- Email: [email protected]
Renewable jet fuel
Available online at www.sciencedirect.com
ScienceDirect Renewable jet fuel Pauli Kallio, Andra´s Pa´sztor, M Kalim Akhtar and Patrik R Jones Novel strategies for sustainable replacement of finite fossil fuels are intensely pursued in fundamental research, applied science and industry. In the case of jet fuels used in gas-turbine engine aircrafts, the production and use of synthetic bio-derived kerosenes are advancing rapidly. Microbial biotechnology could potentially also be used to complement the renewable production of jet fuel, as demonstrated by the production of bioethanol and biodiesel for piston engine vehicles. Engineered microbial biosynthesis of medium chain length alkanes, which constitute the major fraction of petroleum-based jet fuels, was recently demonstrated. Although efficiencies currently are far from that needed for commercial application, this discovery has spurred research towards future production platforms using both fermentative and direct photobiological routes. Addresses Bioenergy Group, University of Turku, Tykisto¨katu 6A, 6krs, 20520 Turku, Finland Corresponding author: Jones, Patrik R ([email protected])
Current Opinion in Biotechnology 2014, 26:50–55 This review comes from a themed issue on Plant biotechnology Edited by Birger Lindberg Møller and R George Ratcliffe
0958-1669/$ – see front matter, # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.copbio.2013.09.006
Introduction The pursuit for sustainable and renewable fuel production systems is a global challenge of great environmental, social and economic importance. Jet fuels constitute a large sector for the consumption of fossil fuels with a market value of $207 billion in 2012 (accounting for 33% of operating expenses at $110.0/barrel Brent of oil) [1] and steadily rising. Biotechnology could potentially make an important contribution to renewable jet fuel production as already demonstrated in the production of bioethanol and biodiesel for piston engine vehicles. Here we survey the current literature regarding possibilities for renewable production of jet fuels with a particular attention on alkanes — the dominant chemical class found in current jet fuels.
What is jet fuel? Jet fuel is a generic name for aviation fuels used in gasturbine engine powered aircrafts. Traditionally jet fuel (or ‘kerosene’) corresponds to the kerosene distillation Current Opinion in Biotechnology 2014, 26:50–55
fraction of crude oil (150–2758C) (Figure 1a) which is a complex blend of up to >1000 different chemical compounds. The main components are linear and branched alkanes and cycloalkanes (Table 1) with a typical carbon chain-length distribution of C6–C16 [2,3]. Since the development of the first jet-powered aircrafts in the late 1930s, the two main operational standards are Jet A used in the US and Jet A-1 used widely elsewhere in the world [2] (Tables 1 and 2). The composition of jet fuels has always been a compromise between the cost (availability of suitable raw material and the requirement for processing) and performance (propulsion properties, safety, and engine-friendliness) — with very little emphasis on the environmental impact. Jet fuels differ from traditional combustion engine fuels in their physicochemical properties because of enginespecific technical requirements and operational conditions. The relative proportion of the various hydrocarbon constituents determines the so-called bulk properties of the jet fuel (Table 2) including energy content, combustibility, density and fluidity. These attributes can be modified by altering the ratio of hydrocarbons with different molecular weights and geometries. For example, the low-temperature flow properties and volumetric energy content of linear alkanes are poor, but compensated by the branched alkanes and aromatic compounds in the jet fuel mixture, respectively [3]. Other important factors such as fuel stability, lubricity, corrosivity and cooling characteristics are influenced by minor components in the fuel [3]. These minor components in kerosene-based fuels include sulphur, oxygen and nitrogen hetero-compounds which are minimized to, for example, enhance the combustion properties and reduce the environmental impact. At the same time, specialized chemical additives such as antioxidants, metal deactivators or biocides are supplemented in the parts per million concentration range to improve or preserve important fuel properties such as those listed above [2,3].
Existing technologies for renewable jet fuel production Currently there are two promising renewable alternatives that can substitute for petroleum-based jet fuels: Bio Derived Synthetic Paraffinic Kerosene (Bio-SPK) [4] and Fischer–Tropsch Synthetic Paraffinic Kerosene (FT-SPK) [5], produced from renewable oils and biomass, respectively [6]. Bio-SPK is produced by transesterification of triacylglycerols and fatty acids extracted from plants, algae or recycled sources followed by hydrocracking and hydroprocessing which generates alkanes of desired length, saturation level and branching. FT-SPK www.sciencedirect.com
Renewable jet fuel Kallio et al. 51
Figure 1
(b) hω
(c)
Generation of starting materials
Solar Energy
Transport
(a) Photosynthesis/ Microbial biocatalysis Multi-step industrical processing
Operational Jet Fuel
Crude oil processing Current Opinion in Biotechnology
Representation of three alternative routes to produce aviation jet fuel discussed in this study. (a) Jet fuel traditionally corresponds to the kerosene distillation fraction of petroleum. (b) Synthetic paraffinic kerosenes, Bio-SPK and FT-SPK, produced from biomass or bio-oils of microbial, plantderived or recycled origin can be used as renewable jet fuel replacements. The process involves the production/collection of the initial substrate material, followed by multi-step industrial processing to generate the final fuel. (c) Alkanes, the main components of jet fuels, can also be produced via specific microbial biosynthetic pathways from fatty acid precursors of different lengths. Currently these have only been demonstrated at conceptual level, but could provide a future platform for the direct production of renewable fuels from biomass or directly from sunlight and CO2.
is obtained through pyrolysis of biomass into synthetic gas (syngas), Fischer–Tropsch synthesis of longer chain alkanes, hydroprocessing and separation. Though the chemical compositions of SPKs and petroleum-based fuels are clearly different (Table 2), they are still very Table 1 Chemical composition of jet fuel Composition (wt%)
Jet A-1
Bio-SPK
FT-SPK
n-Alkanes Iso-alkanes Monocyclic alkanes Polycyclic alkanes Alkyl benzenes Other hydrocarbons
19.6 29.9 20.3 7.3 14.1 8.7
10 90 S.P. S.P. 0 0
2.7 42.8 13.8 29.1 11.2 0.4
Chemical composition of commercial Jet A-1 jet fuel [7], Bio-SPK fuel from jatropha and FT-SPK fuel [7] (wt%); Bio-SPK [4]; S.P. — ‘small percentage’. www.sciencedirect.com
similar in their key technical properties (Table 1) and performance in modern jet aircrafts [4,6,7]. The industrial synthesis of SPK has been well established even at large scale, and the products have been successfully evaluated by major commercial airlines in a 1:1 blend with petroleum-derived kerosene [4,6]. Although the development and use of the technology is advancing rapidly in many countries [6], these systems rely on separate infrastructure for the preparation/collection and transport of the starting materials, and the need for multi-step industrial processing to obtain the final fuel (Figure 1b).
