1. 1212 Vitamin Production (NRM) A vitamin is an organic compound required as a nutrient in tiny amounts by an organism. Vitamins are classified by their biological activity, not their structure. Vitamins have diverse biochemical functions, including function as: 1. a precursors for enzyme cofactor biomolecules (coenzymes) (e.g. B complex vitamins), 2. hormones (e.g. vitamin D), 3. antioxidants (e.g. vitamin E), 4. mediators of cell signaling and regulators of cell and tissue growth and differentiation (e.g. vitamin A). Vitamin Production (NRM)

2. 3 Vitamins may be grouped as follows : The fat-soluble vitamins Vitamin A Vitamin D Vitamin E Vitamin K The water-soluble vitamins Choline Folacin (folic acid) Niacin (nicotinic acid) Panthotenic acid Riboflavin Thiamin Pyridoxine Cobalamin Ascorbic acid Vitamin Production (NRM)4 The discovery of vitamins and their sources Year of discoveryVitaminSource 1909 1912 1918 1920 1922 1926 1929 1931 1934 1936 1941 Vitamin A (Retinol) Vitamin B 1 (Thiamin)* Vitamin C (Ascorbic acid) Vitamin D (Calciferol) Vitamin B 2 (Riboflavin)* Vitamin E (Tocopherol) Vitamin B 12 (Cobalamine)* Vitamin K (Phyllochinone) Vitamin B 5 (Pantothenic acid)* Vitamin B 7 (Biotin) Vitamin B 6 (Pyridoxine)* Vitamin B 3 (Niacin) Vitamin B 9 (Folic acid)* Cod liver oil Rice bran Lemons Cod liver oil Eggs Wheat germ oil Liver Luzerne Liver Rice bran Liver * also produced by microbes

3. Vitamin Production (NRM)5 Vitamin B 12 (Cyanocobalamin) The term cobalamin is all of them contain cobalt. Corrin is the base (central) structure of cobalamin,, composed of a tetrapyrrole ring (four pyrrole units). Cobalamin can be considered in 3 parts: 1. a central corrin ring 2. a lower ligand (benzimodazole) 3. an upper ligand Vitamin Production (NRM)6 Natural forms of cobalamin depending on the upper ligand are: 1. Adenosylcobalamin (coenzyme B 12, AdoCbl) 2. Methylcobalamin (MeCbl) 3. Hydroxycobalamin (OHCbl) Cyanocobalamin (Vitamin B 12 ) is the industrially produced stable cobalamin form, which is a synthetic compound not found in nature.

4. The biosynthesis of cyanocobalamin is intricate and confirmed to certain members of the prokaryotic world- members of the Archaea and certain eubacteria. Animals, humans, and protists require cobalamin but apparently do not synthesize it, whereas plant and fungi are thought to neither synthesize nor use it. Humans require cobalamin between 1-2  g per day. Cobalamin is anti-pernicious anaemia factor. Cobalamin is mainly found in animal products, such as meat, poultry, fish, egg, and milk. The cobalamin- producing bacteria often live in bodies of water and soil, and animals get cobalamin by eating food contaminated with these microorganisms. 7878 Vitamin Production (NRM) The biosynthesis of cobalamin requires somewhere around 70 enzyme-mediated steps involving more than 30 genes for its complete de novo synthesis. In 1993 the Everest Cobalamin was conquered, meaning that all the intermediates on the biosynthetic pathway in Pseudomonas denitrificans were isolated and their structures determined. A genetically engineered highly effective cobalamin- producing strain of P. denitrificans has a productivity that reaches 300 mg/L and accounts for 80% of the cobalamin production in the world. Vitamin Production (NRM)

