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Metabolic Engineering IIMetabolic Engineering II
Bi7430 Molecular Biotechnology
Dhamankar , H. (2011) Curr.Opin.Struct.Biol., 21:1
General workflow of ME project
computational
and
experimental
tools
and Properties
Dhamankar , H. (2011) Curr.Opin.Struct.Biol., 21:1
General workflow of ME project
computational
and
experimental
tools
and Properties
2
Dhamankar , H. (2011) Curr.Opin.Struct.Biol., 21:1
General workflow of ME project
computational
and
experimental
tools
and Properties
Dhamankar , H. (2011) Curr.Opin.Struct.Biol., 21:1
General workflow of ME project
computational
and
experimental
tools
and Properties
E x p e r i m e n t a l ( g e n e t i c ) t o o l s f o r ME
Me t a b o l i c l o a d ( yi e l d v s . v i a b i l i t y o f h o s t )
ME o f b i o s yn t h e t i c ( a n a b o l i c ) p a t h wa ys - e x a m p l e s
ME o f b i o d e g r a d a t i o n ( c a t a b o l i c ) p a t h wa ys - e x a m p l e s
L i m i t a t i o n s a n d p e r s p e c t i v e s o f ME
D i s c u s s i o n
Outline
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EXPERIMENTAL TOOLS ARE APPLIED HAND IN HAND WIT H T HEORET ICAL
TOOLS
experimen tals tools = genetic tools (recombinant DNA technology)
production of transgenic organisms
engineering input on level of:
gene expression (DNA/RNA): gene knockout , gene down/ up-regul ati on,
heterologous expression, codon optimization, chromosomal integration of
gene(s)
protein: protein engineeri ng, proximity of enzymes (substrat e channeling)
small molecules: cof actor balancing
Experimental tools for ME
Boyle, P.M. (2012) Metabolic Engineering, 14:223
“Parts and pipes” in ME
optimization of gene(s) expressionoptimization of gene(s) expression
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Bacterial operone
+1
P TRBS RBSRBS A B C
mRNA
ATG stop ATG stop ATG stop
-35 -10
Gene knock-out
gene(s) of interest is deleted f rom the chromosom e – permanen t state
widely used technique based on phage λ Red recombinase (E. coli)
analogous method used in yeast
www.genebridges.com
Gene down/up regulation
T RANSCRIPT IO NAL AND POST-T RANSCRIPT IO NAL LEVEL
promoter engineering (lac, trc, T5, T7; inducible vs. constitutiv e)
engineeri ng of intergenic regions (mRNA stability, ribosome binding)
Santos, C.N.S. (2008) Curr.Opin.Chem.Biol., 12:168
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LEVEL of T RANSLAT IO N
RNA interferen ce - homologous RNA sequences (gene knock-down)
expression of one or more genes is reduced – transien t state
engineering of ribosome binding sites (RBS calculator1 )
Shine-Dalgarno sequence (consensus sequence AGGAGG)
1Salis, H.M. (2009) Nature Biotechnology, 27: 946
Gene down/up regulation
Codon optimization
SYNT HESIS OF GENES FOR HET EROLOGOUS EXPRESSION
commercial gene synthesi s (Inv itrogen, GeneScript , DNA 2.0)
7.9 Kč/bp, up to 3.5 kbp
codon optimization, GC content optimization
subcloning in v ector of choice
before after
Expression from plasmids
HET EROLOGOUS GENE EXPRESSION FROM PLASMIDs
important characteri stics of each plasmid: copy number, origin of
replication (ORI), promoter, selection marker, multi-cloning sites
(MCS), tags or leading sequences
commerci al vectors (pBAD Inv itrogen, pET Merck)
DUET vectors (deriv ativ es of pET) – suitable f or heterologous
expression of whole metabolic pathways.
