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Secondary meLabollLes from

planL cell culLures

W @ypes
r|mary lnclude baslc molecules for susLenance of llfe processes and
absence of whlch resulL ln lmmedlaLe deaLh eg Molecules llke
CarbohydraLes llplds are needed for growLh developmenL or
reproducLlon eLc
Secondary lnclude secondary producLs havlng mlnor role ln llfe
processes buL ofLen have ecologlcal roles eg aLLracLanLs of polllnaLors and
chemlcal defenses agalnsL mlcrobes lnsecLs and hlgher predaLors
W bsence of SM does noL resulL ln lmmedlaLe deaLh buL raLher ln longLerm lmpalrmenL of Lhe
organlsms survlvablllLy fecundlLy or aesLheLlcs or perhaps ln no slgnlflcanL change aL all
W Pumans use some of Lhese compounds as medlclnes flavorlngs or recreaLlonal drugs
W Secondary meLabollLes can be classlfled on Lhe basls of
Chemlcal sLrucLure (for example havlng rlngs conLalnlng a sugar)
ComposlLlon (conLalnlng nlLrogen or noL)
@helr solublllLy ln varlous solvenLs or Lhe paLhway by whlch Lhey are synLheslzed (eg
phenylpropanold whlch produces Lannlns)
W slmple classlflcaLlon lncludes Lhree maln groups
@he Lerpenes (made from mevalonlc acld composed almosL enLlrely of carbon and hydrogen)
henollcs (made from slmple sugars conLalnlng benzene rlngs hydrogen and oxygen)
nlLrogenconLalnlng compounds (exLremely dlverse may also conLaln sulfur)
W lanL cell bloLechnology has evolved as a promlslng new area wlLhln Lhe fleld of bloLechnology
focuslng on Lhe producLlon of planL secondary meLabollLes lor mosL compounds of lnLeresL eg
morphlne qulnlne vlnblasLlne aLroplne scopolamlne and dlgoxln
W lanL cell and Llssue culLures can be esLabllshed rouLlnely under sLerlle condlLlons from explanLs
such as planL leaves sLems rooLs merlsLems eLc for boLh Lhe ways for mulLlpllcaLlon and exLracLlon
of secondary meLabollLes
W @he varlous beneflLs of producLlon of valuable naLural producLs by cell culLures are
Lase Lo be carrled ouL Lhrough ouL Lhe year
Cvercomlng rlsk of crop fallure due Lo naLural hazards
Cvercomes Lhe danger of exLlncLlon of some specles due Lo mass exLracLlon
W Cell culLures noL only provlde Lhe means for de novo synLhesls buL also acL as blofacLorles for
bloconverslon of low value compounds Lo hlgh value producLs
W Cf varlous planL producLs produced by planL Llssue culLure pharmaceuLlcals have aLLalned mosL
W !apan and Cermany have maxlmum conLrlbuLlon Lo Lhese
W CrowLh and producLlon of secondary meLabollLes are lnversely relaLed boLh ln whole
planL and cell culLures as producLlon of SM occurs ln laLe sLaLlonary phase when medlum
geLs depleLed of some of lLs consLlLuenLs
W SMs made ln
baLches (small Lo medlumscale)
bloreacLors (largescale producLlon)
W CrowLh lnhlblLlon ls assoLlaLed wlLh cyLodlfferenLlaLlon and lnducLlon of enzymes for
secondary meLabollsm So ln such cases dual culLure sysLems are preffered
L lnvolves
W blomass producLlon ln a medlum
opLlmum for cell prollferaLlon and
W Lransfer of healLhy cells Lo a
dlfferenL (producLlon medlum)
whlch ls noL favourable for growLh
buL for producL yleld
Jas flrsL used for producLlon of
lndole from cototootbos toseos by
Zenk eL al
nd Lhen for shlkonln producLlon
from tbospetmom etytbtotboo
