Cover
Title Page
Copyright
List of Contributors
Preface
Chapter 1: Vitamins, Biopigments, Antioxidants and Related Compounds: A Historical, Physiological and (Bio)technological Perspective
1. 1.1 Historical Aspects of the Search for Vitamins
2. 1.2 Vitamins: What's in a Name
3. 1.3 Physiological Functions of Vitamins and Related Compounds
4. 1.4 Technical Functions of Vitamins and Related Compounds
5. 1.5 Production and Application of Vitamins and Related Factors
6. 1.6 Outlook
7. References
Part I: Water-Soluble Vitamins
Chapter 2: Industrial Production of Vitamin B₂ by Microbial Fermentation
1. 2.1 Introduction and Historical Outline
2. 2.2 Occurrence in Natural/Food Sources
3. 2.3 Chemical and Physical Properties; Technical Functions
4. 2.4 Assay Methods and Units
5. 2.5 Biological Role of Flavins and Flavoproteins
6. 2.6 Biotechnological Synthesis of Riboflavin
7. 2.7 Strain Development: Genetic Modifications, Molecular Genetics and Metabolic Engineering
8. 2.8 Fermentation Process
9. 2.9 Downstream Processing
10. 2.10 Chemical Synthesis
11. 2.11 Application and Economics
12. References
Chapter 3: Vitamin B₃, Niacin
1. 3.1 Introduction
2. 3.2 History
3. 3.3 Occurrence in Nature/Food Sources
4. 3.4 Chemical and Physical Properties
5. 3.5 Vitamin B₃ Deficiency Disease (Pellagra)
6. 3.6 Methods Used for Determination of Vitamin B₃
7. 3.7 Synthesis
8. 3.8 Downstream Processing of Nicotinic Acid
9. 3.9 Reactive Extraction
10. 3.10 Physiological Role of Vitamin B₃ (Niacin)
11. 3.11 Safety of Niacin
12. 3.12 Toxicity of Niacin
13. 3.13 Derivatives of Niacin
14. 3.14 Application in Cosmetics, Food and Feed
15. 3.15 Future Prospects
16. References
Chapter 4: Pantothenic Acid
1. 4.1 Introduction and Historical Outline
2. 4.2 Occurrence in Natural Food Sources and Requirements
3. 4.3 Physiological Role as Vitamin or as Coenzyme
4. 4.4 Chemical and Physical Properties
5. 4.5 Assay Methods
6. 4.6 Chemical and Biotechnological Synthesis
7. 4.7 Application and Economics
8. References
Chapter 5: Folate: Relevance of Chemical and Microbial Production
1. 5.1 Introduction
2. 5.2 Folates: Chemical Properties and Occurrence in Food
3. 5.3 Biosynthesis
4. 5.4 Physiological Role
5. 5.5 Bioavailability and Dietary Supplements
6. 5.6 Chemical and Chemoenzymatic Synthesis of Folic Acid and Derivatives
7. 5.7 Intestinal Microbiota, Probiotics and Vitamins
8. 5.8 Folate Production by Lactic acid Bacteria
9. 5.9 Folate Production by Bifidobacteria
10. 5.10 Conclusions
11. References
Chapter 6: Vitamin B₁₂ – Physiology, Production and Application
1. 6.1 Introduction and Historical Outline
2. 6.2 Occurrence in Food and Other Natural Sources
3. 6.3 Physiological Role as a Vitamin or Coenzyme
4. 6.4 Chemical and Physical Properties
5. 6.5 Assay Methods
6. 6.6 Biotechnological Synthesis
7. 6.7 Downstream Processing; Purification and Formulation
8. 6.8 Application and Economics
9. 6.9 Conclusions and Outlook
10. References
Chapter 7: Industrial Fermentation of Vitamin C
1. 7.1 Introduction and Historical Outline
2. 7.2 Occurrence in Natural/Food Sources
3. 7.3 Physiological Role of Asc
4. 7.4 Chemical and Physical Properties
5. 7.5 Assay Methods
6. 7.6 Industrial Fermentation of Asc
7. 7.7 Application and Economics
8. 7.8 Outlook
9. References
Chapter 8: Direct Microbial Routes to Vitamin C Production
1. 8.1 Introduction and Scope
2. 8.2 Principles of Direct L-Ascorbic Acid Formation: The Major Challenges
3. 8.3 Direct L-Ascorbic Acid Formation via 1,4-Lactones
4. 8.4 Direct L-Ascorbic Acid Formation via 2-Keto Aldoses
5. 8.5 Outlook
6. Acknowledgement
7. References
Part II: Fat Soluble Vitamins
Chapter 9: Synthesis of β-Carotene and Other Important Carotenoids with Bacteria
1. 9.1 Introduction
2. 9.2 Carotenoids: Chemical Properties, Nomenclature and Analytics
3. 9.3 Natural Occurrence in Bacteria
4. 9.4 Biosynthesis of Carotenoids in Bacteria