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DUET vector system (T 7 promoter, variabili ty in copy number)
www.merckmillipore.cz
Expression from plasmids
gene 3
gene 2
gene 1
1:2(3):2(3)
Gene order and configuration of genes in operone influence their
expression.1
pseudo-operon monocistron ic
Xu, P. (2012) ACS Synthetic Biology, DOI: 10.1021/sb300016b
operon
promoter
RBS
terminator
Expression from plasmids
1Lim, H.N. (2011) PNAS, 108:10626
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HET EROLOGOUS GENE EXPRESSION FROM CHROMOSO ME
expression f rom chromosome is advantageo us (higher stability, no
antibiotic markers)
methods for integration: homologous recombination (recA, λ Red),
transpositi on (Tn5 and Tn7-based v ectors)
integration of single genes or whole synthetic operones
subsequent duplication or multiplicati on of insertions
Expression from chromosome
SYNT HET IC OPERON DESIGN (sof tware GeneDesigner 2.0)
+1
P TRBS RBSRBS dhaA hheC echA
mRNA
ATG stop ATG stop ATG stop
Expression from chromosome
INSERT ION OF DESIRED GENE(S) BY T N-5 BASED T RANSPOSIT IO N
(non-specifi c).
de Lorenzo, V. (1990) Journal of Bacteriology, 172: 6568
selection of positive clones on LB agar plates with respective antibiotic
Expression from chromosome
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Balancing gene expression VI
HET EROLOGOUS GENE EXPRESSION FROM CHROMOSO ME
multiplication of insertions: Chemicall y Inducible Chromoso mal
Evolution (CIChE)1
1Tyo, K.E.J. (2009) Nature Biotechnology, 27:760
protein levelprotein level
Substrate channelling
Synthetic protein scaffolding made of bacterial dockerins and
cohesins from cellulosome (Clostridiu m, Bacteroid es).
protein scaff olding used f or increasing proximity of 3 glycolytic
enzymes producing f ructose-6-phosphat e1
1You, C. (2012) Angewandte Chemie, 51:1
cohesins
dockerins
pathway
enzymes
substrate product
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small molecules levelsmall molecules level
Cofactor balancing
cof actors play a critical role especially in redox
reactions (NAD(H), NADP(H))
natural pathways (e.g. glycolysis) of ten employ
oxidoreductases
cof actor recycling and balancing is essential
solution: enzyme mediated cofactor recycling
through ov erexpression of NAD+ kinase,
transhydrogenases or dehydrogenases
simultaneously with knock-out s of genes encoding
enzymes f rom competing pathways
NADP+
NADPH
metabolic load1metabolic load1
1Glick, B.R. (1995) Biotechnology Advances, 13:247
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Metabolic load
overexp ressio n of foreign proteins results in decrease of viability
of host cell
rich LB medium minimal medium
Metabolic load
induction
overexp ressio n of foreign proteins results in decrease of viability
of host cell
45 %
YIELD VS. VIABILIT Y OF CELL
1) STAT IC CONT ROL: static balancing of production of pathway
enzymes - lev els of enzymes remain unchanged throughout the
whole cultiv ation (most of the standard techniques mentioned abov e)
2) DYNAMI C CONT ROL: engineering of a dynamic response of host
organism on metabolic load and toxicity of pathway component s –
lev els of enzymes f luctuate during cultiv ation (challenge f or f uture
applications of ME)
Metabolic load
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Dynamic control engineering
Dynamic Sensor-Regul ato r System (DSRS)1
Production of biodiesel f rom f atty acid ethyl ester in E. coli.
1Zhang, F. (2012) Nature Biotechnology, 30:354
ME of biosynthetic pathways
ME APPLIED IN ORDER TO IMPROVE (ESTABLISH) PRODUCT ION OF:
biof uels (ethanol, butanol, H2 , f atty acids deriv ed esters)
natural and non-nat ural alcohols
natural and non-nat ural amino acids
f atty acids
peptides and proteins
secondary metabolites: antibiotics, isoprenoi ds (artemisinin, taxol)
oligo and polysacharides (biodegradabl e polymers)
commodity chemicals (1,3-propanedi ol)
and many others.. .
