Production of Secondary metabolites.pptx

secondary metabolite

1. Module - III P R ODUCTION OF S E CON D A R Y ME T ABOLIT ES

2. T opics • The p r oduction of se c ondary m e t abol i t es of h i gh c omme r cial v alue l i k e • A n tibiot i cs: P en i cill i n V , Amp i cill i n sodium salt, St r e p t o m y cin. • P r oduction of c omme r cial v i t amins l i k e Vi t amin B12, Vi t amin E, Vi t amin B. • P r oduction of Bio e thano l.

3. The p r oduction of se c ondary m e t aboli t es • Se c ondary m e t abol i t es a r e p r oduced i n i d iophase, sec r e t ed when dep l e tion of 1 or mo r e nutr i e n t s is c aused i n med i um • Th e y a r e n ot r equ i r ed f or their g r owth, but i n t err u p t m e t abol i sm of t h ei r ho s t o r g anis m & m a y e v en ki l l. • Ex: M i c r ob i a l or i gi n – t o xi n s(p r o t eins), a n tib i otics e t c.; Pla n t cel l s – al k aloi d s, esse n tia l oi l s, p i gme n ts e t c. • Th e y c an c o n t r ol other o r g anisms l i k e M . O , i n sects, pe s ts, fl i es e t c. Ex: bt t o xi n is a p r o t ei n cr y st al th a t i n t err u p t the m e t abol i sm of ho s ti n g o r g anism(fl i es, mosqui t os, be e tles, t erm i t es e t c.) & ki l l. • M y c o t o xin s (non-a n ti g enic) of cer t ain fungi a r e t o xic t o human, anima l s & p l a n ts & a r e seen i n milk , c orn, peanut, c o t t on seeds, almond, figs, sp i ces e t c. De p en d i n g on c once n t r a tion th e y m a y be mu t a g en i c- c a r cino g en i c , t e r a t o g en i c, oe s t eo g en i c e t c. c ausi n g r educed immu n e r esponse & other d i so r de rs

4. A n tibiotics P r oduction • Antibiotics also h a v e appli ca tions as f eed additi v es, g r owth s timulants . pe s ticides & wider agricultu r al uses. • P enicillin w as the fi r s t major antibiotic f r om a mic r obial sou r ce t o be c omme r cialised . • Antibiotics sold t od a y a r e made either by to t al chemical s ynthesis or b y a c ombin a tio n of mic r obial f ermen t a tion & subseque n t chemical modific a tion. Choice is based on e c onomics . • The mic r obial f ermen t a tio n p r oduces the basic acti v e molecul e a t r el a ti v ely low c ost & th r ough chemical modific a tio n the the r apeutic e f f ects c an be inc r eased . • All these skills h a v e been d e v eloped in individual c ompanies as t r ade sec r e ts . E ach c ompa n y has its own unique s t r ain & its own unique f ermen t a tion & e xt r action t echnologies: t o m a ximise the g ene r a tion p r operties of these s t r ains. • Hence, actual p r ocess c annot be described, only g ene r alised c onsensus p r ocess is described • Since the s ynthesis P . W of mo s t antibiotic is known, the s t r ain imp r o v ement is done either by UV or chemical induced mu t a tion ( T able)

