Genetic Engineering for Biofuels
Production
WSE 573 Spring 2013
Greeley Beck
INTRODUCTION
Alternative transportation fuels are needed in the United States because of
oil supply insecurity, oil price increases, and the need to lower carbon dioxide
emissions that contribute to global warming. The Energy Independence and
Security Act of 2007 mandates the production of 136 billion liters of biofuel by
2022, with 79 billion liters coming from advanced biofuels, such as cellulosic
ethanol and biodiesel.i Although researchers have been working aggressively to
commercialize technologies for the production of these advanced biofuels, progress
has been slow. One useful tool in making these production processes more efficient
and cost effective is genetic engineering. By manipulating the genomes of microbes,
researchers can create novel enzymes and metabolic pathways that can facilitate the
production of fuels and chemicals from biomass.
GENETIC ENGINEERING FOR BIOFUEL PRODUCTION
Biochemical conversion pathways are one of the most popular strategies for
creating fuels and chemicals from lignocellulosic biomass today. These methods
generally require two main steps. First, the cellulose and hemicellulose in the
biomass must be broken down into their constituent sugars. In lignocellulosic
biomass, the cellulose is contained within a matrix of hemicellulose and lignin. In
order to effectively degrade the biomass polymers, they must first be separated
from this natural entanglement with some pretreatment process.ii Enzymes can
then access the cellulose and hemicellulose polymers and break them down into
sugars. In the second step, the biomass sugars are then fed to microbes that ferment
them into ethanol or other chemicals of interest. Genetic engineering can improve
both of these steps in the biochemical conversion of biomass to fuel. The
deconstruction of the biomass polymers is often done with cellulase enzymes that
can be improved by biochemical engineering. Additionally, the fermentation
process can be improved using synthetic biology to manipulate the metabolic
pathways of the fermentative microbes.
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ENZYME FUNCTION AND SYNTHESIS
Many of the genetic engineering techniques relevant for biofuel production
involve the synthesis and optimization of enzymes. It will be useful, therefore, to
begin this report with a review of what enzymes do and how they are made.
An enzyme is simply a protein molecule that functions as a catalyst, helping
some specific chemical reaction take place. Initially, enzymes bind with their
particular substrate at a specific location, known as the active site. Once bound
together, this enzyme-substrate complex works to orient the substrate molecule in a
way that facilitates a chemical reaction. Figure 1 shows a schematic for the
enzymatic hydrolysis of sucrose.iii
First, the sucrose substrate binds at
the active site of the enzyme. This
allows a water molecule to react
with the sucrose, which is then
converted into glucose and fructose.
Many cellular processes rely on this
type of enzyme-catalyzed reaction
and enzymes are therefore essential
for all living organisms.
Enzymes are made within the
cell in the same way that all protein
Figure 1. Sucrose Enzymatic Hydrolysis.
molecules are produced. The DNA of
the cell has specific sequences of three letter codons that contain the genetic
instructions for enzyme synthesis. First, the DNA codon sequence is copied into an
mRNA sequence, in a process called transcription. The mRNA sequence is identical
to the DNA sequence, except that all the “T” nucleotide codon letters have been
converted to a “U” nucleotide. The mRNA sequence is then translated into a series
of amino acids that will form the protein. This translation process can be seen in
detail in Figure 2.iv tRNA is the molecule that translates the language of the three
letter codons into the corresponding amino acids. Using these tRNAs, the ribosome
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reads the mRNA codon sequence
and attaches consecutive amino
acids to form the protein
polypeptide chain. It is this
sequence of amino acids that gives
the protein molecule its structure
and function.
A clip from the PBS
program, DNA: The Secret of Life,
shows an animation outlining this
synthesis process from DNA to
Figure 2. Protein Translation.
protein:v
http://www.youtube.com/watch?v=D3fOXt4MrOM
Once the series of amino acids has been synthesized by the ribosome, the
protein folds into sheets and coils, with the polypeptide sequence dictating this
macromolecular structure. The function of the enzyme depends on the structure of
the active site and, thus, the structure-determining sequence of amino acids will also
govern enzyme functionality. By changing the codons within DNA, genetic
engineers are able to manipulate this important sequence of amino acids and, in
doing so, they can design enzymes with improved properties.
DIRECTED EVOLUTION
One of the major technologies that enzyme engineers use today to advance
biofuel production is directed evolution. Natural evolution occurs when mutations
are created during DNA replication and the sequence of genetic codons is altered.