ME of biosynthetic pathways
Current limitations of biosynthesi s using engineered organisms:
missing standards
low productiv ity (low activ ity of enzymes, side reactions, limits of
host organism s)
non-competitiv e economy of the biosynt hetic proceses
application of GMO (ethics)
low stability of GM construct s (ev olution)
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ME of biosynthetic pathways
EXAMPLE No.1: Engineering of E. coli K12 towards production of
1,3-propan edio l1
commodity chemical (solv ents, adhesiv es, resins, detergent, textile)
synthetic pathway
15 years, 575 scientists (DuPont+G enencor)
1,3-PD produced at rate 3.5 g/L-h, titer of 135 g/L
1) introduction of genes f rom 3 organism s: E.coli (yghD), S.cerevisiae
(dar1, gpp2), K. pneumoniae (dhaB1-B3)
2) deletion of glycerol kinase (glpK), glycerol dehydrogenase (gldA)
3) change of uptake system (deletion of PEP-dependent system and
introduction of ATP-coupled uptake)
1Nakamura, C.E. (2003) Current Opinion in Biotechnology, 14:454
glycolysis
glpk + gldA
S. cerevisiae
K. pneumoniae
ME of biosynthetic pathways
EXAMPLE No.2: Engineering of E. coli towards production of taxol
precurso r (isoprenoid)
taxol – anticancer drug (Taxus brevifolia), precursor taxadiene
complicated chemical synthesi s (35 – 50 steps), low yield
synthetic pathway consist s of nativ e upstream module (8 genes) and
heterologous downstream module (2 genes f rom Taxus)
combinatori al approach: multivari ete-modu lar engineering – search
f or optimally balanced pathway in combinatori al space
combination of 5 plasmids, 3 promoters and 2 pathway modules
minimization of cumulation of toxic intermediate indol
15,000-fold improvement of taxadiene production, titer of 1 g/L
1Ajikumar, P.K. (2010) Science, 330:70
ME of biodegradation pathways
ME of biodegradation pathways f or biodegradation of toxic
compounds in industry, biosensing and in situ bioremedi ation.
host organism s: bacteria (mostly improv ement of natural strains
isolated f rom contaminat ed sites) and plants (phytobioremedi ation)
phenomena of toxicity and adaptation of bacteria (enzymes) towards
anthropogeni c substrat es
paraoxone, toluene, DCE, TCP, lindane
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ME of biodegradation pathways
Current limitations of biodegradati on using engineered organisms:
low competiti vn ess of engineered strains (diff erent conditions in lab
and in the env ironment)
decreased viability of host organism s due to metabolic load and
high toxicity of subst rat es and pathway intermediates
application of GMO (ethics)
limited number of “successf ul stories”
→ ME of biodegradation pathways is challenging
low stability of GM construct s
ME of biodegradation pathways
EXAMPLE: Synthetic pathway for biodegradation of 1,2,3trichloropro
pane (T CP)
TCP – anthropogeni c compound, industrial use, emerging pollutant
no natural strain capable of TCP utilization (lack of dehalogenati ng
enzyme)
TCP pathway
Project workflow:
1989 – description of pathway f or utilization of halogenat ed alcohols
f rom Agrobact erium radiobact er AD1 (HheC, EchA)
1997 – description of haloalkane dehalogenase DhaA (Rhodococcus sp.)
1999 – heterologous expressi on of dhaAwt in A.radiobact er AD11
2002 – heterologous expressi on of dhaAM2 in A.radiobact er AD12
ultimate goal: bacteriu m utilizing T CP as a single carbon source
PROBLEMS:
low v iability of construct s (TCP toxicity, low expression of enzymes)
cumulation of toxic pathway intermediates
low conv ersion of TCP to glycerol (3.6 mM/10 days)
1,2Bosma ,T. (1999 and 2002) Applied Environmental Microbiology, 65:4575 and 68:3582
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TCP pathway
2009 - construction of DhaA311 (32-times improved activity with T CP)
rational design - computer modelling f or selection of hot spots
directed ev olution - saturation mutagenesi s in pre-def ined positions
1Pavlová, M. (2009) Nature Chemical Biology, 5:727
2009 – now: applied principles of metabolic engineering :
gene synthesi s and codon optimization f or E. coli
