Production of advanced biofuels in engineered E. coli

COCHBI-1053; NO. OF PAGES 8

Available online at www.sciencedirect.com

Production of advanced biofuels in engineered E. coli Miao Wen, Brooks B Bond-Watts and Michelle CY Chang Commercial fermentation processes have long taken advantage of the synthetic power of living systems to rapidly and efficiently transform simple carbon sources into complex molecules. In this regard, the ability of yeasts to produce ethanol from glucose at exceptionally high yields has served as a key feature in its use as a fuel, but is also limited by the poor molecular properties of ethanol as a fuel such as high water miscibility and low energy density. Advances in metabolic engineering and synthetic biology allow us to begin constructing new high-flux pathways for production of next generation biofuels that are key to building a sustainable pipeline for liquid transportation fuels. Addresses Departments of Chemistry and Molecular & Cell Biology, University of California, Berkeley, CA 94720-1460, USA Corresponding author: Chang, Michelle CY ([email protected])

Current Opinion in Chemical Biology 2013, 17:xx–yy This review comes from a themed issue on Energy Edited by Stephen Mayfield and Michael D Burkart

S1367-5931/$ – see front matter, # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cbpa.2013.03.034

Introduction Living systems offer a unique solution to targeted chemical synthesis, providing a diverse array of transformations to build inexpensive and sustainable routes to the production of fine and commodity chemicals from simple renewable carbon sources like glucose [1–6]. One major area in which microbial synthesis has been tapped is the production of liquid transportation fuels from renewable plant biomass carbon sources. In this case, the domestication of yeasts throughout human history has allowed the establishment of strains that produce ethanol at near quantitative yield from glucose, which make it the dominant biofuel to date on the basis of the direct relationship between yield and net economic, energy, and carbon balance for fuels [7,8]. However, the low energy density and high water miscibility of ethanol create major limitations in developing a sustainable biofuel pipeline [9–12]. Thus, the development of microbial strains with the ability to produce drop-in gasoline or diesel additives and replacements, — such as longer chain alcohols, esters, and alkanes, — would allow us to begin stepping beyond these issues (Figure 1a). www.sciencedirect.com

A major roadblock in the development of new fermentation processes is that naturally occurring microbial hosts that combine production of desirable gasoline and diesel targets with the efficiency of yeast fermentation have yet to be identified. In this regard, advances in metabolic engineering and synthetic biology offer the opportunity to engineer pathways for the production of advanced biofuel targets in a well-studied and industrially relevant host [13–16]. One challenge is that the metabolic logic underlying the design of high-flux pathways for these advanced targets is quite different from the production of ethanol, which has several advantageous features for high yield. First, ethanol is made directly from pyruvate as a fermentation product that balances the redox requirements of glycolysis (Figure 1b). As a result, its production is required for survival of the host under anaerobic conditions, allowing glycolysis to continue as the sole source of ATP, which generates a selective pressure for quantitative yield from glucose and limits the spread of carbon through the metabolic network. Furthermore, the first step in its biogenesis is the decarboxylation of pyruvate, creating an irreversible sink to drive the pathway equilibrium to completion and immediately commit carbon to its production. In contrast, next-generation fuels are assembled from building blocks derived from pathways that extend beyond glycolysis, which makes their biosynthesis more complex as this aspect greatly expands both the number of metabolic transformations and competing pathways involved (Figure 1b). In addition, the biosynthetic pathways for the production of these advanced targets require many more steps than for ethanol and therefore more components to manage in order to engineer high flux to the end product. In this context, we will discuss the construction of several different engineered pathways for the production of advanced biofuel targets in Escherichia coli.

