Application of Enzymes and Microbes for the Industrial Production of Vitamins and Vitamin-Like Compounds

3.50 Application of Enzymes and Microbes for the Industrial Production of Vitamins and Vitamin-Like Compounds D Laudert and H-P Hohmann, Biotechnology R&D, DSM Nutritional Products, Basel, Switzerland © 2011 Elsevier B.V. All rights reserved.

3.50.1 3.50.2 3.50.3 3.50.4 3.50.5 3.50.6 3.50.7 3.50.8 3.50.9 3.50.10 3.50.11 3.50.12 References

Introduction and Scope Riboflavin – Vitamin B2 Niacin – Vitamin B3 R-Pantothenic Acid and R-Panthenol – Vitamin B5 and Provitamin B5 Biotin – Vitamin B7 Cobalamin – Vitamin B12 L-Ascorbic Acid – Vitamin C Phylloquinones and Menaquinones – Vitamin K Coenzyme Q10 Pyrroloquinoline Quinone L-Carnitine Outlook

Glossary vitamins An organic compound with essential physiological functions beyond supplying carbon and energy is considered as a vitamin for a particular organism, if the compound cannot be synthesized in sufficient amounts by the organism itself under certain living conditions. The compound, therefore, has to be provided by dietary intake. Thirteen vitamins are presently universally recognized. Essential fatty acids or amino acids fall by convention not under the term vitamin. chemical organic synthesis The purposeful execution of chemical reactions to transform chemical substances into desired products, for example, a vitamin. Chemical reactions requiring no input of energy or a catalyst are spontaneous. Nonspontaneous reactions can be facilitated upon the input of energy, for example, heat or light, or by the application of a catalyst. In the organic synthesis such catalysts are

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acids, bases, transition metals, or soluble organometallic compounds. vitamins by biotechnology Biochemical synthesis involves the use of enzymes as catalysts to facilitate chemical reactions. Biotechnological production of vitamins means the development of appropriate enzymic catalysts and their application in vitamin production processes. Due to the high specificity of the enzymes, a series of biochemical reaction steps can be executed in one single compartment, in particular within a living cell, allowing the production of complex organic molecules, such as vitamins, from simple carbon sources, for example, glucose. genetic engineering The purposeful alteration of an organism’s genetic material by in vitro recombined or synthesized DNA to provide the organism with desired traits, for example, the ability to produce a vitamin in much higher amounts, than the unmodified strain is able to do.

3.50.1 Introduction and Scope The term ‘vitamin’, originally a neologism ingeniously coupling the chemical term amine, that is, a nitrogen-containing organic compound, with the Latin word vita meaning life, became part of the everyday language, where it is generally used to indicate healthy and necessary components of the human diet. One of the first molecules isolated, which belonged to the group of the chemical compounds later termed vitamins, was thiamine. Thiamine was identified as the missing factor in polished white rice causing the nutritional deficiency syndrome beriberi. Based on the misconception that all essential microcomponents of the human diet contain amino groups, such as thiamine, the Polish biochemist Funk coined the term vitamine in 1912. However, soon thereafter L-Ascorbic acid or vitamin C was identified as another essentially micronutrient. Individuals on a diet with insufficient L-ascorbic acid will suffer from scurvy. L-Ascorbic acid turned out to be a carbohydrate without an amine function. Nevertheless, the term persisted however not as ‘vitamine’, but shortened to vitamin. The 13 compounds now recognized as vitamins were isolated and chemically characterized between 1910 and 1940. In most cases, the chemical synthesis succeeded soon after the chemical structure was revealed. In the wake of the elegant laboratory synthetic schemes, which were developed to finally proof the presumed chemical structure and demonstrate the highly developed status of organic synthesis in the past century, elaborate synthesis routes suited for large-scale industrial production were developed by researchers of companies such as Hoffmann-La Roche of Switzerland,

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BASF of Germany, and Takeda of Japan. Large-scale vitamin production became important not only for the fortification of human food, but also for the supply of high-quality feed preparations for farm animals. With few exceptions, industrial vitamin production was done by chemical synthesis until the 1980s. In cases where fermentation processes came into play, for example, for vitamin B12 (cobalamin) or during vitamin C (L-ascorbic acid) production, the underlying production strains were natural isolates, whose productivities were improved by classical means, that is, random mutagenesis and selection procedures. With the advance of genetic engineering techniques, the targeted and purposeful alteration of the genetic make-up of chosen production strains became possible to better utilize cellular pathways for chemical transformation, energy transduction, and supramolecular assembly. Consequently, industrial vitamin B2 (riboflavin) production switched around the year 2000 almost completely to microbial processes based on genetically engineered production strains. Effective vitamin B5 (D-pantothenic acid) fermentation processes based on recombinant strains were developed as well, but the chemical pantothenic acid synthesis comprising for some time now a biocatalytic step to afford the optical active molecule prevailed. During the industrial production of vitamin B3 (comprising both nicotinic acid and nicotinamide), a biocatalytic step converts a nitrile precursor molecule to the amide. Due to its complexity, chemical vitamin B7 (D-biotin) production should have already been replaced by a microbial production technology. In fact, efforts have been ongoing for decades to come up with a competitive biotechnological D-biotin process. However, this has been so far without success, mainly because the final biosynthesis step has not been successfully engineered. For the other vitamins, fermentative routes or synthetic schemes involving biocatalytic steps have been reported in the academic and patent literature, but they are far away from industrial realization yet. For the important vitamins E and A, this might not change for a long time, but for industrial production of some of the other vitamins, we can expect that biotechnology will make more and more inroads in the foreseeable future. The focus of this article is on those vitamins whose production involves fermentative processes or biocatalytic process steps. Only those processes or process steps are considered that are already industrially realized or advanced to a certain extent toward this direction. Not included are processes that look according to the available public data more like proof-of-concept studies delivering biotechnological processes with performances far beyond any commercial relevance. Within the scope are also some vitamin-like compounds used for food and feed supplementation for which biotechnological production steps are applied at large scale. This article is intended as a succinct, nevertheless concise overview of biotechnological operations during the production of vitamins and some vitamin-like compounds. The nutritional significance of these compounds is addressed briefly, as it is done for their biosynthesis, the genes involved, and the regulation of the expression of these genes. In line with the encyclopedic nature of this article, the reference list is kept very short with only two or three key references for each vitamin production process. Further information can be retrieved from these references, but also from the web using appropriate keywords. As for the scientific and technical matters, economic figures referenced here are taken exclusively from publically available sources, mainly from the Chemical Economics Handbook – Vitamins published by SRI Consulting [1]. The figures are not cross­ checked with ongoing market monitoring at DSM Nutritional Products and are therefore not endorsed by this company. Throughout the text either the classical vitamin nomenclature (vitamin B1, B2, B3, etc.) or the chemical names are used in parallel.

3.50.2 Riboflavin – Vitamin B2 In the late 1920s, nutritional scientists distinguished between two components of the vitamin B complex, which was discovered in 1917 in extracts of Brewer’s yeast. One component was designated as vitamin B1 or the antineuritic factor, the other as vitamin B2 or the rat antipellagra factor. Whereas vitamin B1 turned out to be a unique chemical entity, that is, thiamine, originally identified in and isolated from rice bran, vitamin B2 was recognized to consist of several different components including a yellow, intensively fluorescing compound designated riboflavin. Riboflavin was the first vitamin isolated from the vitamin B2 complex in 1933 by Kuhn, György, and Wagner. Thereafter, pantothenic acid (vitamin B5) and vitamin B6 (pyridoxine, pyridoxal) were identified as the other main vitamins in the vitamin B2 complex. It was also recognized that the vitamin B2 complex preventing a pellagra-like condition in rats was not identical to the human antipellagra factor (see Section 3.50.3). Today the term vitamin B2 refers to riboflavin only. Riboflavin had already been isolated 50 years earlier by Blyth from whey without recognizing its nutritional function. Kuhn, Weygand, and Karrer determined the structure of the yellow pigment and proved it by chemical synthesis in 1933 and 1934. Riboflavin is biosynthesized in plants and in many microorganisms. Vegetables and milk are major sources of the vitamin in human nutrition. Riboflavin serves as the precursor molecule for the biosynthesis of flavin mononucleotide (FMN) by phosphorylation of riboflavin and flavin adenine dinucleotide (FAD) by adenylation of FMN. FMN and FAD are the cofactors of the broad class of flavoenzymes coupling the two-electron oxidation of many organic substrates to the one-electron transfers of the respiratory chain. At present over 4000 tons of riboflavin are industrially produced each year, meanwhile exclusively by fermentation. About 70% of this material is used as feed additive in the form of free-flowing, spray-dried granules or microgranules. The remaining 30% is required for the fortification of foods, such as breakfast cereals, pastas, sauces, processed cheese, fruit drinks, vitamin-enriched milk products, baby formulas, and clinical infusions. Due to fierce price competition, the riboflavin market is consolidated to a high degree with only a few producers remaining. Market leader is DSM Nutritional Products from Switzerland producing the vitamin in a plant in southern Germany. BASF from Germany moved its riboflavin production facilities to South Korea some years ago. The main Chinese producer is Hubei Guangji

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Ribulose-5-P H

OH

H

O

H

HCOOH

CH 3 H

OH

H

H

OH

RibA (DHBP syn)

CH2O P

O OH CH2O P

6,7-Dimethyl-8­ ribityllumazine

O

O

O H 2N

N N PPP

O

O H

H

H

OH

H OH

RibA (cycloII) RibD phosphatase

NH NH 2

N

GTP

NADPH

HCOOH NADP+ NH3

NH

HN H

H

OH

H

OH

H

OH

RibH

NH

O

N H

H

N

N

Riboflavin biosynthesis

H

H

OH

H

OH

H

CH 2OH

N

H

O

OH CH 2OH

RibE O N NH

Riboflavin by chemical synthesis H

O

H

OH

Ribose H

OH

H

OH

N

Riboflavin

CH 2OH

N

H

H

H

OH

H

OH

H

O

OH CH2OH

O

NH2

3,4-Dimethyl aniline

H2N O

Barbituric acid NH

N H

O

Figure 1 Biosynthesis of riboflavin in prokaryotes starting from one molecule of guanosine triphosphate (GTP) and two molecules of ribulose 5-phosphate. RibA, bifunctional protein comprising cyclohydrolase II and 3,4-dihydroxy-2-butanone 4-phosphate synthase domains; RibD, bifunctional protein comprising 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone-5-phosphate deaminase and reductase domains; RibH, 6,7-dimethyl-8­ ribityllumazine synthase; RibE, riboflavin synthase.

from Hubei Province. Shanghai-located Desano discontinued operation of their chemical production plant and might come up with a fermentation plant soon. Riboflavin biosynthesis [2] in prokaryotes starts from guanosine triphosphate (GTP) and ribulose-5-phosphate in a 1:2 molar ratio, respectively (Figure 1). The hydrolytic opening of the imidazole ring of GTP (RibA cyclohydrolase II reaction) is followed by (1) deamination of the resulting pyrimidinone to afford a pyrimidinedione (RibD deaminase reaction); (2) reduction of the ribosyl side chain (RibD reductase reaction); and (3) dephosphorylation of the resulting ribityl side chain (phosphatase reaction). 6,7-Dimethyl-8-ribityl lumazine (DMRL), the direct biosynthetic precursor of riboflavin, is synthesized by adding the 4-carbon moiety 3,4-dihydroxy-2-butanone-4-phosphate derived from ribulose-5-phosphate to the pyrimidinedione intermediate (RibA DHBP synthase reaction and RibH reaction, respectively). Finally, two molecules of DMRL are converted in a dismutation reaction to riboflavin and the pyrimidinedione intermediate of the pathway (RibE reaction). In the fungal riboflavin biosynthetic pathway, the ribosyl side chain is first reduced and then the imidazole ring is deaminated. The first microbial riboflavin production processes that were developed in the 1940s used Eremothecium ashbyi and Ashbya gossypii, two natural riboflavin-overproducing yeast species, as production strains. These processes turned out to be economically inferior to the upcoming chemical processes in the 1950s. A riboflavin production process based on Candida famata, another natural riboflavin-overproducing fungus, was abandoned after several years of industrial usage by Coors Brewing Company, USA, and Archer Daniels Midland Company, USA. Merck Sharp and Dohme, USA, returned to A. gossypii as host for riboflavin production in the early 1970s and developed a process that was later purchased by BASF. BASF implemented the Ashbya process at industrial scale and after several years during which the chemical and the microbial processes were used in parallel abandoned the chemical route. The vitamin is now exclusively produced in a fermentation plant in Gunsan, South Korea.