5. Co n t… Fi r s t a n tibiotic f ound – P enicillin - β-lac t am f amily • In 1929, Al e x ander F l eming dis c o v e r ed it f r om P enicillium no t atu m which ac t ed on St r ep t o c oc c ai. It bind t o p r o t eins of cel l w al l s of M . O & c ollapse s ynthesis of PL t o kill. • It is an o r g anic acid, dissol v ed in o r g anic sol v ents, dryness lose activit y . But dry po w de r in the f orm of salts of P enicillin w as cu r a tive . • Dur i ng W or l d W ar II, the demand inc r eased & Amer i c a p r oduced mo re • P enicillin (bac t ericidal) h a ving β-lac t am r i ng inhibit the f orm a tion of peptide c r oss-lin k age s in the final s t ages of bac t erial cell w al l s ynthesis , but deg r aded b y β-lac t amase sec r eting mic r obes . • The r e f o r e it is inef f ect i ve a g ain s t S t ap h ylo c o c cus a u r eu s f or which Cl ox acilli n & Fl o x acilli n a r e used . • Natu r al P enicillin types di f f er in R-g r oups in β-lac t am r i ng • P enicillin G & V a r e active a g ain s t G r am + c occi • The b r ead spectrum ampicillin & am o xicillin e x t end activity a g ain s t G r am-ne g a tive bac t erial s t r ains such as Haemophilus influrn z ae, E. c oli & Pro t eus microbilis in f ect i ons. • Am o xicillin is used in c ombination with cl a vulanic acid, a po t ent inhibi t or of β-lac t amas e t o e x t end use of antibiotics. • Azlocilli n & ti c a r cilli n a r e used f or p seudomonas in f ect i ons. • P enicillin G & V a r e f ermen t ed p r oducts f r om the imp r o ved fungus, P eniciliu m chrysogenum . • The bulk of P enicillin G & V is now used as s t arting ma t erial f or the p r oduction of the active β-lac t am nucleus , [6-A P A – 6-aminopenicillinamic acid & 7-ADCA - 7- aminodesace t o x y cephalospho r ani c acid] • The y a r e use d f or chemi c al s ynthesis of semi- s ynthetic P enicillin – Acid s t able, & e f f ect i ve in the r apy • 7-ADCA i s also used f or s y n thesis of o r al b r oad spectrum β- lac t am f amily Cephalospor i n s – ce f ad r o xil, cepha t e xi n e t c. which a r e r esi s t ant t o β-lac t amase • Appli c a tions: 38%-human medicine, 12%- V e t erinary Medicine, 43%-S t arting ma t erial f or p r oduction of semi- s ynthetic P enicillin

6. P enicillin Bio s y n thesis P .W • N a tu r al penicillin a r e f ormed f r om which only ben z yl penicillin c an be isol a t ed . H o w e v e r , the desi r ed penicillin c an be o b t ained b y adding sui t able side chain p r ecu r so r i n t o the medium. • Such penicillin a r e c alled as semi- s y n thetic penicillin. • P enicillin-G & P enicillin-V a r e g ene r ally p r oduced c omme r ciall y . • When c ompa r ed t o n a tu r al penicillin, semi- s y n thetic penicillin h a v e imp r o v ed cha r ac t e r s viz, acid s t abilit y , r esi s t ance t o plasmid or ch r omosomally c oded β- lac t amases, e xpanded a n timic r obial e f f ecti v eness & a r e the r e f o r e, e x t ensi v ely used in the r apy

7. A n tibiotics P enicillin V P r oduction • Inoculum – Dry Spo r es of P . chr y sogenu m a r e used f or inoculation • Medium : O r g anic – S o y a meal, peanut meal , Y ea s t e xt r act, c asein, be e f e xt r act, e t c. F .A s timulate p r oduction. D e fined media also c an be used ( T able). • F erme n t ation: ( Fi r s t p r oduct of Indu s trial f ermen t ation): F or o p timum p r oduction, the cultu r e is g r o wn on a ba t ch medium of c orn s teep liquor or s o y flour plus mine r als , & f ed c arbo h y d r ate as a c om s yrup . Add buf f ering agents; pH-6.8-7.4, T emp. 25° C , du r ation-7d a y s . In addition, the p r ecu r sor & ammonium sulphate a r e added to main t ain criti c al c oncent r ations of these c ompone n ts needed f or the bio s y n thesis of the penicillin • R eco v ery: P enicillin is r e c ov e r ed by sol v e n t e xt r actio n (n-butyl ac e t ate, or m e t h ylisobuty l k e tone ) at an acidic pH at temp. below 10 o C to minimis e both chemi c al & enzym a tic b r ea k d o wn of P enicillin in whole b r oth or clear filt r ates. • Depending on the natu r e of the my celiu m the solids c an be r em ov ed by s tring or ult r a filt r ation s & it c an be t r e a ted , dried & used as soil c onditioner • The penicillin-rich sol v e n t c an be t r e a ted with acti v ated c arbon to r em ov e pigme n ts & other impurities (Ex: Cha r c oal r em ov e py r ogens- f e v er c ausing agent) • The penicillin r e c ov e r ed as the po t assium or sodium sal t by adding K or Na ac e t ates • Further impurities c an be r em ov ed by w ashing the r e c ov e r ed salts with a dry solve n ts such as isop r opanol or n-bu t anol, s terili z ed by heat or cr y s t alli z ed • But P enicillin V is st able t o acid & can be p r ecipi t a t ed di r ectly f r om clear filt r ates at pH of 2 . Di r ect p r ecipi t ation r educes the use of o r g anic sol v e n ts , which c an h a v e positi v e c o s t & e n vi r onmen t al impacts .