Production of short-chain alcohols from branched amino acids The addition of two carbons to ethanol to make a C4 alcohol greatly reduces its solubility in water (7 g/L) while also increasing its energy density [13]. Thus, C4 and other short-chain alcohols are currently regarded as the next-generation biofuel to replace ethanol. Besides the energetic benefits, they can also be blended at high percentage with gasoline to meet future government mandates or perhaps even be used as a drop-in fuel for traditional combustion engines [17,18]. Isobutanol has been observed as trace products of microbial amino acid metabolism through the Erlich degradation pathway for 2-keto acids, which are converted to alcohols in two steps (Figure 2a) [19,20]. Like for ethanol production, this Current Opinion in Chemical Biology 2013, 17:1–8

Please cite this article in press as: Wen M, et al.: Production of advanced biofuels in engineered E. coli, Curr Opin Chem Biol (2013), http://dx.doi.org/10.1016/j.cbpa.2013.03.034

COCHBI-1053; NO. OF PAGES 8

2 Energy

(a)

(b)

OH

OH

OH

glycolysis

Figure 1

glucose

PPP

2 NADH, 2 ATP O

n-butanol

O

isobutanol

2

pyruvate

acetaldehyde

- 2 CO2

O_R

n

fatty acid esters

OH

O

branched amino acids

malonyl-CoA

n-butanol fatty acids isoprenoids

SCoA

acetyl-CoA oxaloacetate

isoprenoids

SCoA

O

O

alkanes

fatty alcohols

O _

n

2 x ethanol

H

O

O n

2 NAD+ 2 NADH O

_ - 2 CO2

2 ethanol

xylose

2 NAD+, 2 ADP

branched alcohols malate

citrate isocitrate

TCA cycle

fumarate succinate

α-ketoglutarate succinyl-CoA Current Opinion in Chemical Biology

The production of advanced biofuels. (a) Structures of advanced fuel targets compared to ethanol. (b) Pathways to advanced biofuels from building blocks derived from central metabolism.

Figure 2

(a)

O

(b) alsS

ilvC

ilvD

O

O O

O O

O

2

O

O

HO

O

NH kdc

O

HO

O

pyruvate

OH

adh

2-ketoisovalerate

O H

OH

O O

oxaloacetate

isobutanol

O O

O

O

O O

O

NH

- CO

O

threonine

2-ketobutyrate

OH

propionaldehyde

n-propanol

NH OH

O O

butyraldehyde

keto acid (n + 1)

(c)

H O

alcohol (n + 1)

O

iterative

O

leuABCD

_ O

LeuABCD

keto acid (n)

H

O O

O

isobutyraldehyde

ilvA HO

NH

isoleucine

CO

SCoA

O

O

O

O

2-ketoisocaproate O

acetolactate 2,3-dihydroxyisovalerate 2-ketoisovalerate

threonine

O O

LeuABCD

O

valine

O

O

O

-CO

CO

SCoA O

n-butanol

outside inside

O

Asp Ala

Asn

2-ketoglutarate

Ile, Leu,Val

2-ketovalerate Ser, Thr, Cys, Gly

pyruvate keto butyrate

Glu

fuels 2-ketoglutarate

inside

KMV, KIC, KIV

NH

NH fuels

Arg, Pro, Gln X

glutamate

X

glutamine

outside NH

NH

NH

Current Opinion in Chemical Biology

The production of alcohols from amino acid metabolism. (a) Construction of pathways for isobutanol, n-butanol, and n-propanol from amino acid metabolism. (b) The design of a ‘‘+1’’ pathway for linear alcohols inspired by branched chain amino acid biosynthesis. (c) Engineering nitrogen metabolism to produce alcohols from protein hydrolysates (blue, nitrogen flow; grey, carbon flow). Current Opinion in Chemical Biology 2013, 17:1–8

www.sciencedirect.com

Please cite this article in press as: Wen M, et al.: Production of advanced biofuels in engineered E. coli, Curr Opin Chem Biol (2013), http://dx.doi.org/10.1016/j.cbpa.2013.03.034

COCHBI-1053; NO. OF PAGES 8

Production of advanced biofuels in engineered E. coli Wen, Bond-Watts and Chang 3