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Over the years the production capabilities of the A. gossypii host strain has been continuously improved. Wild-type A. gossypii, originally isolated as a severe but today negligible cotton pathogen, produces 2 mg riboflavin per gram biomass, possibly for light protection of its spores. Adjustment of process parameters allows a productivity of up to 100 mg g−1 biomass even by wild-type A. gossypii. To increase the metabolic flux through the riboflavin pathway, additional copies of the genes encoding riboflavin biosynthetic enzymes were introduced into the genome of A. gossypii. For that purpose, the A. gossypii rib genes were cloned and a suitable A. gossypii genetic system for transformation and gene expression was elaborated. Further strain improvements were obtained by selection of antimetabolite-resistant mutants, for example, mutants resistant to itaconate, an inhibitor of isocitrate lyase. Itaconate-resistant mutants might have gained an increased flux through the anaplerotic glyoxylate shunt providing malate from acetyl-CoA, which is derived from β-oxidation of fatty acids. An optimized metabolic flux through the glyoxylate shunt could be of relevance for the A. gossypii riboflavin process that uses vegetable oils as fermentation carbon sources. The oils are present in the fermentation broth as osmotically inactive emulsified triglyceride droplets and rigid fat particles allowing a high nutrient load. Upon starvation A. gossypii secretes a lipase hydrolyzing the triglycerides in the culture medium into free fatty acids, which are then ingested by the fungus. The lipase in the broth is inactivated within minutes presumably due to aggregation at the lipid/water interface. Furthermore, elevated concentrations of free fatty acids interfere with lipase secretion. Both regulated lipase secretion and inactivation result in a steady-state equilibrium of lipase activity in the fermentation broth, such that just the amount of fatty acids that is taken up by the fungus is produced from the triglycerides. Riboflavin-overproducing A. gossypii stores significant amounts of the product as intracellular crystals. A heating step after completion of the main fermentation run induces self-lysis of the production strain and liberates riboflavin from the biomass. In addition, heating of the fermentation broth and slow cooling over several hours promote growth of the riboflavin crystals in the broth facilitating the separation of the crystals from the biomass by decantation. Further purification is attained by resuspension of the riboflavin-containing precipitate in diluted aqueous acids, heating, and decantation. At Roche Vitamins, now DSM Nutritional Products, the development of a microbial riboflavin production strain started from Bacillus subtilis Marburg 168, which is not a natural riboflavin-overproducing species. However, deregulated, riboflavin-secreting mutants can be easily obtained. Such a mutant designated RB50 was isolated, which later turned out to express a mutant riboflavin kinase with drastically reduced FMN forming activity, leading to low FMN levels in the cell. Since FMN, but not riboflavin acts as an effector molecule triggering the riboswitch-based riboflavin repression system in B. subtilis, the mutants overproduce and secrete riboflavin. RB50 contains also mutations affecting the regulation of purine biosynthesis. Genetic engineering was then applied to further enhance rib gene expression by making use of strong, constitutive promoters and by increasing the rib gene dosage. Several rounds of traditional mutagenesis and selection for deep yellow colonies completed the development of the first generation DNP production strain. Meanwhile a second-generation strain is employed that features an increased supply of ribulose-5-P and ribose-5-P, which are important building blocks for riboflavin biosynthesis. For this purpose, the gene encoding wild-type transketolase, a key enzyme in the pentose phosphate pathway, was replaced by a mutant allele with drastically reduced activity. A 48 h fed-batch fermentation protocol was developed with glucose as the growth limiting substrate. During the fermentation, riboflavin is secreted from the production strain and crystallizes in the fermentation broth. The long needle-shaped crystals can be easily recovered and separated from the biomass by centrifugation. An acid treatment of the recovered riboflavin crystals at elevated temperatures followed by intensive washing resulted in a 96% pure product. The food/pharma-grade riboflavin of over 99% purity is obtained after recrystallization of the 96% product. Since the year 2000, DSM Nutritional Products produces riboflavin exclusively by the B. subtilis-based microbial process in southern Germany. Riboflavin overexpressing and secreting B. subtilis strains were also developed at the Russian Institute for Genetics and Selection of Industrial Microorganisms, Moscow. In fact B. subtilis VNIIGenetika 304 containing the plasmid pMX45 was the first riboflavin production strain and maybe even the first production strain for a small organic molecule at all, which was obtained by a genetic engineering program. The host strain was deregulated with regard to the purine and the riboflavin biosynthetic pathway. The plasmid pMX45 contained a 10kbp EcoRI fragment comprising the entire rib operon of B. subtilis driven from its natural promoter. With VNIIGenetika 304/pMX45 4.5 g l–1 riboflavin was produced from a total of 100 g l–1 saccharose supplied during a 25-h fermentation run. The co-occurrence of a chromosomal and episomal copy of the rib operon in B. subtilis production strains provided with pMX45 gave rise to plasmid instability. Genetically stable riboflavin production strains were obtained using an integrative vector construct, which targeted the entire rib operon of Bacillus amyloliquefaciens in proximity to the pur operon of a B. subtilis production strain. The resulting strain Y32 produced 3 g l–1 riboflavin during a 72-h shake flask fermentation, but should have the potential to produce considerably more riboflavin under industrial fed-batch fermentation conditions. It is presumed that riboflavin-producing companies from China employ B. subtilis production strains with a genetic make-up similar to that of Y32. Purine analogue-resistant Corynebacterium ammoniagenes mutants that were transformed with plasmids encoding the C. ammoniagenes rib operon proved efficient riboflavin production strains at Kyowa Hakko of Japan. Strain KY13628, which was provided with plasmid pFM67 encoding the entire C. ammoniagenes rib operon driven by the strong C. ammoniagenes promoter P54-6, accumulated 5 g l–1 riboflavin during a 72 h aerobic fermentation run at 32°C with ammonia as pH-titrant and nonlimiting glucose supply. The activities of ribA-encoded cyclohydrolase II and ribE-encoded riboflavin synthase in protein extracts of the strain were 0.011 and 2.8 mU mg–1, respectively. With an optimized ribosomal binding site in front of ribA the activity of cyclohydrolase II was increased threefold and 15.3 g l–1 riboflavin was produced at 4.8% (w/w) yield on glucose. Despite the attractive published productivity, these C. ammoniagenes production strains were probably never employed for commercial riboflavin manufacturing.

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Riboflavin is the paradigm vitamin whose industrial production process switched completely from chemistry to biotechnology. The latter is superior with regard to economic efficiency. But also the advantages for the environment have been demonstrated for both, the Ashbya and the Bacillus process, in several ecological footprint studies. For more details on microbial riboflavin production, the reader is referred to [3].

3.50.3 Niacin – Vitamin B3 The term niacin is an acronym derived from nicotinic acid and vitamin and refers to both nicotinic acid and its nicotinamide derivative. The latter is used as building block for the biosynthesis of the coenzymes nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP). These compounds are used as indispensable cofactors by about 200 different classes of dehydrogenases. NAD functions mainly in catabolic reactions that generate energy by biological oxidation of carbohydrates, proteins, and fatty acids. NADP is involved mainly in anabolic reactions to build up cell mass. Strictly speaking, niacin is not a vitamin, because the amino acid tryptophan can be converted to nicotinic acid in humans provided that an adequate dietary supply of tryptophan, an essential amino acid for humans, is available. The daily requirement of niacin depends on the quantity of tryptophan in the diet and a conversion factor of 1 niacin equivalent (NE) for 60 mg tryptophan was established. The recommended dietary allowance for adults is 14 and 16 mg NEs for women and men, respectively. Niacin occurs widely in nature and concentrations in dietary sources vary from 1 mg kg–1 in whole milk to 150 mg kg–1 in veal liver. Nicotinic acid is the form present in food of plant origin, whereas in animal products nicotinamide predominates. Nicotinic acid was first synthesized in 1867 by oxidative degradation of nicotine. Not before 1937, it was demonstrated that nicotinic acid cures human pellagra, a nutritional deficiency disease with symptoms like dermatitis, diarrhea, and dementia. More than 25 000 and 3000 tons of nicotinic acid and nicotinamide, respectively, are produced mainly for animal feed applications per annum worldwide. Lonza AG of Switzerland produces nicotinic acid in their Swiss production site in Visp, whereas the manufacturing site for the amide is in China, Guangdong Province. Other companies producing vitamin B3 in China include Nantong Reilly Chemical Co. and Nanjing Red Sun Group Co., both in Jiangsu Province. A good part of the commercial nicotinic acid production proceeds via oxidation of 2-methyl-5-ethylpyridine (MEP) with nitric acid, which is required in sub-stoichiometric amounts since the resulting nitric oxide is air oxidized and reused (Figure 2). MEP itself is produced by liquid-phase condensation of paraldehyde and ammonia [4]. An alternative starting material for nicotinic acid and the more preferred one for nicotinamide is 3-picoline, which is supplied by the condensation of acetaldehyde, formaldehyde, and ammonia in the gas phase. The main product of this reaction is pyridine, which explains why major pyridine producers are also active in the vitamin B3 business. A route to 3-picoline that is decoupled from pyridine

Acetaldehyde Ammonia Formaldehyde

Gas-phase reaction

Hydrogenation

NC

CN

NH2

H2 N

2-Methylglutaronitrile

Intramolecular

condensation

Dehydrogenation N H

N

N

3-Methylpiperidine

3-Picoline

Pyridine

2-Methylpentanediamine

Gas-phase aminoxidation C

N

3-Cyanopyridine N O

Ammonia Paraldehyde

Liquid-phase condensation

Oxidation with nitric acid N

2-Methyl-5-ethylpyridine

C

N

Nicotinic acid

Nitrilase catalyzed or chemical

Nitrile hydratase catalyzed O C

OH

NH2

N

Nicotine amide

Figure 2 Production routes for nicotinic acid and nicotinamide starting from different raw materials. Nitrilase and nitrile hydratase convert 3-cyanopyridine into nicotinamide and nicotinic acid, respectively.

