CH391L/S13/Metabolic Engineering and Thermophiles

Metabolic Engineering

Metabolic Engineering is changing and optimizing metabolic pathways in an organism to increase production of a chemical product. This can include the addition of enzymatic steps catalyzed from enzymes encoded by exogenous genes. With economically viable feedstocks metabolic engineering has become a relevant process by which companies can create sustainable chemicals and pharmaceuticals.

Metabolic engineering was first done by chemical mutagenesis of organisms and screening for organisms with increased production of the desired metabolite. Eventually, with increased knowledge of metabolic pathways and genetic engineering techniques in the 1990s the constraints on a metabolic pathway and production of a desired metabolite could be more easily relieved[4]. Modern techniques combine genetic engineering, directed evolution, and systems biology to improve yields in metabolic engineering. Genetic engineering advances in recombineering techniques and cloning help expedite creation of production organisms. Directed evolution has been used at both the protein and organismal level to modulate activity/specificity of enzymes and produce more robust organisms for chemical production. Systems biology has helped in the modeling of reactions in the complex chemical process involved in metabolic engineering, comparative genomics, and functional genomics. A particularly valuable systems biology technique called flux balance analysis, which is "a mathematical method for simulating metabolism in genome scale reconstructions of metabolic networks," has been particularly useful (Example). Bernhard Palsson, an innovator in systems biology, co-founded a metabolic engineering company based on flux balance analysis [5] (software link[6])

Examples of Metabolic Engineering

• Pharmaceuticals - Arteminisin - is an antimalarial drug that has been produced in yeast by the Keasling lab and Amyris.
• Petrochemical Replacements - 1,4-Butanediol and Propanediol - These are chemicals typically made by crude oil processing and now have been shown to be made through metabolic engineering.
• Biofuels - [7] - Metabolic engineering has been used to improve yields in cellulosic ethanol production and for algal biofuels.

Government and Metabolic Engineering

The U.S. Department of Energy (DOE) through the Bioenergy Technologies Office is heavily involved in research and funding of metabolic engineering projects for biofuels and bioproducts [8]. Their website is a good reference for the basics of the field [9].

Biomassfeedstocks, products, and applications [1]

Thermophiles

Organisms on earth are found to live between temperatures of −15°C to 122°C [10] [11]. Thermophiles are a type of extremophile that thrive in high temperature. Thermophiles are broken down into two groups "obligate' thermophiles (require high temperatures for growth) and faculative thermophiles (can live at high or low temperatures). Thermophiles are best known in biology as the source for the enzymes used in PCR, these organisms were isolated in hot springs and deep sea hydrothermal vents.

Terms for organisms based on their temperature preference

• Psychrophile grow down to −15°C to +10°C
• Mesophile grow typically between 20 and 45 °C
• Thermophile grow between 45 and 122 °C
• Hyperthermophile optimal temperatures are above 80°C

Thermophiles produce some of the bright colors of Grand Prismatic Spring, Yellowstone National Park [2]

Use of thermophiles in metabolic Engineering

Thermophiles have distinct advantages and disadvantages in large scale production of biomolecules. Using thermophiles for enzymatic reactions are typically better than their mesophile counterparts because performing enzymatic reactions at high temperatures allows higher substrate concentrations, fewer risks of microbial contaminations, and often higher reaction rates. In addition, many hyperthermophiles use exotic metabolic pathways not seen in typical mesophiles, such as using sulfur instead of oxygen in cellular respiration[12]. The downsides to working with such organisms include: harder to culture, difficult genetic transformations, lack of selectable markers, different G/C content, smaller amount of studied "parts" to choose from, and a general smaller amount of knowledge about the organisms. The majority of research in metabollic engineering of thermophiles has been on cellulosic ethanol production. This involves the breakdown of Hemicellulose and Cellulose.

Cell wall structure of plant cells [3]

Example model thermophilic organisms

• Sulfolobus solfataricus
• Thermus aquaticus
• Pyrococcus furiosus
• Thermosynechococcus elongatus
• Geobacillus stearothermophilus

Examples of thermophiles in metabolic Engineering

Metabolic Engineering of Thermophilic Bacillus licheniformis for Chiral Pure D-2,3-Butanediol Production

Another BDO, 2,3-Butanediol, is also a potential fuel and a platform chemical. Organisms that natively produce the chemical are pathogenic and can only form the product with fermentations at 37°C. To transform the Bacillus licheniformis they had to use a protoplast fusion method. They were able to utilize xylose as a feedstock at 50°C to create 2,3-Butanediol. This will be helpful to utilize lignocellulose substrates as higher temperatures are helpful because of higher rates of degradation and fewer enzymes are need to be added [13].

Metabolic engineering of a thermophilic bacterium to produce ethanol at high yield

In this paper the authors used Thermoanaerobacterium saccharolyticum and made knockouts in the genes for acetate kinase, phosphate acetyltransferase, and L-lactate dehydrogenase. Their strain was able to produce high yields of ethanol as the only measurable fermentation byproduct. Cellulase, an enzyme added exogenously for the breakdown of cellulose, was reduced 2.5 fold when compared to the process in Saccharomyces cerevisiae at 37°C to produce the same yields. The organism was transformed by selection on Erythromycin. They mention that these hemicellulolytic thermophilies along with cellulolytic thermophiles like Clostridium thermocellum can be used for the entire process of cellulosic ethanol production [14].

Exploiting microbial hyperthermophilicity to produce an industrial chemical, using hydrogen and carbon dioxide

In this paper the authors discuss using hydrogen gas to reduce carbon dioxide directly into products as opposed to using organic feedstocks. They transformed Pyrococcus furiosus with five genes from Metallosphaera sedula of the carbon fixation cycle. With hydrogen gas and carbon dioxide they were able to produce 3-hydroxypropionic acid (a DOE top target chemical)[15].

Hydrocarbon Recovery & Conversion

Microbial enhanced oil recovery projects by Dupont [16] and synthetic genomics [17].

Syngas

Syngas is a mixture of CO and H2, projects are ongoing to use organisms that can convert these gases into larger carbon compounds [18].

Notable tools and resources for metabolic engineering

• KEGG pathways - Wiring diagrams of molecular interactions, reactions, and relations
• Cell Designer - modeling tool for biochemical networks
• fiatflux - 13C metabolic flux analysis

IGEM connection

Team Alberta 2012 (http://2011.igem.org/Team:Alberta) focused on making Neurospora crassa, which naturally breaks down cellulose. They were able to up-regulatie fatty acid synthesis and inhibit beta-oxidation. This created a fatty acid overproducer. Achievements page(http://2011.igem.org/Team:Alberta/Achievements/Overview).

Also, some team in texas metabollically engineered E. coli to grow on caffeine by introducing the N-demethylation pathway from Pseudomonas putida CBB5. (http://2012.igem.org/Team:Austin_Texas/Caffeinated_coli)