particular pathway has the advantage that it can also be driven forward by an irreversible decarboxylation. Work from both academic [21] and industrial groups [22] has shown that this pathway can be exploited to produce a broad range of products by engineering endogenous amino acid metabolism as well as the degradation of 2keto acids. The initial insertion of the Erlich degradation pathway consisting of a broad specificity 2-keto acid decarboxylase (KDC) and alcohol dehydrogenase (ADH) resulted in the production of 1-propanol, 1-butanol, isobutanol, 2methyl-1-butanol, 3-methyl-1-butanol, and 2-phenylethanol in E. coli [21]. On the basis of feeding experiments, the limited availability of 2-keto acid precursors was identified as the pathway bottleneck. Isobutanol titers were therefore improved by overexpressing genes converting pyruvate to 2-ketoisovalerate (alsS and ilvCD), the precursor to isobutanol (Figure 2a). Upon deletion of the gene encoding pyruvate-formate lyase to decrease competition for pyruvate, the engineered E. coli host was able to produce 22 g/L of isobutanol at a yield of 86% of the theoretical maximum [21]. In addition to isobutanol, n-butanol and n-propanol can be produced from 2-ketovalerate by engineering its production from the threonine production pathway (Figure 2a) [23]. The high efficiency of this pathway opens the door for expanded engineering applications, such as new host organisms [24,25] or alcohol products [26]. Since the chain length of the alcohols derived from this pathway is limited by the carbon number in the amino acid pathways, a ‘‘+1’’ pathway derived from the chain extension in leucine biosynthesis has been used to increase the size of the 2-keto acid precursors for alcohol production (Figure 2b) [27]. Another interesting advantage of using amino acid precursors for fuel production is that these pathways provide an alternative use for the protein hydrolysates resulting from microbial fermentations. In order to access these substrates, the carbon skeleton must be released from the amino acid rather than being trapped within nitrogen metabolism by engineering nitrogen flux [28]. To start, amino acids were denitrified by introducing three different transamination and deamination cycles, thereby releasing high levels of ammonia (Figure 2c). To divert carbon flux into alcohol synthesis, a metabolic driving force was created by blocking ammonia from re-entry into cell metabolism. By creating this sink, a mixture of alcohols can be produced at high titers (4.0 g/ L) and yields (56% of theoretical yield) from protein sources.

Production of short-chain alcohols from acetyl-CoA In addition to the production of isobutanol from amino acids, n-butanol can be synthesized directly from acetylCoA in an analogous fashion to fatty acid biosynthesis www.sciencedirect.com

(Figure 3a). n-Butanol is naturally produced by Clostridium acetobutylicum as part of its native fermentation pathways as a mixture of acetone, n-butanol, and ethanol (ABE), a process which was first industrialized in the 1920s [29]. In an effort to improve industrial production, this pathway was transplanted directly into yeast and E. coli but was limited to n-butanol production yields of <1% to 10% [15,30–32], which is significantly lower than the yield in the native butanologenic host (45%). These results suggest that the enzymatic pathway from C. acetobutylicum is limited in its ability to produce n-butanol at high flux in heterologous hosts, with studies implicating the enoyl-CoA reduction step as the bottleneck in the pathway [31,33]. The dependence of n-butanol production on the enoylCoA reduction step led our group to further evaluate its role in controlling pathway flux [33]. Interestingly, E. coli native metabolism does not appear to react with butyryl-CoA while feeding of crotonyl-CoA in the presence of the heterologously expressed pathway lead to reversion to its precursor, 3-hydroxybutyryl-CoA. These observations indicate that the enoyl-CoA reduction may be particularly important for drawing carbon away from the reversible and non-committed steps early in the pathway (PhaA, Keq  10 5; Crt, Keq  10 1) and that an effectively irreversible step was required at this node to drive the pathway equilibrium to completion. Since the thermodynamics of the enoyl-CoA reduction step could not be changed, we utilized a kinetic trap approach instead by replacing the native enoyl-CoA reductase (butyryl-CoA dehydrogenase, Bcd) from C. acetobutylicum with trans-enoyl-CoA reductase (Ter), a mechanistically distinct enzyme with a high kinetic barrier to the back reaction related to its use of a single NADH co-factor [33], leading to 1500-fold amplification in titer, compared to our original pathway design, to >4 g/L (41% of theoretical yield). To evaluate the contribution of Ter as a kinetic trap, we took advantage of its ability to biochemically distinguish trans-butenoyl-CoA and cis-butenoylCoA as substrate. Although the catalytic efficiency of Ter with respect to cis-butenoyl-CoA is 105-fold lower compared to trans-butenoyl-CoA, the n-butanol titer only drops by 10-fold with the incorrect stereochemistry, which demonstrates that the kinetic trapping behavior appears to be the key feature of Ter for increasing flux through the synthetic pathway. This effective irreversibility can be further used to amplify n-butanol titers through anaerobic production in engineered E. coli strains with fermentation pathways knocked out, leading to product titers of 30 g/L under fed-batch conditions [34]. n-Butanol can also be produced by engineering the native metabolism of E. coli and deregulating pathways already present in the host. In this regard, the native boxidation machinery has been engineered to proceed in reverse to synthesize rather than degrade fuel-like molecules [35] (Figure 3b). As such, this pathway is quite Current Opinion in Chemical Biology 2013, 17:1–8