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starts from 2-methylpentanediamine, which reacts intramolecularly to 3-methylpiperidine. After dehydrogenation to 3-picoline, gas-phase ammoxidation leads to 3-cyanopyridine. Under the catalyzing activity of nitrile hydratases, 3-cyanopyridine specifically reacts to nicotinamide. Nicotinic acid production is negligible. For commercial application, whole cell biocatalysts expressing nitrile hydratases are used, for example, Rhodococcus rhodochrous J1 [5] possibly in an immobilized form to facilitate a continuous process. Other Rhodococcus strains such as GF270 and GF376 show improved biocatalytic characteristics, for example, heat stability. Bacteria of the genera Amycolatopsis and Actinomadura have the ability to rapidly and specifically convert 3-cyanopyridine to nicotinamide, as well [6]. Nitrilases in contrast to nitrile hydratases catalyze the complete hydrolysis of nitriles to the corresponding carbonic acids. It is unclear whether nitrilase-based biocatalysts are industrially applied for nicotinic acid production from 3-cyanopyridine. This compound as well as 3-picoline can be converted to nicotinic acid in a purely chemical hydrolysis or oxidation reaction, respectively.

3.50.4 R-Pantothenic Acid and R-Panthenol – Vitamin B5 and Provitamin B5 A water-soluble, acidic compound stimulating the growth of certain Saccharomyces cerevisiae strains was described and partially characterized by Williams in 1933. He chose the name pantothenic acid derived from the Greek word ‘pantothen’ meaning ‘from everywhere’ for this compound, because it was found in a wide range of biological materials. The chemical structure, an amide between R-pantoate and β-alanine was elucidated in 1938 also by Williams. A year later, pantothenic acid was recognized as the last missing component of the so-called vitamin B2 complex (see Section 3.50.2) confirming its nutritional function as a vitamin in mammals. One year later, a method to synthesize and crystallize the molecule was established. Pantothenic acid or vitamin B5 is mainly used as a building block for coenzyme A biosynthesis, which occupies a central role in the metabolism of all cells. Dietary deficiencies of pantothenic acid are extremely rare because it is widely distributed in plants, animals, and microbes in the form of coenzyme A. The recommended daily dietary allowance for adults is 5 mg. Rich food sources are organ meats like veal liver (7.9 mg/ 100 g) or Brewer’s yeasts (7.2 mg/100 g), but also eggs (1.6 mg/100 g), milk (0.35 mg/100 g), vegetables (0.2–0.6 mg/100 g), and whole grain cereals (1.0 mg/100 g). Pantothenic acid is used in multivitamin preparations and added as supplement to a variety of foods such as breakfast cereal, beverages, and baby and dietetic foods. As pantethine, a derivative of pantothenic acid, it is used in pharmaceutical applications to lower cholesterol and triglycerides level. The bulk of industrially produced pantothenic acid, however, goes into animal feed preparations. R-panthenol the alcohol analogue of pantothenic acid is converted after ingestion into vitamin B5 and is thus a provitamin of vitamin B5. The main application of panthenol is in personal care as a humectant, emollient, and moisturizer in shampoos and hair conditioners. In ointments, panthenol has good skin penetration and together with allantoin it is used for treatment of minor skin lesions. The current world demand of R-pantothenate and R-panthenol is more than 10 000 and 2000 tons, respectively. The commercially important calcium salt of R-pantothenic acid and R-panthenol is produced by the condensation of R-pantolactone with β-alaninate or 3-amino-1-propanol (Figure 3). β-Alaninate is derived from acrylonitrile or acrylic acid and ammonia. 3-Amino-1-propanol production starts also from acrylonitrile or from ethylene oxide. Hydroxypropionitrile is in both cases a process intermediate. The industrial process for R-pantolactone starts from isobutyraldehyde, formaldehyde, and hydrogen cyanide via the intermediates 3-hydroxy-2,2-dimethylpropanal and 2,4-dihydroxy-3,3-dimethylbutyronitrile. Acidic hydrolysis of the latter affords racemic pantolactone in high yield. For optical resolution, a biocatalytic step based on stereoselective hydrolysis has gained industrial importance. Developed by Daiichi Fine Chemical of Japan in the late 1980s, the racemate is contacted with an R-selective lactonase from a Fusarium sp., which leads to fast hydrolysis of the R-lactone to R-pantoate, while the S-stereoisomer stays widely untouched [7]. Extractive separation of both compounds is followed by lactonization of R-pantoate in the aqueous phase and its purification and crystallization. The S-pantolactone in the organic phase is racemized by thermal treatment and subjected to a new optical separation round. A similar production route using a Fusarium sp. as biocatalyst for R-selective ring opening might be taken by various Chinese producers, such as Xinfu Pharmaceutical Co., Zhejiang Province and Xinfa Pharmaceutical Co., Shandong Province [8]. Various Aspergillus spp. express lactonases that like the Fusarium enzyme rapidly hydrolyze R-pantolactone to R-pantoate with high specificity. Optical separation of the pantolactone racemate has also been described with S-selective lactonases. In a simplified process concept published by DSM Nutritional Products R-pantolactone is obtained biocatalytically with high enantiomeric excess from 3-hydroxy-2,2-dimethylpropanal and cyanide followed by acidic hydrolyzation. As biocatalysts Prunus amygdalus hydroxy nitrile lyase isoenzymes are used. To avoid any spontaneous, that is, racemic nitrile formation with a negative effect on the enantiomeric excess, the reaction has to be carried out at low pH. The required acid stable biocatalyst with good activity at low pH could be obtained by enzyme engineering of the P. amygdalus hydroxy nitrile lyase isoenzyme V [9]. Significant efforts have been devoted to a fermentative route to R-pantothenic acid starting from a sustainable carbohydrate feed stock. Biosynthesis of pantothenic acid starting from the common metabolic intermediates α-ketoisovalerate and aspartate is well understood. The products of the panB and panE genes are involved in the conversion of α-ketoisovalerate to R-pantoate. The panD gene product is a pyruvoyl-dependent decarboxylase converting aspartate to β-alanine. Finally, the ATP-dependent panC gene product combines the two pantothenic acid precursors into the final product. Fermentation processes based on Escherichia coli, Corynebacterium glutamicum, and B. subtilis have been reported. The E. coli process developed by Takeda of Japan delivers a respectable 66 g l–1 R-pantothenate in a 72-h fermentation run, but requires β-alanine as a co-substrate. The fact that in the E. coli

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S-Pantolactone Extraction, racemization R,S-2,4-Dihydroxy-3,3-dimethyl butyronitrile Acidic CN OH hydrolysis

Chemical synthesis Formaldehyde

HO

O R-Lactonase R,S-Pantolactone

Spont cyanylation O

OH

Pyruvate COOH

R-Pantoic acid OH

Acidic lactonization

O O

R-Pantolactone

O

H2N

Acrylonitrile or acrylic acid ammonia

COOH Pyruvate

AlsS

Condensation

COOH

β-Alanine

OH

O HO

O

R-2,4-Dihydroxy-3,3-dimethyl butyronitrile OH Acidic CN hydrolysis C HO N

3-Hydroxy-2,2-dimethyl propanal

Isobutyraldehyde

OH

HO

HNL route

HNL

R-Lactonase route

O

H2C O HO

O O

OH

C N

O

OH

N

CH3

HOOC

COOH O

HO

Aspartate

Biosynthesis

R-Pantothenic acid PanD

NH2

llvC

β-Alanine H2N

O

OH COOH

COOH O

COOH

IlvD

OH

COOH OH

O

COOH PanB

OH

OH PanE

HO

Methylene-THF α-Ketoisovaleric acid

PanC

HO O

O

R-Pantoic acid

Figure 3 Industrial production (top) and biosynthesis (bottom) of R-pantothenic acid. In the chemical route, optical pure R-pantothenic acid is obtained either by the R-lactonase-mediated kinetic resolution of racemic pantoate or by the hydroxynitril lyase catalyzed stereospecific synthesis of R-pantoate. HNL, hydroxy nitrile lyase; AlsS, acetolactate synthase; IlvC, ketol-acid reductoisomerase; IlvD, dihydroxy-acid dehydratase; PanB, 3-methyl-2­ oxobutanoate hydroxymethyltransferase; PanE, ketopantoate reductase; PanC, pantothenate synthase; PanD, aspartate 1-decarboxylase.

process 40% of the molecular mass of pantothenic acid is not provided by the basic carbohydrate fermentation substrate, but originates from the processed chemical β-alanine should not be without disadvantageous economic consequences. The B. subtilis processes developed by OmniGene Inc., USA, shows an even higher productivity with 86 g l–1 R-pantothenate accumulating within 48 h fermentation time. Interestingly, sufficient β-alanine is provided endogenously in this process superseding exogenous β-alanine supply [10]. Standard separation techniques, such as centrifugation, ultrafiltration, ion exchange chromatography, and crystallization, are suited to isolate R-pantothenate from the fermentation broth and provide sufficient purity. A feed grade product might be achieved by simply spray drying the R-pantothenate containing fermentation broth. Despite the high productivity and the straightforward downstream operations, the microbial pantothenic acid fermentation processes have not made it so far to industrial realization. Further efforts are required to come up with even better strains, in which the metabolic flux is mainly piped toward pantothenic acid to ensure a sufficiently high product yield on the consumed carbon source.