5. 9.5 Biotechnological Synthesis of Carotenoids by Carotenogenic and Non-Carotenogenic Bacteria
6. 9.6 Conclusion
7. References
Chapter 10: β-Carotene and Other Carotenoids and Pigments from Microalgae
1. 10.1 Introduction and Historical Outline
2. 10.2 Occurrence in Nature and Food Sources
3. 10.3 Physiological Role as a Vitamin or as a Coenzyme
4. 10.4 Chemical and Physical Properties; Technical Functions
5. 10.5 Assay Methods and Units
6. 10.6 Biotechnological Synthesis
7. 10.7 Chemical Synthesis or Extraction
8. 10.8 Process Economics
9. References
Chapter 11: Microbial Production of Vitamin F and Other Polyunsaturated Fatty Acids
1. Lipid Nomenclature
2. 11.1 Introduction: Essential Fatty Acids
3. 11.2 General Principles for the Accumulation of Oils and Fats in Microorganisms
4. 11.3 Production of Microbial Oils
5. 11.4 Safety Issues
6. 11.5 Future Prospects
7. Acknowledgements
8. References
Chapter 12: Vitamin Q₁₀: Property, Production and Application
1. 12.1 Background of Vitamin Q₁₀
2. 12.2 Chemical and Physical Properties of CoQ₁₀
3. 12.3 Biosynthesis and Metabolic Regulation of CoQ₁₀
4. 12.4 Chemical Synthesis and Separation of CoQ₁₀
5. 12.5 Applications and Economics of CoQ₁₀
6. References
Chapter 13: Pyrroloquinoline Quinone (PQQ)
1. 13.1 Introduction and Historical Outline
2. 13.2 Occurrence in Natural/Food Sources
3. 13.3 Physiological Role as Vitamin or as Bioactive Substance
4. 13.4 Physiological Role as a Cofactor
5. 13.5 Chemical and Physical Properties; Technical Functions
6. 13.6 Assay Methods
7. 13.7 Biotechnological Synthesis
8. 13.8 Strain Development: Genetic Modification, Molecular Genetics and Metabolic Engineering
9. 13.9 Up- and Down-stream Processing; Purification and Formulation
10. 13.10 Chemical Synthesis or Extraction Technology
11. 13.11 Application and Economics
12. References
Part III: Other Growth Factors, Biopigments and Antioxidants
Chapter 14: L-Carnitine, the Vitamin B_T: Uses and Production by the Secondary Metabolism of Bacteria
1. 14.1 Introduction and Historical Outline
2. 14.2 Occurrence in Natural/Food Sources
3. 14.3 Physiological Role as Vitamin or as Coenzyme
4. 14.4 Chemical and Physical Properties
5. 14.5 Assay Methods and Units
6. 14.6 Biotechnological Synthesis of L-Carnitine Microbial Metabolism of L-Carnitine and Its Regulation
7. 14.7 Other Methods for L-Carnitine Production: Extraction from Natural Sources and Chemical Synthesis
8. Acknowledgement
9. References
Chapter 15: Application of Carnosine and Its Functionalised Derivatives
1. 15.1 Introduction and Historical Outline
2. 15.2 Sources and Synthesis
3. 15.3 Physico-Chemical and Biological Properties of Carnosine
4. 15.4 Biotechnological Synthesis of Carnosine Derivatives: Modification, Vectorisation and Functionalisation
5. 15.5 Applications of Carnosine and Its Derivatives
6. References
Chapter 16: Metabolism and Biotechnological Production of Gamma-Aminobutyric Acid (GABA)
1. 16.1 Introduction
2. 16.2 Properties and Occurrence of GABA in Natural Sources
3. 16.3 Metabolism of GABA
4. 16.4 Regulation of GABA Biosynthesis
5. 16.5 Biotechnological Production of GABA
6. 16.6 Physiological Functions and Applications of GABA
7. 16.7 Conclusion
8. Acknowledgement
9. References
Chapter 17: Flavonoids: Functions, Metabolism and Biotechnology
1. 17.1 Introduction
2. 17.2 Structure and Occurrence in Food
3. 17.3 Activity and Metabolism
4. 17.4 Biosynthesis of Flavonoids in Plants
5. 17.5 Biotechnological Production
6. 17.6 Concluding Remarks
7. References
Chapter 18: Monascus Pigments
1. 18.1 Introduction and History of Monascus Pigments
2. 18.2 Categories of MPs
3. 18.3 Physiological Functions of MPs
4. 18.4 Chemical and Physical Properties of MPs
5. 18.5 Assay Methods and Units of MPs
6. 18.6 MPs Producer – Monascus spp.
7. 18.7 Application and Economics of MPs
8. Acknowledgements
9. References
Index
End User License Agreement