Alternative routes towards renewable fuels Bacteria, yeast and algae already have the capacity to produce a multitude of potential precursors and ready-touse fuel molecules like ethanol, alkanes and H2 as part of their native metabolism. The range of product chemistry Current Opinion in Biotechnology 2014, 26:50–55
52 Plant biotechnology
Table 2 Physical properties of jet fuel
Flash point (8C) Autoignition temperature (8C) Freezing point (8C) Density at 158C (kg/L) Specific energy (MJ/kg) Energy density (MJ/L) Sulfur, total mass (%)
Jet A-1
Jet A
42.0 210 47 0.775–0.840 43.2 34.7 0.3
51.1 210 40 0.775–0.840 43.0 35.3 0.3
Bio-SPK
n-Octane
n-Hexadecane
46.5 340 57 0.749 44.3 33.2 0.0001
13.0 220 57 0.730 47.4 34.6 0
135.0 202 18 0.773 47.3 36.6 0
Physical properties of commercial Jet A and Jet A-1 jet fuels [3] in comparison with biological synthetic paraffinic kerosene [4] from jatropha and pure n-alkanes [3].
can be expanded further by modifying existing pathways or by creating synthetic routes via metabolic engineering [8]. Stringent selection and genetic optimization of the host organisms are thereafter required to maximize the productivity for biotechnological applications. The success in such efforts towards commercial application is best exemplified by the development of cellulosic bioethanol [9]. An important future goal is to simplify the fuel production process, and reduce the number of steps (in production, transport, separation and/or processing) by directly generating the desired fuel from sunlight and CO2 instead of proceeding through terrestrial biomass (Figure 1c). For this, photosynthetic aquatic microorganisms such as cyanobacteria and eukaryotic algae provide an attractive platform for the direct conversion of solar energy into engine-ready fuels that preferentially are excreted from the host [10]. Although photosynthetic microbial biosynthesis currently cannot compete with the heterotrophic or chemical production systems in efficiency or cost, the concepts have been established (e.g. [11]), and provide a basis for the development of truly renewable hydrocarbon fuels with minimal impact on available land and nutrient resources for food production.
Potential target compounds used as jet fuel blending components As demonstrated by the comparison of petroleum-derived jet fuels and Bio-SPK (Tables 1 and 2), the alkane mixtures can differ dramatically even though the performance and physicochemical properties are very similar. Rather than mimicking the chemical composition of existing fuels the likely best strategy is to generate blending components which collectively confer the desired physicochemical properties required for jet fuel. A defined blend of selected biofuel components is likely to perform far better than any one single individual chemical and potentially also provides enhanced reproducibility relative to current jet fuels. It would be important for the aviation industry to identify suitable blends in order to steer biotechnologists in the right direction. The most obvious biological end-target would nevertheless be medium chain-length alkanes, as they constitute Current Opinion in Biotechnology 2014, 26:50–55
the bulk of both petroleum and SPK jet fuels. Alkanes also exhibit lower toxicity in comparison to many other fuel alternatives [12,13], at least for cyanobacteria. Although there are several other biosynthetic pathways and approaches which could be exploited for the production of fuel components [14,15] the remainder of this review is focused on alkanes derived from microbial fatty acid biosynthesis.
Fuel precursors from microbial fatty acid biosynthesis Bacterial fatty acid synthase II (FASII) catalyzes one of the pivotal pathways for the production of advanced fuels. This pathway generates fatty acyl-ACP (fatty acyl-Acyl Carrier Protein) precursors that are essential for membrane biosynthesis [16]. The fatty acyl-ACP chain is built up through iterative decarboxylative Claisen condensation reactions followed by the reduction of the keto groups to generate the final saturated carbon chain. One of the key findings relating to the bacterial FAS II pathway is that it can be deregulated by overexpression of a thioesterase, resulting in the over-accumulation of fatty acids [17]. Extensive work by Khosla’s lab led to the development of strains capable of generating fatty acids with a productivity of 4.5 g/L/day under fed-batch fermentation [18]. Further studies have shown that a broad range of fatty acid hydrocarbon chains of different lengths can be generated by simply varying the substrate specificity of the thioesterase [19]. In a variation of the ACPbased FASII pathway known as reverse beta oxidation, formation of the hydrocarbon chain can also proceed via CoA ester intermediates [20]. This pathway has been utilized for the production of medium chain-length fatty acids with titres of 7 g/L [21]. As is clearly evident from these studies, bacterial fatty acid synthesis pathways can be successfully exploited to generate a range of hydrocarbon backbones for the production of precursors and ready fuels.
Fatty acid derived fuel molecules; alkanes For alkane biosynthesis, the released fatty acid intermediate must first undergo enzyme-catalyzed reduction to generate the corresponding aldehyde precursor (Figure 2). The fatty aldehyde can then be converted www.sciencedirect.com
Renewable jet fuel Kallio et al. 53
Figure 2
Figure 3 Carbon source
(1)
Ala134
(b)
(a) O
Host strain & conditions
Enzyme properties
SCoA
KM
t
ca ;K
Val41
Acetyl-CoA Acetyl-CoA
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Alkane production from fatty acids. (1) Native intracellular metabolism generating acetyl-CoA from inorganic and/or organic carbon sources, (2) generation of fatty acyl chains by reverse beta oxidation, (3) generation of fatty acyl chains by the fatty acid synthesis pathway, (4) release of free fatty acids, (5) reduction of fatty acids to fatty aldehydes, (6) alkane generation by aldehyde deformylating oxygenase. Branching of alkanes can occur by petrochemical processing (e.g. hydrocracking and hydro processing) or by modification of the fatty acid pool with b-ketoacyl ACP synthase III [32]. Dashed lines represent multiple reactions; straight lines represent single one-step reactions.
into an alkane by a soluble non-heme diiron enzyme called aldehyde deformylating oxygenase (ADO, previously known as cyanobacterial aldehyde decarbonylase) [22]. This family of enzymes was discovered only recently by genome subtraction analyses of sequenced cyanobacterial strains [23]. The enzyme has subsequently been subjected to intense biochemical characterization by several groups [24–28]. ADO catalyses oxygen-dependent cleavage [26,28] of aldehyde precursors into alkane and formate [29] via a radical-based mechanism [27], displaying www.sciencedirect.com
Current Opinion in Biotechnology
Factors affecting the efficiency of synthetic alkane pathways. (a) Selection of appropriate microbial host and optimizing the culture conditions for biotechnological applications. (b) Structure-based modification for improved enzyme properties, as demonstrated by the change in ADO substrate preference resulting from substitutions in the substrate binding site [30]. (c) Coexpression of associated pathway enzymes for enhanced turnover, such as catalase decomposing H2O2 which otherwise can inhibit ADO [34]. (d) Maximizing the biosynthetic flux towards the end product by optimizing the pathway via, for example, translational tuning [36] or enzyme colocalization [37].