3.50.5 Biotin – Vitamin B7 The history of biotin dates back until 1901 when Wildiers discovered that S. cerevisiae required more than yeast ash, an ammonium salt, and a fermentable sugar for growth as postulated by Pasteur. The missing factor termed bios turned out to be a mixture of several compounds, one of which designated as biotin was isolated and crystallized in 1935 by Kögl. The relevance of biotin as an indispensable nutritional factor for mammals became clear, when György discovered in 1940 that biotin was identical to vitamin H. The latter had been described as a factor present in various foodstuffs and in yeast, which prevented a pellagra-like condition evoked in rats that were fed with high doses of egg white. The capital H of vitamin H stands for the German word ‘Haut’ meaning skin. The adverse component in egg white is avidin with an extremely high affinity to biotin causing biotin starvation in the target animal if overdosed. The chemical structure of biotin was established in 1944 by Kögl in

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Food Ingredients

Europe as well as by du Vigneaud in the United States. The molecule has three asymmetric centers and, thus, can occur in eight stereoisomeric forms, only one of which (the 3aS, 4S, 6aR isomer, D-(+)-biotin) has the full vitamin H activity. In the text that follows, ‘biotin’ stands for this active isomer. Biotin is indispensable as a cofactor for a small group of enzymes catalyzing carboxylation, decarboxylation, and transcarbox­ ylation reactions. These enzymes are (1) acetyl-CoA carboxylase converting acetyl-CoA to malonyl-CoA during fatty acid biosynthesis; (2) methylcrotonyl-CoA carboxylase involved in leucine degradation; (3) propionyl-CoA carboxylase involved in gluconeogenesis starting from lactate, glycerol, or amino acids; and (4) pyruvate carboxylase, forming oxaloacetate from pyruvate during the anaplerotic reaction. In their activated forms these enzymes contain a biotin molecule, covalently attached to specific lysine residues by the enzyme carboxylase synthetase in an ATP-requiring reaction. The adequate daily intake level of biotin for adults is 30 μg from dietary sources. Particularly rich sources for biotin are brewer’s yeast (115 μg/100 g), beef liver (100 μg/100 g), soybeans (60 μg/100 g), and egg yolk (25 μg/100 g). Biotin deficiency in humans is extremely rare because bacteria in the human large intestine produce biotin to a significant amount. Biotin deficiency can occur in individuals with inborn errors of biotin metabolism, in individuals taking certain medications, and occasionally in women during pregnancy. Infant milk formulas, baby foods, breakfast cereals, and dietetic products are frequently enriched with biotin. Biotin is regularly included in multivitamin preparations. It has been demonstrated that protein and amino acid metabolism in fingernail cells and hair roots is activated by biotin. Therefore, healthcare products for hair and skin are supplemented with this vitamin, from which its reputation as the ‘beauty vitamin’ originates. However, from the more than 80 metric tons of biotin produced worldwide per annum by far the bulk part is used for the preparation of poultry, swine, ruminants, and fish feed products. Industrial biotin production is carried out by a chemical process, which was devised by Goldberg and Sternbach in the late 1940s and later adapted by Gerecke. The lengthy, multistep process including an optical separation step is, despite the various process improvements achieved over the years, still very costly. Therefore, industrial researchers are prompted to look for alternative microbial routes. Biosynthesis of biotin starts from activated derivatives of pimelic acid (Figure 4). In B. subtilis pimelic acid is supplied by cytochrome P450 depending BioI via in-chain cleavage of fatty acids. Probably, C14 myristic acid is cleaved between C7 and C8 followed by the consecutive formation of alcohol, threo-diol, and aldehyde intermediates. Pimeloyl-CoA is derived by the BioW catalyzed reaction. Very recently it has been shown that in E. coli the pimeloyl moiety is synthesized by a modified fatty acid pathway, involving BioC and BioH and resulting in pimeloyl-ACP (acetyl accepting protein). Conversion of pimeloyl-CoA or pimeloyl-ACP to dethiobiotin via the bioF, bioA, and bioD gene products is effective. Abundant substrates and conventional biosynthetic reactions are involved in these steps. The final step, however, the BioB catalyzed sulfur recruitment and thiophane ring formation, becomes a significant bottleneck upon attempts to enhance the metabolic flux through the biotin pathway. The intriguing BioB reaction has been elucidated in some detail recently. It became clear that a 5′-desoxyadenosyl radical mechanism activates the nonfunctionalized C1 and C4 positions of dethiobiotin involving the Fe4S4 iron sulfur cluster of BioB. Sulfur is recruited from an Fe2S2 iron sulfur cluster that is present in BioB as well. It was speculated that BioB is used as a reactant rather than a catalyst. In vivo experiments, however, demonstrated that biotin synthase is catalytic, but that catalysis puts the protein at risk of proteolytic destruction. Furthermore, 5′-deoxyadenosine, a co-product of the BioB reaction, is a potent biotin synthase inhibitor in vitro and in vivo. Several attempts have been made in the past to breed biotin overproduction strains. B. subtilis strains were constructed at OmniGene and Roche Vitamins containing multiple copies of the biotin biosynthetic genes under the control of a strong phage promoter. It was recognized that in B. subtilis the amino donor required for the conversion of 7-keto-8-aminopelargonic acid to 7,8-diaminopelargonic acid is not methionine as in E. coli, but lysine. Upon lysine co-feeding, the engineered B. subtilis strains converted pimelic acid quite effectively to dethiobiotin, but further conversion to biotin was rather poor [11]. The highest biotin titer reached in a microbial process so far is 500 mg l–1 in addition to 100 mg l–1 dethiobiotin in a 10-day fed-batch fermentation developed in the mid-1990s at Tanabe Seiyaku, Japan [12]. A Serratia marcescens mutant resistant to various antimetabolites (acidomycin, ethionine, S-2-aminoethylcysteine) was employed as production strain. The expression of the biotin pathway genes was enhanced by their ectopic expression from a broad host range plasmid comprising a 7.2 kb DNA fragment, the S. marcescens bio operon. The still too low product titer, the protracted fermentation time, and safety concerns with S. marcescens prevented the process from industrial realization. The recent insights into the BioB catalyzed reaction, however, might provide novel clues how to engineer this peculiar reaction for higher efficiency, which is the key requirement for a superior microbial biotin process.

3.50.6 Cobalamin – Vitamin B12 The name vitamin B12 refers to a group of physiologically active cobalt-containing molecules also called cobalamins. In the 1920s, Whipple was the first to describe a nutritional factor present in liver that cured anemia in dogs. Later Minot and Murphy identified this factor as iron but coincidentally discovered that another substance present in liver cured pernicious anemia, a then fatal human disease if not treated by the consumption of large amounts of liver or a concentrated liver juice. For their initial work on vitamin B12, Whipple, Minot, and Murphy were awarded with the 1934 Nobel Prize in Medicine. The antiperniziosa

O

O

O

2 rounds

fatty acid synthase

O

BioC HO

ACP

CH3O

Malonyl-ACP

ACP

Methylester

O

O

O

CH3O

O

HO

ACP

ACP

Pimeloyl-ACP

Methylester

O

O

BioH

NH2

Alanine

Pimeloyl supply in

E. coli, S.marcescens

COOH

HO

BioF

CoA

CO2

Malonyl-CoA

N from lys or met

H2N

BioA COOH

7-Keto-8-aminopelargonic acid CO2

Pimeloyl supply in B. subtilis

CHO

Alanine

O

H2N

O

S from Fe2S2 of BioB

NH2

CO2

HN

HN

NH

NH

BioB

BioD COOH

7,8-Diaminopelargonic acid

O

COOH

Dethiobiotin

S

COOH

Biotin

BioF

NH2 COOH

HOOC

HOOC

BioI Myristic acid

OH

O

HOOC

OH BioI, O 2?

COOH

Pimelic acid

BioW

CoAS

C

COOH

Pimeloyl-CoA

Figure 4 Biosynthesis of biotin starting from malonyl-ACP in Escherichia coli or myristic acid in Bacillus subtilis. BioC, methyltransferase; BioH, carboxylesterase; BioF, 7-keto-8-aminopelargonic acid synthase; BioA, adenosylmethionine-7-keto-8-aminopelargonic acid aminotransferase; BioD, dethiobiotin synthetase; BioB, biotin synthase; BioI, cytochrome P450 enzyme (multiple-step oxidative cleavage); BioW; pimeloylCoA ligase.

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Food Ingredients

factor recognized as a vitamin and designated B12 was not isolated until 1948. Its chemical structure was revealed by X-ray crystallography in 1955 by Hodgkin, for which she was awarded with the 1964 Nobel Prize in Chemistry. Cobalamins turned out to be molecules of enormous structural complexity containing a ring-contracted porphinoid derived from uroporphyrino­ gen III with a cobalt ion ligated at the center. A dimethylbenzimidazole group forms the lower ligand. The upper ligand can be an adenosyl, methyl, or hydroxy group giving rise to adenosyl, methyl, or hydroxy cobalamin, respectively. Industrially produced very stable cyanocobalamin, which is converted after ingestion to one of the natural cobalamins, has a cyano group as upper ligand. Almost 20 years after the chemical structure of vitamin B12 was elucidated, the total chemical synthesis with more than 70 synthetic steps was achieved in 1972 by Eschenmoser and Woodward, without doubt a paramount achievement in the art of organic synthesis. In humans only two metabolic reactions are dependent on cobalamin. One is a mutase reaction converting L-methylmalonylCoA to succinyl-CoA during the assimilation of odd-numbered fatty acids. The other is the methionine synthase reaction during which a methyl group is transferred from methyltetrahydrofolate to homocysteine to form methionine. In both cases cobalamin is used as an indispensable cofactor of the enzymes. Rich sources of vitamin B12 in the human diet are organ meats such as beef liver (60 μg/100 g), fish (~8 μg/100 g), or eggs (3 μg/100 g). The recommended dietary daily allowance is 2–3 μg for adults. Recent surveys point to a high proportion of inadequate vitamin B12 intake, even in industrialized countries, and the need of nutritional supplementation, especially for vegan and elderly people. Only some bacteria and archaea can synthesize cobalamins de novo. Among enteric bacteria, two pathways, an aerobic pathway as in Pseudomonas denitrificans and an anaerobic pathway as, for example, in Propionibacteria or Salmonella typhimurium, have been identified for the initial corrin ring formation and the insertion of cobalt (Figure 5). Given the complex structure of vitamin B12, it is not surprising that its biosynthesis is highly intricate. More than 20 biochemical reactions are involved considering only the steps from uroporphyrinogen III, a common intermediate also used for heme and chlorophyll biosynthesis, to the cobalamins [13]. Since an industrial production process based on the protracted and tedious 70-step chemical synthesis is obviously economically not feasible, large-scale vitamin B12 production always relied on fermentation employing species of Bacillus, Methanobacterium, Propionibacterium, or Pseudomonas. The world production for applications in animal and human nutrition in the range of 30 tons yr−1 is low, but a selling price of several thousand Euro per kilogram attracted a number of Chinese producers, such as CSPC Huarong Pharmaceutical Company, NCPC Victor, Yufeng Bioengineering, all Hebei Province, and Duowei Pharmaceutical Company, Ningxia Hui Autonomous Region, into the market that was dominated by French Sanofi-Aventis. As a result, the market is currently characterized by severe production overcapacities and concomitant price pressure. Whereas until the 1990s, both Propionibacterium- and P. denitrificans-based industrial production processes coexisted, nowadays vitamin B12 is almost exclusively produced by the latter. Fermentation raw materials that serve as carbon sources in fed-batch fermentation runs are starch hydrolysates or molasses. Glycine betaine present in significant amounts in sugar beet molasses or

CONH2

Processed corrin ring (cobric acid)

CONH2 CH3

CH3

CONH2

H2NOC H3C

N

X: Methyl or 5 deoxyadenosyl or hydroxy or cyano

N

X

Co+

H3C N

N

CH3

H2NOC

CH3 CH3

1-Amino 2-propanol

O

HO

CH3

NH

CONH2 CH3

O

O

HO

P

N

CH3

N

CH3

Dimethylbenzimidazole

O

O OH

Figure 5 Chemical structure of methyl-, adenosyl-, hydroxyl-, or cyanocobalamin.