5. Flow chart for production of Vitamin B 12 from P. denitrificans P. denitrificans Inoculum cultivation Preculture Production culture Inoculum cultivation on agar slant with medium contain sugar beet molasses, yeast extract, etc. Preculture in erlenmeyer flask with medium the same as for inoculum cultivation, without agar Production in erlenmeyer with medium contain sugar beet molasses, yeast extract, etc. Cobalt and 5,6-dimethyl benzimidazole must be added as supplemen. Betaine is assumed to cause an activation of biosynthesis or an increase in membrane permeability. Sugar beet molasses is used as a low-cost betaine source. 9 10 Vitamin Production (NRM) Vitamin B 12 from Propionibacterium shermanii or P. freudenreichii These strains are used in a two stage process with added cobalt. In a preliminary anaerobic phase (2-4 days), 5’-deoxyadenosyl- cobinamide is mainly produced. In a second, aerobic phase (3-4 days) the biosynthesis of 5,6- dimethylbenzimidazole to produce 5’-deoxyadnosylcobalamine (coenzyme B 12 ) Isolation and Purification Cells are lysed by heat treatment at 80-120 0 C for 10-30 minutes at pH 6.5-8.5. The cells on lysis release various cobalamin. The obtained of cobalamin is converted into cyanocobalamin. The purification of the product is done using adsorption method for substances like amberlite, alumina, silanized silica gel follwed by elution with water-alcohol or water-phenol mixtures. Vitamin Production (NRM)

6. 11 Vitamin B 2 (Riboflavin, Lactoflavin) Riboflavin (6,7-dimethyl-9-(D- 1’-ribityl)-isoalloxazine is an alloxazine ring linked to alcohol derived from the pentose sugar ribose. The isoalloxazine ring acts as a reversible redox system. Riboflavin has an essential role in the oxidative mehanism in the cell. Vitamin Production (NRM)12 Vitamin Production (NRM) Riboflavin is a water-soluble yellow-orange fluorescent pigment, heat-stable in neutral or acid solution, but heating in alkaline solutions may destroy it. It is easily destroyed by light, especially ultraviolet. Humans require cobalamin between 1 mg per day. Deficiency causes ariboflavinosis, characterized by cracked skin and eye problems including blurred vision. Riboflavin is present in milk as free riboflavin, but is present in other foods (liver, heart, kidney, eggs, or leafy vegetables) as part of flavoproteins which contain the protestic groups FMN (flavin mononucleotide) or FAD (flavin adenin dinucleotide). The current world production of riboflavin is about 2,400 ton of which 75% is for feed additive and the remaining for food and pharmaceuticals.

7. Vitamin Production (NRM)13 Riboflavin is produced industrially by several processes: 1. 2. chemical synthesis for pharmaceutical use (20% of world wide production) biotransformation of glucose to D-ribose and subsequent chemical conversion to riboflavin (about 50% of world wide production) 3. direct fermentation (30% of world wide production) Riboflavin is synthesized by many microorganisms, including bacteria, yeasts, and fungi, such as: - Clostridium acetobutylicum (97 mg/L) - Candida flareri (567 mg/L). - Ascomycetes: Eremothecium ashbyii (2480 mg/L) Ashbya gossypii (6420 mg/L) constitutive ribo- flavin-synthesizing enzymes Vitamin Production (NRM)14 Two closely related ascomycete fungi, Eremothecium ashbyii and Ashbya gossypii,are mainly used for the industrial production. Yields much higher than 10 g of riboflavin per liter of culture broth are obtained in a sterile aerobic submerged fermentation with a nutrient medium containing molasses or plant oil as a major carbon source. Yeasts (Candida flaeri, C. famata, etc.) and bacteriacan also be used for the practical production. Riboflavin production by genetically engineered Bacillus subtilis and Corynebacteriumammoniagenes which overexpress genes of the enzymes involved in riboflavin biosynthesis reach 4.5 g and 17.4 g, respectively

