Vitamin Production by Microbes
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).
Vitamins may be grouped as follows :
The fat-soluble vitamins The water-soluble vitamins Vitamin A Choline
Folacin (folic acid) Vitamin D Niacin (nicotinic acid)
Panthotenic acid Vitamin E Riboflavin
Thiamin Vitamin K Pyridoxine
Cobalamin Ascorbic acid
- also produced by microbes
Liver Vitamin B 9 (Folic acid)* 1941 Liver Vitamin B 3 (Niacin) 1936 Rice bran Vitamin B 6 (Pyridoxine)* 1934 Liver Vitamin B 7 (Biotin) 1931 Liver Vitamin B 5 (Pantothenic acid)* 1931 Luzerne Vitamin K (Phyllochinone) 1929 Liver Vitamin B 12 (Cobalamine)* 1926 Wheat germ oil Vitamin E ( Tocopherol ) 1922 Eggs Vitamin B 2 (Riboflavin)* 1920 Cod liver oil Vitamin D ( Calciferol ) 1918 Lemons Vitamin C (Ascorbic acid) 1912 Rice bran Vitamin B 1 ( Thiamin )* 1912 Cod liver oil Vitamin A (Retinol) 1909 Source Vitamin Year of discovery
The discovery of vitamins and their sources
V it a m in B (Cya noc oba la m in) 1 2
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)
Natural forms of cobalamin depending on the upper ligand are:
1. Adenosylcobalamin (coenzyme B , AdoCbl)
12
2. Methylcobalamin (MeCbl)
3. Hydroxycobalamin (OHCbl) Cyanocobalamin (Vitamin B ) is the industrially produced
12
stable cobalamin form, which is a synthetic compound not found in nature.
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.
g per day. Humans require cobalamin between 1-2 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. 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.
Flow chart for production of Vitamin B from P. denitrificans
12 P. denitrificans
Inoculum cultivation on agar slant with medium contain sugar beet molasses, yeast extract, etc.
Inoculum cultivation Preculture in erlenmeyer flask with medium the same as for inoculum cultivation, without agar
Production in erlenmeyer with medium contain Preculture 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 Production culture membrane permeability.
Sugar beet molasses is used as a low-cost betaine source.
Vitamin B from Propionibacterium shermanii or P. freudenreichii 12 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 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.
V it a m in B (Ribofla vin, La c t ofla vin)
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.
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).
Riboflavin is produced industrially by several processes: 1. chemical sy nthesis for pharmaceutical use (20% of world wide production)
2. of glucose to D-ribose and
biotransformation
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) constitutive ribo-
Ashbya gossypii (6420 mg/L) flavin-sy nthesizing
Produc t ion by fe rm e nt a t ion 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
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.
3. Phase three the synthesis of cell bound riboflavin in the form FAD and FMN.
-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.
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 cobalamin between 1.5-2.0 mg per day.
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
Produc t ion proc e sse s for -c a rot e ne using Bla k e sle a t rispora
B. t. (+)
B. t .(-) Production is induced by trisporic
acids (act as (+) –gamones/sexual hormones).
Culture on Culture on agar slant agar slant
-carotene synthesis is Activator of -ionon.
isoniazid , in combination with Preculture Preculture
The addition of purified kerosene to the medium doubles the yield.
Mixed preculture
The addition of antioxidant to -carotene increase the stability of within the cells.
Production culture
Cre a t ion of nove l c a rot e noid biosynt he t ic pa t hw a ys in E. c oli. Novel carotenoid structures are in red; red arrows indicate in vitro evolved gene functions
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) 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.