Available online at www.sciencedirect.com Metabolic engineering of microorganisms for biofuels production: from bugs to synthetic biology to fuels Sung Kuk Lee1,2, Howard Chou1,2,3, Timothy S Ham1,4, Taek Soon Lee1,2 and Jay D Keasling1,2,3,5 The ability to generate microorganisms that can produce biofuels similar to petroleum-based transportation fuels would allow the use of existing engines and infrastructure and would save an enormous amount of capital required for replacing the current infrastructure to accommodate biofuels that have properties significantly different from petroleum-based fuels. Several groups have demonstrated the feasibility of manipulating microbes to produce molecules similar to petroleum-derived products, albeit at relatively low productivity (e.g. maximum butanol production is around 20 g/L). For cost- effective production of biofuels, the fuel-producing hosts and pathways must be engineered and optimized. Advances in metabolic engineering and synthetic biology will provide new tools for metabolic engineers to better understand how to rewire the cell in order to create the desired phenotypes for the production of economically viable biofuels. Addresses 1 Joint BioEnergy Institute, Emeryville, CA 95608, USA 2 Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA 3 Department of Bioengineering, University of California, Berkeley, CA 94720, USA 4 Department of Computational Biosciences, Sandia National Laboratories, Albuquerque, NM 87185, USA 5 Department of Chemical Engineering, University of California, Berkeley, CA 94720, USA Corresponding author: Keasling, Jay D (Keasling@berkeley.edu) Current Opinion in Biotechnology 2008, 19:556���563 This review comes from a themed issue on Chemical biotechnology Edited by Huimin Zhao and Wilfred Chen Available online 10th November 2008 0958-1669/$ ��� see front matter # 2008 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2008.10.014 Introduction Alternative transportation fuels are in high demand owing to concerns about climate change, the global petroleum supply, and energy security [1,2]. Currently, the most widely used biofuels are ethanol generated from starch (corn) or sugar cane and biodiesel produced from veg- etable oil or animal fats [3 ]. However, ethanol is not an ideal fuel molecule in that it is not compatible with the existing fuel infrastructure for distribution and storage owing to its corrosivity and high hygroscopicity [1,4 ]. Also, it contains only about 70% of the energy content of gasoline. Biodiesel has similar problems (URL: http:// www.bdpedia.com/biodiesel/alt/alt.html): it cannot be transported in pipelines because its cloud and pour points are higher than those for petroleum diesel (petrodiesel), and its energy content is approximately 11% lower than that of petrodiesel. Furthermore, both ethanol and bio- diesel are currently produced from limited agricultural resources, even though there is a large, untapped resource of plant biomass (lignocellulose) that could be utilized as a renewable source of carbon-neutral, liquid fuels [5]. Microbial production of transportation fuels from renew- able lignocellulose has several advantages. First, the production is not reliant on agricultural resources com- monly used for food, such as corn, sugar cane, soybean, and palm oil. Second, lignocellulose is the most abundant biopolymer on earth. Third, new biosynthetic pathways can be engineered to produce fossil-fuel replacements, including short-chain, branched-chain, and cyclic alco- hols, alkanes, alkenes, esters and aromatics. The devel- opment of cost-effective and energy-efficient processes to convert lignocellulose into fuels is hampered by signifi- cant roadblocks, including the lack of genetic engineering tools for native producer organisms (non-model organ- isms), and difficulties in optimizing metabolic pathways and balancing the redox state in the engineered microbes [6]. Furthermore, production potentials are limited by the low activity of pathway enzymes and the inhibitory effect of fuels and byproducts from the upstream biomass processing steps on microorganisms responsible for pro- ducing fuels. Recent advances in synthetic biology and metabolic engineering will make it possible to overcome these hurdles and engineer microorganisms for the cost- effective production of biofuels from cellulosic biomass. In this review, we examine the range of choices available as potential biofuel candidates and production hosts, review the recent methods used to produce biofuels, and discuss how tools from the fields of metabolic engin- eering and synthetic biology can be applied to produce transportation fuels using genetically engineered micro- organisms. Liquid fuels and alternative biofuel molecules An understanding of what makes a good fuel is important in order to retool microorganisms to produce more useful alternative biofuels. The best fuel targets for the near Current Opinion in Biotechnology 2008, 19:556���563 www.sciencedirect.com

