1212 vitamin production (nrm) a vitamin is an organic compound required as a nutrient in tiny...

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Vitamin Production (NRM)

A vitamin is an organic compound required as a nutrient intiny amounts by an organism.

Vitamins are classified by their biological activity, not theirstructure.

Vitamins have diverse biochemical functions, includingfunction 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 andtissue growth and differentiation (e.g. vitamin A).


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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 discovery Vitamin Source

1909

1912

1912

1918

1920

1922

1926

1929

1931

1931

1934

1936

1941

Vitamin A (Retinol)

Vitamin B1 (Thiamin)*

Vitamin C (Ascorbic acid)

Vitamin D (Calciferol)

Vitamin B2 (Riboflavin)*

Vitamin E (Tocopherol)

Vitamin B12 (Cobalamine)*

Vitamin K (Phyllochinone)

Vitamin B5 (Pantothenic acid)*

Vitamin B7 (Biotin)

Vitamin B6 (Pyridoxine)*

Vitamin B3 (Niacin)

Vitamin B9 (Folic acid)*

Cod liver oil

Rice bran

Lemons

Cod liver oil

Eggs

Wheat germ oil

Liver

Luzerne

Liver

Liver

Rice bran

Liver

Liver

* also produced by

microbes


Vitamin B12 (Cyanocobalamin)

The term cobalamin isall of them containcobalt.

Corrin is the base(central) structure ofcobalamin,, composedof a tetrapyrrole ring(four pyrrole units).

Cobalamin can beconsidered in 3 parts:

1. a central corrin ring

2. a lower ligand(benzimodazole)

3. an upper ligand


Natural forms of cobalamin depending on the upperligand are:

1. Adenosylcobalamin (coenzyme B12, AdoCbl)2. Methylcobalamin (MeCbl)3. Hydroxycobalamin (OHCbl)

Cyanocobalamin (Vitamin B12 ) is the industrially producedstable cobalamin form, which is a synthetic compound notfound in nature.

The biosynthesis of cyanocobalamin is intricate andconfirmed to certain members of the prokaryotic world-members of the Archaea and certain eubacteria.

Animals, humans, and protists require cobalamin butapparently do not synthesize it, whereas plant and fungiare 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 asmeat, poultry, fish, egg, and milk. The cobalamin-producing bacteria often live in bodies of water and soil,and animals get cobalamin by eating food contaminatedwith these microorganisms.

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The biosynthesis of cobalamin requires somewhere around70 enzyme-mediated steps involving more than 30 genes forits complete de novo synthesis.

In 1993 the Everest Cobalamin was conquered, meaningthat all the intermediates on the biosynthetic pathway inPseudomonas denitrificans were isolated and their structuresdetermined.

A genetically engineered highly effective cobalamin-producing strain of P. denitrificans has a productivity thatreaches 300 mg/L and accounts for 80% of the cobalaminproduction in the world.


Flow chart for production of Vitamin B12 from P. denitrificans

P. denitrificans

Inoculum cultivation

Preculture

Production culture

Inoculum cultivation on agar slant withmedium contain sugar beet molasses, yeastextract, etc.

Preculture in erlenmeyer flask with medium thesame as for inoculum cultivation, without agar

Production in erlenmeyer with medium containsugar beet molasses, yeast extract, etc. Cobaltand 5,6-dimethyl benzimidazole must be addedas supplemen. Betaine is assumed to causean activation of biosynthesis or an increase inmembrane permeability.

Sugar beet molasses is used as a low-costbetaine source.

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Vitamin B12 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 B12)

Isolation and Purification

Cells are lysed by heat treatment at 80-120 0C for 10-30 minutes at pH6.5-8.5. The cells on lysis release various cobalamin. The obtained ofcobalamin is converted into cyanocobalamin.

The purification of the product is done using adsorption method forsubstances like amberlite, alumina, silanized silica gel follwed by elutionwith water-alcohol or water-phenol mixtures.


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Vitamin B2 (Riboflavin, Lactoflavin)

Riboflavin (6,7-dimethyl-9-(D-1’-ribityl)-isoalloxazine is analloxazine ring linked toalcohol derived from thepentose sugar ribose.

The isoalloxazine ring acts asa reversible redox system.

Riboflavin has an essentialrole in the oxidative mehanismin the cell.



Riboflavin is a water-soluble yellow-orange fluorescentpigment, heat-stable in neutral or acid solution, but heatingin alkaline solutions may destroy it. It is easily destroyed bylight, especially ultraviolet.

Humans require cobalamin between 1 mg per day.Deficiency causes ariboflavinosis, characterized by crackedskin and eye problems including blurred vision.

Riboflavin is present in milk as free riboflavin, but is presentin other foods (liver, heart, kidney, eggs, or leafyvegetables) as part of flavoproteins which contain theprotestic groups FMN (flavin mononucleotide) or FAD (flavinadenin dinucleotide).

The current world production of riboflavin is about 2,400 tonof which 75% is for feed additive and the remaining for foodand pharmaceuticals.


Riboflavin is produced industrially by several processes:1.