8. Co n t…Di f f e r e n t P enicillin p r oduction • The desi r ed P enicillin is p r oduced b y adding di f f e r e n t phe n yl ac e tic acid deri v a ti v es in the medium. • Ex: high phe n yl lactic acid supplied b y c orn s t eep liquor g ene r a t e P enicillin G • But it is t o xic t o the P .chrysogenu m mould & hence it is f ed a t di f f e r e n t c once n t r a tions • Out of ma n y r el a t ed n a tu r al peni c illin li k e G, V , F , e t c., mo s t impor t a n t a r e P enicillin G ( Ben z yl) & V(Phen o xym e t h yl) • P enicillin G is v e r s a tile due t o appli c a tion in ring e xpansion • The 6-Aminopenicillinamic acid(6-A P A) is p r oduced b y h y d r ol y sis of P enicillin G & V salts using immobili z ed P enicillin amidases (H y d r ol y sis is g r e a t er f or V than G ), pH-7-8 b y addition of c au s tic or ammonium h y d r o xide • 6-A P A is r e c o v e r ed b y p r ecipi t a tion a t pH-4 in the p r esence of immiscible sol v e n ts & the p r ecu r sor acid is r e-c y cled f or P enicillin p r oduction

9. Semi- s y n thetic P enicillin P r oduction by Chemi c al modifi c a tion • 1000 ’ s of Semi- s y n th e tic P enicill i n h a v e been o b t ained by chemi c al means f r om 6-Aminopenicill i namic acid (6-A P A-it ’ s a P enicill i n w i thout an acyl g r oup) by acyl a tion p r ocess. • P enicill i n G f or e x ample co n v er t ed t o 6-A P A & phe n ylac e ti c acid. • The 6-A P A is then e thical l y acyl a t e d w i th an app r opri a t e side chain t o p r oduce a semi- s y n th e tic penicil l in. • Ma n y of them a r e mar k edly superior t o P enicill i n G f or clinical use • Acyl a tion is done w i th deri v a ti v e of carb o xyl i c acids (Ex: Acid chlor i de or m i x ed an h y dride), carried out in o r g anic sol v e n ts under an h y d r ous condit i on (O r g anic am i ne – Ex: tri e t h y l am i ne is added) • Al t ern a ti v el y , its done in aqueous solution i n ac e t one- w at er mi x tu r e in p r esence of sodium bi c arbon a te • F ormed se m i- s y n th e tic P enicill i n is iso l at ed by di s tributing as its f r ee acid i n t o w at er i m m i scible o r g anic sol v e n ts whi l e the salts & i m puriti e t s i nt o w at e r.

10. P enicill i n p r oduction • Carbon sou r ce is g ene r ally supplied in the f orm of lactose. Glu c ose, suc r ose, gl y ce r ol and sorbitol c an also be empl o y ed as c arbon sou r ce. Nit r ogen sou r ce is g ene r ally supplied in the f orm of ammonium sulph at e or ammonium ac e t at e or ammonium nit r at e. Abundan t f ormation of m y celium and spo r es t a k es place when a medium c on t ains c orn- st eep liquor be c ause it c on t ains impor t ant amino acids r equi r ed f or m y celial g r owth. • P o t assium, phosphorus, magnesium, sulphu r , zinc and c opper a r e supplied in the f orm of salts. P o t assium and phosphorus a r e supplied in the f orm of po t assium di h yd r o g en phosph at e, magnesium, i r on and c opper a r e supplied in the f orm of sulph at es. All these elements m a y be p r ese n t in c orn s t eep liquo r. • P enicillin-F and penicillin-K a r e the n a tu r ally p r oduced penicillin s s ynthesi z ed by P . no t a tu m and P . chrysogenum, r especti v el y , in the absence of p r ecu r so r . But, if phe n ylac e tic acid is supplied in the medium P . chrysogenu m p r oduces penicillin-G in st ead of penicillin-K. Similarl y , desi r ed s ynth e tic penicillin s c an be ob t ained by adding the medium with sui t able p r ecu r so r. • T r eatme n t of Crude Ext r act: • The r esul t ed sodium penicillin is t r e a t ed with cha r c oal t o r em o v e py r ogens ( f e v er c ausing sub s t ances). It is als o , som e times, st erili z ed t o r em o v e bacteria by using Seitz filte r . Then, the sodium penicillin is p r epa r ed in cry s t alline f orm by cry s t alli z a tion. It m a y be pac k ed as po w der in s t erile v ials or p r epa r ed in the f orm of t abl e ts or in the f orm of s yrups f or o r al usage. The pharmaceuti c al g r ade m a y be used in the p r oduction of semi s ynth e tic penicillin. • Uses of P enicillin: • 1. Mo s t of the penicillin ’ s a r e acti v e a g ain st G r am-positi v e bacteria, in which th e y inhibit the cell w all s ynthesis leading t o the de a th of bac t eria. • 2. Used the r apeuti c ally in the t r e a tment of in f ectious diseases of humans c aused by G r am (+) positi v e bacteria.

