MeLagenomlcs for manlpulaLlon of CuL mlcro flora

Speaker
Arumbaka Sudheer 8abu
uCn
nu8l karnal
Introduction
W W Metagenom|cs Metagenom|cs ls a rapldly growlng fleld of
research LhaL alms aL sLudylng unculLured
organlsms Lo undersLand Lhe Lrue dlverslLy of
mlcrobes Lhelr funcLlons cooperaLlon and
evoluLlon ln envlronmenLs such as soll waLer
anclenL remalns of anlmals ot tbe Jlqestlve
system of oolmols ooJ bomoos %uson et ol
2009)
0tug0nomics
@he Lerm ''meLagenomlcs'' was colned by andelsman et
ol
@he Lerm ''meLagenomlcs'' was colned by andelsman et
ol
lL ls deflned as Lhe appllcaLlon of modern genomlcs
Lechnlques Lo Lhe sLudy of communlLles of mlcroblal
organlsms dlrecLly ln Lhelr naLural envlronmenLs
bypasslng Lhe need for lsolaLlon and laboraLory culLlvaLlon
of lndlvldual specles %Chen achLer 2003)
lL ls deflned as Lhe appllcaLlon of modern genomlcs
Lechnlques Lo Lhe sLudy of communlLles of mlcroblal
organlsms dlrecLly ln Lhelr naLural envlronmenLs
bypasslng Lhe need for lsolaLlon and laboraLory culLlvaLlon
of lndlvldual specles %Chen achLer 2003)
Rum0n 0tug0nomics
8umlnanLs recrulL and reLaln a dlverse collecLlon of bacLerla
archaea proLozoa and fungl LhaL can produce energy and proLeln
yleldlng nuLrlenLs from planL blomass %lrklns and ?u 2006)
8umlnanLs recrulL and reLaln a dlverse collecLlon of bacLerla
archaea proLozoa and fungl LhaL can produce energy and proLeln
yleldlng nuLrlenLs from planL blomass %lrklns and ?u 2006)
lnLeracLlon among Lhe mlcrobes resulLs ln synerglsm leadlng Lo
beLLer feed degradaLlon
lnLeracLlon among Lhe mlcrobes resulLs ln synerglsm leadlng Lo
beLLer feed degradaLlon
Ma[orlLy of Lhese mlcrobes %03) are nonculLurable whlch
makes Lhe sysLempoorly undersLandable
Ma[orlLy of Lhese mlcrobes %03) are nonculLurable whlch
makes Lhe sysLempoorly undersLandable
8esulLs wlLh LradlLlonal manlpulaLlon Lechnlques are noL always
repeaLable
8esulLs wlLh LradlLlonal manlpulaLlon Lechnlques are noL always
repeaLable
AppIicutions of rum0n 0tug0nomics
CreaLlon of genomlc sequence daLabase of rumen mlcrobes
ldenLlflcaLlon of novel enzymes/mlcrobes from rumen
ldenLlflcaLlon of unculLured meLhanogens
ulfferenLlaLlng and quanLlLaLlve deLermlnaLlon of rumen blomass
NkC 2007
TruditionuI Vs 0tug0nomic upprouch
NkC 2007
St0ps in 0tug0nomic Strut0gy
Sampllng
unA exLracLlon and
preparaLlon
Clonlng and LransformaLlon
Llbrary screenlng
uownsLream analysls and explolLaLlon
W|od|vers|ty of env|ronment
W 1ype of Lnr|chment to be
used
WAmount of b|omass ava||ab|e
WSamp|e |ntegr|ty
WSequence ana|ys|s and
annotat|on
- |so|at|on of gene(s) of
|nterest
- act|v|ty man|pu|at|on
opt|m|zat|on
Vector propert|es Insert s|ze
compat|b|||ty host range etc
- S|ze of ||brary Cata|og|ng
Storage
Screen|ng v|a Ck
or hybr|d|zat|on
- Iunct|ona|
screen|ng
- Use of p|atforms
such as m|croarrays
- Iunct|ona|
screen|ng |n mu|t|p|e
hosts
- Mo|ecu|ar we|ght of
extracted DNA
-Leve| of pur|f|cat|on
necessary
- Ach|ev|ng appropr|ate
s|ze range of DNA
fragments
Issues and Dec|s|ons Issues and Dec|s|ons
kowa|chuk et o/ 2007
@echnlques and Approaches applled Lo access
ML@ACLnCMLS