List of Illustrations

Chapter 2: Industrial Production of Vitamin B₂ by Microbial Fermentation
1. Figure 2.1 Chemical structure of riboflavin and the two coenzymes derived from riboflavin, FMN and FAD. FMN is formed from riboflavin by the addition of a phosphate group derived from adenosine triphosphate. FAD is formed from FMN after addition of an adenosine monophosphate group derived from a second molecule of adenosine triphosphate.
2. Figure 2.2 Biosynthesis of riboflavin and flavocoenzymes. RIB1: GTP cyclohydrolase II; RIB7: 2,5-Diamino-6-ribosylamino-4-(3H)-pyrimidinone-5′-phosphate reductase; RIB2: 2,5-Diamino-6-ribitylamino-4-(3H)-pyrimidinone-5′-phosphate deaminase; RIB3: 3,4-dihydroxy-2-butanone-4-phosphate synthase; RIB4: 6,7-dimethyl-8-ribityllumazine synthase; RIB5: riboflavine synthetase; FMN1: riboflavin kinase; FAD1: Flavin adenine dinucleotide synthetase.
3. Figure 2.3 Modified genes to increase riboflavin production in A. gossypii. ICL1, isocitrate lyase; MLS1, malate synthase; GLY1, threonine aldolase; SHM2, serine hydroxymethyltransferase; BAS1nBAS2, Myb-related transcription factors; PRS, phosphoribosyl pyrophosphate synthetases; ADE4, phosphoribosyl pyrophosphate amidotransferase; RIB, genes of the Rib pathway; and VMA1, vacuolar ATPase.
Chapter 3: Vitamin B₃, Niacin
1. Figure 3.1 Chemical structure of nicotinic acid and nicotinamide.
2. Figure 3.2 Chemical synthesis of starting materials required for nicotinic acid manufacture.
3. Figure 3.3 Synthesis of nicotinic acid from 2-methyl-5-ethylpyridine.
4. Figure 3.4 Ammoxidation of 3-picoline and hydrolysis of cyanopyridine to niacinamide and nicotinic acid.
5. Figure 3.5 Reactions in the gas-phase oxidation of picoline to nicotinic acid.
6. Figure 3.6 Pathways of nicotinic acid and NAD synthesis. (Modified from Greenbaum and Pinder, 1968.) (NMN): Nicotinic acid mononucleotide, NMN: Nicotinamide mononucleotide, PRPP: 5-phosphoribosyl 1-pyrophosphate, PPi: Pyrophosphates.
7. Figure 3.7 Biocatalytic route for synthesis of nicotinamide and nicotinic acid.
8. Figure 3.8 Nicorandil.
9. Figure 3.9 Chemical structure of inositol hexaniacinate (European Food Safety Authority (EFSA), 2009).
Chapter 4: Pantothenic Acid
1. Figure 4.1 Chemical structure of pantothenic acid.
2. Figure 4.2 Chemical structure of coenzyme A (CoA).
3. Figure 4.3 Chemical structure of calcium hopantenate.
4. Figure 4.4 Chemical structure of pantothenol.
5. Figure 4.5 Comparison of enzymatic and chemical resolution processes for DL-PL (Shimizu et al., 2001).
6. Figure 4.6 Pathway of biosynthesis of CoA from pantothenate (Combs, 2008).
7. Figure 4.7 The CoA biosynthetic pathway and its key players in bacteria and mammals. ATP, adenosine triphosphate; ADP, adenosine diphosphate; CO₂, carbon dioxide; PPi, pyrophosphate (Lopez Martinez, Tsuchiya and Gout, 2014).
8. Figure 4.8 Biosynthesis pathway of pantothenic acid in Corynebacterium glutamicum ATCC 13032 (Hüser et al., 2005).
Chapter 5: Folate: Relevance of Chemical and Microbial Production
1. Figure 5.1 Structures of folic acid (FA), tetrahydrofolic acid (THF) and of its native, biologically active derivatives.
2. Figure 5.2 Pathway of de novo bacterial biosynthesis of folate. GTP, guanosine triphosphate; DHPP, 6-hydroxymethyl-7,8-dihydropterin pyrophosphate; pABA, para-aminobenzoic acid; DHP, dihydropteroate; DHF, dihydrofolate and THF, tetrahydrofolate.
3. Figure 5.3 Structures of one-carbon derivatives of THF and example of reactions in which they are involved: (a) transfer of a C₁ moiety and reduction to DHF; (b) transfer of a formyl group (GAR, glycinamide ribonucleotide transformylase) and (c) transfer of a methyl (MS, homocysteine S-methyl transferase or methionine synthase).
4. Figure 5.4 Chemical synthesis of FA.
5. Figure 5.5 Chemoenzymatic synthesis of (6S)-methylTHF (7) and (6S)-5-formylTHF (9).
6. Figure 5.6 Chemical methods for the synthesis of (6S)-methylTHF (7) and (6S)-5-formylTHF (9).
Chapter 6: Vitamin B₁₂ – Physiology, Production and Application
1. Figure 6.1 Structure of vitamin B₁₂. The DMBI group (green) is coordinated with the cobalt at the lower ligand (α) and linked to the corrin ring (red) by an amino-propanol-ribosyl side chain (base-on conformation). At the upper ligand (β), an R group is linked to the cobalt. (a) 3D structure: the planar corrin ring and the orientation of the ligands, (b) the different upper-or β-ligand occurring in food.
2. Figure 6.2 Absorption, cellular uptake and cofactor roles in human metabolism of vitamin B₁₂. B₁₂: active B₁₂ form; Co: cobalt and oxidation state; HC: haptocorrin; IF: intrinsic factor; AdoB₁₂: 5′-deoxyadenosylcobalamin; MeB₁₂: methyl-cobalamin; THF: tetrahydrofolate; TC: transcobalamin (modified from Green and Miller, 2014; Randaccio et al., 2010).
Chapter 7: Industrial Fermentation of Vitamin C
1. Figure 7.1 Vitamin C (L-ascorbic acid).
2. Figure 7.2 Tadeus Reichstein.
3. Figure 7.3 The Reichstein process for vitamin C synthesis.
4. Figure 7.4 The interaction relationship between K. vulgare and the companion strain.
5. Figure 7.5 The two-step fermentation process for Asc production.
6. Figure 7.6 Chemical conversion of 2-keto-L-gulonic acid to vitamin C.
Chapter 8: Direct Microbial Routes to Vitamin C Production
1. Figure 8.1 Structures of D-glucose, L-ascorbic acid and 2-keto-L-gulonic acid. The stereocenters at C₅ and C₄ of L-ascorbic acid and 2-keto-L-gulonic acid are indicated by one or two asterisks, respectively. The corresponding C₂ and C₃ positions in D-glucose, relevant for L-Asc synthesis routes involving ‘carbon inversion’ (natural pathway in animals, industrial synthesis route) are similarly indicated.
2. Figure 8.2 Conceptual outline of direct routes from D-glucose to L-Asc. The final oxidation step leading to L-Asc will be at C₂ for 1,4-lactone intermediates (such as L-gulonic acid 1,4-lactone), or C₁ for 2-keto aldose intermediates (such as L-sorbosone). Both types of intermediates have different propensities for different types of ring structures. 1,4-lactones are prevalent, while for the 2-keto aldoses, the corresponding 1,4-hemiacetals are not formed to appreciable extend. L-Asc formation from 2-keto aldoses may proceed via 1,5-hemiacetals, but an additional re-arrangement step to L-Asc is required.
