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20 Φεβ 2013 (πριν από 4 χρόνια και 7 μήνες)

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Conference Session B3

Paper #3121


University of Pittsburgh

Swanson School of Engineering

1

April 13, 2013

DNA AND METABOLIC PATHWAY MANIPULATION IN
ESCHERICHIA COLI

FOR FUEL PRODUCTION


Justine Fuga (
jmf149@pitt.edu
), Victoria Collier (
vmc8@pitt.edu
)


Abstract

This

paper
presents the production of
petrochemical substitutes and biofuel elements by
microorganisms as one potential solution

to replenish the
dwindling petroleum supply
. We chose to focus this paper
specifically
on fue
l element production

by Escherichia coli
for
the following reasons. First, much research has already
been collected on this organism’s DNA and metabolic
pathways and has shown that E. coli is a successful
microbial host for fuel

element synthesis
. Second
, E. coli’s
ability to be easily manipulated an
d its numerous metabolic
pathways enable this organism t
o produce several

biofuels
[1].
DNA cloning and splicing are the first steps needed to
transform E. coli into a biofuel producing bacterium.

Metabolic engineering is then used to manipulate the new
me
tabolic pathways inserted into E. coli’s native DNA to
optimize productivities, yields and titers. This paper will
demonstrate how genetic and metabolic engineering are
applied to E. coli to produce three types of biofuel e
lements:
fatty acids
, hydrogen an
d alochols
.
This application has the
potential to provide
a completely renewable and sustainable
source of fuel

that would encourage further development
and use of biofuel engines and hydrogen fuel cells
.
These
innovations are less expensive to use and
less destructive

to
the environment, but they are not currently being used by
enough people to
make a significant improvement

to the
economy or environment
. This breakthrough in the
bioengineering world will
assist

the United States in
becoming

a more sust
ainable society that is less dependent
on foreign, nonrenewable fossil fuels
.


Key

W
ords

biofuel
,
genetic engineering, metabolic
engineering, recombinant DNA, redox reaction, plasmid,
metabolic pathway intermediate


BIOFUEL PRODUCTION USING
BIOENGINEERING



In this section, we will introduce the topic of our paper.
This section gives an overview and highlights the points that
we will go into more detail about later in this paper. It will
lay a foundation of genetic and metabolic engineering in
order to
facilitate the readers’ understanding when an in

depth description of these processes are later explained.
Also, this section serves as an introduction for the
information that will make up
the body of this paper and lays
out
the organization of that info
rmation.


A Dependent World



This section

will emphasize why a change to the fuel
industry is necessary and what consequences we may
face if there is no modification in the way we consume
fuel. We will accentuate these points with the use of
statistics. This information will express why DNA and
m
etabolic pathway manipulation in
E. coli

for fuel
production, a potential solution to this problem, is a
significant innovation.


E. coli
: An Ideal Host



This section shows the benefits of using
E. coli
for
genetic and metabolic engineering, and there
fore, is
important to our topic because it shows why we chose
E.
coli

as our host of concern in this paper. We will explain
how previous research on this host has already shown that it
is a very effective contender for fuel production. Also, we
will explai
n how
E. coli
’s wide range of useful metabolic
pathways and easily manipulated genes make this host a
great candidate for biofuel production
.


GENETIC ENGINEERING: THE
PROCESS EXPLAINED



In this section on genetic engineering, we will
descr
ibe DNA
cloning and splicing in addition to

the
recombinant DNA process. We will go into detail on
isolating a gene of interest, identifying and removing
plasmids, the bonding of donor DNA and host DNA, the
recombinant
DNA’s introduction in
to the host cell, and
re
combinant DNA replication. This information is
relevant to our topic because it explains what process is
necessary to obtain a fuel producing bacterium.


METABOLIC ENGINEERING:
MANIPULATING
E. COLI



This section will address the role of metabolic
eng
ineering in modifying
E. coli

for the production of
three types of biofuels: fatty acids, hydrogen and
alcohols. In addition, the procedure for creating and
optimizing the metabolic pathways necessary for
production of these particular fuel elements will be
discussed. This section is
relevant to the topic of the
paper because the general procedure that will be
described is applied to manipulate
E. coli
to synthesize
each type of biofuel discussed in the preceding
subsections.