2.

chemical synthesis for pharmaceutical use (20% ofworld wide production)biotransformation of glucose to D-ribose and

subsequent chemical conversion to riboflavin (about50% of world wide production)

3. direct fermentation (30% of world wide production)

Riboflavin is synthesized by many microorganisms, includingbacteria, 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-synthesizingenzymes


Two closely related ascomycete fungi, Eremothecium ashbyiiand Ashbya gossypii,are mainly used for the industrialproduction.

Yields much higher than 10 g of riboflavin per liter of culturebroth are obtained in a sterile aerobic submerged fermentationwith a nutrient medium containing molasses or plant oil as amajor carbon source. Yeasts (Candida flaeri, C. famata, etc.)and bacteriacan also be used for the practical production.

Riboflavin production by genetically engineered Bacillus subtilisand Corynebacteriumammoniagenes which overexpress genesof the enzymes involved in riboflavin biosynthesisreach 4.5 g and 17.4 g , respectively

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Production by fermentation of Ashbya gossypii

About 30% of the world industrial riboflavin output is produced by directfermentation with A. gossypii and up to can produce riboflavin up to 15 g/Lafter 10 days to be the maximum yield.

The hypae can accumulate large amounts of riboflavin released from thecells by heat treatment (1 h, 1200C, pH 4.5) the mycelium is separtedand discarded riboflavin is then further purified.

The fermentation is conducted in four phases:

1. Phase one (the initial rapid growth of A. gossypii) glucose isutilized and pyruvic acid accumulates.

2. Phase two (the production phase) the level of the pyruvatereduces, ammonia in the medium accumulates.

the synthesis of cell bound riboflavin in the form FAD3. Phase threeand FMN.

autolysis of the cells release of free riboflavin intoVitamin Production (NRM)

4. Phase fourmedium.


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-LaRoche, Fuji/Daiichi (Japan)]. They have many pharmaceuticaland feed/food applications.Recent chemical and molecular biology studiesrevealed that 1-deoxy-D-xylulose and 4-hydroxy-L-threonine are the precursorsfor thebiosynthesis of pyridoxine, but its complete biosyntheticpathway isnot known in detail.

Screening for vitamin B6 producers among microorganismsfound severalpotential strains, such as Klebsiella sp.,Flavobacterium sp., Pichia guilliermondii, Bacillussubtilis, and Rhizobium meliloti.

-Carotene (Provitamin A)

Carotenoids are not just another group of naturalpigments but substances with very special andremarkable properties that no other groups ofsubstances possess.

They perform important functions in nature, includinglight-harvesting, photoprotection, protective and sex-related coloration patterns in many animal species andas precursors of vitamin A in vertebrates.

They may serve protective roles as well against age-related diseases in humans, being implicated in theprevention or protection against serious human healthdisorders such as cancer and heart disease.

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Carotenoids are found in many animal and planttissues, but originate exclusively from plants ormicrobes.

-carotene is converted into vitamin A in theintestinal mucous membrane and is stored in theliver as the palmitate ester.

Humans require between 1.5-2.0 mg per day.



Structures of several carotenoids that can be produced by fermentation

Carotenoids are highly unsaturatedisoprene derivatives.

The conjugated double bondsystem determines the photo-chemical properties and chemicalreactivity that are the basis ofcarotenoid biological functions.

Only compounds with the -iononestructure (the ring structure foundat each end of the -carotenemolecule) are effective asprovitamin A.

Two molecules of vitamin A can beformed from -carotene.

Only one molecule of vitamin A canbe formed from - and -carotene.


Production processes for -carotene usingBlakeslea trispora

B. t. (+)

Culture onagar slant

Preculture

B. t .(-)

Culture onagar slant

Preculture

Mixed preculture

Production culture

Production is induced by trisporicacids (act as (+) –gamones/sexualhormones).

Activator of -carotene synthesis isisoniazid, in combination with -ionon.

The addition of purified kerosene tothe medium doubles the yield.

The addition of antioxidant toincrease the stability of -carotenewithin the cells.

Creation of novel carotenoid biosynthetic pathways in E.coli. Novel carotenoid structures are in red; red arrows indicatein vitro evolved gene functions

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Identification of a novel carotenoid oxygenase leads to thesynthesis of novel oxygenated carotenoid structures byrecombinant E. coli. Directed evolution of this enzymes createsnovel E. coli color phenotypes.

http://www.cbs.umn.edu/BMBB/faculty/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 entirecarotenoid biosynthetic pathway has been fully described.

Synechococcus sp. PCC 7002 produces seven carotenoidsthat accumulate to significant amounts during standardexponential growth: -carotene, zeaxanthin, cryptoxanthin,echinenone, hydroxy-echinenone, myxoxanthophyll, and anewly discovered aromatic carotenoid, synechoxanthin.

Synechoxanthin, c,c-caroten-18,18’-dioic acid, is the firstaromatic carotenoid to bedocumented in cyanobacteria.

1212 vitamin production (nrm) a vitamin is an organic compound required as a nutrient in tiny...

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