W MosL useful modlflcaLlon ln growLh medlum for use ln SM producLlon are
8educLlon or elemlnaLlon of 24u or oLher phyLohormones
8educLlon ln phosphaLe levels
ncrease ln sucrose level or alLeraLlon ln C/n raLlo
W @he opLlmum medla for dlfferenL SM producLlon by a cell llne ls llkely Lo be dlfferenL
W Ior A[ma||c|n Ior 1ryptam|ne
hosphaLe absenL hosphaLe hlgh levels 04 mM
nlLraLe 2mM nlLraLe near zero
mmonlum absenL mmonlum mM
Clucose CpLlmum conc 300mM Clucose 00mM
W n some cases fuslon of growLh and producLlon sLeps ls posslble ln Lhe same medlum by a blL
alLeraLlons ln medlum eg n alkalold producLlon from C roseus SmlLh eL al suggesLed a slngle
sLage process wlLh medla glucose concenLraLlon lncrease by 6
W Growth regu|ators effecL growLh and dlfferenLlaLlon and Lhus secondary meLabollsm
W n general hlgh uxln level (24u) whlch sLlmulaLes dedlfferenLlaLlon and prollferaLlon of cells
erduces SM
W Lg 8erberln producLlon @ moos
CrowLh medlum wlLh 24u
roducLlon medlum wlLh n and 8 (uxln and CyLoklnln comblned)
W C generally lnhlblLs Secondary meLabollsm Lg Pas no effecL on CaLaranLlne producLlon buL
very rare example of lLs poslLlve effecL on SM producLlon ls berberln producLlon from copts
jopoono whlch ls aLLrlbuLed Lo C lnduced sLarch synLhesls lnhlblLlon decreased nlLreogen upLake
and upLake and Lurnover of sucrose
W @he pn of medlum ls shown Lo enhance permeablllLy of Lhe cell membrane and Lhus helps ln Lhe
release of lnLracellular alkalolds
W L|ght ls lmporLanL regulaLory facLor for producLlon of alkalolds ln Llssue culLures Lx n Croseus
llghL effecLs a[mallcln/serpenLlne accumulaLlon raLlo CaLaranLhlne producLlon ls compleLely
rapressed ln Lhe absence of llghL
W Gaseous env|ronment malnly avallblllLy of oxygen and carbon dloxlde play lmporLanL role ln SM
producLlon Lx ncreased lnlLlal oxygen mass Lransfer shorLens Lhe Llme for serpenLlne
accumulaLlon wlLhouL alLerlng flnal yleld
W @he explanLs used Lo lnlLlaLe Llssue culLures are hlghly
heLerogenous wlLh regard Lo Lhe meLabollc producLlve of lLs
consLlLuenLs cells whlch lncrease furLher ln culLure condlLlons
lnduclng geneLlc and eplgeneLlc changes
W @he producLlvlLy of an heLerogenous culLure ls average Lo Lhe
producLlvlLy of hlgh yleldlng and low yleldlng sLralns
W So selecLlon and clonlng of P?L ls done Lo effecLlve for
lmprovemenL of producLlon of SM
W lor lsolaLlon of sLable llnes repeaLed selecLlons are Lo be
madeand only lf Lhe producL of a cell llne remalns sLable over
several selecLlon cycles a Lrue varlanL could be ldenLlfled
5Lc1lON Ol 851 cLON5
W Screenlng for hlgh yleldlng llnes for coloured compounds ls
done by selecLlng Lhe mosL coloured areas of cell clumps
W Cells wlLh hlghly fluroscenL compound can be selecLed on
basls of fluroscence by flow cyLomeLry
W lor colourless compounds speclflc acLlons of squashed cells or
lmmunologlcal LesL of exLracL may help for besL clones
W Ma[or steps are
Se|ect|on of h|gh y|e|d|ng ||nes
In|t|at|on of cu|ture
Screen|ng for best produc|ng
var|ants(resonab|y stab|e ma|n|y)
Cu|ture estab||shment of these ||nes
Irom estab||shed cu|tures of these
||nes better y|e|d|ng subc|ones are
roduct|on of SM us|ng them
W L||c|tors are compounds LhaL Lrlgger Lhe formaLlon of secondary meLabollLes