Application of Enzymes and Microbes for the Industrial Production of Vitamins and Vitamin-Like Compounds

593

supplied as pure solution is indispensable for high productivity. The stimulating effect of glycine betaine on vitamin B12 biosynth­ esis, which includes eight methylation reactions, is commonly attributed to the replenishing of the C1 precursor pool during glycine betaine assimilation, which is initiated by betaine homocysteine methyltransferase (BHMT). However, the amount of glycine betaine consumed during a typical vitamin B12 fermentation run exceeds the methyl group demand for B12 produced during the run by several 100-fold. Furthermore, it has been demonstrated that BHMTase defective P. denitrificans mutants are still able to overproduce vitamin B12. The formerly used Propionibacterium-based processes did not require glycine betaine co-feeding. In summary, the accurate molecular explanation for the glycine betaine effect might still await elucidation. The vitamin B12 fermentation medium provides, in addition to the carbon substrate, glycine betaine, and the other common fermentation substrates cobalt ions, dimethylbenzimidazole, and sometimes 3-amino-2-propanol, which are all components of the cobalamin molecule. Another peculiarity of B12 fermentation runs is the effect of the oxygen supply regime. Under oxygen-limiting (not anaerobic) fermentation conditions, the yields on the carbon source and probably on betaine, the two major cost drivers, are increased significantly over oxygen excess fermentation conditions. Under optimal fermentation conditions, around 200 mg l–1 vitamin B12 predominantly in the form of adenosylcobalamin accumulates in the fermentation medium during 7-day runs [14]. The downstream steps to isolate and purify vitamin B12 comprise filtration, cyanide treatment, chromatography, extraction, and crystallization, yielding cyanocobalamin in high purity. Elucidation of the vitamin B12 biosynthesis pathway in P. denitrificans and cloning of the 22 cobalamin biosynthetic genes of the organism allowed researchers at Rhone Poulenc to construct genetically engineered production strains, presumably providing a technological advantage in terms of productivity and yield on raw materials over strains solely obtained by classical random mutagenesis and selection methods [15, 16].

3.50.7

L-Ascorbic

Acid – Vitamin C

The Ebers Papyrus from 1550 BC, an important medical document of ancient Egypt, describes already the symptoms of scurvy, such as collagen instability causing loss of teeth, bleeding of all mucous membranes, and others. A common disease among mariners and discoverers during the age of exploration, it became clear at the beginning of the twentieth century that scurvy results from the lack of a nutritional factor in the human diet first designated as antiscorbutic factor, later known as vitamin C or ascorbic acid. Using guinea pigs suffering from scurvy (see below) as experimental model, vitamin C was isolated from lemon juice and crystallized in 1931 by King, Svirbely, and Szent-Györgyi. Its chemical structure was elucidated 2 years later by Haworth. Vitamin C turned out to be a weak 6-carbon sugar acid with a pentagonal ring structure. The exocyclic carbon 5 has L-configuration. Szent-Györgyi and Haworth were awarded the 1937 Nobel Prizes in Medicine and Chemistry, respectively, for their research work on vitamin C. Vitamin C acts as an indispensable cofactor of various oxidases involved in the biosynthesis of collagen, carnitine, and hormones such as adrenaline. Its involvement in the formation of hydroxylated proline and lysine residues of pro-alpha chains of collagen, which serve as cross-linking points within the collagen triple helix and between adjacent triple helixes, is the reason why in its absence defective, unstable collagen fibrils are formed leading to the scurvy symptoms. The high antioxidant power makes vitamin C an effective radical scavenger, another important physiological function of the molecule. L-Ascorbic acid biosynthesis starts from D-glucose, but follows different routes in plants and animals. L-Galactono-1,4-lactone, the ultimate precursor of the plant pathway, is reached via an inversion of the D-glucose carbon skeleton (C1 of glucose becomes C6 in ascorbic acid). In animals, L-gulono-1,4-lactone is obtained by three epimerization reactions. All plants and most animal species are prototroph for vitamin C. Humans, higher primates, guinea pigs, and a small number of other animals require vitamin C supplementation of their diet due to a defect in the gene encoding L-gulono-1,4-lactone oxidase, the last enzyme in the animal synthesis pathway. Fungi of the genera Zygomycetes, Ascomycetes, and Basidiomycetes synthesize D-erythroascorbate, a C5 analogue of ascorbate, from D-arabinose via an inversion pathway. With about 110 000 tons per year, L-ascorbic acid is by far the most bulk produced vitamin. Chinese competitors entering the vitamin C market in the early 1990s caused a ruinous competition. DSM Nutritional Products from Switzerland (formerly Roche Vitamins), remaining as the sole Western producer, positioned itself in the premium segment of the vitamin C market. Chinese companies such as Weisheng Pharma or North China Pharmaceutical Company, both in Hebei Province or Jiangshan Pharmaceutical Co., Jiangsu Province, became the leading producers for the bulk market. In contrast to other vitamins only 10% of the annual ascorbic acid production is used for feed applications, whereas the main outlet is the pharmaceutical (50%), food (25%), and beverages industry (15%). Pharmaceutical applications are either based on the stimulation of collagen synthesis, exploited among others in cosmetic products, or on the health benefits reported, for example, prevention of flu, heart diseases, and cancer. The food and beverages industry takes advantage of the antioxidant capacity of L-ascorbic acid to extend durability, prevent discoloration, and to protect flavor and nutrient contents of their products. Industrial production of vitamin C started out initially by extraction from fruit. The first chemical process based on L-xylosone was carried out in 1933. Until 1934, the famous Reichstein-Grüssner process was developed. Starting with a catalytic hydrogenation of D-glucose to D-sorbitol, the resulting sugar alcohol is oxidized to L-sorbose using Gluconobacter strains as biocatalysts. The subsequent chemical steps including acetonization, oxidation, deacetonization, and rearrangement of the intermediate 2-keto-L-gulonic acid (2KGA) deliver L-ascorbic acid (Figure 6). Today 2KGA is produced mainly by a two-stage microbial process developed in China in the late 1970s and early 1980s [17, 18]. As in the Reichstein process, L-sorbose is provided by Gluconobacter oxidation of D-sorbitol. In a second

Catalytic hydrogenation

‘inversion’ of C-skeleton

HC O H HO H H

OH H OH OH

HO H H

Oxidation with Ketogulonicigenium

CH2OH

CH2OH

OH

HO

H

O

H

HO

H OH H

CH2OH H

Oxidation with G. oxidans

OH

H

OH

HO

CH2OH

CH2OH

D-Glucose

D-Sorbitol

CH2OH D-Sorbitol

HO

H

H HO

OH H CH2OH

L-Sorbose

H HO H HO

Oxidation with Ketogulonicigenium O O

HO

H

HO

OH H CH2OH

L-Sorbosone

H HO

Chemical rearrangement O O H OH H CH2OH

2-Keto-L-gulonic acid

H

CH2OH OH O

O

H HO L-Ascorbic

OH acid

Figure 6 Industrial production of L-ascorbic acid. In a two-stage fermentation process, D-sorbitol derived from D-glucose hydrogenation is oxidized via L-sorbose and L-sorbosone to 2-keto-L-gulonic acid. The latter is chemically rearranged to L-ascorbic acid. For simplicity, the open-chain formulas are drawn as Fischer projections.

Application of Enzymes and Microbes for the Industrial Production of Vitamins and Vitamin-Like Compounds

595

fermentation step, replacing the chemical oxidation steps, 2KGA is obtained via L-sorbosone employing Ketogulonicigenium strains, in Chinese literature frequently referred to as Gluconobacter oxidans. For efficient oxidation, co-cultivation with a helper strain, for example, Bacillus megaterium, is mandatory but the underlying molecular mechanism is still unclear. The execution of the process in two steps is necessary to keep Ketogulonicigenium apart from D-sorbitol. This is important, since otherwise unwanted D-glucose and its oxidation products would be formed. Finally, like in the Reichstein process, 2KGA is converted via the methyl ester to L-ascorbic acid. The two-stage process can achieve up to 130 g l–1 2KGA with a yield above 80% on D-sorbitol. The reduced number of process steps compared to the Reichstein-Grüssner process lowering the process complexity, reduced investment, reduced energy, and lower chemical consumption should provide the two-stage process with a clear cost advantage. In a more recent process variant developed at DSM Nutritional Products, the three oxidation reactions from D-sorbitol to 2KGA are performed in one process step facilitated by a mixed culture of Gluconobacter suboxydans IFO 3255 and DSM No. 4025 in a sophisticated, nevertheless robust process regime preventing side product formation. The co-cultivation with a helper strain required in the two-stage process can be omitted [19]. A single strain process for 2KGA based on genetically engineered G. oxidans strains was developed as well, but is not commercially exploited, probably because the space-time yield of the mixed culture processes is not matched. Alternative microbial routes to 2KGA starting from D-glucose via 2,5-diketo-D-gluconate have been developed employing Erwinia and Corynebacterium spp. in a two-step tandem process or genetically engineered Erwinia sp. expressing a gene for a Corynebacterium 2,5-diketo reductase. These processes did not make it to industrial realization. All vitamin C bioprocesses developed to an industrial stage so far result in the formation of the precursor compound 2KGA, which is converted in a costly chemical rearrangement step to the final product. Obviously, numerous attempts have been made to design microbial routes directly forming vitamin C. One approach tried to utilize the biosynthetic capacity of Baker’s yeast, which can synthesize the 5-carbon vitamin C analogue D-erythroascorbic acid from D-arabinose via D-arabino-1,4-lactone involving D-arabinose dehydrogenase and D-arabinono-1,4-lactone oxidase. Indeed, S. cerevisiae strains genetically engineered for overexpression of D-arabinose dehydrogenase and D-arabinono-1,4-lactone oxidase produced minor amounts of L-ascorbic acid starting from L-galactose, which should be supplied from a combination of epimerase and isomerase reactions starting from the Reichstein intermediate L-sorbose. In a second approach, microalgae synthesizing vitamin C according to the plant pathway via L-galactono-γ-lactone yielded up to 2 g l–1 of the vitamin mainly associated with the biomass. Although D-glucose was the fermentation substrate, the reported low productivity renders a commercial application of the microalgae system rather unlikely. A promising direct route to vitamin C might have been opened by the discovery of dehydrogenases present in Ketogulonicigenium and Gluconobacter spp. that convert L-sorbosone, the partially oxidized biosynthetic intermediate of microbial 2KGA processes, into vitamin C [20, 21].

