Metagenomics and pathway engineering

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22 Οκτ 2013 (πριν από 3 χρόνια και 11 μήνες)

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Genteknik och molekylärbiologi BB1110
Genteknik och molekylärbiologi BB1110
Föreläsning 15:
Föreläsning 15:
Metagenomics
Metagenomics
&
&
pathway engineering
pathway engineering
25/11-2009
25/11-2009
Patrik Samuelson
Patrik Samuelson

Less than 1% of microbes are readily available by
cultivation

Accordingly, the vast majority of organisms cannot be
studied by traditional techniques

Inevitably many enzymes will be overlooked that could
otherwise facilitate more efficient bioconversions
Microbes
in the
environment
: the
uncultivated majority
The methodological approach of accessing the total genomic DNA and
thus the metabolic potential contained within a microbial community
This can be achieved in two
fundamenatally
different ways:
-
sequence-based
+ readily performed using PCR-based or hybridization-based
procedures
- genes obtained are limited to those having homologies to the probe
sequence and may not allow us to obtain novel genes
-
function-based
+ straightforward way to obtain genes having a desired function
- often problematic due to biased and

insufficient expression of
anonymous genomic fragments
Metagenomics
Metagenomic
gene
discovery
Stable isotope-probing
(SIP) and
BrdU enrichment
Fig. 12.2 Biotechnology: Applying the genetic revolution
BrdU
=
5-bromo-2-deoxyuridine
Supressive subtractive hybridization
Fig. 12.3 Biotechnology: Applying the genetic revolution
Novel antibiotics discovered
in a
metagenomic library
Fig. 12.1 Biotechnology: Applying the genetic revolution
Function-based approaches
for
metagenomics
Fig. 12.4 Biotechnology: Applying the genetic revolution
Functional
screening of
metagenomic libraries
The probability of identifying a certain gene depends on
multiple factors such as:

the host-vector system

size of the target gene

its abundance in the source
metagenome

the assay method

the efficiency of
heterologous
gene expression
Substrate-induced
gene expression (SIGEX) screening
Uchiyama

T et al

Nat
Biotechnol

2005,
23:
88-93
From sugars to
ethanol
Fig. 13.1 Biotechnology: Applying the genetic revolution
Pathway engineering for
xylose
utilization
Fig. 13.2 Biotechnology: Applying the genetic revolution
The
XylA
protein (
xylose isomerase
) converts
xylose
to
xylulose
, and
XylB
(
xylulose kinase
)
phosphorylates
this to form
xylulose
5-phosphate
Pathway engineering for
xylose
utilization-reactions
Fig. 13.3 Biotechnology: Applying the genetic revolution
The
XylA
protein (
xylose isomerase
) converts
xylose
to
xylulose
, and
XylB
(
xylulose kinase
)
phosphorylates
this to form
xylulose
5-phosphate
tkt =
transketolase
;
tal
=
transaldolase
Enzymatic breakdown of starch by amylases
Fig. 13.4 Biotechnology: Applying the genetic revolution
Cellulose degradation
Fig. 13.5 Biotechnology: Applying the genetic revolution
Similar reactions of
naphtalene
and
indole
Fig. 13.8 Biotechnology: Applying the genetic revolution
Engineered indigo pathway
Fig. 13.8 Biotechnology: Applying the genetic revolution
Biosynthesis of
beta-lactam
antibiotics
Fig. 13.18 Biotechnology: Applying the genetic revolution
Engineered pathway to 7-aminocephalosporanic acid (7-ACA)
Fig. 13.19 Biotechnology: Applying the genetic revolution
Polyketide
pathway
Fig. 13.20 Biotechnology: Applying the genetic revolution
tetracyclins
&
macrolides
(e.g. erythromycin)
Modular PKT pathway for erythromycin
Fig. 13.21 Biotechnology: Applying the genetic revolution
Engineering
polyketides
by changing the initiator
molecule
Fig. 13.22 Biotechnology: Applying the genetic revolution
Example of natural product that have been
successfully expressed in
heterologous
hosts
Evolution of efficient pathways for degradation of
anthropogenic chemicals
Anthropogenic

chemicals are widely used in agriculture,
industry, medicine and military operations.
Examples:
-

pesticides (
atrazine
, pentachlorophenol (PCP),
dichlorodiphenyltrichloroethane (DDT)
-

explosives (trinitrotoluene (TNT))
-

solvents (trichloroethylene)
-

dielectric fluids (polychlorinated biphenyls (PCBs)
Microbial enzymes that act on anthropogenic compounds arise
from promiscuous activities of previously existing enzymes
-

detoxification
-

complete degradation
Two pathways for biodegradation of anthropogenic pollutants
a)
Pseudomonas
b)

S.
chlorphenolicum
Copley SD
Nat.
Chem
.
Biol
.
5:
559-566 (2009)
Engineering microbes with enhanced
degradative
abilities
New approaches:


assembly of

pathways using enzymes from multiple organisms


directed evolution of inefficient enzymes


genome shuffling to

improve microbial fitness under challenging
conditions
Examples of promiscuous enzymatic activities
Copley SD
Nat.
Chem
.
Biol
.
5:
559-566 (2009)
Binding of
ligands
to the active site of
o-
succinylbenzoate
synthase
(a) The product of the normal reaction,
o-
succinylbenzoate
. (b) A
promiscuous substrate,
N-
acetylmethionine
Copley SD
Nat.
Chem
.
Biol
.
5:
559-566 (2009)
Pathways for degradation of 4-nitrotoluene
Copley SD
Nat.
Chem
.
Biol
.
5:
559-566 (2009)
An engineered pathway for degradation of
paraoxon
Copley SD
Nat.
Chem
.
Biol
.
5:
559-566 (2009)
Balancing pathway flux
Two principal
stategies
:


modulating the expression levels of individual
enzymes (promoter strength, RBS strength,
copy nr etc)


improving turnover activities of rate-limiting
enzymes by directed evolution
Synthetic metabolic pipelines
AtoB= acetoacetyl-CoA thiolase
(from E. coli)
HMGS =
hydroxy-methylglutaryl-CoA synthase
(from yeast)
HMGR =
hydroxy-methylglutaryl-oA reductase
(from yeast)
Dueber
J.E. et al
Nat.
Biotechnol
.
27:
753-759 (2009)
Enhancement of
mevalonate
production is scaffold
dependent
Dueber
J.E. et al
Nat.
Biotechnol
.
27:
753-759 (2009)