3. Figure 8.3 Top: Erythroascorbic acid biosynthesis in S. cerevisiae. Bottom: L-Ascorbic acid biosynthesis in recombinant S. cerevisiae provided with heterologous genes encoding (i) GDP-D-mannose-3,5-epimerase, (ii) GDP-L-galactose-phosphorylase and (iii) L-galactose-1-phosphatase. Enzymes encoded by the heterologous genes are marked in italic. The recombinant pathway taps into the abundant GDP-D-mannose pool of the host strain.
4. Figure 8.4 Direct production of L-Asc via L-galactono-1,4-lactone obtained from pectin in Aspergillus niger as single microorganism. Knockout of gaaB encoding L-galactonic acid dehydratase and overexpression of gaaA encoding D-galacturonic acid reductase re-directs the flux towards L-galactono-1,4-lactone. The synthesis route to L-Asc is completed by heterologous expression of L-galactono-1,4-lactone dehydrogenase from Acerola (marked by italics).
5. Figure 8.5 Direct production of L-Asc via L-gulono-1,4-lactone from starch by combinations of chemical and biocatalytic reaction steps. Chemical conversion of starch to L-gulono-1,4-lactone is followed by biocatalytic oxidation to L-Asc by any of the known L-gulono-1,4-lactone oxidoreductases.
6. Figure 8.6 A possible novel biocatalytic route from D-sorbitol directly to L-Asc. Correct stereo conformation is obtained as in the Reichstein-Grüssner and the 2-KGA fermentation process by inversion of the carbon skeleton of D-glucose to L-sorbose via D-sorbitol. The key enabling elements of the direct route to L-Asc are (i) oxidation of L-sorbose to L-sorbosone at C₁ without further oxidation to 2-KGA and (ii) oxidation of L-sorbosone at C₁ by specific L-Asc-forming dehydrogenase that provides 2-keto-L-gulonic acid as 1,4-lactone instead of the free acid. This reaction presumably proceeds from the 1,5-hemiacetal isomer of L-sorbosone as substrate and entails an additional re-arrangement step to the 1,4-lactone.
7. Figure 8.7 Structure of the Sndhak homologous enzymes soluble glucose dehydrogenase (sGdh) from Acinetobacter calcoaceticus and aldose sugar dehydrogenase (Asd) from Escherichia coli. The course of the polypeptide backbone is indicated in cartoon representation. The bound cofactor PQQ and substrate β-D-glucose are shown in stick representation. The overall architecture of both enzymes is highly similar, but sGdh is distinguished by a set of five strongly extended loops (indicated), which form a rim around the substrate binding site. The drawings were prepared with the Maestro molecular graphics software (Schrödinger, LLC, New York, NY, 2014) using the coordinates with the pdb access codes 1CRU for sGdh and 2G8S for Asd. The glucose in the Asd structure is added for illustration based on the position of glucose in the sGdh structure.
8. Figure 8.8 Routes to L-Asc and 2-KGA from the prevalent L-sorbosone isomers. Oxidation of L-sorbosone to 2-KGA generally proceeds from the 2,6-pyranose form (Ssdh, mSndh, cSndh). The 1,4 furanose form would be predestined for resulting in L-Asc upon oxidation, but has only very minor abundance and is therefore not shown. Oxidation of L-sorbosone to L-Asc presumably proceeds via the 1,5-pyranose form with subsequent transesterification (Sndhai, Sndhak), but it may also yield 2-KGA by-product upon hydrolysis. The specificity of Sndhai and Sndhak to the anomers of L-sorbosone 1,5-pyranose is discussed in Figure 8.9. SsdB presumably uses the same 1,5-pyranose form of L-sorbosone as substrate, but yields exclusively 2-KGA upon hydrolysis of the intermediate.
9. Figure 8.9 Structural match of the putative natural substrates of Sndhak (β-D-glucose, β-D-xylose) and Sndhai (myo-inositol, α-D-xylose) to the 1,5-pyranose isomers of L-sorbosone. The numbering of the carbon atoms is indicated, as is the hydrogen at C₁ which is abstracted as hydride upon oxidation (Oubrie et al., 1999b). The structures are drawn to result in the same orientation of this catalytically critical hydrogen at C₁. Note that the assignment of the anomers as α and β is inverted in L-sorbosone compared to D-glucose and D-xylose due to the change of the stereochemistry of the anomeric reference atom C₅.
Chapter 9: Synthesis of β-Carotene and Other Important Carotenoids with Bacteria
1. Figure 9.1 Synthesis of isoprenoid precursors dimethylallyl-pyrophosphate (DMAPP) and isopentenyl-pyrophosphate (IPP) via the methyl-D-erythritol-4-phosphate (MEP) pathway.
2. Figure 9.2 Biosynthesis pathway of β-carotene. Dimethylallyl-pyrophosphate (DMAPP); isopentenyl-pyrophosphate (IPP), geranyl-pyrophosphate (GPP); farnesyl-pyrophosphate (FPP) and geranylgeranyl-pyrophosphate (GGPP).
3. Figure 9.3 Pathway of the synthesis of C₄₀ carotenoids by recombinant Escherichia coli. The central precursor substrates IPP and DMAPP can be formed via MEP and/or mevalonate pathway. G3P, glyceraldehyde-3-phosphate; DXP, 1-deoxyxylulose-5-phosphate; MEP, 2-C-methyl-erythritol-4-phosphate; IPP, isopentenyl-pyrophosphate; DMAPP, dimethylallyl-pyrophosphate; GPP, geranyl-pyrophosphate; FPP, farnesyl-pyrophosphate and GGPP, geranylgeranyl-pyrophosphate.
4. Figure 9.4 Scheme of the astaxanthin biosynthesis pathway in recombinant E. coli, proceeding from β-carotene (1) to astaxanthin (10). W β-carotene ketolase (CrtW), Z β-carotene hydroxylase (CrtZ), (2) β-cryptoxanthin, (3) echinenone, (4) 3′-hydroxyechinenone, (5) 3-hydroxyechinenone, (6) zeaxanthin, (7) canthaxanthin, (8) adonixanthin and (9) adonirubin.
Chapter 11: Microbial Production of Vitamin F and Other Polyunsaturated Fatty Acids
1. Figure 11.1 (a) Structures of the principal essential fatty acids and major polyunsaturated fatty acids. (b) Structure of a triacylglycerol (sometimes but incorrectly termed triglyceride), where R₁, R₂ and R₃ are the long carbon chains (alkyl groups) of fatty acids. These may be the same or different.