The new genes lead to the production of novel proteins, which can be advantageous
or harmful to the new organism. Natural selection will then favor useful mutations
while the deleterious ones die out. The directed evolution technique mimics this
process in the lab. Figure 3 outlines the basic steps involved directed evolution.vi
First, mutations must be induced in the gene that codes for the enzyme of interest.
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Once a library of these gene
mutants has been created, they
are then cloned into a DNA
expression vector. These vectors
are then inserted into bacteria
cells that produce the enzyme.
The bacterial colonies can then
be screened for a certain
property, selecting for those
mutants that produced an
improved enzyme. The
screening criteria can range from
enzyme activity or productivity
Figure 3. Directed Evolution Process.
to temperature or acidity tolerance. Automated robotic screening allows for the
selection of the best mutants among millions of mutant strains. The improved gene
can then be recycled through the process to become further evolved.
MUTAGENESIS
The libraries of gene mutants necessary for directed evolution can be created
in several ways. To induce many mutations randomly within the gene of interest,
error-prone polymerases can be used in the standard polymerase chain reaction
(PCR). In the following clip, DNAInteractive.org succinctly explains gene
amplification using PCR:vii
http://www.youtube.com/watch?v=2KoLnIwoZKU
Error-prone PCR functions in the same way, but the polymerases used have low
fidelity, causing them to create many mutations within the gene of interest in
addition to amplification.viii This method of mutagenesis often requires many
rounds of directed evolution to produce useful results because the induced
mutations are located randomly within the gene of interest.
Another method, known as site-directed saturation-mutagenesis, uses
structural information to selectively mutate at a specific location in the enzyme.ix
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Mutations are integrated within the primers that anneal the polymerases to the
gene of interest for PCR. Using this method, researchers are able to concentrate
mutations at the active site of the enzyme where they are likely to be the most
useful.
Significant advances in enzyme functionality can be achieved using a third
mutagenesis process called DNA shuffling. This method uses in vitro DNA
recombination to exchange the functional domains of homologous enzymes. Figure
4 outlines this process for shuffling
together two endoglucanase
enzymes.x The two genes are
initially cut up at several random
locations. The fragments of both
genes are then recombined to
create new mutant chimeras that
contain large stretches of genetic
material from both enzymes. The
mutant genes are then cloned into
bacteria and advantageous
properties are selected for in a
high-throughput screening process.
The major benefit of using this
method is that enzyme activity can
be substantially improved. By
recombining large portions of
Figure 4. DNA Shuffling of Two Endoglucanase Genes.
multiple enzymes, mutant
chimeras can potentially contain
more than one active site, which can greatly enhance the activity of the enzyme.
CODEXIS ENGINEERING OF CELLULASE ENZYMES
Codexis, Inc. is one company that uses these directed evolution technologies
to improve biochemical conversion pathways for biofuels production.xi Using site6
directed mutagenesis and DNA shuffling techniques, the company has created
optimized cellulase enzyme packages that degrade cellulose into its constituent
sugars. These CodeXyme cellulase packages are designed to utilize a wide variety of
feedstock including corn stover, corncobs, sugarcane bagasse, cane straw, wheat
straw and rice straw. They have been shown to convert 75-85% of the glucan and
xylan contained in the feedstock to C6 and C5 sugars with an enzyme loading of 1015g of enzyme per kg of glucan. The company currently sells two separate
CodeXyme cellulase packages – CodeXyme 4 and CodeXyme 4X. The biomass must
be pretreated in order for the enzymes to effectively access the biopolymer
substrates. These pretreatment conditions often adversely affect the enzymes and,
thus, the enzymes must be evolved for tolerance in those environments. The
CodeXyme 4 package has been screened for acidity tolerance and is therefore
optimized for dilute acid pretreatments. The CodeXyme 4X package has been
evolved for heat tolerance, so it is suitable for hydrothermal pretreatments.
One of the major advantages of using this directed evolution technology to
create better enzymes is that the enzymes continue to evolve and are improved over
time. Figure 5 clearly demonstrates this effect in the Codexis cellulase enzymes.xii
Figure 5. 4 Year Improvement in CodeXyme Cellulase Packages.
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By selecting for mutant strains with enhanced activity and improved production,
Codexis has increased the sugar yield of their enzymes more than 10 fold while
decreasing the manufacturing cost more than 3 fold in only four years.
