Genetic Engineering for Biofuels Production

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. 2

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 molecules are produced. The DNA of Figure 1. Sucrose Enzymatic Hydrolysis. 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 3

Figure 2. Protein Translation. 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 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. 4

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 5

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 site- 6

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 10-15g 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. 7

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 8

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. 9

i Sissine, F. (2007) Energy Independence and Security Act of 2007: A Summary of Major Provisions. Congressional Research Service, Washington, D.C. ii United States Department of Energy (2009) Bioenergy Research Centers, An Overview of the Science, 2009. iii http://www.tokresource.org/tok_classes/biobiobio/biomenu/enzymes/ iv 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/ 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 viii 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. ix 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. x http://www.nature.com/nrmicro/journal/v2/n7/box/nrmicro925_bx2.html xi http://www.codexis.com/ xii http://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 xvi 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. 10