2. Figure 11.2 Biochemical conversions of fatty acids by fatty acid desaturases (DS) and elongases leading to the production of long-chain polyunsaturated fatty acids of the n-3, n-6 and n-9 series. The reactions are found in some microorganisms and animals, except for the Δ12 and Δ15 desaturases (indicated by *); hence, linoleic acid (LIN) and alpha-linolenic acid (ALA) are regarded as ‘essential’ fatty acids. In plants, polyunsaturated fatty acids up to ALA and GLA are produced and, in some species, stearidonic acid (STA) but no longer.
3. Figure 11.3 A suggested scheme to account for the biosynthesis of long-chain polyunsaturated fatty acids in marine microorganisms, and particularly in Schizochytrium sp. Other thraustochytrids, using a polyketide synthase (PKS) system (see Metz et al., 2001). The pathway begins with the condensation of acetate, attached to an acyl carrier protein (ACP) and malonyl-ACP [derived from acetyl-CoA using acetyl-CoA carboxylase (ACC)] to give acetoacetyl-ACP. This reaction is carried out by ketosynthetase (KS), sometimes known as condensing enzyme. Acetoacetyl-ACP is then reduced by a ketoreductase (KR) to 3-hydroxybutyryl-ACP, which loses water by a dehydratase (DH) reaction to give crotonyl-ACP, which is reduced by an enol reductase (ER) to give butyryl-ACP. Then follows a further addition of malonyl-ACP and the same sequence of reactions, involving KS, KR and DH, up to the formation of 6 : 1(trans-2)-ACP. In conventional fatty-acid biosynthesis, this intermediate would be reduced (by ER) to 6 : 0 and the sequence would be repeated a further five times to give 16 : 0 (see Figure 11.2). For the PKS sequence, the 6 : 1(trans-2) intermediate might be isomerised to 6 : 1(cis-3), which undergoes condensation with malonyl-ACP leading to the formation of 8 : 2(Δ2, Δ5); then follows seven further cycles of condensation with retention of four of the seven double bonds to give DHA as the final product. As Schizochytrium spp. and related organisms (but not Crypthecodinium cohnii) also synthesise docosapentaenoic acid (DPA) of the ω-6 series, a branched pathway is postulated to occur as the ration of DHA to DPA remains virtually constant under all conditions (Hauvermale et al., 2006)
4. Figure 11.4 Pathway of biosynthesis of fatty acids from glucose in oleaginous microorganisms possessing malic enzyme for NADPH generation. (Adapted from Ratledge, 2014.) Numbers in parentheses indicate the required stoichiometry for the conversion of glucose to 1 mol fatty acyl-CoA. ACC, acetyl-CoA carboxylase; ACL, ATP:citrate lyase; CS, citrate synthase; EMP reactions of the Embden–Meyerhof–Parnas pathway; FAS, fatty acid synthase; ICDH, isocitrate dehydrogenase; MDH, malate dehydrogenase; ME, malic enzyme (NADP-dependent); PC, pyruvate carboxylase; PDH, pyruvate dehydrogenase and G3P, glycerol 3-phosphate. ? = unspecified reactions, probably using glucose-6-dehydrogenase and 6-phosphogluconate dehydrogenase. It should be noted that when the concentration of AMP falls after N limitation, the activity of ICDH is greatly diminished so that there is little activity of the tricarboxylic acid cycle in the mitochondrion. The requirement of ICDH for AMP is indicated by +. Overall conversion: 4.5 glucose + CoA + 9 NAD⁺ + 7 NADPH + 17 ATP → C₁₈-fatty acyl-CoA + 9 CO² + 9 NADH + 7 NADP⁺ + 17 ADP + 17P_i.
5. Figure 11.5 Photomicrographs of microorganisms used for the production of SCOs. (a) Mucor circinelloides similar to that used for production of GLA-SCO (from Professor Yuanda Song and Mr X. Tang, Jiangnan University, China). (b) Crypthecodinium cohnii used for the production of DHA (from Dr Casey Lippmeier, DSM USA). (c) Mortierella alpina used for the production of ARA (from Dr C. Lippmeier, DSM USA). (d) Schizochytrium sp. used for the production of DHA (from the collection of photographs of the late Dr Kirk Apt and provided by Dr C. Lippmeier, DSM USA). (e) Yarrowia lipolytica used for the production of EPA-rich oil – the large droplet in the centre of the yeast is triacylglycerol material
Chapter 12: Vitamin Q₁₀: Property, Production and Application
1. Figure 12.1 Chemical structure of CoQ₁₀.
2. Figure 12.2 Biosynthetic pathways of CoQ₁₀ proposed in most prokaryotes (a) and eukaryotes (b).
3. Figure 12.3 Flowchart for CoQ₁₀ separation by solvent extraction and HPLC.
4. Figure 12.4 Flowchart for CoQ₁₀ separation by solvent extraction and silica gel column.
5. Figure 12.5 Flowchart for CoQ₁₀ separation by solvent extraction and its analysis.
6. Figure 12.6 Flowchart for CoQ₁₀ separation by solvent extraction.
Chapter 13: Pyrroloquinoline Quinone (PQQ)
1. Figure 13.1 Structure of PQQ and its redox activity. PQQ is normally in oxidised state under aerobic conditions and accepts one electron in a semiquinone form or two electrons in a reduced form.
2. Figure 13.2 Proposed mechanism for the ligand-independent activation of epidermal growth factor receptor (EGFR) signalling through redox cycling of PQQ. (a) When epidermal growth factor (EGF) binds to EGFR, phosphorylation of the cytosolic domain occurs, then signalling pathway starts through sequential phosphorylation via activation of extracellular signal-regulated kinase 1/2 (ERK 1/2). (b) The activated (phosphorylated) EGFR is dephosphorylated by protein tyrosine phosphatase 1B (PTP1B), which has catalytic cysteinyl thiol. (c) PQQ is incorporated into the cytoplasm and reduced by reductants existing in cytosol to become PQQH₂, which is sequentially oxidised by O₂ to produce H₂O₂ (redox cycling). Substantial amount of H₂O₂ is accumulated, and the catalytic cysteinyl thiol in PTRP1B is oxidised and inactivated. Inhibition of PTRP1B leads to accumulation of phosphorylated (activated) EGFR which is derived by auto-phosphorylation without EGF binding at low level. Thus, signalling pathway is activated in the ligand-independent manner.
3. Figure 13.3 Several adducts of PQQ with nucleophilic compounds. IPQ (glycine): glycine adduct of PQQ, and it is also obtained after reaction with other amino acids. IPQ (other AA): adduct of PQQ reacted with amino acids other than glycine and the side chain is represented as R. PQQ + ROH: adduct of PQQ with alcohols (ROH). PQQ + RNH₂: adduct of PQQ with amines (RNH₂). PQQ + 2H₂O: adduct of PQQ with two molecules of water in alkaline conditions.