catalytic activity with fatty aldehydes ranging from C4 up to C18 [30]. The reaction is dependent on electrons that can be supplied by either chemical or proteinaceous (ferredoxin) electron acceptor–donors [23,24]. To date, there are only few reports of engineered alkaneproduction systems [23,31,32], and in all cases the yield has been abysmally low. Given that engineered strains produce fatty alcohols from the same aldehyde precursors to a titre above 300 mg/L of culture [21,31] the problem appears to lie at least in part with the low conversion efficiency from aldehyde to alkane. While it is still unclear whether there are yet unidentified enzymes or cofactors needed for maximal activity of ADO, fundamental research continues to provide insight into the primary bottlenecks and approaches to overcome these limitations (Figure 3). Many of the issues can be attributed to the poor kinetic properties of ADO with high KM and low kcat towards the aldehyde substrates, especially for shorter chain-lengths [30]. This poses a potential problem due to competing endogenous reactions such as those catalyzed by highly efficient aldehyde reductases [33], and emphasizes the importance of host strain selection for biotechnological applications (Figure 3a). A complementary Current Opinion in Biotechnology 2014, 26:50–55
54 Plant biotechnology
approach is to engineer the enzyme properties by structure-based active-site modification (Figure 3b), as demonstrated by amino acid substitutions that altered the substrate binding and selectivity of ADO [30]. The local enzyme environment (biosynthetic context, Figure 3c) is also important, and can be modulated by the introduction of additional enzyme components in the pathway. For example, addition of a catalase [34] was shown to enhance ADO turnover by alleviating the inhibitory effects of H2O2 formed in a side-reaction between the electron donor ferredoxin and O2, the second substrate of ADO [35]. Typically, synthetic pathways lack natural evolutionary optimization for optimal performance because the genes have been forced together from heterologous sources. To solve this, the interplay between the consecutive enzymatic steps in the alkane pathways could potentially be improved by translational tuning via RBS optimization [36] or by enzyme colocalization [37] (Figure 3d). The microbial fatty acid biosynthesis pathways mainly yield linear end-products, which alone do not meet the required jet fuel specifications. Further metabolic engineering to introduce geometrical diversification, including branching [38], cyclization [39] or introduction of double bonds [40], may therefore be of interest. The utility of such a concept was shown in a recent study, in which keto-acids, arising from the metabolism of amino acids, were directed through the FAS II machinery of Escherichia coli to form branched alkanes [32].
Conclusions There is a need to explore new opportunities for the production of renewable jet fuel. Currently there are efficient ways to produce renewable synthetic kerosenes from oils and biomass, though they are dependent on the production and collection of biomass as substrate, as well as multi-step processing. Future alternatives may be more competitive if the number of steps required for the conversion of primary substrates into the final fuel is reduced. On the basis of this concept, we have discussed possibilities for microbial production systems in which simple sugars, or ultimately sunlight and CO2, can be directly converted into alkane-based engine-ready jet fuel blending components. Currently, the microbial platforms for hydrocarbon fuel production simply do not offer the productivity, yields or titres for economic feasibility. For the most part these systems have only been demonstrated at a proof-of-concept level and the barrier to commercialization is further exacerbated by the challenge of up-scaling and fuel isolation/purification. Meanwhile, the price of petroleum is likely to increase as the supply of crude oil diminishes, and advances in fundamental and applied biotechnological research will provide new and increasingly competitive alternatives for the inevitable shift towards a sustainable supply of jet fuel. Current Opinion in Biotechnology 2014, 26:50–55
Acknowledgements The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7-ENERGY-2010-1) under grant agreement no. 256808 and European Research Council under the European Union’s Seventh Framework Programme (FP7/2007-2013)/ European Research Council Grant Agreement 260661.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
Platts I: Fuel Price Analysis. IATA: http://www.iata.org/ publications/economics/fuel-monitor/Pages/price-analysis.aspx.
2.
Maurice LQ, Lander H, Edwards T, Harrison WE III: Advanced aviation fuels: a look ahead via a historical perspective. Fuels 2001, 80:747-756.
3.
Hemighaus G, Boval T, Bacha J, Barnes F, Franklin M, Gibbs L, Hogue N, Jones J, Lesnini D, Lind J et al.: Aviation Fuels Technical Review. Chevron Corporation; 2006. A comprehensive and detailed review by the Chevron Corporation on aviation jet fuels and aviation gasolines, their specifications, composition and properties. Despite the early year of release (2006) the publication provides an important foundation for the comparison of jet fuels with the current approaches for sustainable alternatives. 4.
Kinder JD, Rahmes T: Evaluation of Bio-Derived Synthetic Paraffinic Kerosenes (Bio-SPK). The Boeing Company; 2009.
5.
Kaneko TFD, Makino E, Gray D, Tamura M: Coal liquefaction. Ullmann’s Encyclopedia of Industrial Chemistry. Wiley-VCH Verlag GmbH & Co. KGaA; 2000.
Steele P, Pearce B, Rı´os A, Nash P, Massat F, Young N, Bann A, Morgan D, Lakeman M, Scott T et al.: Powering the Future of Flight. Air Transport Action Group; 2012. In this publication Air Transport Action Group (ATAG), a global organization representing the entire commercial aviation sector, evaluates the current status and future of sustainable biofuels in aviation industry. The authors provide insight into the goals and viable approaches taken to enable the production and use of sustainable biofuels, supported by a selection of case studies on related infrastructures and international collaboration.
6.
7.
van der Westhuizen R, Ajam M, De Coning P, Beens J, de Villiers A, Sandra P: Comprehensive two-dimensional gas chromatography for the analysis of synthetic and crudederived jet fuels. J Chromatogr A 2011, 1218:4478-4486. A quantitative and qualitative chemical comparison of synthetic and petroleum-derived jet fuels, which served as a basis for the approval of Fischer–Tropsch technology for the production of renewable hydrocarbon fuel replacements.
8.
McEwen JT, Atsumi S: Alternative biofuel production in nonnatural hosts. Curr Opin Biotechnol 2012, 23:744-750.
9.
Geddes CC, Nieves IU, Ingram LO: Advances in ethanol production. Curr Opin Biotechnol 2011, 22:312-319.