W 1hey can be ab|ot|c or b|ot|c
W Natura| or 8|ot|c ellclLors lnclude polysaccharldes such as fungal exLracLs pecLln and
chlLosan Lg
ChlLosan ellclLs by many fold lncrease of anLhraqulnone producLlon ln obo okooe nell
Conldla of I Joblos when added Lo C arboreum lncreases Lhe yleld of Cosslplum fro 3
0 g/l Lo 300 g/l ln 3 days
W Ab|ot|c e|||c|tors lnclude lnorganlc or organlc compounds uv lrradlaLlon also lnduce producL
accumulaLlon ln culLured cells Lg
ddlLlon of naCl and kCl causes almosL 200 lncrease ln caLaranLhlne accumulaLlon
@he producLlon of dlmerlc alkalolds by shooL culLure of C roseus can be lnduced by
lrradlaLlon wlLh nearulLravloleL llghL
W @lme of appllcaLlon of ellclLor ls crlLlcal for yleld of SM by culLured cells
W MosL of Lhe culLures respond Lo elllclLaLlon only durlng growLh phase
W LlllclLor LreaLmenL afLer a culLure has sLarLed Lo accumulaLe Lhe lnduclble compound does
noL enhance or accleraLe lLs producLlon
W SM producLlon by cell culLure shows
Plgh producLlon cosL due Lo slow growLh of planLs
Low producL yleld
CeneLlc lnsLablllLy of selecLed llnes
Low shear reslsLance
nLracellular accumulaLlon of producL
W Some of Lhese problems are reduced by lmmoblllsaLlon of cells
W n Lhls Lechnlque cells are conflned wlLhln a reacLor sysLem prevenLlng Lhelr enLry lnLo
Lhe moblle phase whlch carrles Lhe subsLraLe and Lhe producL
W mmoblllsaLlon ls only relevanL ln producLlon process lnvolvlng 2 sLages
llrsL 8lomass producLlon by suspenLlon culLure
Second producL formaLlon by lmmoblllsed cells
W Advantages of |mmob|||zed ce|| cu|ture are
Lnables prolonged use of blomass
2 Cell denslLy of Lhe bloreacLor can be lncreased whlch enables Lhe use of
W 8educed medlum cosL
W use of small reacLor
W Low equlpmenL lnsLallaLlon cosL
W Slmpllfles uownsLream processlng
Slmple deslgn bloreacLor can be used as enLrapped cells are proLecLed agalnsL
4 L uncouples growLh and producL formaLlon whlch allows producL opLlmlsaLlon
wlLhouL effecLlng growLh
3 @he non dlvldlng lmmoblllsed cells are less prone Lo geneLlc changes rovldes
sLable producLlon raLes
6 L mlnlmlses fluld vlscoslLy
7 L even promoLes SM secreLlon ln some cases
W @he besL deslgn of reacLor used depends on Lhe meLhod of lmmoblllzaLlon used llke
LnLrapmenL of cells ln gel (agarose alglnaLe agar eLc ) or seml permeable membrane
llxlng on surface of lnerL supporL (llber glass maL)
W Surface lmmoblllzaLlon promoLes naLural Lendency of cells Lo aggregaLe lmprovlng synLhesls
and accumulaLlon of SM
W speclal beneflL of surface lmmoblllzaLlon ls absence of any physlcal resLrlcLlon Lo mass
Lransfer beLween culLure medlum and blomass surface
W n lmmoblllzed sysLem whlch could malnLaln vlable cells over exLended perlod of Llme and
releases bulk producL ln exLracellular medlum ln sLable form can drasLlcally reduce
producLlon cosL
W L|m|tat|ons of an |mmob|||zed ce|| systems are
L ls llmlLed Lo sysLems where producLlon ls decoupled from cell growLh
@he lnlLlal blomass needs Lo produced ln suspenslon culLure
@he producL secreLlon ln medlum ls lmperaLlve
Jhen secreLlon occurs Lhere may be exLracellular degradaLlon of producLs
Jhen Cel enLrapmenL ls used lL lnLroduces an addlLlonal dlffuslon barrler