Victoria Collier

Justine Fuga




2

Fatty Acids



This section will discuss the modificatio
n of
E. coli

for the synthesis of fatty acid based biofuels.
One
challenge encountered with engineering
E.
coli

for fatty
acid biofuel production is the presence of competing
fermentation and c
ellular respiration pathways. Specific
details about this probl
em will be discussed, followed by
a description of genetic and metabolic engineering
solutions to this problem. This section will conclude by
presenting the benefits of using fatty acid
derived
biofuels, such as their
availability.

This
information is
impo
rtant because it describes in detail the challenges,
solutions and benefits unique to metabolically
engineering fatty acid derived biofuels produced by
E.
coli.


Hydrogen



This section will discuss the modification of
E. coli

for the synthes
is of hydr
ogen

biofuels.

E. coli
’s native
pathway for producing hydrogen is hindered by the
organism’s need for oxygen. Further details about this
challenge will be discussed in this section, followed by a
description of the modifications needed to eliminate this
pr
oblem.

This section will conclude by presenting the
be
nefits of using hydrogen biofuel
s, such as the
inexpensive feedstock required for its synthesis.

This
information is important because it describes in detail the
challenges, solutions and benefits uniqu
e to
metaboli
cally engineering hydrogen

biofuels produced
by
E. coli.


Alcohols



This section will discuss the modification of
E. coli

for the synthesis of alcohol based biofuels.
E.
coli
’s
natural production of alcohols, particularly ethanol,
produces numerous undesirable byproducts. The details
surrounding this metabolic engineering challenge will be
discussed. Following this will be a thorough explanation
of solutions to this problem, n
amely
replacing
E. coli
’s
native ethanol production pathway with another
microorganism’s
ethanol production
pathway.

To
conclude, this section will present the benefits of using
alcohol based biofuels, such as their versatility.
This

information is importa
nt because it describes in detail the
challenges, solutions and benefits unique to
metabolically engineering alcohol derived biofuels
produced by
E. coli.


ETHICAL CONCERNS



This section describes the controversy of the ethics of
DNA cloning and manip
ulation. We will go into detail
on why some argue that humans have no right to
manipulate the DNA of living organisms. Also, we will
describe why others oppose this view and claim that
DNA cloning and manipulation of
E. Coli

is completely
moral. This secti
on is important to our paper in general
because it gives insight to a completely different aspect
of our topic. This section is not concerned with how or
why
E. Coli

is genetically and metabolically engineered
for fuel production like the rest of this pape
r, but it asks
the question, “Should it be?”


AN INDEPENDENT NATION



In this conclusion, we will explain how the use of
genetic and metabolic engineering to manipulate
E. coli

can directly impact the United States. We will go into
detail how our count
ry’s economy and environment
would benefit from this application of genetic and
metabolic engineering. This section is important because
it reinforces our purpose for writing this paper.


REFERENCES


[1]

T.

Liu

and C.

Khosla
. (December 2010). “
Genetic
Engineering of
Escherichia coli

for Biofuel Production
.”
Annual Reviews: A Nonprofit Scientific Publisher.
(Online
Article).
http://www.annualreviews.org/doi/full/10.1146/annurev
-
genet
-
102209
-
163440?url_ver=Z39.88
-
2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%3dpubm
ed&


This article from a peer reviewed, scientific journal would
support
our paper because it outlines the benefits of using
E.
coli

for genetic engineering, explaining our choice for our
host of concern. Benefits include the ease of manipulating
E.
Coli’
s

genes and its wide range of useful metabolic
pathways. In addition, t
he

article explains how
E. Coli’
s
frequent use in experimentation has shown great success
with genetic engineering.


ADDITIONAL SOURCES


C. Trinh, F. Srienc. (2009). “Metabolic Engineering of
Escherichia coli for Efficient Conversion of Glycerol to
Ethanol.”

Applied and Environmental Microbiology
. (Online
article).
http://aem.asm.org/content/75/21/6696 p. 6696
-
6750

This
peer reviewed article, from a professional journal that
focuses on env
ironmental microbiology,
highlights

the
author’s findings on the efficiency and effects

of
E. coli
’s
synthesis of ethanol by aerobic metabolic pathway. This
article demonstrates a particular focus on ethanol production
from glycerol, a popular feedstock fo
r the production of
biodiesel. This article will aid in our argument of how
E. coli

is a well
-
qualified candidate for biofuel production.