@echnlques and Approaches applled Lo access
ML@ACLnCMLS
Shotgun stud|es
roduct or act|v|ty
dr|ven stud|es
hy|ogenet|c and |arge
|nsert metagenom|c
approaches
Uses mass genome sequenc|ng
fo||owed by scaffo|d reconstruct|on
and gene annotat|on
Des|gned to Search Spec|f|c
m|crob|a| act|v|t|es and the
genes encod|ng them
L|nk|ng the genome
|nformat|on w|th hy|ogenet|c
markers of m|crob|a| groups
of |nterests
kowa|chuk et o/ 2007
Metagenom|cs (g DNA) Metagenom|cs (g DNA)
Genome enr|chment
]Amp||f|cat|on(SSnSI
etc)
Genome enr|chment
]Amp||f|cat|on(SSnSI
etc)
Sub[ected to DNA
sequenc|ng( random
shotgun sequenc|ng
yrosequenc|ng etc)
Sub[ected to DNA
sequenc|ng( random
shotgun sequenc|ng
yrosequenc|ng etc)
Metatranscr|ptom|cs
(m kNA)
Metatranscr|ptom|cs
(m kNA)
Genes enr|chment (
keverse 1ranscr|pt|on
Ck DLA)
Genes enr|chment (
keverse 1ranscr|pt|on
Ck DLA)
Genes poo|] Genes
c|on|ng and
express|on
Genes poo|] Genes
c|on|ng and
express|on
c DNA ||brar|es]
metagenom|c genes
c DNA ||brar|es]
metagenom|c genes
erblvore
Gastro |ntest|na|
samp||ng
In v|tro enr|chment
(SI rDU |abe||ng
substrate se|ect|on
etc)
Lxtract|on and
pur|f|cat|on of
m|crob|a| metagenome
An overv|ew of An|ma| GI
metagenom|c ana|ys|s
Data
generat|on
and ana|ys|s
Iunct|on
dr|ven
ana|ys|s
Sequence
dr|ven
ana|ys|s
SI stab|e |sotope prob|ng rDUSbromo2deoxyur|d|ne
SSn suppress|on subtract|ve hybr|d|zat|on DLA d|fferent|a| express|on ana|ys|s S|ngh et o/ 2008
Iunct|on Dr|ven Ana|ys|s Iunct|on Dr|ven Ana|ys|s
Sequence Dr|ven Ana|ys|s Sequence Dr|ven Ana|ys|s
Cene clonlng and expresslon
Cenome / gene
sequence
ldenLlflcaLlon
robeAsslsLed gene
ldenLlflcaLlon
Clonlng and expresslon
of genes of lnLeresL
Ident|f|cat|on of roducts
eed /food processlng and
blo lndusLry appllcaLlons for
susLalnable producLs
novel mlcrobes and mlscproducLs
%proLelns anLlmlcroblals pepLldes
amlno aclds eLc
S|ngh et o/ 2008
Pondom Sequencing Approoch
Clones are randomly sequenced Clones are randomly sequenced
ln presence of reference genome comparlson ls
done
ln presence of reference genome comparlson ls
done
lf reference genome ls noL Lhere Lhen
clones are sorLed on Lhe basls of
lf reference genome ls noL Lhere Lhen
clones are sorLed on Lhe basls of
C+C conLenL C+C conLenL
Codon usage Codon usage
@hus represenL
mlcroblal
communlLy and
characLerlsLlc of
Lhe sampled
envlronmenL
8uL no @axonomlc
or hylogeneLlc
AfflllaLlon
%orgefed Sequencing Approoch
Clones obLalned are screened for Lhe presence of byloqeolc oocbots (165
tkNA)
1bls ls ofteo fltst ste lo lotqe metoqeoomlc tojects
llMl1A1lON
lnefflclenL ln explorlng unknown genes
uaLa ls ln numerous sequenced fragmenLs
8eassembled lnLo compleLe genome
Mlcroblal lnLeracLlons paLhways are noL explalnable
uncfionoI 8osed Approoch
Ident|f|cat|on of constructed c|ones
express|ng a des|red tra|t |n a surrogate host
nerinio n/i |s most preferred surrogate
It |s a qu|ck |dent|f|cat|on approach for the
usefu| act|v|ty
1h|s approach does not requ|re the gene of
|nterest to be recogn|zed by sequence
ana|ys|s
Besides E. ca// ,s host, Act/namycetes group h,;e been successfully used.