Please cite this article in press as: Wen M, et al.: Production of advanced biofuels in engineered E. coli, Curr Opin Chem Biol (2013), http://dx.doi.org/10.1016/j.cbpa.2013.03.034

COCHBI-1053; NO. OF PAGES 8

4 Energy

Figure 3

(a)

atoB/phaA O

hbd O

crt OH

O

SCoA

SCoA

acetyl-CoA

acetoacetyl-CoA

O SCoA

hydroxybutyryl-Co A

ter

adhE2

O

O SCoA

crotonyl-CoA

butyryl-CoA O

(b)

OH

SCoA

butanol

O SCoA

R

3-ketoacyl-CoA FadB

O

YqeF FadA FadI

FabB FabJ

SCoA

O SCoA OH R

O

O SCoA

R

3-hydroxylacyl-CoA

SCoA

fuels

acyl-CoA

FadB FadJ

FadE

FadB

YdiO O R

SCoA

enoyl-CoA Current Opinion in Chemical Biology

The production of n-butanol from acetyl-CoA. (a) A synthetic pathway for the production of n-butanol on the basis of the clostridial pathway. (b) nButanol production by enabling reversal b-fatty acid oxidation. The outside cycle (black) depicts enzymes possibly involved in n-butanol synthetis. The inner cycle (grey) depicts enzymes in the b-fatty acid oxidation pathway.

similar in its reaction chemistry to the pathways inspired by C. acetobutylicum described above, which are also ATP-independent on the basis of the use of a thiolase to avoid the utilization of malonyl-CoA. An active pathway was constructed by manipulating b-oxidation pathway regulators and catabolic repressors, and overexpressing an endogenous aldehyde dehydrogenases. Titers were further improved by overexpression of various enzymes in the pathway, increasing n-butanol titer to 2 g/L. It is interesting to note that the catalytic activity at each node along the n-butanol pathway is Current Opinion in Chemical Biology 2013, 17:1–8

approximately the same, which suggests that the buildup of pathway intermediates is prevented. In combination with the thermodynamic driving force from diffusion of butanol into bulk media, the carbon is pulled into the engineered n-butanol pathway despite the reversibility of first thiolase-dependent reaction.

Production of diesel targets from acetyl-CoA Beyond gasoline targets, longer-chain products in the diesel range (C12–C20) provide the largest benefit for increasing energy density and reducing the energetic cost www.sciencedirect.com

Please cite this article in press as: Wen M, et al.: Production of advanced biofuels in engineered E. coli, Curr Opin Chem Biol (2013), http://dx.doi.org/10.1016/j.cbpa.2013.03.034

COCHBI-1053; NO. OF PAGES 8

Production of advanced biofuels in engineered E. coli Wen, Bond-Watts and Chang 5

Figure 4 (a)

O _

O O_R

R_OH

O

O

ACP O malonyl-ACP

n fatty acid ester O

O n SCoA fatty acyl-CoA

n H fatty aldehyde

n OH fatty alcohol

O

ACL

O

ACP n 3-keto acyl-ACP FabB FabG FabF NADPH

O

TE

n OH fatty acid

OH

ACP n 3-hydroxyl acyl-ACP

n ACP acyl-ACP

- CO

O

FabI NADH

FabA FabZ O

n alkane

ACP n enoyl-ACP

atoB

(b)

