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5. Metabolic Engineering II5. Metabolic 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
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 M E
M e 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 )
M E 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 w a ys - e x a m p l e s
M E 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 w a 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 M E
D i s c u s s i o n
Outline
EXPERIMENTAL TOOLS ARE APPLIED HAND IN HAND WITH THEORETICAL
TOOLS
experimentals tools = genetic tools (recombinant DNA technology)
production of transgenic organisms
engineering input on level of:
gene expression (DNA/RNA): gene knockout, gene down/up-regulation,
heterologous expression, codon optimization, chromosomal integration of
gene(s)
protein: protein engineering, proximity of enzymes (substrate channeling)
small molecules: cofactor balancing
Experimental tools for ME
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Boyle, P.M. (2012) Metabolic Engineering, 14:223
“Parts and pipes” in ME
optimization of gene(s) expressionoptimization of gene(s) expression
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 from the chromosome – permanent state
widely used technique based on phage λ Red recombinase (E. coli)
analogous method used in yeast
www.genebridges.com
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Gene down/up regulation
TRANSCRIPTIONAL AND POST-TRANSCRIPTIONAL LEVEL
promoter engineering (lac, trc, T5, T7; inducible vs. constitutive)
engineering of intergenic regions (mRNA stability, ribosome binding)
Santos, C.N.S. (2008) Curr.Opin.Chem.Biol., 12:168
LEVEL of TRANSLATION
RNA interference - homologous RNA sequences (gene knock-down)
expression of one or more genes is reduced – transient 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
SYNTHESIS OF GENES FOR HETEROLOGOUS EXPRESSION
commercial gene synthesis (Invitrogen, GeneScript, DNA 2.0)
7.9 Kč/bp, up to 3.5 kbp
codon optimization, GC content optimization
subcloning in vector of choice
before after
Expression from plasmids
HETEROLOGOUS GENE EXPRESSION FROM PLASMIDs
important characteristics of each plasmid: copy number, origin of
replication (ORI), promoter, selection marker, multi-cloning sites
(MCS), tags or leading sequences
commercial vectors (pBAD Invitrogen, pET Merck)
DUET vectors (derivatives of pET) – suitable for heterologous
expression of whole metabolic pathways.
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DUET vector system (T7 promoter,variability 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 monocistronic
Xu, P. (2012) ACS Synthetic Biology, DOI: 10.1021/sb300016b
operon
promoter
RBS
terminator
Expression from plasmids
1Lim, H.N. (2011) PNAS, 108:10626
HETEROLOGOUS GENE EXPRESSION FROM CHROMOSOME
expression from chromosome is advantageous (higher stability, no
antibiotic markers)
methods for integration: homologous recombination (recA, λ Red),
transposition (Tn5 and Tn7-based vectors)
integration of single genes or whole synthetic operones
subsequent duplication or multiplication of insertions
Expression from chromosome
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SYNTHETIC OPERON DESIGN (software GeneDesigner 2.0)
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P TRBS RBSRBS dhaA hheC echA
mRNA
ATG stop ATG stop ATG stop
Expression from chromosome
INSERTION OF DESIRED GENE(S) BY TN-5 BASED TRANSPOSITION
(non-specific).
de Lorenzo, V. (1990) Journal of Bacteriology, 172: 6568
selection of positive clones on LB agar plates with respective antibiotic
Expression from chromosome
Balancing gene expression VI
HETEROLOGOUS GENE EXPRESSION FROM CHROMOSOME
multiplication of insertions: Chemically Inducible Chromosomal
Evolution (CIChE)1
1Tyo, K.E.J. (2009) Nature Biotechnology, 27:760
protein levelprotein level
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Substrate channelling
Synthetic protein scaffolding made of bacterial dockerins and
cohesins from cellulosome (Clostridium, Bacteroides).
protein scaffolding used for increasing proximity of 3 glycolytic
enzymes producing fructose-6-phosphate1
1You, C. (2012) Angewandte Chemie, 51:1
cohesins
dockerins
pathway
enzymes
substrate product
small molecules levelsmall molecules level
Cofactor balancing
cofactors play a critical role especially in redox
reactions (NAD(H), NADP(H))
natural pathways (e.g. glycolysis) often employ
oxidoreductases
cofactor recycling and balancing is essential
solution: enzyme mediated cofactor recycling
through overexpression of NAD+ kinase,
transhydrogenases or dehydrogenases
simultaneously with knock-outs of genes encoding
enzymes from competing pathways
NADP+
NADPH
time for break
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metabolic load1metabolic load1
1Glick, B.R. (1995) Biotechnology Advances, 13:247
YIELD VS. VIABILITY OF CELL
1) STATIC CONTROL: static balancing of production of pathway
enzymes - levels of enzymes remain unchanged throughout the
whole cultivation (most of the standard techniques mentioned above)
2) DYNAMIC CONTROL: engineering of a dynamic response of host
organism on metabolic load and toxicity of pathway components –
levels of enzymes fluctuate during cultivation (challenge for future
applications of ME)
Metabolic load
Metabolic load
overexpression of foreign proteins results in decrease of viability
of host cell
rich LB medium minimal medium
Metabolic load
induction
overexpression of foreign proteins results in decrease of viability
of host cell
45 %
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Dynamic control engineering
Dynamic Sensor-Regulator System (DSRS)1
Production of biodiesel from fatty acid ethyl ester in E. coli.