4. Figure 13.4 PQQ backbone structure is derived from two amino acids, glutamate and tyrosine.
5. Figure 13.5 Illustrated scheme of PQQ biosynthesis. Six (K. pneumoniae) or seven (M. extorquens AM1) genes are shown to be involved in synthesis. PqqA has conserved glutamate and tyrosine residues and is believed to be the starting material of PQQ. PqqB is thought to be involved in transport of PQQ to periplasm or in introduction of quinone moiety. PqqC is the enzyme in the last step of the PQQ synthesis, producing PQQH₂ from AHQQ which is the biosynthetic intermediate and accumulated in the mutant disrupted in pqqC. PqqD forms a fusion protein with PqqC or PqqE in some genomes and was recently shown to be able to form a complex with PqqA. PqqF and PqqG are homologous to zinc proteases and are believed to cleave four peptide bonds in PqqA to release AHQQ or a precursor of AHQQ.
Chapter 14: L-Carnitine, the Vitamin B_T: Uses and Production by the Secondary Metabolism of Bacteria
1. Figure 14.1 Biotransformation of trimethylammonium compounds in E. coli. Crotonobetaine is transformed into L-carnitine by carnitine dehydratase activity. D- and L-carnitine are transformed through carnitine racemase activity. Crotonobetaine reductase activity leads to the production of the by-product γ-butyrobetaine.
2. Figure 14.2 Genetic organisation of carnitine-metabolising genes in E. coli. Two structural operons are expressed from the same promoter/operator region. Carnitine-metabolising enzymes are encoded by caiTABCDE operon. The fixABCX operon-encoded proteins are necessary for crotonobetaine reduction. A seventh ORF located at the 3′-end of the cai operon, caiF, encodes for a transcriptional regulator which specifically regulates the expression of cai and fix operons.
3. Figure 14.3 Metabolism of trimethylammonium compounds in Escherichia coli. Trimethylammonium compounds γ-butyrobetaine, crotonobetaine and L-carnitine are activated to form the corresponding thioesters, γ-butyrobetainyl-CoA, crotonobetainyl-CoA and L-carnitinyl-CoA. The reaction proceeds at the expense of ATP hydrolysis (1, betaine:CoA ligase, CaiC). Crotonobetainyl-CoA is reduced to γ-butyrobetainyl-CoA (2, crotonobetainyl-CoA reductase, CaiA). Crotonobetainyl-CoA is enantiospecifically hydrated to L-carnitinyl-CoA (3, carnitinyl-CoA dehydratase, CaiD). The coenzyme A moiety is exchanged from products to substrates (4, crotonobetainyl-CoA:L-carnitine coenzyme A transferase, CaiB). See the main text for details.
Chapter 15: Application of Carnosine and Its Functionalised Derivatives
1. Figure 15.1 Molecular structure of carnosine.
2. Figure 15.2 First chemical synthesis of carnosine (Baumann and Ingvaldsen, 1918).
3. Figure 15.3 Chemical synthesis of carnosine by Curtius method (Sifferd and Du Vignaud, 1935).
4. Figure 15.4 Chemical synthesis of carnosine (Cherevin et al., 2007).
5. Figure 15.5 Use of bacterial β-aminopeptidase for the synthesis of carnosine.
6. Figure 15.6 N-derivatives of carnosine.
7. Figure 15.7 Derivatives of carnosine at the carboxylic moiety.
8. Figure 15.8 Analogues of carnosine.
9. Figure 15.9 Enzymatic process of N-oleoyl carnosine synthesis (Husson et al., 2011).
10. Figure 15.10 Rhodamine – labelled POPC in STED multiphoton (a) and 3D reconstruction of DPPC – liposomal membrane by atomic force microscopy (b).
Chapter 16: Metabolism and Biotechnological Production of Gamma-Aminobutyric Acid (GABA)
1. Figure 16.1 The structure of GABA.
2. Figure 16.2 The metabolism pathway of GABA in bacteria. The pathway consists of biosynthesis and catabolic pathway, as well as export and uptake systems of GABA. The biosynthesis pathway is catalysed by L-glutamate decarboxylase (GAD). The catabolic pathway consists of two enzymatic steps. They are catalysed by GABA transaminase (GABA-T) and succinic semialdehyde dehydrogenase (SSADH). The export and uptake of GABA are carried out by Glu/GABA antiporter (GadC) and GABA permease (GabP). GadC mediates the exchange of intracellular GABA with the extracellular Glu. GabP transports GABA into cytoplasm. Other abbreviation: TCA, tricarboxylic acid cycle.
3. Figure 16.3 Compartmentation of GABA metabolism in Arabidopsis. The key enzymes involved are coloured blue. GAD is located in the cytosol, but GABA-T and SSADH are located in mitochondria. Two transporters, GAT1 (GABA transporter 1) and GABP (GABA permease), are located at the plasma and mitochondrial membrane, respectively. GLYR1 and GLYR2 (glyoxylate reductase isoforms 1 and 2) are located at cytosol and plastid, respectively.
4. Figure 16.4 Overall structure of GadC. (a) Overall structure of E. coli full-length GadC. TM1–TM10 are rainbow-coloured, with TM1 in blue and TM10 in red. TM11 and TM12 are shown in grey. The C-terminal fragment (C-plug) is coloured magenta. (b) The C-plug of GadC blocks an otherwise inward-open conformation. Two perpendicular views of GadC are shown. The C-plug blocks access to the negatively charged substrate-binding cleft (inset).
5. Figure 16.5 Representation of the E. coli genome map with the location of the gene loci relating to GABA biosynthesis and regulation. Genes encoding GAD (red), Glu/GABA antiporter (blue), additional membrane proteins (orange), multi-drug exporters (grey), acid-stress periplasmic chaperons (magenta), LuxR-like transcriptional regulators (light green), AraC-like transcriptional regulators (dark green) and small regulatory RNAs (yellow). The origin of each arrow is where transcripts start.
Chapter 17: Flavonoids: Functions, Metabolism and Biotechnology
1. Figure 17.1 Basic structure and numbering of flavonoids: phenylchromane (a) and chalcone (b) forms.
2. Figure 17.2 Flavonoid biosynthetic pathways. Enzyme abbreviations are described in the text.
Chapter 18: Monascus Pigments
1. Figure 18.1 The chemical structures of 1,4,6-trihydroxynaphthalene and the complex formed by 1,4,6-trihydroxynaphthalene with N-glucosylrubropunctamine or N-glucosylmonascorubramine.
2. Figure 18.2 Organisation of MP biosynthetic gene clusters from M. pilosus, M. ruber and M. purpureus, with the putative cluster gene from T. stipitatus (Balakrishnan et al., 2014a, with kind permission from Elsevier).