Victoria Collier

Justine Fuga




3

“Genetic engineering a comprehensive overview.”
Biology
Questions and Answers.

(Online article).
http://www.biology
-
questions
-
and
-
answers.com/genetic
-
engineering.html

This article is an academic website sponsored by “Biology
Questions and Answers” which gives information on genetic
eng
ineering that is explained in an easily comprehensible
way. It defines genetic engineering, restriction enzymes,
plasmids, and DNA ligase while also explaining the
techniques of recombinant DNA technology for E. coli. This
information would be useful to ou
r paper to lay a foundation
of the basic terms involved in genetic engineering.


J. Clomburg, R, Gonzolez. (2009). “Biofuel production in
Escherichia coli: the role of metabolic engineering and
synthetic biology.”
Applied Microbiology and
Biotechnology.

(O
nline article).
http://link.springer.com/article/10.1007%2Fs00253
-
010
-
2446
-
1

p. 419
-
434.

This professional peer
-
reviewed article

provides detailed
descriptions about the metabolic

pathways
needed for
production alcohol, fatty acid

and hydrogen based biofuels
.
These descriptions include
the challenges thus far
encountered with production of these fuels along with
metabolic engineering solutions to these challenges.
Information in th
is article will help support our argument
that metabolic engineering is a viable solution to the
diminishing petroleum supply crisis.


J. McEwen, S. Atsumi. (2012). “Alternative biofuel
production in non
-
natural hosts.”
Current Opinion in
Biotechnology.

(Online article).
http://www.sciencedirect.com/science/journal/09581669

p.
744
-
750.

This article, from a profe
ssional journal specializing in
biotechnology
, outlines multiple
alternative fuels that can be
produced by various microorganisms, briefly summarizes
the major enzymes and metabolic pathways needed to
produce such fuels, and differentiates the origins of the
recombinant DNA constructed in each microorganism. This
articl
e will provide details needed for us to describe the
relationship between genetic and metabolic engineering in
prokaryotic alternative fuel production.


L. Bergeron. (2011). “E. coli could convert sugar to
biodiesel at ‘an extraordinary rate,’ say Stanford

researchers.”
Stanford Report.
(Online article).
http://news.stanford.edu/news/2011/november/khosla
-
ecoli
-
biodiesel
-
111011.html


This

academic article, from Sta
nford University’s Stanford
Report
, describes in detail the challenges associated with
engineering E. coli to produce fatty acid biofuel elements
and the benefits and difficulties related to different types of
biofuels. This article will provide informatio
n needed to aid
in our presentation of possible solutions to difficulties in
metabolically engineering E. coli for commercial fuel
production purposes.


“Recombinant DNA (rDNA): Technology, Plasmids, and
Resources.”
Resource Sharing Educational Resources.
(Online article).
http://www.thefreeresource.com/recombinant
-
dna
-
rdna
-
technology
-
plasmids
-
and
-
resources

This article from “thefreesource” online encyclope
dia has an
informational purpose. This source gives a step
-
by
-
step
breakdown of the recombinant DNA process. It goes into
detail on isolating a gene of interest, identifying and
removing plasmids, the bonding of donor DNA and host
DNA, the recombinant DNA’
s introduction into the host
cell, and rDNA replication. This information would be
relevant to our paper when explaining the fundamental
process of genetic engineering.


R. Kalscheuer , A. Steinbüchel , and T. Stölting.
(2006, June
26). “
Microdiesel:
Esche
richia coli

engineered for fuel
production
.”
Microbiology
. (Online Article).
http://mic.sgmjournals.org/content/152/9/2529.full

This source is an academic journal written by microbiology
professors for the purpose of sharing the authors’ research
and results of experimentation. This article contains
particularly useful information on biofuels keeping a
balanced carbon dioxide cycle and reducing emissions. It
also explains how biofuels wou
ld be advantageous because
they are good for engines, non
-
hazardous, and
biodegradable. This information will contribute to our paper
by highlighting the benefits of biofuels.