3.50.8 Phylloquinones and Menaquinones – Vitamin K A then unknown nutritional deficiency disorder leading to hypodermic bleedings in chicken upon administration of a cholesterolfree diet was discovered by Dam in the early 1930s. The factor missing in this chicken diet was designated antihemorrhagic vitamin or vitamin K after the first letter of the German word for blood clotting ‘Koagulation’. Isolated in 1929 from lucerne, Dam and Doisy determined the structure of vitamin K and synthesized the compound at lab scale in 1934. For their contributions to the under­ standing of blood clotting, especially the role of vitamin K, Dam and Doisy received the 1943 Nobel Prize in Medicine. Vitamin K denotes for 3-substituted 2-methyl-1,4-naphthoquinone derivatives that naturally occur in two forms, phylloquinones and menaquinones. Phylloquinone (vitamin K1) occurring in plants contains a phytyl group at position 3, whereas menaquinone (vitamin K2), synthesized by various microbes, contains a polyisoprenyl side chain at position 3 of the naphthoquinone ring. Depending on the microbial source the isoprenyl side chain consists from 6 to 13 isoprenyl units. Menadione (vitamin K3) without any isoprenyl side chain does not occur in nature, but can be converted to vitamin K2 in the animal gut and is therefore used for animal nutrition purposes. The reduced hydroquinone form of vitamin K acts as cofactor for γ-glutamyl carboxylase forming γ-carboxyglutamate residues in various plasma proteins. In humans 14 γ-carboxylated proteins have been discovered so far, all of them being involved in blood coagulation (prothrombin, factors VII, IX, X, and proteins C, S, and Z), vascular metabolism, or bone metabolism (osteocalcin, matrix Gla protein). The daily requirement of vitamin K is 90–120 μg for women and men. The best dietary sources for vitamin K1 are green leafy vegetables such as cabbage (70 μg/100 g), lettuce (110 μg/100 g), broccoli (160 μg/100 g), or spinach (300 μg/100 g). The most important source for vitamin K2 is the bacterial flora colonizing the jejunum and ileum sections of the human and animal small intestines. Vitamin K deficiency is uncommon in healthy adults but occurs in individuals with liver disease, gastrointestinal disorders, fat malabsorption, or after prolonged antibiotic treatment. Since human milk contains only low concentrations of vitamin K, the compound is routinely administered to newborns in many countries to avoid hemorrhagic diseases. New clinical studies suggest that menaquinone optimizes the calcium binding to the bone structure diminishing, thus, the development of osteoporosis and simultaneously interfering with the calcification of the arteries reducing, thus, the risk of cardiovascular diseases. Based on these new findings, vitamin K is frequently used in food supplements or for food fortification. The vitamin is available in straight forms in tablets and capsules or in multivitamin preparation.

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Food Ingredients

Chorismate

Isochorismate

OH HOOC

2-Succinyl-benzoylCoA

OH HOOC

O

O

COOH

COOH

O

Shikimate pathway MenF

MenD, YtxM, MenC, MenE

SCoA O

COOH Biosynthesis of vitamin K2 in B. subtilis

MenB OH COOH 1,4-Dihydroxy-2-naphtoate

Chemical synthesis of vitamin K1 and K3 O

OH

OH

2-Methyl naphthalene

MenA

Menadiol monoester Menadione vitamin K3 O

PPO Heptaprenyl-PP O

OCOR 6 2-Demethyl-menaquinone

Acetylene HO Isophytol 3

MenH

O

O O

3

O

6

Phylloquinone vitamin K1

Menaquinone vitamin K2 O

Figure 7 Chemical synthesis of vitamins K3 and K1 (phylloquinone, lower left) and biosynthesis of vitamin K2 (menaquinone, upper right). MenF, isochorismate synthase; MenD, SHCHS-synthase; YtxM, DHNA-CoA-lyase; MenC, 2-succinyl-benzoate-synthase; MenE; 2-succinyl-CoA-ligase; MenB, DHNA-CoA-ligase; MenA, DHNA-polyisoprenyl-transferase; MenH, methyltransferase.

Vitamin K1 is industrially produced by chemical synthesis condensing isophytol and menadiol monoester in a Friedel-Crafts­ type reaction (Figure 7). Menadiol monoester is obtained from 2-methylnaphtalene via menadione. The starting material for isophytol is acetylene. The world market for vitamin K1 is in the range of 6 metric tons per year with main applications in pharmaceuticals (70%), food (20%), and cosmetics (10%). Manufactures for vitamin K1 are DSM Nutritional Products and several Chinese companies. Increased customer demand for natural products generated a market for vitamin K2, which is derived from microbial sources. In bacteria menaquinones are constituents of the respiratory chain and play an important role in the electron transport. The majority of Gram-positive bacteria utilize only menaquinones, whereas most Gram-negative bacteria utilize ubiquinone under aerobic and menaquinone under anaerobic conditions. The biosynthetic pathway leading to menaqui­ none has been studied in detail [22] for E. coli and B. subtilis. In the latter, menaquinone biosynthesis starting from the common metabolite chorismate and polyprenyl pyrophosphate is accomplished by eight enzymes that are encoded at map position 269° (menF, menD, ytxM, menB, menE, menC), 337.5° (menA), and 203.5° (menH) of the B. subtilis chromosome. The reaction steps entail ring closure and aromatization to form a naphtohydroquinone, attachment of the polyisoprenyl chain, and finally the methylation in the position 2. Bacteria overproducing menaquinone to some extent are Flavobacterium spp. and lactic acid bacteria like Lactococcus lactis. Most of the menaquinone currently available in the market is extracted from natto, a traditional Japanese diet, which is prepared in a solid-state fermentation process with Bacillus natto from steamed soybeans. B. natto is almost identical to B. subtilis. B. subtilis menaquinone-7 production strains were obtained by NTG- or UV-mutagenesis, followed by selection for resistance against toxic menaquinone antimetabolites, such as menadione, diphenylamine, or 1-hydroxy-2-naphtoic acid [23, 24]. The reported produc­ tivities of such mutant strains are in the range of 10–20 mg menaquinone-7 per liter of broth in submerged fermentations. Used as starter strains for natto production 1–2 mg of vitamin K2 is found in 100 g natto. The vitamin can be extracted from natto with isopropyl alcohol and further purified by absorption chromatography, molecular distillation, steam distillation, and chromato­ graphy. The world largest supplier for menaquinone-7 extracted from natto is J-Oil Mills from Japan. B. subtilis GN13/72 from Gnosis SpA of Italy accumulate about 1.5% vitamin K2 in the dry cell mass after 142 h submerged batch fermentations with dextrose or glycerol as carbon source [25]. With 11 g l–1 dry biomass produced, vitamin K2 accumulates to 167 mg l–1 of fermentation broth. Details on the way how GN13/72 was obtained have not been published. Gnosis has launched a menaquinone-7 product obtained from B. subtilis for nutraceutical applications.

Application of Enzymes and Microbes for the Industrial Production of Vitamins and Vitamin-Like Compounds

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3.50.9 Coenzyme Q10 Coenzyme Q (CoQ, ubiquinone) plays a critical role in the mitochondrial respiration of many eukaryotic cells. Procaryotes use CoQ for electron transfer during respiration as well. CoQ collects the electrons generated by various dehydrogenases (NADH dehydrogenase, succinate dehydrogenase, glycerol-3-phosphate dehydrogenase, and acyl-CoA dehydrogenase) and delivers them to the cytochrome bc1 complex (complex III). In addition CoQ is a potent antioxidant. The vital role of CoQ in biological energy transfer was first described in the chemiosmotic theory by Mitchell who was awarded the 1978 Nobel Prize in Chemistry. CoQ was isolated independently by Crane in the United States and Morton in England in 1957. Its chemical structure, a 2,3-dimethoxy-5­ methyl-1,4-benzoquinone with a prenyl side chain in position 6, was elucidated 1 year later. Various kinds of CoQs distinguished by the length of the isoprenoid side chain are found in microorganisms, plants, and animals. CoQ10 (decaprenyl side chain) is the most abundant CoQ in human mitochondria, whereas S. cerevisiae and E. coli use CoQ6 (hexaprenyl side chain) and CoQ8 (octaprenyl side chain), respectively. Human cells have the ability to synthesize CoQ10 de novo, therefore, the compound is not a vitamin sensu strictus, but a good part of the CoQ10 in the human body is derived from food intake. Good food sources of CoQ10 are organ meats with high-energy demand such as heart or muscle of pork, beef, and chicken (3100 μg/100 g). Vegetables and dairy products contain relatively low level of CoQ10 (300–400 μg/100 g). However, no dietary reference intakes or recommended daily allowance have yet been set for CoQ10. CoQ10 deficiency can result from impaired CoQ10 biosynthesis, decreased dietary intake, increased turn over of CoQ10 caused for instance by oxidative stress, or any combination of these factors. After reaching adulthood, CoQ10 levels are decreasing in human tissues, presumably due to declining endogenous synthesis. For people with a mean plasma concentration below 0.8 μg ml–1, supplementation with a CoQ10 preparation is advised to maintain good health. CoQ10 has been studied for its role in heart health, antiaging, and cognitive health. In particular, its role in delaying the onset of Parkinson’s disease attracted a great deal of attention. There are indications that people suffering from angina, heart attack, and hypertension benefit from CoQ10 supplementation. Primarily due to numerous pharmaceutical applications and its increased popularity as supplement in food, beverages, and personal care products, the market size for CoQ10 reached an estimated volume of 150 tons in 2005. North America has a market share of 55%, followed by Asia Pacific with 35% and Europe with 10%. During biosynthesis of CoQ10 decaprenyl diphosphate from the terpenoid biosynthetic pathway and 4-hydroxybenzoate derived from chorismate are condensed followed by several ring modification steps, including decarboxylation, methylation, hydroxylation, and methyl transfer (Figure 8). Eukaryotes synthesize the isoprenoid building blocks generally via the mevalonate pathway, while most, but not all eubacteria utilize the deoxyxylulose pathway. Most of the CoQ10, which is currently available on the market, is manufactured in Japan, the main players being Kaneka, Mitsubishi, Nisshin, and Asahi [26]. New CoQ10 producers from China, India, and Korea have entered the market recently or are on the verge of doing so. Kaneka Corporation, the world’s largest manufacturer of CoQ10 with plants in Japan and Texas, produces CoQ10 by fermentation with a yeast strain, possibly Schizosaccharomyces pombe. This fission yeast biosynthesizes and uses as redox carrier CoQ10 instead of CoQ6, the hexaprenyl CoQ of S. cerevisiae. The other Japanese companies use bacterial production strains, possibly Rhodobacter sphaeroides derivatives, or a semisynthetic route. In the latter, the CoQ10 side chain is derived from solanesol extracted from tobacco plants. This nine-isoprenoid long alcohol is first chemically elongated to decaprenol. During the final step, the decaprenol is reacted with 2,3-dimethoxy-5-methyl-p-benzoquinone to produce CoQ10. With this method, both trans and cis forms of CoQ10 are formed, but subsequent purification steps reduce the cis-isomer content to below 0.5%. The academic and patent literature on microbial CoQ10 production focuses on fermentation processes with bacteria, particu­ larly Agrobacterium tumefaciens and Rhodobacter (also designated as Rhodopseudomonas) sphaeroides [27]. Improved mutants of these species contained 5–10 mg CoQ10 per gram dry cell mass after around 100 h fermentation runs. Limited air supply stimulated CoQ10 accumulation probably interfering with presumed negatively feedback regulation. Further improved production strains were obtained by transforming A. tumefaciens with a recombinant expression vector for the A. tumefaciens dps and dxs genes. CoQ10 production of a wild-type R. sphaeroides strain, naturally recruiting the isoprenoid building blocks via the deoxyxylulose pathway, was stimulated upon transformation with the mevalonate pathway encoding genes from Paracoccus zeaxanthinifaciens [28]. Conventionally, CoQ10 is obtained by solvent extraction and crystallization after termination of the fermentation run. Recently, a fermentation–extraction coupled process with high specific production (32.5 mg CoQ10 per gram dry cell weight) has been published. The process productivity, however, was low with 43 mg l–1 produced during 38 h fermentation time.