List of Tables

Chapter 1: Vitamins, Biopigments, Antioxidants and Related Compounds: A Historical, Physiological and (Bio)technological Perspective
1. Table 1.1 Discovery years of vitamins and their original source
2. Table 1.2 List of vitamins by generic descriptor, with some of their vitamers including active forms
3. Table 1.3 Water-soluble vitamins and their corresponding coenzymes
4. Table 1.4 Survey of the vitamins with main food sources, deficiency diseases, Recommended Dietary Allowance (RDA) and overdose diseases
5. Table 1.5 Physiological functions of vitamin C and fat-soluble vitamins
Chapter 3: Vitamin B₃, Niacin
1. Table 3.1 Niacin in food and feedstuffs (milligram per kilograme on dry mass basis)
2. Table 3.2 Chemical and physical properties of nicotinamide
3. Table 3.3 Chemical and physical properties of nicotinic acid
4. Table 3.4 Microorganisms used as source of enzymes or whole cell biocatalysts for synthesis of nicotinic acid
5. Table 3.5 Enzymes that require NAD and NADP as coenzyme
Chapter 4: Pantothenic Acid
1. Table 4.1 Adequate Intake of pantothenic acid in humans according to life stage groups
2. Table 4.2 Main functions of CoA (coenzyme A) and ACP (acyl carrier protein) in cell metabolism
3. Table 4.3 Pantothenic acid and its naturally occurring derivatives (Shimizu and Kataoka, 1999)
4. Table 4.4 Production of the intermediates in CoA (coenzyme A) biosynthesis by Brevibacterium ammoniagenes (Shimizu and Kataoka, 1999)
5. Table 4.5 Percentage mean change from baseline for serum lipids at months 1–4 (Yang et al., 2014)
Chapter 5: Folate: Relevance of Chemical and Microbial Production
1. Table 5.1 Genes and enzymes for the biosynthesis of DHPP, THF-polyglutamate, chorismate and pABA predicted from the sequenced genomes of genus Bifidobacterium (BLAST, www.genome.jp/kegg/pathway.html)
Chapter 7: Industrial Fermentation of Vitamin C
1. Table 7.1 Vitamin C content in different fruits and vegetables.
2. Table 7.2 Application of Asc and its derivatives
Chapter 9: Synthesis of β-Carotene and Other Important Carotenoids with Bacteria
1. Table 9.1 Overview on prefixes and their structures for carotenoid nomenclature
2. Table 9.2 Examples of native bacterial strains that produce industrially important carotenoids
3. Table 9.3 Synthesis of industrially important carotenoids by recombinant bacteria expressing a heterologous and/or modified native carotenoid-biosynthesis pathway
4. Table 9.4 E. coli genes, which showed increased formation of carotenoids upon overexpression or disruption
Chapter 11: Microbial Production of Vitamin F and Other Polyunsaturated Fatty Acids
1. Table 11.1 Fatty acid profiles of fungi and plants used for GLA production
2. Table 11.2 Commercially available microbial oils containing docosahexaenoic acid (DHA)
3. Table 11.3 Oils derived from Mortierella alpina containing arachidonic acid (ARA)
4. Table 11.4 Fatty acid profiles of oils from Yarrowia lipolytica and Schizochytrium sp. that contain high levels of EPA
5. Table 11.5 EPA contents of photosynthetically grown algae
6. Table 11.6 A comparison of fatty acid profiles from Nannochloropsis oculata (Almega PL oil) and krill oil (from Kagan et al., 2013)
Chapter 12: Vitamin Q₁₀: Property, Production and Application
1. Table 12.1 CoQ₁₀ contents of diverse food sources
2. Table 12.2 CoQ₁₀ contents of processed food sources
3. Table 12.3 Specific CoQ₁₀ contents produced from different cell types
4. Table 12.4 Major enzymes involved in CoQ₁₀ biosynthesis
5. Table 12.5 Specific CoQ₁₀ contents produced from chemically modified mutants
6. Table 12.6 Specific contents of CoQ₁₀ produced from various fermentation systems
Chapter 14: L-Carnitine, the Vitamin B_T: Uses and Production by the Secondary Metabolism of Bacteria
1. Table 14.1 Chromatographic methods for the analysis of L-carnitine and its derivatives
2. Table 14.2 Mass spectrometry (MS) methods of analysis of L-carnitine and its derivatives
3. Table 14.3 Enzymatic methods
Chapter 16: Metabolism and Biotechnological Production of Gamma-Aminobutyric Acid (GABA)
1. Table 16.1 GABA metabolic genes and their encoded proteins in several species
Chapter 17: Flavonoids: Functions, Metabolism and Biotechnology
1. Table 17.1 Basic skeletons of the main flavonoid classes
Chapter 18: Monascus Pigments
1. Table 18.1 MP constituents isolated from Monascus-fermented products
2. Table 18.2 Purification and analysis of MPs by HPLC
3. Table 18.3 Identification methods of MP compounds
4. Table 18.4 Information on Monascus genomes

Preface

Vitamins, provitamins and related compounds belong to the few chemicals that evoke a positive appeal to most people; even for a layman, the term vitamin sounds synonymous to vitality, health, physical and mental strength, fitness, well-being and so on. Indeed, each one of us needs his/her daily intake of vitamins, which should normally be provided by a balanced and varied diet. However, even today, this is not always the case. Current food habits or preferences, food availabilities, as well as food processing, cooking or preservation methodologies and technologies do not always assure a sufficient balanced natural daily vitamin supply to a healthy individual, let alone to a sick or stressed human being. Today, modern society is seldom confronted with the notorious avitaminoses of the past in the Western World, but they do still occur frequently in overpopulated, war-ridden, poverty- or famine-struck regions in many parts of the World. Apart from their in vivo nutritional–physiological roles as essential growth factors and coenzymes for human beings, animals, plants and microorganisms, vitamins and related compounds are increasingly being introduced as food and as feed additives, as medical–therapeutical agents, as health-promoting aids, and also as technical aids, for example, as antioxidants or biopigments. Today, an impressive number of processed foods, feeds, cosmetics, pharmaceutical and chemical formulations contain extra vitamins or vitamin-related compounds, and single and multivitamin preparations are commonly taken or prescribed. These considerations point towards an extra need for vitamin supply, other than those provided from microbial, plant and animal food sources. Most added vitamins and related compounds are indeed now industrially prepared via chemical synthesis, extraction technologies and/or biotechnological routes, such as fermentation and/or biocatalysis. This volume focusses on the use of industrial biotechnological principles and bioprocesses for the production of vitamins and related compounds such as biopigments and antioxidants.