LIMI1A1ICN
Many genes are noL expressed ln any parLlcular hosL bacLerla
selecLed for clonlng
Many genes are noL expressed ln any parLlcular hosL bacLerla
selecLed for clonlng
Success requlres falLhful LranscrlpLlon and LranslaLlon of
genes of lnLeresL
Success requlres falLhful LranscrlpLlon and LranslaLlon of
genes of lnLeresL
eLerologous expresslon remalns a barrler eLerologous expresslon remalns a barrler
Construction of m0tug0nomic Iibrury
W Sma|| |nsert ||brar|es %0 kb) L coll ls Lhe hosL
sLraln %enne et ol)
W Large |nsert
W Cosmld llbrarles 23 Lo 33 kb mosLly ln Lhe pWL3
vecLor of hosL SLraLagene %LnLcheva et ol 200)
W osmld llbrarles of 40 kb%8e[a et ol2002)
W octetlol ArLlflclal Chromosome %8AC) upLo 200
kb %8e[a et ol2000)
W 5ttetomyces llvlJoos ooJ some CramnegaLlve
bacLerla are currenLly belng developed as hosLs
R0c0nt PhyIog0n0tic studi0s of rum0n microbiom0
W MosLly focused on domesLlcaLed rumlnanL
anlmals especlally dalry caLLle
1echn|ques used
434 pyrosequenclng
6S r8nA bacLerlal Lagencoded Lx ampllcon
pyrosequenclng %b@LA)
9itto et o/ 2010
ConLd
PhyIog0n0tic unuIysis of UmbgI3A
MeLagenomlc llbrary of unculLured bacLerla from
bovlne rumen was consLrucLed
Sequenclng of a 43 kb unA fragmenL revealed a beLa
glucosldase gene ombql3A wlLh an C8 of 36 bp
encodlng a producL whlch shared 63 ldenLlLy and 7
slmllarlLy wlLh a beLaglucosldase
Dmbql3A conLalned a domaln of glycosyl hydrolase
famlly 3 and 3C
SLudy lndlcaLed ombql3A may come from a specles of
cytoboqo
hao Cuang Cun2006
0tug0nomic Iibrury of Sout rum0n microfIoru
IalldaLed daLa wlll be used Lo screen clones wlLh dlfferenL enzymaLlc acLlvlLles
such as cellulases xylanases and amylases
8acLerlal dlverslLy deLermlned by maLchlng Lhe above llbrary wlLh resLrlcLlon
enzyme random clonlng and sequenclng of 6S runA clones Lechnlques
lnserLs ranged from 3 kb
rom LoLal unA by dlrecL exLracLlon a meLagenomlc llbrary was consLrucLed
Cunha 200
Rum0n phyIog0n0tic unuIysis of ubuIus bubuIis
lndlcaLed dlverse bacLerlal populaLlon ln Lhe rumen
hy|ogenet|c ana|ys|s showed I|rm|cutes phy|um (674) Cytophaga I|ex|bacter actero|des
subd|v|s|on (174) roteobacter|a (87) c|one ADSC10S to the Synerg|stetes group (22)
ollowlng C8 ampllflcaLlon 6S runA llbrary was consLrucLed
CommunlLy unA exLracLed from rumen fluld
?ebron200
Metagenom|c ana|ys|s of Surt| buffa|o (uba|us buba||s) rumen
S|ngh et a| 2011
D|ets
A100 roughage
7S roughage
CS0 roughage
D2S roughage
A|| samp|es were
dom|nated by
actero|detes]Ch|orob|
I|rm|cutes and
roteobacter|a
FunctionuI div0rsity
W MosL of Lhe meLagenomlc sLudles of Lhe rumen
have been on glycosyl hydrolases
W 22 clones wlLh dlsLlncL acLlvlLles were ldenLlfled
ln Lhe llbrary and characLerlzed
2 esLerases
endo34glucanases
cyclodexLrlnase
Sequence analysls of Lhe of Lhe above reLrleved
enzymes lndlcaLed LhaL 36 of Lhem were novel
errer et ol 2007
N0Iy id0ntifi0d g0n0s/0nzym0s huving uniqu0
uttribut0s
Lnzyme]gene Act|v|ty Author
Laccase avlng excepLlonally hlgh
afflnlLy and acLlvlLy
8eloqul eL al 2006
Amylase WlLh broad subsLraLe
range p
hu eL al 2007
ybrld glycosyl
hydrolase
WlLh enhanced acLlvlLy hao eL al 20
Cene 8l3 Lncodlng polyphenol
oxldases
8eloqul eL al 2006
novel gene 8uCelA acLlvlLy agalnsL xylan and
