4 Terpenoids	238
	9.5.2 Plant Hormones	239
	9.5.3 Miscellaneous Substrates (Alkaloids, Benzoxazinoids, Furanones, and Xenobiotics)	239
	9.6 Glucoside Production by Whole Cell Biocatalysts	240
	9.6.1 Production System	240
	9.6.2 Types of Whole-Cell Biocatalysts	247
	9.6.3 Process Optimization	247
	9.6.3.1 Vector Conveyed Process Optimization	248
	9.6.3.2 UGT Optimization	248
	9.6.3.3 Host Genome Optimization – Metabolic Engineering	249
	9.7 Recent Applications of Glycosyltransferases for Production of Small Molecule Glycosides	252
	9.7.1 Glycosyltransferases and Glycosylation in Product for Consumer Consumption	252
	9.7.2 Glycorandomization	254
	9.7.3 Glycosyltransferases and Their Role in Cancer Therapies	254
	9.8 Conclusions and Future Prospects	255
	References	256
	Part III: Analytical and Legal Aspects	269
	10: Authenticity Control of Natural Products by Stable Isotope Ratio Analysis	270
	10.1 Introduction	270
	10.2 Isotope Ratio Mass Spectrometry – IRMS	272
	10.2.1 Notations in IRMS	272
	10.2.2 Elemental Analyzer-IRMS (EA-IRMS) and Gaschromatography-IRMS (GC-IRMS)	273
	10.2.3 Liquid Chromatography-IRMS (LC-IRMS)	275
	10.2.4 Site-Specific Natural Isotope Fractionation-Nuclear Magnetic Resonance Spectroscopy (SNIF-NMR)	275
	10.3 Practical Applications of Stable Isotope Analysis on Miscellaneous Natural Products	276
	10.3.1 Phenylpropanoids (Vanillin)	276
	10.3.2 Terpenes (Monoterpenes and Tetraterpenes)	278
	10.3.3 Polyphenols (Resveratrol)	279
	10.3.4 Alkaloids (Caffeine)	280
	10.4 Conclusions	280
	References	280
	11: Natural or Synthetic? The Legal Framework in the EU for the Production of Natural Flavouring Ingredients	283
	11.1 Introduction	283
	11.2 General Conditions	284
	11.3 Scope of the Regulation	284
	11.4 Definitions	285
	11.4.1 Definitions of Flavourings in General	285
	11.4.2 Definition of Natural Flavouring Substance	286
	11.4.3 Definitions of Flavouring Preparations	290
	11.5 Processes for the Production of Natural Flavouring Ingredients	294
	11.5.1 EFFA Considerations on the Permitted Order of the Various Processes	294
	11.5.2 Biotechnology for the Production of Natural Flavouring Ingredients	295
	11.5.3 Further Considerations on the Production of Natural Flavouring Substances and Flavouring Preparations According to the EFFA Guidance Document	296
	11.6 Analytical Methods to Assess Authenticity	297
	11.7 Labelling of Flavourings (B2B)	298
	11.7.1 General Labelling Requirements	298
	11.7.2 Labelling of Natural Flavourings	299
	11.7.3 Considerations on Different Interpretations of the 95/5-­Rule (Art. 16(4))	302
	11.8 Safety Evaluation of Flavourings and Their Inclusion in the EU Union List	303
	References	306
	Index	308