HMGS

O

O

tHMGR O

O

SCoA

SCoA

acetyl-CoA

acetoacetyl-CoA

O

SCoA

O

5-phosphomevalonate

OH

O

HMG-CoA

O OP

OH

_

mevalonate idi

MDV1

OH

O

O

OH O

_

ERG8

_

ERG12

OH

_

OPP

OPP

O

5-pyrophosphomevalonate

ispA

IPP

bis

OPP DMAPP

chemical reduction

OPP farnesyl pyrophosphate

bisabolene

bisabolane Current Opinion in Chemical Biology

The production of biodiesel from acetyl-CoA. (a) Biosynthesis and tailoring of fatty acids (TE, thioesterase; ACL, acyl-CoA ligase). (b) Engineering the isoprenoid pathway for the production of biodiesels.

for product separation. Two major metabolic routes to hydrocarbons of this chain length have been engineered from fatty acid biosynthesis and the isoprenoid pathway, both of which utilize the acetyl-CoA building block to build the backbone (Figure 1b). However, each pathway offers different advantages with respect to fuel production in terms of yields and product diversity. Fatty acid esters derived from plant oils are already in use today as biodiesels, but are costly to produce from an agricultural perspective [36]. Thus, microbial fermentation provides an option with greater potential for longterm sustainability. Overall, the engineering of fatty acidderived biodiesels breaks down into two phases, biosynthesis of fatty acids and their tailoring to produce the desired product (Figure 4a). Fatty acid biosynthesis is highly regulated at several different points because the intermediate acyl-ACP is primarily converted to membrane phospholipids. One of the most important control www.sciencedirect.com

points is the feedback inhibition by fatty-acyl ACPs of acetyl-CoA carboxylase (ACCase), which catalyzes the ATP-dependent carboxylation of acetyl-CoA to form malonyl-CoA as the first committed step in fatty acid biosynthesis [37]. Two approaches have been taken to increase the malonyl-CoA pool by overexpressing the ACCase [38] as well as an acyl-ACP thioesterase to release the free fatty acid product [38,39]. Since free fatty acids are degraded back to acetyl-CoA, the b-oxidation pathway is also blocked by deletion of either fadD or fadE. The productivity of these engineered strains has been found to increase the free fatty acid content of E. coli by 20–40-fold to produce 1200–1500 mg/L of product at yields that are 6–14% of the theoretical limit [38,39]. Although these genetic modifications substantially increase the free fatty acid content, additional cell-free [40] and in vitro [41] studies of fatty acid biosynthesis have the potential to provide new insight into additional strategies to amplify fatty acid titers and yields. Current Opinion in Chemical Biology 2013, 17:1–8

Please cite this article in press as: Wen M, et al.: Production of advanced biofuels in engineered E. coli, Curr Opin Chem Biol (2013), http://dx.doi.org/10.1016/j.cbpa.2013.03.034

COCHBI-1053; NO. OF PAGES 8

6 Energy

A variety of strategies have been explored for converting fatty acids into different biofuels, such as alcohols, esters, and alkanes (Figure 1a). As fatty acid esters are already currently in use, initial efforts have focused on engineering their production in vivo rather relying on post-fermentation chemical processing. In this regard, a pathway for the production of fatty acid ethyl esters (FAEE) has been incorporated into fatty acid overproduction strains by insertion of an acyl-CoA ligase and a broad-specificity acyltransferase [42,43] along with two enzymes to provide ethanol for transesterification (Figure 4a) [39]. Since extension of the pathway to the FAEE drops productivity twofold to 700 mg/L, it is likely that metabolic imbalance related to rapid accumulation of ethanol and secretion of free fatty acids is limiting in this system. Therefore, a dynamic sensor-regulator system was designed to tune the expression of the ethanol and FAEE production modules in response to acyl-CoA availability. Upon introduction of a FadR-based biosensor, the extracellular accumulation of free fatty acid decreased and led to an overall increase of FAEE production from 9.4% to 28% of the maximum theoretical yield [44]. To expand the promise of utilizing engineered E. coli for biodiesel production on an industrially relevant scale, a consolidated bioprocess has been developed to allow the FAEE-producing E. coli to use cellulose and hemicellulose from ionic liquid-pretreated switchgrass as carbon source at 80% of estimated theoretical yield [45]. Beyond fatty acid esters, the fermentation product that would most closely reproduce diesel is a mixture of alkanes. Alkanes have been reported in a diversity of microorganisms, including cyanobacteria, and are found to contain an odd number of carbons. Since heptadecane is the most abundant alkane reported, it appears that alkanes are derived from the ‘‘n 1’’ decarbonylation of aldehydes. The two genes involved in this process were found in Synechococcus elongatus by comparative genome analysis and used to engineer the alkane production in E. coli [46]. In conjunction with the identification of a gene involved in ‘‘n + 1’’ pathways [47], these discoveries provide promise for our ability to access deoxygenated hydrocarbons through this pathway. Isoprenoids are another class of hydrocarbons made in biological systems that could serve as biodiesels. It has been demonstrated that E. coli is able to produce sesquiterpene at high titer (27 g/L) when a heterologous mevalonate pathway is introduced to bypass the regulation of native deoxyxylulose-5-phosphate pathway the (Figure 4b) [48]. Because of the modularity of isoprenoid biosynthesis, this strain can also be utilized for the production of any C15 product by simply changing the terpene synthase. In this regard, bisabolane, the hydrogenated product of bisabolene, has been demonstrated to be a biosynthetic alternative to D2 diesel fuel (Figure 4b) [49]. Since terpene synthases catalyze the cyclization of FPP with loss of pyrophosphate, they have been found to Current Opinion in Chemical Biology 2013, 17:1–8