3.50.10 Pyrroloquinoline Quinone Pyrroloquinoline quinone (PQQ, also know as methoxatin) was discovered in 1964 by Hauge and its structure was determined by Kennard 15 years later. In addition to flavins and nicotinamides, PQQ acts as the third redox cofactor in bacteria. PQQcontaining enzymes called quinoproteins have an unusual structure with radial symmetry sometimes referred to as 6- or 8-bladed propeller structure. Examples of quinoproteins are methanol dehydrogenase in bacteria that grow on methane or methanol, ethanol dehydrogenase from Acetobacter, or polyol dehydrogenase from Gluconobacter. PQQ is present in common foods, as in tea, papayas, kiwi fruits, and in B. natto fermented soybeans. Mice that are fed with chemically defined diets deprived of PQQ often show signs of reproductive failure and compromised neonatal growth indicating a metabolic or nutritional role for PQQ in mammals. Nutritional intervention trials on human volunteers providing evidence for PQQ’s beneficial effects on men have not

598

Food Ingredients

Erythrose-4-P Pyruvate

Acetyl-CoA O

OH

COOH SCoA

PO O

OH

O

Glyceraldehyde-3-P

Pyruvate

Mevalonate OH

COOH

HOOC

COOH

CHO PO

Dxs

OH

OH

O

CH2 O OH

OH

COOH

Chorismate

OPP

OP O

Isopentenyl-PP COOH

OH

1-Deoxy-D-xylulose-5-P

Dps PPO 9

Decaprenyl-PP

OH

4-Hydroxy benzoate

COOH OOH

O CH3

O H3C H3C 9

OH

O 9

O

3-Decaprenyl-4-hydroxybenzoate

Ubiquinone-10 CoQ10

2,3-Dimethoxy-5-methyl­ 4-benzoquinone Solanesol (nonaprenol)

O

Decaprenol

CH3

O

OH

H3C

OH 8

9

H3C

O O

Figure 8 Biosynthesis (top) of coenzyme Q (CoQ10) by condensation of 4-hydroxybenzoate derived from chorismate with isoprenoid building blocks derived from the mevalonate pathway or the deoxyxylulose pathway. CoQ10 can be synthesized from solanesol extracted from tobacco plants (bottom).

been published so far, but studies on human fibroblast cultures demonstrated that PQQ enhances cell growth and proliferation when added to cell cultures [29]. PQQ was prominently announced recently as a new vitamin, the first since 55 years, after the alleged discovery that a mammalian enzyme involved in the degradation of lysine used PQQ as a prosthetic group. However, the proposition of dietary PQQ being the prosthetic group of a mammalian enzyme was seriously questioned [30], leaving the question open whether PQQ is a vitamin sensu strictus. Developed by Mitsubishi Gas Chemical of Tokyo, Japan, PQQ-containing products were recently introduced in the human dietary supplement market by Maypro Group, NY, after acceptance by the US Food and Drug Administration as a new dietary ingredient in 2008. The product claims are on antioxidative activity. Probably all of the genes encoding enzymes involved in PQQ biosynthesis are known from various methylotrophic and nonmethylotrophic microorganisms since the early 1990s. Nevertheless, their exact functional assignment within the course of the reactions toward PQQ is incomplete [31]. The first step of PQQ biosynthesis is the linkage of glutamate and tyrosine at the 9 and 9′ position catalyzed by the pqqE gene product (Figure 9). Remarkably, PqqE does not recruit the free amino acids, but as members of the PqqA polypetide chain. Depending on the organism PqqA is 24–39 amino acids long comprising the two PQQ precursor amino acids, separated by a linker of three amino acids, in the middle of the polypeptide chain. The pqqA gene is the first gene of the pqq operon that also contains pqqE and, depending on the organism, two to five additional genes including pqqF involved in the removal of the surplus amino acids and the trimming of the 9- to 9′-linked glu-tyr dipeptide to mature PQQ. Biosynthesis of metabolic compounds from polypeptide chains is not uncommon in nature, the most prominent example might be the ACV

599

Application of Enzymes and Microbes for the Industrial Production of Vitamins and Vitamin-Like Compounds

O Val-Thr-Leu COOH

COOH N H 9 9′

N H

9

9′ Tyr

Glu

PqqE

COOH

(aa)x N-terminal

PqqA

O

(aa)x N-terminal

H2N

COOH HN

9

9

PqqF

9′ NH2

NH OH

COOH

OH

(aa)y C-terminal

HO

NH HO O

O

O Val-Thr-Leu

(aa)y C-terminal

O

PqqC, ?

9′ HOOC

N

O

OH O

PQQ

Figure 9 Biosynthesis of pyrroloquinoline quinone (PQQ). PqqE, coenzyme PQQ synthesis protein E; PqqF, coenzyme PQQ synthesis protein F; PqqC, pyrroloquinoline quinone synthase.

tripeptide precursor of penicillins, but in contrast to PqqA these precursor peptides are produced by non-ribosomal polypeptide synthases. Organic chemical synthesis of PQQ is possible, but consists of many steps and requires extensive removal of isomers and various other byproducts. Considering the polypeptide origin of PQQ, it is surprising that many methanol-utilizing bacteria excrete the cofactor into the cultivation medium. The Hyphomicrobium sp. TK0441 was selected by Mitsubishi during a broad survey for PQQ producing bacteria and cultivation conditions were developed for high PQQ excretion [32]. Upon cultivation under iron-limited conditions, PQQ accumulated up to 1 g l–1 during a protracted 15 days fermentation run. Methanol supplied into the fermentor to keep a concentration of 1–2 g l–1 was used as carbon source. Isolation from the fermentation broth and partial purification is achieved by ion exchange chromatography.

3.50.11

L-Carnitine

L-Carnitine (R-3-hydroxy-4-trimethylaminobutyrate) is a vitamin-like compound that naturally occurs in animal and vegetable tissues, as well as in microorganisms. The name is derived from the Latin word carnis for meat, from where the compound was initially isolated by Russian scientists in 1905. L-Carnitine can be synthesized by humans and animals from lysine (see the section below) in sufficient amounts to meet the daily requirements. However, in some individuals L-carnitine biosynthesis is impaired, making a dietary L-carnitine intake mandatory. Main food sources of L-carnitine are red meat such as sheep (200 mg/100 g), beef (80 mg/100 g), pork (30 mg/100 g), but also milk (1–2 mg/100 g), asparagus, wheat, and rice (1–2 mg/100 g). In addition to the ability to shuttle short-chain organic acids from the inside of the mitochondria to the cytosol, the main function of L-carnitine is to bind and transport long-chain fatty acids into mitochondria where they are dissimilated. Correspondingly L-carnitine is involved in energy metabolism of heart, liver, muscle, brain, and adipose tissues. Further roles in sperm maturation, the immune system and connecting tissue have been established. Due to its participation in fatty acid dissimilation, L-carnitine is mainly applied as ingredient of weight management products. Moreover, applications such as sports nutrition, infant nutrition, cardiovascular health, male fertility, brain performance, or nutrition of vegetarians and elderly are claiming effectiveness of L-carnitine. These claims lead to new applications and increased demand of the compound. In addition to applications in human nutrition, L-carnitine is applied as feed additive for companion and farm animals to improve fat and energy metabolism. The global annual production of L-carnitine is about 3000 tons. Lonza who pioneered the L-carnitine manufacturing technology and the L-carnitine market operates production plants in Kolin, Czech Republic, and Nasha, Guangzhou Province. More than 10 Chinese producers are active in production of L-carnitine as well. Several mammalian proteins including actin, calmodulin, cytochrome c, histones, and myosin contain N6-trimethyl-lysine (TML) residues, which serve as biosynthetic precursors for carnitine. N6-methylation occurs posttranslationally catalyzed by specific methyltransferases with S-adenosylmethionine as cosubstrate. After lysosomal hydrolysis of these proteins, TML is released and serves as substrate for TML dioxygenase (TMLD; EC 1.14.11.8) to afford 3-hydroxy-TML. Aldolytic cleavage of 3-hydroxy-TML catalyzed by a specific aldolase (HTMLA; EC 4.1.2.‘X’) yields 4-trimethylaminobutyraldehyde and glycine. Dehydrogenation of the former by a dehydrogenase (TMABA-DH; EC 1.2.1.47) results in the formation of 4-butyrobetaine. Finally, butyrobetaine is hydroxylated in the position 3 by 4-butyrobetaine dioxygenase (BBD; EC 1.14.11.1) affording L-carnitine (Figure 10) [33]. Given the remarkable nature of the L-carnitine biosynthetic precursor compound, it is obvious that a large-scale industrial production process cannot be designed according to the natural route. At Lonza L-carnitine is obtained biocatalytically from achiral butyrobetaine derived from trimethylamine and 1,4-butyrolactone [34]. The latter is obtained through partial hydrogenation of the commodity chemical maleic anhydride. For conversion of butyrobetaine to L-carnitine, a Gram-negative, aerobic bacterium designated HK4 with taxonometric relationship to Agrobacterium and Rhizobium meliloti is used. HK4 was isolated by its ability to grow on crotonobetaine as sole carbon and nitrogen source. The strain assimilated butyrobetaine, L-carnitine, and also glycine betaine. The substrate spectrum suggested that butyrobetaine assimilation in HK4 followed the β-oxidation route of fatty acids. To prevent complete assimilation, an HK4 mutant designated HK13 was selected that lost its ability to grow on butyrobetaine, crotonobetaine, and L-carnitine, but growth on betaine glycine was not impaired. In the HK13 mutant, the β-oxidation-like pathway of butyrobetaine assimilation was obviously halted right after the hydration step. During cultivation with glycine betaine as

600

Food Ingredients

Maleic anhydride O

O

O

Partial hydrogenation Trimethylamine H3C

N

1,4-Butyrolactone O O Hydrolysis of N 6-trimethyl-lysine (TML) containing proteins

CH3 Several chemical steps

CH3

N 6-Trimethyl-lysine

Butyrobetaine H3C

CH3

H3C

OH

N+ CH3

NH2

CH3 N+

COOH

CH3

O

O Dioxygenase

BcoA Butyrobetainyl-CoA H3C

CH3

H3C

SCoA

N+ CH3

NH2

CH3 +

N

COOH

CH3

O

O NH2

Aldolase

BcoC

Glycine COOH

Crotonobetainyl-CoA H3C

CH3

SCoA

N+ CH3

H3C

O

H2O

N+ CH3

BcoD

CH3 CH3 OH

Butyrobetaine

SCoA

N+

O Dehydrogenase

L-Carnityl-CoA H3C

CH3

H3C

O

CH3

Unknown thioesterase Next step of β -oxidation blocked in L-carnitine production strains

CH3

OH

N+ O

Dioxygenase H3C

CH3

OH

N+ CH3 OH

O

L-Carnitine

Industrial production

Biosynthesis

Figure 10 Biosynthesis of L-carnitine (right) and industrial production route (left). BcoA, 4-butyrobetainyl-CoA-synthetase; BcoC, 4-butyrobetainyl-CoA­ dehydrogenase; BcoD, crotobetainyl-CoA-hydrolase.

assimilable carbon source butyrobetaine present in the medium as second substrate is taken up by HK13 and converted to L-carnitine, which is excreted and accumulates in the medium. Random mutagenesis and selection procedures resulted in improved production strains. The butyrobetaine utilization genes of HK4, that is, bcoA, bcoC, and bcoD, have been cloned offering a genetic engineering approach for further improved strains. In fed-batch fermentation runs optimized HK13 derivatives convert butyrobetaine in very high yield to L-carnitine. The product isolation and purification process consists of cell separation, electrodialysis for salt removal, activated carbon treatment, drying, and recrystallization.