Industrial biotechnology encompasses the exploitation of the genetic and biochemical machinery of useful microorganisms (bacteria, fungi, yeasts and microalgae) and of higher cells for the synthesis of bulk and fine chemicals (including vitamins and related factors), pharmaceuticals, enzymes, biomaterials and energy, using renewable resources rather than fossil ones. Two main types of microbiology-based enabling technologies are involved: fermentation-based technologies and enzyme-based technologies. Fermentation technology relates to the directed and controlled mass production of microbial or higher cells, their enzymes and/or their metabolites. Enzyme technology or biocatalysis deals with the use of microbial or higher cells for their enzyme systems (produced via fermentation processes) to catalyse desirable chemical chiral reactions. Both technologies were initially often rescued only when chemical processes failed to be successful or were uneconomical. Nowadays, they are often the first-choice technologies for several reasons: they are based on renewable resources, deliver simple as well as very complex molecules directly in a desirable chiral form and in an economically favourable way, and they are considered in the society as clean, sustainable and re-usable technologies. Industrial microbiology has its foundations based on knowledge of basic sciences and of technologies as well. It has always been a cornerstone of ‘microbial biotechnology’, even before this name was coined. Indeed, the discipline has attracted the interest of scientists and bioengineers for decades, but new developments in science, in technology, in industry and in society have made it an even more fascinating and indispensable field of research and application. Scientific breakthroughs in high-throughput screening methodologies, in molecular genetics of industrial microbial strains, in systems (micro)biology, in directed evolution, metabolic engineering and modelling, but equally in enzyme and cell engineering, in novel culture techniques, rapid sampling and sensor methodologies, in bioreactor design and in downstream processing, all have contributed to the growing interest and use and impact of industrial microbiology and biotechnology in the industry. The design-based engineering of industrial microbial strains is still hampered by incomplete knowledge of cell biochemistry, metabolic regulation and cell biology. Advances in systems biology technologies and in synthetic (micro)biology can now also contribute to fill this gap. Equally, microbial enzymes are increasingly being used in industry and are further optimised as to their characteristics for practical use in large-scale biocatalytic reactions; basic and applied studies of enzyme and protein engineering and of enzyme technology are essential here. Protein engineering of microbial enzymes is now an important tool to overcome the limitations of natural enzymes as useful biocatalysts; combination of directed evolution and rational protein design using computational tools has become significant to create even novel enzymes, expanding their application potential in industry. The asymmetric biocatalysis with microbial enzymes and cells has now achieved high efficiency, enantioselectivity and yield, such that – for a wide variety of chiral products, including vitamins, biopigments, antioxidants and related compounds – biocatalysis has become a preferred production alternative in organic synthesis and in the chemical industry for fine as well as bulk chemicals.

All the aforementioned developments have justified the timely publishing of a comprehensive book on industrial biotechnology of current vitamin production, biopigments, antioxidants and related compounds. Eighteen comprehensive chapters, all written by renown experts, focus on all aspects, from historical to the latest developments in both fields, fermentation science and enzyme technology, as applied to (pro)vitamins, biopigments, antioxidants and related compounds. So far, such information is scattered widely in the scientific literature; for some compounds, only secrecy and sparse data are available. Some well-known vitamin compounds that are produced currently only chemically are deliberately not covered in this biotech-focussed volume, including B₁, B₆, B₇, D, E and K. For some of these molecules, biotechnological processes are being developed, although, indeed, not competitive as yet with chemical synthesis. Other published volumes cover only one or a few specific vitamin compounds or deal mainly with chemical synthesis, nutritional, biochemical, pharmaceutical or medical aspects.

This volume also aims at demonstrating the broad potential of industrial microbiology and biotechnology to produce these chemically quite complex molecules and its impact on society; it may awake the mind of the researchers – also in other fields of science and technology – to speed up the introduction of these clean biotechnologies in the industry and their products in society!

The help of several colleagues and friends in suggesting potential authors for difficult-to-get chapters has been invaluable to assemble a comprehensive volume. We want to mention especially Dr. Hans-Peter Hohmann, DSM Nutritional Products, Basel, Switzerland; Em. Prof. Yoshiki Tani, Faculty of Agriculture, Kyoto University, Japan; Dr. Hideo Kawabe and Dr. Hideharu Anazawa, Japan Bioindustry Association, Japan and Prof. K. Matsushita, Yamaguchi University, Japan; and Em. Prof. Colin Ratledge, Department of Biological Sciences, University of Hull, UK.

The positive interaction with all the contributing authors is highly appreciated as well. The editors are very much indebted to the staff of Wiley-VCH, Verlag GmbH & Co, Weinheim, Germany, especially to Dr. Reinhold Weber, Dr. Andreas Sendtko and Mrs. Lesley Fenske, who were extremely helpful at the different stages from the conception to the birth of this book! Most gratitude goes to our respective wives, Mireille and Ines, who could only have withstood our mental absence, strengthened with multivitamin preparations, although our sole vitamin shot was their encouraging and moral support during this biotechnological enterprise!

Belgium
Spain
2016

Erick J. Vandamme
José L. Revuelta

Preface

Part I
Water-Soluble Vitamins

Part II
Fat Soluble Vitamins

Part III
Other Growth Factors, Biopigments and Antioxidants