METABOLIC ENGINEERING IN FERMENTATIVE MICROBES
Genetic engineering can also be used to improve the fermentation step in
biofuel production. Synthetic biology involves the design, synthesis and
introduction of new genetic programming to organisms for new biological
functions.xiii This strategy is used to reengineer the metabolic pathways of the
fermentation microbes that convert biomass sugar into products. Codexis has used
their genetic engineering technologies to introduce a non-native pathway for the
fermentation xylose in yeast.xiv The primary sugar resulting from the
deconstruction of cellulose and hemicellulose is glucose, but xylose is also produced
in significant amounts. Currently, xylose is not converted to ethanol by the yeast
used in today’s first generation ethanol production. Thus, the conversion efficiency
of a biochemical process using cellulose and hemicellulose feedstock can be
substantially increased by also utilizing the xylose sugars. Metabolic engineering
can also be used to design organisms that will secrete novel chemicals of interest.
LS9 is one company that has engineered a microbe that will secrete long chain fatty
acids that can be used as biodiesel.xv Researchers at the University of California, Los
Angeles were also recently able to design a biosynthetic pathway in Escherichia coli
to produce the industrial chemicals n-helptanol and 3-phenylpropanol.xvi This
marked the first time these chemicals had been produced directly from glucose in E.
coli. Using genetic engineering to design microbes that produce useful secretions
has the potential to sustainably produce chemical feedstock, not only for energy but
also other chemical industries.
ECONOMICS
Bioenergy programs are often criticized because they are dependent on
government subsidies to remain economically viable. Certainly, the facilities
performing the actual conversion of biomass into fuel are dependent on these
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government incentives. However, the cellulase enzymes for biomass deconstruction
and the propriety fermentation microbes are often purchased from other
companies. Thus, the industry doing the genetic engineering is usually separate
from the biofuel facility itself. For example, Codexis and Novozymes – two of the
largest producers of cellulase enzymes – sell their products to biofuels companies
rather than using them themselves. In this way, they avoid the large capital
investment necessary to create their own production facilities. Although they
certainly benefit from government funding to biofuels programs, many of the
genetic engineering companies are not entirely dependent on government subsidies.
This is largely due to the fact that genetic engineering technologies are relevant in
other industries as well. Enzymes are used in many chemical industries and
improvement of these enzymes is always useful. Thus, directed evolution
technologies for enhanced enzyme performance can be used to create products for
use outside of biofuels production.
CONCLUSIONS
Genetic engineering technologies will certainly play a large role in the effort
to produce chemicals and fuels in an ecologically friendly manner. Advances in
protein synthesis technologies permit the design and directed evolution of enzymes
specifically tailored for industrial applications. Improved biomass deconstruction
enzymes will facilitate the degradation of biomass into sugars and synthetic biology
will increase the performance of fermentation microbes. Like the enzymes they
create, these technologies will evolve in the future, becoming more efficient and
likely gaining more widespread implementation.
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Sissine, F. (2007) Energy Independence and Security Act of 2007: A Summary of
Major Provisions. Congressional Research Service, Washington, D.C.
i
United States Department of Energy (2009) Bioenergy Research Centers, An
Overview of the Science, 2009.
ii
iii
http://www.tokresource.org/tok_classes/biobiobio/biomenu/enzymes/
http://limbiclab.com/2012/12/02/a-crash-course-in-dna-amino-acids-andproteins-how-the-code-of-life-produces-the-stuff-that-makes-you/
iv
v
http://www.youtube.com/watch?v=D3fOXt4MrOM
vi
http://www.rsc.org/chemistryworld/Issues/2004/July/rational.asp
vii
http://www.youtube.com/watch?v=2KoLnIwoZKU
Cirino, P. C., Mayer, K. M., & Umeno, D. (2003). Generating mutant libraries using
error-prone PCR. In Directed Evolution Library Creation (pp. 3-9). Humana Press.
viii
Zheng, L., Baumann, U., & Reymond, J. L. (2004). An efficient one-step site-directed
and site-saturation mutagenesis protocol. Nucleic acids research,32(14), e115-e115.
ix
x
http://www.nature.com/nrmicro/journal/v2/n7/box/nrmicro925_BX2.html
xi
http://www.codexis.com/
xiihttp://www.codexis.com/pdf/CodeXymeProductLaunchPresentation_March2013.
pdf
xiii
http://www.codexis.com/pdf/5-HowCodexisTechnologyWorks.pdf
xiv
http://www.codexis.com/pdf/3-CodexisApplicationsBiofuels.pdf
xv
http://www.ls9.com/technology/technology-overview
Marcheschi, R. J., Li, H., Zhang, K., Noey, E. L., Kim, S., Chaubey, A., ... & Liao, J. C.
(2012). A synthetic recursive “+ 1” pathway for carbon chain elongation. ACS
chemical biology, 7(4), 689-697.
xvi
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