3.50.12 Outlook A quarter century ago, genetic engineering techniques started to radically change the way of developing microorganisms into production strains, which constitute the core of every biotechnological process. These techniques provided the means to assemble within the genome of existing microorganisms genetic parts into functional relationships, comparable to the assembly of

Application of Enzymes and Microbes for the Industrial Production of Vitamins and Vitamin-Like Compounds

601

mechanical parts into machines. The genetic parts were the genes together with the required expression elements, which after they have been expressed in the host cell into catalytically active enzymes, act in a coordinated fashion to convert the available or provided educts into the desired products. The basis for the genetic design was the enormous biochemical knowledge gathered by that time about the way how the monomeric constituents of living matter are biosynthesized from simple carbon and nitrogen sources. The concept designated as metabolic pathway engineering or, nowadays synthetic biology, aroused much euphoria anticipating that the production of vitamins, so far mainly in the realms of chemical synthesis, would sooner or later switch to biotechnological methods. This happened indeed, but only for vitamin B2. Several reasons account for this not too overwhelming track record. The chemical vitamin processes are extremely well developed with regard to both economic and ecologic efficiency, at least when operated by Western companies. In addition, frequently the chemical processes are carried out in depreciated, never­ theless well-maintained plants. They are simply hard to beat under economic aspects. But an ineffective biotechnological process will be inferior to its chemical counterpart with regard to ecologic and sustainability aspects as well. When the era of recombinant biotechnology was proclaimed, people were aware of the biochemical reactions of the cellular central metabolism and the specialized routes toward amino acids, nucleotides, lipids, and others. These pathways mainly follow conventional chemical reaction mechanisms. The pathways toward vitamins, however, turned out to involve peculiar and intricate steps catalyzed by enzymes with slow reaction kinetics, at least as far as they are determined in vitro. Striking examples are the BioB reaction of the biotin pathway, already mentioned above, or the ThiC and the PdxY reactions of the vitamin B1 and B6 pathway, respectively. In the case of riboflavin biosynthesis, which involves pretty slow enzymes as well, the problem was solved by brut force overexpression of the enzymes. This simplistic approach failed in the other cases. To make the biotechnological production of vitamins a real success story, a full and detailed understanding of the molecular details of their biosynthesis and the identification of all relevant functions within the cell, contributing directly and indirectly to the synthesis of these molecules, is of foremost importance. This requires further serious biochemical studies. But also the concepts of systems biology and the holistic approaches provided by the various ‘omics’ methods should become instrumental here. Recombinant approaches of production strain development are frequently supported by classical mutagenesis and selection campaigns. Whole genome sequencing of the individual members from the strain lineages thus emerging provide the means to correlate the performance of these individuals to single-nucleotide polymorphisms they carry in their genome. Particularly, the latter might become the method of choice to identify novel, not anticipated genetic engineering targets. For realization of the genetic design of superior production strains novel, affordable DNA synthesis methods are available, such as the chemical DNA synthesis based on phosphoramidites without the need for an initial template DNA. These methods are fast and highly reliable, providing genes, which are codon optimized for the selected host strain, together with the most suited transcription and translation signals. Accelerated by the overriding macro trend toward a sustainable economy based on renewable resources, application of these new technologies might pave the way toward economically sound biotechnological processes for all vitamins. After the next quarter century, we might see the vitamins used for food and feed supplementation being produced from renewable carbohydrates and delivered by fermentation factories.

References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16]

[17] [18] [19]

SRI_Consulting (2005) Chemical Economics Handbook – Vitamins. http://www.sriconsulting.com. Fischer M and Bacher A (2005) Biosynthesis of flavocoenzymes. Natural Product Reports 22(3): 324–350. Hohmann H-P and Stahmann K-P (2010) Biotechnology of riboflavin production. Comprehensive Natural Products II Chemistry and Biology, vol. 7, pp. 115–139. Oxford: Elsevier. Chuck R A Catalytic Green Process for the Production of Niacin. http://www.lonza.com/group/en/company/news/publications_of_lonza.html Nagasawa T and Yamada H (1989) Microbial transformations of nitriles. Trends in Biotechnology 7: 153–158. Robins KT and Nagasawa T (2003) Process for Preparing Amides. Switzerland: Lonza AG. EP1002122. Kataoka M, Shimizu K, Sakamoto K, et al. (1995) Optical resolution of racemic pantolactone with a novel fungal enzyme, lactonohydrolase. Applied Microbiology and Biotechnology 43: 974–977. Tang Y-X, Sun Z-H, Hua L, et al. (2002) Kinetic resolution of dl-pantolactone by immobilized Fusarium moniliforme SW-902. Process Biochemistry 38(4): 545–549. Glieder A, Liu Z, Pscheidt B, et al. (2009) R-HNL Random Variants and Their Use for Preparing Optically Pure, Sterically Hindered Cyanohydrins. Netherlands: DSM IP Assets B.V. EP2092060. Hermann T, Patterson TA, Pero JG, et al. (2003) Processes for Enhanced Production of Pantothenate. Ludwigshafen: BASF AG (DE). EP1370670. Van Arsdell SW, Perkins JB, Yocum RR, et al. (2005) Removing a bottleneck in the Bacillus subtilis biotin pathway: BioA utilizes lysine rather than S-adenosylmethionine as the amino donor in the KAPA-to-DAPA reaction. Biotechnology and Bioengineering 91(1): 75–83. Sakurai N, Imai Y, Masuda M, et al. (1994) Improvement of a d-biotin-hyperproducing recombinant strain of Serratia marcescens. Journal of Biotechnology 36(1): 63–73. Warren MJ, Raux E, Schubert HL, et al. (2002) The biosynthesis of adenosylcobalamin (vitamin B12). Natural Product Reports 19(4): 390–412. Li KT, Liu DH, Chu J, et al. (2008) An effective and simplified pH-stat control strategy for the industrial fermentation of vitamin B(12) by Pseudomonas denitrificans. Bioprocess and Biosystems Engineering 31(6): 605–610. Blanche F, Cameron B, Crouzet J, et al. (2003) Methods of Increasing the Production of Cobalamins Using Cob Gene Expression. United States: Rhone-Poulenc Biochimie. US6656709. Crouzet J, Levy-Schil S, Cameron B, et al. (1991) Nucleotide sequence and genetic analysis of a 13.1-kilobase-pair Pseudomonas denitrificans DNA fragment containing five cob genes and identification of structural genes encoding Cob(I)alamin adenosyltransferase, cobyric acid synthase, and bifunctional cobinamide kinase-cobinamide phosphate guanylyltransferase. Journal of Bacteriology 173(19): 6074–6087. Bremus C, Herrmann U, Bringer-Meyer S, et al. (2006) The use of microorganisms in l-ascorbic acid production. Journal of Biotechnology 124(1): 196–205. Yin G-L, Tao Z-X, Yu L-H, et al. (1980) Studies on the production of vitamin C precursor 2-keto-L-gulonic acid from L-sorbose by fermentation. Acta Microbiologica Sinica 20(3): 246–251. Hoshino T, Ojima S, and Sugisawa T, et al. (1994) Fermentation Process for Producing 2-keto-L-gulomic Acid. Netherlands: DSM IP Assets. EP0518136.

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[20] Berry A, Lee C, Mayer AF, et al. (2010) Polypeptides and Encoding Polynucleotides for Microbial Production of L-ascorbic Acid and Associated Methods. Heerlen, NL: DSM IP Assets B.V. US7700723. [21] Miyazaki T, Sugisawa T, and Hoshino T (2006) Pyrroloquinoline quinone-dependent dehydrogenases from Ketogulonicigenium vulgare catalyze the direct conversion of L-sorbosone to L-ascorbic acid. Applied and Environmental Microbiology 72(2): 1487–1495. [22] Meganathan R (2001) Biosynthesis of menaquinone (vitamin K2) and ubiquinone (coenzyme Q): A perspective on enzymatic mechanisms Vitamins & Hormones. In: Gerald Litwack TB (ed.) Cofactor Biosynthesis, pp. 173–218, New York, NY: Academic Press. [23] Sato T, Yamada Y, Ohtani Y, et al. (2001) Production of menaquinone (vitamin K2)-7 by Bacillus subtilis. Journal of Bioscience and Bioengineering 91(1): 16–20. [24] Tsukamoto Y, Kasai M, Kakuda H, et al. (2001) Construction of a Bacillus subtilis (natto) with high productivity of vitamin K2 (menaquinone-7) by analog resistance. Bioscience, Biotechnology, and Biochemistry 65(9): 2007–2015. [25] Benedetti A, Daly, S, Xaiz R, et al. (2008) A process for the preparation of vitamin K2, Gnosis S.p.A. EP1803820. [26] Soft_Gel_Technologies (2010). Overcoming Bioavailability Challenges Associated with Coenzyme Q10 Supplementation: The CoQsol-CF™ Solution. http://www.nutrilearn.com/softgel/coqsol.html [27] Jeya M, Moon H-J, Lee J-L, et al. (2010) Current state of coenzyme Q10 production and its applications. Applied Microbiology and Biotechnology 85: 1653–1663. [28] Berry A, Hümbelin M, and Lopez-Ulibarri R (2008) Improved Production of Coenzyme Q-10. Netherlands: DSM IP Assets. EP1641931. [29] Rucker R, Chowanadisai W and Nakano M (2009) Potential physiological importance of pyrroloquinoline quinone. Alternative Medicine Review 14(3): 268–277. [30] Anthony C(2010) PQQ Is Not a Vitamin. http://www.chris-anthony.co.uk/ [31] Puehringer S, Metlitzky M, and Schwarzenbacher R (2008) The pyrroloquinoline quinone biosynthesis pathway revisited: A structural approach. BMC Biochemistry 9(1): 8. [32] Urakami T, Yashima K, Kobayashi H, et al. (1992) Production of pyrroloquinoline quinone by using methanol-utilizing bacteria. Applied and Environmental Microbiology 58(12): 3970–3976. [33] Vaz FM and Wanders RJ (2002) Carnitine biosynthesis in mammals. Biochemical Journal 361(Pt 3): 417–429. [34] Meyer H-P and Robins KT (2005) Large scale bioprocess for the production of optically pure L-Carnitine. Monatshefte für Chemie/Chemical Monthly 136: 1269–1277.