11. Co n t… • Inoculum Production: • The microorga n ism which is used in a fermentation process is called as the inoculum. A high yielding strain of P. chrysogenu m is generally employed as inoculum. • A strain of the fungus is sub-cultured from stock culture for inoculum developmen t . Spores from primary source are suspended in water or in a dilute solution of a nonto x ic wetting agent such as 1:10000 sodium lauryl sulfate. The spores are then added to flasks or bottles of wheat bran plus nutrient solution and these are incubated for five to seven days at 24°C so as to provide heavy sporulation. The entire process is repeated several times in order to have more sporulation. • The resulting spores are used directly to inoculate inoculum tanks or stirred fermenters. The incubation temperature is maintained at 24-27°C for 2 days with agitation and aeration in order to facilitate heavy mycelial growth, which may be added to a second or even a third stage fermentat i on. • The resulting inoculum which is employed in a production tank is tested both by micros c opic examination and by sub-culturing method. Many sporulation media have been designed to obtain large number of spores. The one developed by Moyer and Coghill (1946) is mos t extensively used and given below • Inoculation: • Introductio n of pure inoculum into the production tanks or fermenters is called as inoculation. • This is done by any one of the following three methods: • 1. Dry Spores may be used as Inoculum: • Since the spores of P. chrysogenu m are hydrophobic, either spores are blown deep into the medium or a wetting agent such as sodium lauryl sulphate is used. • 2. Suspension of Ungerminate d Spores: • This suspension is made by using 1:10000 sodium lauryl sulfat e solution. This suspensio n is fed to the fermenter by suitable techniques like spray guns or pipettes. This is followed by agitation and aeration of the fermentation medium in order to achieve equal and uniform distribution of the spores in the entire medium. • 3 . Feeding the fermentation tanks with pre-germinated spores or mycelial pellets which are prepared by the germination of spores. Pellets are generally fed to the fermentation medium after two or three days of spore inoculation. • Fermenters with a capacity of 40,000 to 2 lakhs liter s are generally employe d for the production of penicillin. Due to difficulties with the oxygen supply larger tanks are not employed. Some manufacturer’s use of Wald h of fermenters or air lift fermenters, but this is only possible in mutants which generate low visco s i t y. Depending upon the production strain, the operational temperature is maintained between 25°-27°C. A typical flow chart for penicillin production is given in Fig. 6.5.

12. Co n t…. • P enicillin yields with time a r e linear f r om app r o xima t ely 48 t o 96 hou r s. The final penicillin yield is in the r an g e of 3 t o 5% which la r g ely depends upon the amount of c arbo h yd r at e c onsumed during f ermen t a tion p r ocess, which is app r o xima t ely equal t o 1500 in t ern a tional units per millilite r . S yl v e s t er and Coghill (1954) h a v e e s tima t ed th a t t o p r oduce 1000 g allons of f ermen t ed cultu r e, which is c apable of yielding 2.2-2.7 kg of penicillin by the subme r g ed cultu r e m e thod r equi r es app r o xima t ely 227 kg of nutrients, 3400 kg of s t eam, 45460 l t of w at e r , 1000 kWh of electricity and 7075 m 3 of ai r. • Medium: • Carbon sou r ce is g ene r ally supplied in the f orm of lactose. Glu c ose, suc r ose, gl y ce r ol and sorbitol c an also be empl o y ed as c arbon sou r ce. Nit r ogen sou r ce is g ene r ally supplied in the f orm of ammonium sulph at e or ammonium ac e t at e or ammonium nit r at e. Abundan t f ormation of m y celium and spo r es t a k es place when a medium c on t ains c orn- st eep liquor be c ause it c on t ains impor t ant amino acids r equi r ed f or m y celial g r owth. • P o t assium, phosphorus, magnesium, sulphu r , zinc and c opper a r e supplied in the f orm of salts. P o t assium and phosphorus a r e supplied in the f orm of po t assium di h yd r o g en phosph at e, magnesium, i r on and c opper a r e supplied in the f orm of sulph at es. All these elements m a y be p r ese n t in c orn s t eep liquo r. • P enicillin-F and penicillin-K a r e the n a tu r ally p r oduced penicillin s s ynthesi z ed by P . no t a tu m and P . chrysogenum, r especti v el y , in the absence of p r ecu r so r . But, if phe n ylac e tic acid is supplied in the medium P . chrysogenu m p r oduces penicillin-G in st ead of penicillin-K. Similarl y , desi r ed s ynth e tic penicillin s c an be ob t ained by adding the medium with sui t able p r ecu r so r.