10. Rosgaard L, de Porcellinis AJ, Jacobsen JH, Frigaard NU, Sakuragi Y: Bioengineering of carbon fixation, biofuels, and biochemicals in cyanobacteria and plants. J Biotechnol 2012, 162:134-147. 11. Lan EI, Liao JC: ATP drives direct photosynthetic production of 1-butanol in cyanobacteria. Proc Natl Acad Sci U S A 2012, 109:6018-6023. 12. Ka¨ma¨ra¨inen J, Knoop H, Stanford NJ, Guerrero F, Akhtar MK, Aro EM, Steuer R, Jones PR: Physiological tolerance and stoichiometric potential of cyanobacteria for hydrocarbon fuel production. J Biotechnol 2012, 162:67-74. 13. Ozaki K, Ohta A, Iwata C, Horikawa A, Tsuji K, Ito E, Ikai Y, Harada K: Lysis of cyanobacteria with volatile organic compounds. Chemosphere 2008, 71:1531-1538. 14. Kung Y, Runguphan W, Keasling JD: From fields to fuels: recent advances in the microbial production of biofuels. ACS Synth Biol 2012, 1:498-513. www.sciencedirect.com
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15. Peralta-Yahya PP, Zhang F, del Cardayre SB, Keasling JD: Microbial engineering for the production of advanced biofuels. Nature 2012, 488:320-328. 16. Chan DI, Vogel HJ: Current understanding of fatty acid biosynthesis and the acyl carrier protein. Biochem J 2010, 430:1-19. 17. Cho H, Cronan JE: Defective export of a periplasmic enzyme disrupts regulation of fatty acid synthesis. J Biol Chem 1995, 270:4216-4219. 18. Liu T, Vora H, Khosla C: Quantitative analysis and engineering of fatty acid biosynthesis in E. coli. Metab Eng 2010, 12:378-386. 19. Jing F, Cantu DC, Tvaruzkova J, Chipman JP, Nikolau BJ, Yandeau-Nelson MD, Reilly PJ: Phylogenetic and experimental characterization of an acyl-ACP thioesterase family reveals significant diversity in enzymatic specificity and activity. BMC Biochem 2011, 12:44. The authors screen the chain-length specificity of a wide selection of thioesterases, identifying several new enzymes with potential utility for biofuel production. 20. Inui H, Miyatake K, Nakano Y, Kitaoka S: Fatty acid synthesis in mitochondria of Euglena gracilis. Eur J Biochem 1984, 142: 121-126. 21. Dellomonaco C, Clomburg JM, Miller EN, Gonzalez R: Engineered reversal of the b-oxidation cycle for the synthesis of fuels and chemicals. Nature 2011, 476:355-359. 22. Krebs C, Bollinger JM, Booker SJ: Cyanobacterial alkane biosynthesis further expands the catalytic repertoire of the ferritin-like ‘di-iron-carboxylate’ proteins. Curr Opin Chem Biol 2011, 15:291-303. 23. Schirmer A, Rude M, Li X, Popova E, del Cardayre S: Microbial biosynthesis of alkanes. Science 2010, 329:559-562. A pivotal study in which the authors discover and characterize a new family of enzymes responsible for alkane biosynthesis in cyanobacteria. 24. Das D, Eser BE, Han J, Sciore A, Marsh EN: Oxygen-independent decarbonylation of aldehydes by cyanobacterial aldehyde decarbonylase: a new reaction of diiron enzymes. Angew Chem Int Ed Engl 2011, 50:7148-7152. 25. Eser BE, Das D, Han J, Jones PR, Marsh EN: Oxygenindependent alkane formation by non-heme iron-dependent cyanobacterial aldehyde decarbonylase: investigation of kinetics and requirement for an external electron donor. Biochemistry 2011, 50:10743-10750. 26. Li N, Chang WC, Warui DM, Booker SJ, Krebs C, Bollinger JM: Evidence for only oxygenative cleavage of aldehydes to alk(a/ e)nes and formate by cyanobacterial aldehyde decarbonylases. Biochemistry 2012, 51:7908-7916. 27. Paul B, Das D, Ellington B, Marsh EN: Probing the mechanism of cyanobacterial aldehyde decarbonylase using a cyclopropyl aldehyde. J Am Chem Soc 2013, 135:5234-5237. 28. Li N, Nørgaard H, Warui DM, Booker SJ, Krebs C, Bollinger JM: Conversion of fatty aldehydes to alka(e)nes and formate by a cyanobacterial aldehyde decarbonylase: cryptic redox by an unusual dimetal oxygenase. J Am Chem Soc 2011, 133: 6158-6161. 29. Warui DM, Li N, Nørgaard H, Krebs C, Bollinger JM, Booker SJ: Detection of formate, rather than carbon monoxide, as the
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stoichiometric coproduct in conversion of fatty aldehydes to alkanes by a cyanobacterial aldehyde decarbonylase. J Am Chem Soc 2011, 133:3316-3319. 30. Khara B, Menon N, Levy C, Mansell D, Das D, Marsh EN, Leys D, Scrutton NS: Production of propane and other short-chain alkanes by structure-based engineering of ligand specificity in aldehyde-deformylating oxygenase. Chembiochem 2013, 14:1204-1208. The authors describe the first work where structure-guided engineering is used in order to modify the chain-length specificity of an aldehyde deformylating oxygenase (formerly aldehyde decarbonylase). The chain-length preference is shifted from medium (C12–C18) to short (C4–C8) chain-lengths. In addition, the nature of the unknown ligand identified in structures of ADO is identified and one engineered variant does not accumulate the ligand. 31. Akhtar MK, Turner NJ, Jones PR: Carboxylic acid reductase is a versatile enzyme for the conversion of fatty acids into fuels and chemical commodities. Proc Natl Acad Sci U S A 2013, 110:87-92. 32. Howard TP, Middelhaufe S, Moore K, Edner C, Kolak DM, Taylor GN, Parker DA, Lee R, Smirnoff N, Aves SJ et al.: Synthesis of customized petroleum-replica fuel molecules by targeted modification of free fatty acid pools in Escherichia coli. Proc Natl Acad Sci U S A 2013, 110:7636-7641. 33. Rodriguez GM, Atsumi S: Isobutyraldehyde production from Escherichia coli by removing aldehyde reductase activity. Microb Cell Fact 2012, 11:90. 34. Andre C, Kim SW, Yu XH, Shanklin J: Fusing catalase to an alkane-producing enzyme maintains enzymatic activity by converting the inhibitory byproduct H2O2 to the cosubstrate O2. Proc Natl Acad Sci U S A 2013, 110:3191-3196. The authors discover an important limiting factor for the functionality of cyanobacteria aldehyde deformylating oxygenase, H2O2, and demonstrate a solution that improves the specific activity. 35. Misra HP, Fridovich I: The generation of superoixide radical during the autoxidation of ferredoxins. J Biol Chem 1971, 246:6886-6890. 36. Zelcbuch L, Antonovsky N, Bar-Even A, Levin-Karp A, Barenholz U, Dayagi M, Liebermeister W, Flamholz A, Noor E, Amram S et al.: Spanning high-dimensional expression space using ribosome-binding site combinatorics. Nucleic Acids Res 2013, 41:e98. 37. Dueber JE, Wu GC, Malmirchegini GR, Moon TS, Petzold CJ, Ullal AV, Prather KL, Keasling JD: Synthetic protein scaffolds provide modular control over metabolic flux. Nat Biotechnol 2009, 27:753-759. 38. Choi KH, Heath RJ, Rock CO: Beta-ketoacyl-acyl carrier protein synthase III (FabH) is a determining factor in branched-chain fatty acid biosynthesis. J Bacteriol 2000, 182:365-370. 39. Grogan DW, Cronan JE: Cloning and manipulation of the Escherichia coli cyclopropane fatty acid synthase gene: physiological aspects of enzyme overproduction. J Bacteriol 1984, 158:286-295. 40. Cahoon EB, Mills LA, Shanklin J: Modification of the fatty acid composition of Escherichia coli by coexpression of a plant acyl–acyl carrier protein desaturase and ferredoxin. J Bacteriol 1996, 178:936-939.