term will be molecules that are already found in or similar to components of fossil-based fuel in order to be compa- tible with existing engines (spark ignition engine for gasoline, compression ignition engine for diesel fuel, and gas turbine for jet fuel). There are several relevant factors to consider when designing biofuel candidates (Table 1). Energy contents, the combustion quality described by octane or cetane number, volatility, freezing point, odor, and toxicity are important factors to consider. In the following section, we will consider several biofuel candidates and their properties. Gasoline and its alternatives Gasoline is a complex mixture of hydrocarbons including linear, branched, and cyclic alkanes (40���60%), aromatics (20���40%), and oxygenates [7]. The carbon number of hydrocarbons in gasoline varies from 4 to 12. Ethanol, the most popular additive to gasoline, has an octane number of 129, but its energy content per gallon is about 70% of that of gasoline. Ethanol also has problems as a fuel owing to high miscibility with water, which makes it difficult to distill from the fermentation broth and to transport through existing pipelines. Recently, n-butanol has received more attention as an alternative gasoline additive. Butanol has two more carbons than ethanol, which results in an energy content of about 40% higher than that of ethanol. The octane number of butanol is 96 [8], which is somewhat lower than that of ethanol but is still comparable to that of gasoline (91���99). Unlike ethanol, butanol is less soluble in water than ethanol. It can also be used not only as an additive to gasoline but also as a fuel by itself in conventional engines (URL: http://www.butanol.com). In general, the octane number increases when the mol- ecule has methyl branching and double bonds. Branched C4 and C5 alcohols are also considered potential gasoline additives. Among them, isobutanol (2-methyl-1-propa- nol) has very similar properties to n-butanol with a higher octane number, and is currently under investigation as a new biofuel target (URL: http://www.gevo.com). Other short chain alcohols, such as isopentanol (3-methyl-1- butanol or isoamyl alcohol) and isopentenol (3-methyl- 3-buten-1-ol or 3-methyl-2-buten-1-ol) are also attractive gasoline fuel additives. Their octane numbers range slightly above 90, and they have higher energy contents than butanol. These alcohols can be produced from the isoprenoid biosynthetic pathway [9] or by transformation of amino acids as reported recently [4 ]. Branched, short- chain alkanes such as isooctane are a good gasoline replacement, but the biological production of these mol- ecules would require significant changes to existing meta- bolic pathways and may take significant effort to achieve. Diesel and its alternatives Diesel fuel is also a complex mixture of hydrocarbons including linear, branched, and cyclic alkanes (75%) and aromatics (25%). The carbon number of hydrocarbons in petrodiesel varies from 9 to 23, with an average of 16 (Table 1). Biodiesel is generally composed of fatty acid methyl esters (FAMEs), and is mostly derived from vegetable oil or animal fat. The fatty acids in FAMEs generally have a chain length from 12 to 22, containing zero to two double bonds. Biodiesel has a comparable cetane rating and energy content to petrodiesel and additional advantages, such as higher lubricity and less emission of pollutants. Another source for biodiesel would be isoprenoids, which are naturally occurring branched or cyclic hydrocarbons mostly synthesized in plants. They usually have methyl branches, double bonds, and ring structures, which improve the fluidity at lower tempera- tures but lower cetane ratings. Therefore, linear or cyclic monoterpenes (C10) or sesquiterpenes (C15) are potential targets for biodiesel fuel, especially with complete or Metabolic engineering for biofuels production Lee et al. 557 Table 1 Types of liquid fuels. Fuel type Major components Important property Biosynthetic alternatives Gasoline C4���C12 hydrocarbons Octane numbera Ethanol, n-butanol and iso-butanol Linear, branched, cyclic, aromatics Energy contentb Short chain alcohols Anti-knock additives Transportability Short chain alkanes Diesel C9���C23 (average C16) Cetane numberc Biodiesel (FAMEs) Linear, branched, cyclic, aromatic Low freezing temperature Fatty alcohols, alkanes Anti-freeze additives Low vapor pressure Linear or cyclic isoprenoids Jet fuel C8���C16 hydrocarbons Very low freezing temperature Alkanes Linear, branched, cyclic, aromatic Net heat of combustion Biodiesel Anti-freeze additives Density Linear or cyclic isoprenoids a A measurement of its resistance to knocking. Knocking occurs when the fuel/air mixture spontaneously ignites before it reaches the optimum pressure and temperature for spark ignition. b The amount of energy produced during combustion. The number of C���H and C���C bonds in a molecule is a good indication of how much energy a particular fuel will produce. c A measurement of the combustion quality of diesel fuel during compression ignition. A shorter ignition delay, the time period between the start of injection and start of combustion of the fuel is preferred, and the ignition delay is indexed by the cetane number. www.sciencedirect.com Current Opinion in Biotechnology 2008, 19:556���563