13. A m p icill in • Ampicilli n is the fi r s t b r oad spectrum a n tibioti c used t o p r e v e n t & t r e a t a number of G+ & - bac t erial i n f ections, in r espi r a t ory t r act, urinary t r act, meningitis, salmonellosis e t c. • It is r el a ti v ely harmless which c an be used during p r egnancy & als o used t o p r e v e n t g r oup B St r ep t o c oc c al i n f ection in new born & als o act a g ain s t multidrug r esi s t a n t s t r ains • P enicillin G & V a r e the basic p r ecu r so r s of a wide r an g e of semi- s y n thetic a n tibiotics, e.g., ampicillin. • This is achie v ed b y h y d r ol y sis o f acyl g r oup t o g et 6-Aminopenicillinamic acid (6-A P A) using amidase & further addition of r equi r ed acyl g r ou ps • This is achie v ed b y passa g e th r ough a c olumn of immobili z ed penicillin amidase on a number of supports including c y ano g en b r omide-acti v a t ed Sephad e x G200

14. P r o d u c t i o n : A m p i c i l li n so d i u m s a lt • Modifi c a tion of P enicillin G & V is achi e v ed b y r emoving their n a tu r al acyl g r oup, le a ving 6 A P A t o which other acyl g r ou p s c an be added t o c o n f er n e w p r operties. • P enicillin amidase is usually o b t ained f r om E. c oli. • Ampicillin has been p r oduced by the use of penicillin-G-amidase immobilised b y adsor p tion t o D E AE -cellulos e in a pac k ed bed c olumn a t neut r al pH: • Then the 6-A P A p r oduced is acyl a t e d t o g e t ampi c illin (An h y d r ous f orm) • F or b e t t er qualit y , Ampicillin sodium salt is p r oduced • The sodium salt of ampicillin is p r epa r ed b y f orming a solution of the di e t h ylamin e salt of the ampicillin in m e t h ylene dichloride & r e c o v ering the sodium salt p r ecipi t a t ed upon the addition of a sodium or sodio p r ecipi t a n t. • P r ocess s t e p s f or p r oducing sodiu m ampi c illin in cr y s t alline f orm: • 1. P r eparing the soluble di e t h ylamin e salt of ampicillin b y the r eaction of an h y d r ous ampicillin with di e t h ylamin e in m e t h ylene chloride , adding the r e t o a m e t h ylene chloride solution of sodium 2- e t h ylh e x ano a t e, • 2. The r eaction solution w as cr y s t alli z e d in a cr y s t alli z e r , • 3. The r esulting cr y s t al w as fil t e r ed, w ashed, & dried t o o b t ain the finished p r oduct ampicillin sodium salt. • Then high yields of high purity sodiu m ampi c illin in cr y s t alline f orm is o b t ained b y adding ac e t onitrile t o the mixtu r e .

15. Ca r b o h y d r a t e A n ti b i o t i cs : A m i n og l y c o s i d e s – E x : S t r e p t o m y c in • O v er 100 aminogl y c osid e antibiotics a r e the r e, but only f ew a r e p r oduced indu s trially • Al l r i ng s tructu r es of these antibiotics a r e der i ved f r om glu c ose • They h a ve bac t ericidal activity a g ain s t G r am + & - bac t eria & m y c obac t eria & act by binding t o 30S subunit of ribosome & p r event p r o t ein s ynthesis • Als o , bac t erial r esi s t ance de v eloped f a s t & deri v a t iv es of aminogl y c osides a r e developed • Al l c ause side ef f ect - kidney damage & de a fness. Ex: Use of St r ep t o m y cin t o t r eat TB. P r olonged use c ause neu r o t o xic r eaction & partial hearing . But di h y d r ospect r o m y ci n o ve r c ome it • St r ain imp r o vement c an gi v e less t o xicity & mo r e yield. • M . O used: Al l a r e p r oduced f r om Actino m y ce t e s spp. Ex: St r ep t o m y cin f r om Strep t o m y c es griseus , Gen t a m y cin f r om Mic r omospor a purpura e e t c. • One cultu r e c an p r oduce v ariety of molecules. Ex: K ana m y cin A, B , C or Gen t a m y cin C1,2 e t c. • Chal l enge s a r e the r e t o g et s t r ains which r educe p r oduction of un w an t ed p r oduct s & t o mai n t ain antibiotic types. • St r e p t o m y ci n is a B r oad spectrum a n tibiot ic • The m e t abolic P . W of St r e p t o m y cin bio s y n thesi s s t arts f r om glucose by using ~ 20 en z ymes (Fig.)