Current Opinion in Biotechnology 2014, 26:50–55
ScienceDirect Renewable jet fuel Pauli Kallio, Andra´s Pa´sztor, M Kalim Akhtar and Patrik R Jones Novel strategies for sustainable replacement of finite fossil fuels are intensely pursued in fundamental research, applied science and industry. In the case of jet fuels used in gas-turbine engine aircrafts, the production and use of synthetic bio-derived kerosenes are advancing rapidly. Microbial biotechnology could potentially also be used to complement the renewable production of jet fuel, as demonstrated by the production of bioethanol and biodiesel for piston engine vehicles. Engineered microbial biosynthesis of medium chain length alkanes, which constitute the major fraction of petroleum-based jet fuels, was recently demonstrated. Although efficiencies currently are far from that needed for commercial application, this discovery has spurred research towards future production platforms using both fermentative and direct photobiological routes. Addresses Bioenergy Group, University of Turku, Tykisto¨katu 6A, 6krs, 20520 Turku, Finland Corresponding author: Jones, Patrik R ([email protected])
Current Opinion in Biotechnology 2014, 26:50–55 This review comes from a themed issue on Plant biotechnology Edited by Birger Lindberg Møller and R George Ratcliffe
0958-1669/$ – see front matter, # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.copbio.2013.09.006
Introduction The pursuit for sustainable and renewable fuel production systems is a global challenge of great environmental, social and economic importance. Jet fuels constitute a large sector for the consumption of fossil fuels with a market value of $207 billion in 2012 (accounting for 33% of operating expenses at $110.0/barrel Brent of oil) [1] and steadily rising. Biotechnology could potentially make an important contribution to renewable jet fuel production as already demonstrated in the production of bioethanol and biodiesel for piston engine vehicles. Here we survey the current literature regarding possibilities for renewable production of jet fuels with a particular attention on alkanes — the dominant chemical class found in current jet fuels.
What is jet fuel? Jet fuel is a generic name for aviation fuels used in gasturbine engine powered aircrafts. Traditionally jet fuel (or ‘kerosene’) corresponds to the kerosene distillation Current Opinion in Biotechnology 2014, 26:50–55
fraction of crude oil (150–2758C) (Figure 1a) which is a complex blend of up to >1000 different chemical compounds. The main components are linear and branched alkanes and cycloalkanes (Table 1) with a typical carbon chain-length distribution of C6–C16 [2,3]. Since the development of the first jet-powered aircrafts in the late 1930s, the two main operational standards are Jet A used in the US and Jet A-1 used widely elsewhere in the world [2] (Tables 1 and 2). The composition of jet fuels has always been a compromise between the cost (availability of suitable raw material and the requirement for processing) and performance (propulsion properties, safety, and engine-friendliness) — with very little emphasis on the environmental impact. Jet fuels differ from traditional combustion engine fuels in their physicochemical properties because of enginespecific technical requirements and operational conditions. The relative proportion of the various hydrocarbon constituents determines the so-called bulk properties of the jet fuel (Table 2) including energy content, combustibility, density and fluidity. These attributes can be modified by altering the ratio of hydrocarbons with different molecular weights and geometries. For example, the low-temperature flow properties and volumetric energy content of linear alkanes are poor, but compensated by the branched alkanes and aromatic compounds in the jet fuel mixture, respectively [3]. Other important factors such as fuel stability, lubricity, corrosivity and cooling characteristics are influenced by minor components in the fuel [3]. These minor components in kerosene-based fuels include sulphur, oxygen and nitrogen hetero-compounds which are minimized to, for example, enhance the combustion properties and reduce the environmental impact. At the same time, specialized chemical additives such as antioxidants, metal deactivators or biocides are supplemented in the parts per million concentration range to improve or preserve important fuel properties such as those listed above [2,3].
Existing technologies for renewable jet fuel production Currently there are two promising renewable alternatives that can substitute for petroleum-based jet fuels: Bio Derived Synthetic Paraffinic Kerosene (Bio-SPK) [4] and Fischer–Tropsch Synthetic Paraffinic Kerosene (FT-SPK) [5], produced from renewable oils and biomass, respectively [6]. Bio-SPK is produced by transesterification of triacylglycerols and fatty acids extracted from plants, algae or recycled sources followed by hydrocracking and hydroprocessing which generates alkanes of desired length, saturation level and branching. FT-SPK www.sciencedirect.com
Renewable jet fuel Kallio et al. 51
Figure 1
(b) hω
(c)
Generation of starting materials
Solar Energy
Transport
(a) Photosynthesis/ Microbial biocatalysis Multi-step industrical processing
Operational Jet Fuel
Crude oil processing Current Opinion in Biotechnology
Representation of three alternative routes to produce aviation jet fuel discussed in this study. (a) Jet fuel traditionally corresponds to the kerosene distillation fraction of petroleum. (b) Synthetic paraffinic kerosenes, Bio-SPK and FT-SPK, produced from biomass or bio-oils of microbial, plantderived or recycled origin can be used as renewable jet fuel replacements. The process involves the production/collection of the initial substrate material, followed by multi-step industrial processing to generate the final fuel. (c) Alkanes, the main components of jet fuels, can also be produced via specific microbial biosynthetic pathways from fatty acid precursors of different lengths. Currently these have only been demonstrated at conceptual level, but could provide a future platform for the direct production of renewable fuels from biomass or directly from sunlight and CO2.
is obtained through pyrolysis of biomass into synthetic gas (syngas), Fischer–Tropsch synthesis of longer chain alkanes, hydroprocessing and separation. Though the chemical compositions of SPKs and petroleum-based fuels are clearly different (Table 2), they are still very Table 1 Chemical composition of jet fuel Composition (wt%)
Jet A-1
Bio-SPK
FT-SPK
n-Alkanes Iso-alkanes Monocyclic alkanes Polycyclic alkanes Alkyl benzenes Other hydrocarbons
19.6 29.9 20.3 7.3 14.1 8.7
10 90 S.P. S.P. 0 0
2.7 42.8 13.8 29.1 11.2 0.4
Chemical composition of commercial Jet A-1 jet fuel [7], Bio-SPK fuel from jatropha and FT-SPK fuel [7] (wt%); Bio-SPK [4]; S.P. — ‘small percentage’. www.sciencedirect.com
similar in their key technical properties (Table 1) and performance in modern jet aircrafts [4,6,7]. The industrial synthesis of SPK has been well established even at large scale, and the products have been successfully evaluated by major commercial airlines in a 1:1 blend with petroleum-derived kerosene [4,6]. Although the development and use of the technology is advancing rapidly in many countries [6], these systems rely on separate infrastructure for the preparation/collection and transport of the starting materials, and the need for multi-step industrial processing to obtain the final fuel (Figure 1b).