cloning in pET and DUET v ectors, overexp ressi on
detailed characterization of pathway enzymes (kinetic properties)
characterization and quantif ication of metabolites (GC analysis)
PROOF OF CONCEPT: reconst ructi on of pathway in vitro
TCP pathway
In vitro reconstru ction of T CP pathway (soluble enzymes)
TCP pathway
DhaAwt, HheC, EchA
mixed in ratio 1:1:1
DhaA31, HheC, EchA
mixed in ratio 1:1:1
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2009 – now: applied principles of metabolic engineering :
kinetic model of the pathway
reconst ruction of pathway in vivo (E. coli)
def ined toxicity of TCP and pathway intermediat es f or cell
modular engineering f or balancing of gene expressi on (DUET v ectors)
combinatori al approach: construction of sev eral v ariants of the
pathway and selection of one with the most eff icient conv ersion of TCP
to glycerol
TCP pathway
In vivo reconstru ction of T CP pathway (E. coli, DUET vectors)
TCP pathway
reaction course simulation
DhaAwt, HheC, EchA
in ratio 2.5 : 0.5 : 1
conversion of TCP by E. coli resting cells
DhaA31, HheC, EchA
in ratio 2.5 : 0.5 : 1
Kurumbang ,N.P. et al.(2013) submitted to ACS Synthetic Biology
Current limitations of ME
long way f rom lab scale (ml - L) to industry scale (103 - 105 L)
costly processes (esp. product recov ery and purif ication)
low productiv ity of engineered pathways – requirement at least 100
g/L f or commodity chemicals (1,3-propanedi ol 135 g/L) or 1 g/L f or
pharmaceutical s (taxadiene 1g/L)
complexity of lif e - ev olution
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Perspectives of ME
c a t a l o g o f p o t e n t i a l l y u s e f u l p r o m i s c u o u s a c t i v i t i e s o f k n o w n
e n z y m e s
s c r e e n i n g o f n e w h o s t o r g a n i s m s , p a t h w a y s , e n z y m e s
( m e t a g e n o m e a p p r o a c h v s . s e q u e n c i n g a n d b i o i n f o r m a t i c s )
c o n s t r u c t i o n o f b a c t e r i a l c h a s s e s w i t h m i n i m a l g e n o m e s
i n s i l i c o s c r e e n i n g
d e n o v o d e s i g n o f n e w e n z y m e s ( i n s i l i c o ) a n d g e n e s y n t h e s i s
e n g i n e e r i n g o f i n v i t r o s y s t e m s ( r e d u c t i o n o f c o m p l e x i t y )
f r o m t h e l a b t o t h e r e a l a p p l i c a t i o n s : d e c r e a s i n g t h e c o s t s o f
t h e p r o c e s s e s ( f r o m g e n e s y n t h e s i s t o p r o d u c t p u r i f i c a t i o n )
Discussion
Yadav, V.G. (2012) Metabolic Engineering, 14:233
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Substrate channelling
Synthetic protein scaffolding made of eukaryoti c binding domains.
protein scaff olding used f or tuning of stoichiomet ry of enzymes in
metabolic pathway (AtoB, HMGS, HMGR) f or producti on of mev alonate1
1Dueber, J.E. (2009) Nature Biotechnology, 27:753
Genome-scale ME
Multiplex Automated Genome Engineering (MAGE)1
Simultaneous mutagenesi s of multiple genes in vivo using the pool
of syntheti c oligonucleoti des.
1Wang, H.H. (2009) Nature, 460:894
A B C D E
F G
1 2 3 4
5 6
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Gene knock-out
gene(s) of interest is deleted f rom the chromosom e – permanen t state
widely used technique based on phage λ Red recombinase (E. coli)
analogous method used in yeast
www.genebridges.com Datsenko, K.A. (2000) PNAS, 97:6640
ME of biosynthetic pathways
EXAMPLE No.3: Engineering of E. coli towards production of
levopiramad iene (secondary metabolite, diterpenoid)1
lev oporamidiene – pharmaceutically important compound
plant-deriv ed pathway: Gingko biloba (lps), Taxus chinensis (ggpps)
+ nativ e terpenoid pathway f rom E. coli (4 genes)
combination of protein and metabolic engineering
ov erexpressi on of pathway enzymes, codon optimization, protein
engineeri ng of lev opiramadiene synthase (LPS) and geranylgeranyl
diphosphat e synthase (GGPPS) towards increased specif icity and
activ ity (5x) – E. coli directly used as a in vivo screening system
2,600-fold improvemen t of production, titer of 0.7 g/L (in 168 hrs)
1Leonard E. (2010) PNAS, 107:13654
glycerol