1Zhang, F. (2012) Nature Biotechnology, 30:354
ME of biosynthetic pathways
ME APPLIED IN ORDER TO IMPROVE (ESTABLISH) PRODUCTION OF:
biofuels (ethanol, butanol, H2, fatty acids derived esters)
natural and non-natural alcohols
natural and non-natural amino acids
fatty acids
peptides and proteins
secondary metabolites: antibiotics, isoprenoids (artemisinin, taxol)
oligo and polysacharides (biodegradable polymers)
commodity chemicals (1,3-propanediol)
and many others...
ME of biosynthetic pathways
Current limitations of biosynthesis using engineered organisms:
missing standards
low productivity (low activity of enzymes, side reactions, limits of
host organisms)
non-competitive economy of the biosynthetic proceses
application of GMO (ethics)
ME of biosynthetic pathways
EXAMPLE No.1: Engineering of E. coli K12 towards production of
1,3-propanediol1
commodity chemical (solvents, adhesives, resins, detergent, textile)
synthetic pathway
15 years, 575 scientists (DuPont+Genencor)
1,3-PD produced at rate 3.5 g/L-h, titer of 135 g/L
1) introduction of genes from 3 organisms: 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
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ME of biosynthetic pathways
EXAMPLE No.2: Engineering of E. coli towards production of taxol
precursor (isoprenoid)
taxol – anticancer drug (Taxus brevifolia), precursor taxadiene
complicated chemical synthesis (35 – 50 steps), low yield
synthetic pathway consists of native upstream module (8 genes) and
heterologous downstream module (2 genes from Taxus)
combinatorial approach: multivariete-modular engineering – search
for optimally balanced pathway in combinatorial 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 for biodegradation of toxic
compounds in industry, biosensing and in situ bioremediation.
host organisms: bacteria (mostly improvement of natural strains
isolated from contaminated sites) and plants (phytobioremediation)
phenomena of toxicity and adaptation of bacteria (enzymes) towards
anthropogenic substrates
paraoxone, toluene, DCE, TCP, lindane
ME of biodegradation pathways
Current limitations of biodegradation using engineered organisms:
low competitivness of engineered strains (different conditions in lab
and in the environment)
decreased viability of host organisms due to metabolic load and
high toxicity of substrates and pathway intermediates
application of GMO (ethics)
limited number of “successful stories”
→ ME of biodegradation pathways is challenging
ME of biodegradation pathways
EXAMPLE: Synthetic pathway for biodegradation of 1,2,3trichloropropane
(TCP)
TCP – anthropogenic compound, industrial use, emerging pollutant
no natural strain capable of TCP utilization (lack of dehalogenating
enzyme)
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TCP pathway
Project workflow:
1989 – description of pathway for utilization of halogenated alcohols
from Agrobacterium radiobacter AD1 (HheC, EchA)
1997 – description of haloalkane dehalogenase DhaA (Rhodococcus sp.)
1999 – heterologous expression of dhaAwt in A.radiobacter AD11
2002 – heterologous expression of dhaAM2 in A.radiobacter AD12
ultimate goal: bacterium utilizing TCP as a single carbon source
PROBLEMS:
low viability of constructs (TCP toxicity, low expression of enzymes)
cumulation of toxic pathway intermediates
low conversion of TCP to glycerol (3.6 mM/10 days)
1,2Bosma ,T. (1999 and 2002) Applied Environmental Microbiology, 65:4575 and 68:3582
TCP pathway
2009 - construction of DhaA311 (32-times improved activity with TCP)
rational design - computer modelling for selection of hot spots
directed evolution - saturation mutagenesis in pre-defined positions
1Pavlová, M. (2009) Nature Chemical Biology, 5:727
2009 – now: applied principles of metabolic engineering:
gene synthesis and codon optimization for E. coli
cloning in pET and DUET vectors, overexpression
detailed characterization of pathway enzymes (kinetic properties)
characterization and quantification of metabolites (GC analysis)
PROOF OF CONCEPT: reconstruction of pathway in vitro
TCP pathway
In vitro reconstruction of TCP 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
reconstruction of pathway in vivo (E. coli)
defined toxicity of TCP and pathway intermediates for cell
modular engineering for balancing of gene expression (DUET vectors)
combinatorial approach: construction of several variants of the
pathway and selection of one with the most efficient conversion of TCP
to glycerol
TCP pathway
In vivo reconstruction of TCP 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
Current limitations of ME
long way from lab scale (ml - L) to industry scale (103 - 105 L)
costly processes (esp. product recovery and purification)
low productivity of engineered pathways – requirement at least 100
g/L for commodity chemicals (1,3-propanediol 135 g/L) or 1 g/L for
pharmaceuticals (taxadiene 1g/L)
complexity of life
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 )