Alternative routes towards renewable fuels Bacteria, yeast and algae already have the capacity to produce a multitude of potential precursors and ready-touse fuel molecules like ethanol, alkanes and H2 as part of their native metabolism. The range of product chemistry Current Opinion in Biotechnology 2014, 26:50–55
52 Plant biotechnology
Table 2 Physical properties of jet fuel
Flash point (8C) Autoignition temperature (8C) Freezing point (8C) Density at 158C (kg/L) Specific energy (MJ/kg) Energy density (MJ/L) Sulfur, total mass (%)
Jet A-1
Jet A
42.0 210 47 0.775–0.840 43.2 34.7 0.3
51.1 210 40 0.775–0.840 43.0 35.3 0.3
Bio-SPK
n-Octane
n-Hexadecane
46.5 340 57 0.749 44.3 33.2 0.0001
13.0 220 57 0.730 47.4 34.6 0
135.0 202 18 0.773 47.3 36.6 0
Physical properties of commercial Jet A and Jet A-1 jet fuels [3] in comparison with biological synthetic paraffinic kerosene [4] from jatropha and pure n-alkanes [3].
can be expanded further by modifying existing pathways or by creating synthetic routes via metabolic engineering [8]. Stringent selection and genetic optimization of the host organisms are thereafter required to maximize the productivity for biotechnological applications. The success in such efforts towards commercial application is best exemplified by the development of cellulosic bioethanol [9]. An important future goal is to simplify the fuel production process, and reduce the number of steps (in production, transport, separation and/or processing) by directly generating the desired fuel from sunlight and CO2 instead of proceeding through terrestrial biomass (Figure 1c). For this, photosynthetic aquatic microorganisms such as cyanobacteria and eukaryotic algae provide an attractive platform for the direct conversion of solar energy into engine-ready fuels that preferentially are excreted from the host [10]. Although photosynthetic microbial biosynthesis currently cannot compete with the heterotrophic or chemical production systems in efficiency or cost, the concepts have been established (e.g. [11]), and provide a basis for the development of truly renewable hydrocarbon fuels with minimal impact on available land and nutrient resources for food production.
Potential target compounds used as jet fuel blending components As demonstrated by the comparison of petroleum-derived jet fuels and Bio-SPK (Tables 1 and 2), the alkane mixtures can differ dramatically even though the performance and physicochemical properties are very similar. Rather than mimicking the chemical composition of existing fuels the likely best strategy is to generate blending components which collectively confer the desired physicochemical properties required for jet fuel. A defined blend of selected biofuel components is likely to perform far better than any one single individual chemical and potentially also provides enhanced reproducibility relative to current jet fuels. It would be important for the aviation industry to identify suitable blends in order to steer biotechnologists in the right direction. The most obvious biological end-target would nevertheless be medium chain-length alkanes, as they constitute Current Opinion in Biotechnology 2014, 26:50–55
the bulk of both petroleum and SPK jet fuels. Alkanes also exhibit lower toxicity in comparison to many other fuel alternatives [12,13], at least for cyanobacteria. Although there are several other biosynthetic pathways and approaches which could be exploited for the production of fuel components [14,15] the remainder of this review is focused on alkanes derived from microbial fatty acid biosynthesis.
Fuel precursors from microbial fatty acid biosynthesis Bacterial fatty acid synthase II (FASII) catalyzes one of the pivotal pathways for the production of advanced fuels. This pathway generates fatty acyl-ACP (fatty acyl-Acyl Carrier Protein) precursors that are essential for membrane biosynthesis [16]. The fatty acyl-ACP chain is built up through iterative decarboxylative Claisen condensation reactions followed by the reduction of the keto groups to generate the final saturated carbon chain. One of the key findings relating to the bacterial FAS II pathway is that it can be deregulated by overexpression of a thioesterase, resulting in the over-accumulation of fatty acids [17]. Extensive work by Khosla’s lab led to the development of strains capable of generating fatty acids with a productivity of 4.5 g/L/day under fed-batch fermentation [18]. Further studies have shown that a broad range of fatty acid hydrocarbon chains of different lengths can be generated by simply varying the substrate specificity of the thioesterase [19]. In a variation of the ACPbased FASII pathway known as reverse beta oxidation, formation of the hydrocarbon chain can also proceed via CoA ester intermediates [20]. This pathway has been utilized for the production of medium chain-length fatty acids with titres of 7 g/L [21]. As is clearly evident from these studies, bacterial fatty acid synthesis pathways can be successfully exploited to generate a range of hydrocarbon backbones for the production of precursors and ready fuels.
Fatty acid derived fuel molecules; alkanes For alkane biosynthesis, the released fatty acid intermediate must first undergo enzyme-catalyzed reduction to generate the corresponding aldehyde precursor (Figure 2). The fatty aldehyde can then be converted www.sciencedirect.com
Renewable jet fuel Kallio et al. 53
Figure 2
Figure 3 Carbon source
(1)
Ala134
(b)
(a) O
Host strain & conditions
Enzyme properties
SCoA
KM
t
ca ;K
Val41
Acetyl-CoA Acetyl-CoA
(2)
(3)
O
O
XhoI
NcoI
SCoA
n
ACP n Fatty acyl-ACP
Fatty acyl-CoA
pk138 5.8kb
(4)
ADO
(d) Pathway optimization
(c) Biosynthetic context
(4) O OH
n Fatty acid
(5) O H n Fatty aldehyde
(6)
n-1 n-alkane Current Opinion in Biotechnology
Alkane production from fatty acids. (1) Native intracellular metabolism generating acetyl-CoA from inorganic and/or organic carbon sources, (2) generation of fatty acyl chains by reverse beta oxidation, (3) generation of fatty acyl chains by the fatty acid synthesis pathway, (4) release of free fatty acids, (5) reduction of fatty acids to fatty aldehydes, (6) alkane generation by aldehyde deformylating oxygenase. Branching of alkanes can occur by petrochemical processing (e.g. hydrocracking and hydro processing) or by modification of the fatty acid pool with b-ketoacyl ACP synthase III [32]. Dashed lines represent multiple reactions; straight lines represent single one-step reactions.
into an alkane by a soluble non-heme diiron enzyme called aldehyde deformylating oxygenase (ADO, previously known as cyanobacterial aldehyde decarbonylase) [22]. This family of enzymes was discovered only recently by genome subtraction analyses of sequenced cyanobacterial strains [23]. The enzyme has subsequently been subjected to intense biochemical characterization by several groups [24–28]. ADO catalyses oxygen-dependent cleavage [26,28] of aldehyde precursors into alkane and formate [29] via a radical-based mechanism [27], displaying www.sciencedirect.com
Current Opinion in Biotechnology
Factors affecting the efficiency of synthetic alkane pathways. (a) Selection of appropriate microbial host and optimizing the culture conditions for biotechnological applications. (b) Structure-based modification for improved enzyme properties, as demonstrated by the change in ADO substrate preference resulting from substitutions in the substrate binding site [30]. (c) Coexpression of associated pathway enzymes for enhanced turnover, such as catalase decomposing H2O2 which otherwise can inhibit ADO [34]. (d) Maximizing the biosynthetic flux towards the end product by optimizing the pathway via, for example, translational tuning [36] or enzyme colocalization [37].