8. 15 Production by fermentation of Ashbya gossypii About 30% of the world industrial riboflavin output is produced by direct fermentation with A. gossypii and up to can produce riboflavin up to 15 g/L after 10 days to be the maximum yield. The hypae can accumulate large amounts of riboflavin released from the cells by heat treatment (1 h, 120 0 C, pH 4.5) the mycelium is separted and discarded riboflavin is then further purified. The fermentation is conducted in four phases : 1. Phase one (the initial rapid growth of A. gossypii)glucose is utilized and pyruvic acid accumulates. 2. Phase two (the production phase) the level of the pyruvate reduces, ammonia in the medium accumulates. the synthesis of cell bound riboflavin in the form FAD3. Phase three and FMN. autolysis of the cells release of free riboflavin into Vitamin Production (NRM) 4. Phase four medium. Vitamin Production (NRM)16 Pyridoxine (Vitamin B6) Vitamin B6 compounds, mainly pyridoxineand pyridoxal 5b- phosphate, are exclusivelyproduced by chemical synthesis [ca. 2,500 t aP1; major producers, Takeda, Hoffmann-La Roche, Fuji/Daiichi (Japan)]. They have many pharmaceutical and feed/food applications. Recent chemical and molecular biology studiesrevealed that 1- deoxy-D-xylulose and 4-hydroxy-L-threonine are the precursors for thebiosynthesis of pyridoxine, but its complete biosynthetic pathway isnot known in detail. Screening for vitamin B6 producers among microorganisms found severalpotential strains, such as Klebsiella sp., Flavobacterium sp., Pichia guilliermondii, Bacillus subtilis, and Rhizobium meliloti.

9.  -Carotene (Provitamin A) Carotenoids are not just another group of natural pigments but substances with very special and remarkable properties that no other groups of substances possess. They perform important functions in nature, including light-harvesting, photoprotection, protective and sex- related coloration patterns in many animal species and as precursors of vitamin A in vertebrates. They may serve protective roles as well against age- related diseases in humans, being implicated in the prevention or protection against serious human health disorders such as cancer and heart disease. 17 18 Vitamin Production (NRM) Carotenoids are found in many animal and plant tissues, but originate exclusively from plants or microbes.  -carotene is converted into vitamin A in the intestinal mucous membrane and is stored in the liver as the palmitate ester. Humans require between 1.5-2.0 mg per day. Vitamin Production (NRM)

10. 20 Structures of several carotenoids that can be produced by fermentation Carotenoids are highly unsaturated isoprene derivatives. The conjugated double bond system determines the photo- chemical properties and chemical reactivity that are the basis of carotenoid biological functions. Only compounds with the  -ionone structure (the ring structure found at each end of the  -carotene molecule) are effective as provitamin A. Two molecules of vitamin A can be formed from  -carotene. Only one molecule of vitamin A can be formed from  - and  -carotene. Vitamin Production (NRM) 19 Production processes for  -carotene using Blakeslea trispora B. t. (+) Culture on agar slant Preculture B. t.(-) Culture on agar slant Preculture Mixed preculture Production culture Production is induced by trisporic acids (act as (+) –gamones/sexual hormones). Activator of  -carotene synthesis is isoniazid, in combination with  -ionon. The addition of purified kerosene to the medium doubles the yield. The addition of antioxidant to increase the stability of  -carotene within the cells.

11. Creation of novel carotenoid biosynthetic pathways in E. coli. Novel carotenoid structures are in red; red arrows indicate in vitro evolved gene functions 21 22 Vitamin Production (NRM) Identification of a novel carotenoid oxygenase leads to the synthesis of novel oxygenated carotenoid structures by recombinant E. coli. Directed evolution of this enzymes creates novel E. coli color phenotypes. http://www.cbs.umn.edu/BMBB/fac ulty/csd/HTML/research_isoprenoid.htm (10-6-08) Vitamin Production (NRM)

12. 23 Cyanobacterial carotenoids are tetraterpenoid (C-40) compounds with poly-ene chromophores. There is still no cyanobacterium for which the entire carotenoid biosynthetic pathway has been fully described. Synechococcus sp. PCC 7002 produces seven carotenoids that accumulate to significant amounts during standard exponential growth:  -carotene, zeaxanthin, cryptoxanthin, echinenone, hydroxy-echinenone, myxoxanthophyll, and a newly discovered aromatic carotenoid, synechoxanthin. Synechoxanthin, c,c-caroten- 18,18’-dioic acid, is the first aromatic carotenoid to be documented in cyanobacteria.