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),

Vitamins may be grouped as follows:

Choline

Folacin (folic acid) Niacin (nicotinic acid) Panthotenic acid

Riboflavin Thiamin Pyridoxine Cobalamin Ascorbic acid Vitamin A

Vitamin D

Vitamin E

Vitamin K

Liver

Vitamin B₅(Pantothenic acid)*

1931

Luzerne

Vitamin K (Phyllochinone) 1929

Liver

Vitamin B₁₂ (Cobalamine)*

1926

Wheat germ oil Vitamin E (Tocopherol)

1922

Eggs

Vitamin B₂(Riboflavin)*

1920

Cod liver oil Vitamin D (Calciferol)

1918

Lemons Vitamin C (Ascorbic acid)

1912

Rice bran

Vitamin B₁(Thiamin)*

1912

Cod liver oil Vitamin A (Retinol)

1909

Source Vitamin

Year of discovery

The discovery of vitamins and their sources

* also produced by

V it a m in B_{1 2} (Cya noc oba la m in)

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 B₁₂, AdoCbl) 2. Methylcobalamin (MeCbl)

3. Hydroxycobalamin (OHCbl)

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

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

Flow chart for production of Vitamin B₁₂ 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.

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 0_{C for 10-30 minutes at pH}

V it a m in B₂ (Ribofla vin, La c t ofla vin)

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

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

Riboflavin is produced industrially by several processes: 1. chemical sy nthesis for pharmaceutical use (20% of

world wide production)

2. 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) constitutive

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, 1200_{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.

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.

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 photo-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.

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 .(-)

Culture on agar slant Culture on agar slant Preculture Preculture Mixed preculture

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

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

Identification of a novel carotenoid oxygenase leads to the synthesis of novel oxygenated carotenoid structures by

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