catalytic activity with fatty aldehydes ranging from C4 up to C18 [30]. The reaction is dependent on electrons that can be supplied by either chemical or proteinaceous (ferredoxin) electron acceptor–donors [23,24]. To date, there are only few reports of engineered alkaneproduction systems [23,31,32], and in all cases the yield has been abysmally low. Given that engineered strains produce fatty alcohols from the same aldehyde precursors to a titre above 300 mg/L of culture [21,31] the problem appears to lie at least in part with the low conversion efficiency from aldehyde to alkane. While it is still unclear whether there are yet unidentified enzymes or cofactors needed for maximal activity of ADO, fundamental research continues to provide insight into the primary bottlenecks and approaches to overcome these limitations (Figure 3). Many of the issues can be attributed to the poor kinetic properties of ADO with high KM and low kcat towards the aldehyde substrates, especially for shorter chain-lengths [30]. This poses a potential problem due to competing endogenous reactions such as those catalyzed by highly efficient aldehyde reductases [33], and emphasizes the importance of host strain selection for biotechnological applications (Figure 3a). A complementary Current Opinion in Biotechnology 2014, 26:50–55
54 Plant biotechnology
approach is to engineer the enzyme properties by structure-based active-site modification (Figure 3b), as demonstrated by amino acid substitutions that altered the substrate binding and selectivity of ADO [30]. The local enzyme environment (biosynthetic context, Figure 3c) is also important, and can be modulated by the introduction of additional enzyme components in the pathway. For example, addition of a catalase [34] was shown to enhance ADO turnover by alleviating the inhibitory effects of H2O2 formed in a side-reaction between the electron donor ferredoxin and O2, the second substrate of ADO [35]. Typically, synthetic pathways lack natural evolutionary optimization for optimal performance because the genes have been forced together from heterologous sources. To solve this, the interplay between the consecutive enzymatic steps in the alkane pathways could potentially be improved by translational tuning via RBS optimization [36] or by enzyme colocalization [37] (Figure 3d). The microbial fatty acid biosynthesis pathways mainly yield linear end-products, which alone do not meet the required jet fuel specifications. Further metabolic engineering to introduce geometrical diversification, including branching [38], cyclization [39] or introduction of double bonds [40], may therefore be of interest. The utility of such a concept was shown in a recent study, in which keto-acids, arising from the metabolism of amino acids, were directed through the FAS II machinery of Escherichia coli to form branched alkanes [32].
Conclusions There is a need to explore new opportunities for the production of renewable jet fuel. Currently there are efficient ways to produce renewable synthetic kerosenes from oils and biomass, though they are dependent on the production and collection of biomass as substrate, as well as multi-step processing. Future alternatives may be more competitive if the number of steps required for the conversion of primary substrates into the final fuel is reduced. On the basis of this concept, we have discussed possibilities for microbial production systems in which simple sugars, or ultimately sunlight and CO2, can be directly converted into alkane-based engine-ready jet fuel blending components. Currently, the microbial platforms for hydrocarbon fuel production simply do not offer the productivity, yields or titres for economic feasibility. For the most part these systems have only been demonstrated at a proof-of-concept level and the barrier to commercialization is further exacerbated by the challenge of up-scaling and fuel isolation/purification. Meanwhile, the price of petroleum is likely to increase as the supply of crude oil diminishes, and advances in fundamental and applied biotechnological research will provide new and increasingly competitive alternatives for the inevitable shift towards a sustainable supply of jet fuel. Current Opinion in Biotechnology 2014, 26:50–55
Acknowledgements The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7-ENERGY-2010-1) under grant agreement no. 256808 and European Research Council under the European Union’s Seventh Framework Programme (FP7/2007-2013)/ European Research Council Grant Agreement 260661.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
Platts I: Fuel Price Analysis. IATA: http://www.iata.org/ publications/economics/fuel-monitor/Pages/price-analysis.aspx.
2.
Maurice LQ, Lander H, Edwards T, Harrison WE III: Advanced aviation fuels: a look ahead via a historical perspective. Fuels 2001, 80:747-756.
3.
Hemighaus G, Boval T, Bacha J, Barnes F, Franklin M, Gibbs L, Hogue N, Jones J, Lesnini D, Lind J et al.: Aviation Fuels Technical Review. Chevron Corporation; 2006. A comprehensive and detailed review by the Chevron Corporation on aviation jet fuels and aviation gasolines, their specifications, composition and properties. Despite the early year of release (2006) the publication provides an important foundation for the comparison of jet fuels with the current approaches for sustainable alternatives. 4.
Kinder JD, Rahmes T: Evaluation of Bio-Derived Synthetic Paraffinic Kerosenes (Bio-SPK). The Boeing Company; 2009.
5.
Kaneko TFD, Makino E, Gray D, Tamura M: Coal liquefaction. Ullmann’s Encyclopedia of Industrial Chemistry. Wiley-VCH Verlag GmbH & Co. KGaA; 2000.
Steele P, Pearce B, Rı´os A, Nash P, Massat F, Young N, Bann A, Morgan D, Lakeman M, Scott T et al.: Powering the Future of Flight. Air Transport Action Group; 2012. In this publication Air Transport Action Group (ATAG), a global organization representing the entire commercial aviation sector, evaluates the current status and future of sustainable biofuels in aviation industry. The authors provide insight into the goals and viable approaches taken to enable the production and use of sustainable biofuels, supported by a selection of case studies on related infrastructures and international collaboration.
6.
7.
van der Westhuizen R, Ajam M, De Coning P, Beens J, de Villiers A, Sandra P: Comprehensive two-dimensional gas chromatography for the analysis of synthetic and crudederived jet fuels. J Chromatogr A 2011, 1218:4478-4486. A quantitative and qualitative chemical comparison of synthetic and petroleum-derived jet fuels, which served as a basis for the approval of Fischer–Tropsch technology for the production of renewable hydrocarbon fuel replacements.
8.
McEwen JT, Atsumi S: Alternative biofuel production in nonnatural hosts. Curr Opin Biotechnol 2012, 23:744-750.
9.
Geddes CC, Nieves IU, Ingram LO: Advances in ethanol production. Curr Opin Biotechnol 2011, 22:312-319.