16. P r oduction p r ocess f or S t r e p t o m y cin • P r ocess: • The inoculum is p r oduced w i th Stre p t o m y c es griseus • The medium used usually consi s ts of s o y m eal or s o y flour or corn s yrup th a t can supply glucose a t a sl o w r at e (a m ylase activ i ty is poor in St r e p t o m y ces sp). Salt s of am m onia & phosph at es a r e not used in s t art i ng b a t ch be c ause, glucose, ammonia & phosph a t e in high qua n tities inhibit s t r e p t o m y cin s y n thesi s by F .B r ep r es s ion. Hence, the init i al supply of nit r ogen (NH 3 ) & phosph a t e is also ob t ained f r om s o y meal . • Since St r e p t o m y ces h av e poor a m ylase activ i t y , corn s yrup m a y be used • F erme nta tion conditions f or o p ti m al p r oduction: t e m p. 27-30° C , pH 6.5-7.5, ae r a tion r at e 0.5-1.0 vvm. The du r a tion of f er m e n ta tion p r ocess depends on the s t r ain used, & is b e t w een 6 t o 8 d a y s. • R e c o v ery of St r ep t o m y cin: • St r e p t o m y cin o r other am i nogl y cosides a r e basic in n a tu r e . Th e y c an be r e c o v e r ed by w eak c a tionic e x change r es i ns in an ion - e x change column. • T r e a tme n t w i th acti v at ed carbon is of t en neces s ary t o r em o v e i m purities . • St r e p t o m y cin can be p r ecipi t at ed in the f orm of sulph a t e salt .

17. P r o d u c t i o n o f c o mm e r c i a l V i t a m i ns • Mo s t vi t amins w e r e p r e v iously p r epa r ed f r om animal & pla n t tissues • Mic r oo r g anisms a r e n o w used as sou r ces of a wide r an g e of Vi t amins, including thiamin (vi t amin B1), ribofl a vin (vi t amin B2), p yrid o xine (vi t amin B6), c obalamin (vi t amin B12), biotin, f olic acid, l-as c orbic acid (vi t amin C), β- c a r o t ene (p r o-vi t amin A), e r g o s t e r o l (p r o- vi t amin D2) & pa n t othenic acid. • All pho t og r aphic M . O s y n thesise all Vi t amins & other g r o wth s timul a t o r s r equi r ed f or its g r o wth & hence a r e primary me t aboli t es • Th e y a r e s y n thesised in amou n t th a t is enough f or the cell ’ s need & ne v er accumul a t e it , but cer t ain M . O only (Indu s trially i n t e r e s t ed) e x c r e t e e x cess Vi t amins if it is cultu r ed in highly specified & artificial c onditions . • Such s y n thesise be y ond it ’ s r equi r eme n t, accumul a t e in cultu r e medium & the M . O a r e e xploi t ed f or c omme r cial p r oduction • Ex: Ca r o t ene (P r o-Vit.A) f r om Bla k esle a trispora ; Ribofl a vin(Vit.B2) f r om Ashby a gos s ypii ; L- sarbos e in Vit . C s y n thesis f r om Glu c onobac t e r o xida n ts ; Vit.B12 f r om Bacillus mega t erium, c oagulans, Pseudomonas denitrifi c an s & S trep t o m y c es olivaseous • Th e y a r e c on s titu t ed f r om simple ing r edie n ts in the medium • Som e vi t amins a r e p r oduced b y di r ect f erme n t a tion p r ocesses, whe r eas othe r s, b y biot r an s f orm a tion s or c ombined chemi c al & mic r obiologi c al p r ocesses.

18. Thank y ou.