carboxymeLhylcellulose
%CMC)
Chang Lel 20
S0n0 tcp3A
336Lrlchloro2pyrldlnol %@C) ls a ma[or meLabollLe of Lhe lnsecLlclde
chlorpyrlfos and ls hazardous Lo human and anlmal healLh
336Lrlchloro2pyrldlnol %@C) ls a ma[or meLabollLe of Lhe lnsecLlclde
chlorpyrlfos and ls hazardous Lo human and anlmal healLh
A gene encodlng a @C degradlng enzyme was cloned from a
meLagenomlc llbrary prepared from cow rumen
A gene encodlng a @C degradlng enzyme was cloned from a
meLagenomlc llbrary prepared from cow rumen
@he gene %tc3A) ls 23 kb ln lengLh @he gene %tc3A) ls 23 kb ln lengLh
8ecomblnanL Lcoll harborlng Lhe tc3A gene can degrade @C 8ecomblnanL Lcoll harborlng Lhe tc3A gene can degrade @C
MaLh 200 MaLh 200
Nov0I g0n0 C7-1
W lL encodes acldlc cellulases
W Lxpressed ln coll
W @he purlfled enzyme showed opLlmal acLlvlLy
aL p 43 and sLablllLy ln a broad p range
from p 33 Lo 03
W lLs enzyme acLlvlLy was sLlmulaLed by uL
dlLhloLhrelLol
uuan et ol 200
W lL encodes acldlc cellulases
W Lxpressed ln coll
W @he purlfled enzyme showed opLlmal acLlvlLy
aL p 43 and sLablllLy ln a broad p range
from p 33 Lo 03
W lLs enzyme acLlvlLy was sLlmulaLed by uL
dlLhloLhrelLol
uuan et ol 200
Nov0I 0nzym0 RA,04
8A04 dlscovered from cow rumen meLagenome belonged Lo
alphaamylase famlly
8A04 dlscovered from cow rumen meLagenome belonged Lo
alphaamylase famlly
MalnLalned 0 acLlvlLy aL p 30 and 3 and 73 degrees C MalnLalned 0 acLlvlLy aL p 30 and 3 and 73 degrees C
@he enzyme hydrolysed alphau%4) bonds 3fold fasLer Lhan
alphau%6) bonds Lo yleld malLose and glucose as Lhe maln
producLs and lL exhlblLed LransglycosylaLlon acLlvlLy
@he enzyme hydrolysed alphau%4) bonds 3fold fasLer Lhan
alphau%6) bonds Lo yleld malLose and glucose as Lhe maln
producLs and lL exhlblLed LransglycosylaLlon acLlvlLy
Ierrer et o/ 2007 Ierrer et o/ 2007
Nov0I Iipus0 S0n0s RIipE1 und RIipEZ
8ecovered from a meLagenomlc llbrary of Lhe rumen mlcrobloLa of
Chlnese olsLeln cows
@hermally unsLable and reLalned 60 acLlvlLy over a p range of 633
ave hlgher hydrolyLlc acLlvlLy Loward lauraLe %C
2
) palmlLaLe %C
6
) and
sLearaLe %C

)
lgh speclflclLy for longchaln faLLy acld wlll be useful for manlpulaLlon of
rumen llpld meLabollsm
Llu kal Lang200
ufure Prospecfs
ow mlcrobes and %or) Lhelr secreLed producLs affecL gasLrolnLesLlnal
physlology and lmmune responses ln Lhe hosL
CeneLlcs of synLrophlc lnLeracLlons eg lnLerspecles hydrogen Lransfer or
oLher mlcrobe mlcrobe lnLeracLlons relevanL Lo hosL nuLrlLlon and healLh
robloLlc and %or) prebloLlc research and developmenL
ulscovery of novel enzymes and caLalysLs
CpLlmlze flber uLlllzaLlon
ConcIusion
AppllcaLlon of meLagenomlcs wlll conLrlbuLe much Lo Lhe undersLandlng of Lhe
ecology of rumen organlsm
@he ramlflcaLlon of meLagenomlc daLa for rumen mlcroorganlsms are profound
lncludlng Lhe poLenLlal Lo elucldaLe and overcome Lhe blochemlcal ecologlcal or
physlologlcal processes LhaL are raLe llmlLlng for rumlnal flber degradaLlon
8y llnklng Lhese daLas wlLh anlmal dleLs and lo vltto analysls lL may be posslble Lo
deflne key mlcrobes and Lhelr role ln Lhe flber degradaLlon
Learnlng novel paLhways wlll lead Lo knowledge abouL Lhe currenL nonculLurable
bacLerla Lo Lhen culLure Lhese sysLems