10. Rosgaard L, de Porcellinis AJ, Jacobsen JH, Frigaard NU, Sakuragi Y: Bioengineering of carbon fixation, biofuels, and biochemicals in cyanobacteria and plants. J Biotechnol 2012, 162:134-147. 11. Lan EI, Liao JC: ATP drives direct photosynthetic production of 1-butanol in cyanobacteria. Proc Natl Acad Sci U S A 2012, 109:6018-6023. 12. Ka¨ma¨ra¨inen J, Knoop H, Stanford NJ, Guerrero F, Akhtar MK, Aro EM, Steuer R, Jones PR: Physiological tolerance and stoichiometric potential of cyanobacteria for hydrocarbon fuel production. J Biotechnol 2012, 162:67-74. 13. Ozaki K, Ohta A, Iwata C, Horikawa A, Tsuji K, Ito E, Ikai Y, Harada K: Lysis of cyanobacteria with volatile organic compounds. Chemosphere 2008, 71:1531-1538. 14. Kung Y, Runguphan W, Keasling JD: From fields to fuels: recent advances in the microbial production of biofuels. ACS Synth Biol 2012, 1:498-513. www.sciencedirect.com
Renewable jet fuel Kallio et al. 55
15. Peralta-Yahya PP, Zhang F, del Cardayre SB, Keasling JD: Microbial engineering for the production of advanced biofuels. Nature 2012, 488:320-328. 16. Chan DI, Vogel HJ: Current understanding of fatty acid biosynthesis and the acyl carrier protein. Biochem J 2010, 430:1-19. 17. Cho H, Cronan JE: Defective export of a periplasmic enzyme disrupts regulation of fatty acid synthesis. J Biol Chem 1995, 270:4216-4219. 18. Liu T, Vora H, Khosla C: Quantitative analysis and engineering of fatty acid biosynthesis in E. coli. Metab Eng 2010, 12:378-386. 19. Jing F, Cantu DC, Tvaruzkova J, Chipman JP, Nikolau BJ, Yandeau-Nelson MD, Reilly PJ: Phylogenetic and experimental characterization of an acyl-ACP thioesterase family reveals significant diversity in enzymatic specificity and activity. BMC Biochem 2011, 12:44. The authors screen the chain-length specificity of a wide selection of thioesterases, identifying several new enzymes with potential utility for biofuel production. 20. Inui H, Miyatake K, Nakano Y, Kitaoka S: Fatty acid synthesis in mitochondria of Euglena gracilis. Eur J Biochem 1984, 142: 121-126. 21. Dellomonaco C, Clomburg JM, Miller EN, Gonzalez R: Engineered reversal of the b-oxidation cycle for the synthesis of fuels and chemicals. Nature 2011, 476:355-359. 22. Krebs C, Bollinger JM, Booker SJ: Cyanobacterial alkane biosynthesis further expands the catalytic repertoire of the ferritin-like ‘di-iron-carboxylate’ proteins. Curr Opin Chem Biol 2011, 15:291-303. 23. Schirmer A, Rude M, Li X, Popova E, del Cardayre S: Microbial biosynthesis of alkanes. Science 2010, 329:559-562. A pivotal study in which the authors discover and characterize a new family of enzymes responsible for alkane biosynthesis in cyanobacteria. 24. Das D, Eser BE, Han J, Sciore A, Marsh EN: Oxygen-independent decarbonylation of aldehydes by cyanobacterial aldehyde decarbonylase: a new reaction of diiron enzymes. Angew Chem Int Ed Engl 2011, 50:7148-7152. 25. Eser BE, Das D, Han J, Jones PR, Marsh EN: Oxygenindependent alkane formation by non-heme iron-dependent cyanobacterial aldehyde decarbonylase: investigation of kinetics and requirement for an external electron donor. Biochemistry 2011, 50:10743-10750. 26. Li N, Chang WC, Warui DM, Booker SJ, Krebs C, Bollinger JM: Evidence for only oxygenative cleavage of aldehydes to alk(a/ e)nes and formate by cyanobacterial aldehyde decarbonylases. Biochemistry 2012, 51:7908-7916. 27. Paul B, Das D, Ellington B, Marsh EN: Probing the mechanism of cyanobacterial aldehyde decarbonylase using a cyclopropyl aldehyde. J Am Chem Soc 2013, 135:5234-5237. 28. Li N, Nørgaard H, Warui DM, Booker SJ, Krebs C, Bollinger JM: Conversion of fatty aldehydes to alka(e)nes and formate by a cyanobacterial aldehyde decarbonylase: cryptic redox by an unusual dimetal oxygenase. J Am Chem Soc 2011, 133: 6158-6161. 29. Warui DM, Li N, Nørgaard H, Krebs C, Bollinger JM, Booker SJ: Detection of formate, rather than carbon monoxide, as the
www.sciencedirect.com
stoichiometric coproduct in conversion of fatty aldehydes to alkanes by a cyanobacterial aldehyde decarbonylase. J Am Chem Soc 2011, 133:3316-3319. 30. Khara B, Menon N, Levy C, Mansell D, Das D, Marsh EN, Leys D, Scrutton NS: Production of propane and other short-chain alkanes by structure-based engineering of ligand specificity in aldehyde-deformylating oxygenase. Chembiochem 2013, 14:1204-1208. The authors describe the first work where structure-guided engineering is used in order to modify the chain-length specificity of an aldehyde deformylating oxygenase (formerly aldehyde decarbonylase). The chain-length preference is shifted from medium (C12–C18) to short (C4–C8) chain-lengths. In addition, the nature of the unknown ligand identified in structures of ADO is identified and one engineered variant does not accumulate the ligand. 31. Akhtar MK, Turner NJ, Jones PR: Carboxylic acid reductase is a versatile enzyme for the conversion of fatty acids into fuels and chemical commodities. Proc Natl Acad Sci U S A 2013, 110:87-92. 32. Howard TP, Middelhaufe S, Moore K, Edner C, Kolak DM, Taylor GN, Parker DA, Lee R, Smirnoff N, Aves SJ et al.: Synthesis of customized petroleum-replica fuel molecules by targeted modification of free fatty acid pools in Escherichia coli. Proc Natl Acad Sci U S A 2013, 110:7636-7641. 33. Rodriguez GM, Atsumi S: Isobutyraldehyde production from Escherichia coli by removing aldehyde reductase activity. Microb Cell Fact 2012, 11:90. 34. Andre C, Kim SW, Yu XH, Shanklin J: Fusing catalase to an alkane-producing enzyme maintains enzymatic activity by converting the inhibitory byproduct H2O2 to the cosubstrate O2. Proc Natl Acad Sci U S A 2013, 110:3191-3196. The authors discover an important limiting factor for the functionality of cyanobacteria aldehyde deformylating oxygenase, H2O2, and demonstrate a solution that improves the specific activity. 35. Misra HP, Fridovich I: The generation of superoixide radical during the autoxidation of ferredoxins. J Biol Chem 1971, 246:6886-6890. 36. Zelcbuch L, Antonovsky N, Bar-Even A, Levin-Karp A, Barenholz U, Dayagi M, Liebermeister W, Flamholz A, Noor E, Amram S et al.: Spanning high-dimensional expression space using ribosome-binding site combinatorics. Nucleic Acids Res 2013, 41:e98. 37. Dueber JE, Wu GC, Malmirchegini GR, Moon TS, Petzold CJ, Ullal AV, Prather KL, Keasling JD: Synthetic protein scaffolds provide modular control over metabolic flux. Nat Biotechnol 2009, 27:753-759. 38. Choi KH, Heath RJ, Rock CO: Beta-ketoacyl-acyl carrier protein synthase III (FabH) is a determining factor in branched-chain fatty acid biosynthesis. J Bacteriol 2000, 182:365-370. 39. Grogan DW, Cronan JE: Cloning and manipulation of the Escherichia coli cyclopropane fatty acid synthase gene: physiological aspects of enzyme overproduction. J Bacteriol 1984, 158:286-295. 40. Cahoon EB, Mills LA, Shanklin J: Modification of the fatty acid composition of Escherichia coli by coexpression of a plant acyl–acyl carrier protein desaturase and ferredoxin. J Bacteriol 1996, 178:936-939.
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