act as an irreversible sink that controls product titers. Thus, five bisabolene synthases from plants were screened for their productivity for bisabolene. Upon codon-optimization of the best candidate, bisabolene could be produced at 900 mg/L and isolated for reduction to the biodiesel product. Because of the strong influence of the terpene synthase on productivity, further discovery or engineering efforts to optimize their activity will allow the construction of strains with higher fuel yields.

Conclusions Advanced biofuels offer significant advantages in terms of fuel properties and process design, however these pathways remain challenging to engineer with the efficiency and robust performance of yeast ethanol production. Towards this goal, high-flux pathways for production of both gasoline and diesel range fuels can be constructed with the introduction of a sink or driving force to push the pathway equilibrium to completion. In addition, advances in downstream chemical processing of shorter chain products can allow upgrading of these fuels for biodiesel production [50,51].

Acknowledgements Research in our laboratory is funded by the generous support of the University of California, Berkeley, the Energy Biosciences Institute, the Camille and Henry Dreyfus Foundation, the Arnold and Mabel Beckman Foundation, the Dow Sustainable Products and Solutions Program, the Agilent Foundation, the Hellman Family Foundation, the National Science Foundation, the National Institutes of Health, the Department of Energy, and the Defense Advanced Research Projects Agency.

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.

Glazer AN, Nikaido H: Microbial Biotechnology: Fundamentals of Applied Microbiology. New York: W.H. Freeman and Co.; 1995, .

2.

An Z (Ed): Handbook of Industrial Mycology. New York: Marcel Dekker; 2005.

3.

Keasling JD: Synthetic biology for synthetic chemistry. ACS Chem Biol 2008, 3:64-76.

4.

Escalante A, Calderon R, Valdivia A, de Anda R, Hernandez G, Ramirez O, Gosset G, Bolivar F: Metabolic engineering for the production of shikimic acid in an evolved Escherichia coli strain lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system. Micro Cell Fact 2010, 9:21.

5.

Yadav VG, Stephanopoulos G: Reevaluating synthesis by biology. Curr Opin Microbiol 2010, 13:371-376.

6.

Weeks AM, Chang MCY: Constructing de novo biosynthetic pathways for chemical synthesis inside living cells. Biochemistry 2011, 50:5404-5418.

7.

Lin Y, Tanaka S: Ethanol fermentation from biomass resources: current state and prospects. Appl Microbiol Biotechnol 2006, 69:627-642.

8.

DOE: The Annual Energy Review 2010. Washington, DC: U.S. Department of Energy; 2010, .

9.

Fischer CR, Klein-Marcuschamer D, Stephanopoulos G: Selection and optimization of microbial hosts for biofuels production. Metab Eng 2008, 10:295-304. www.sciencedirect.com

Please cite this article in press as: Wen M, et al.: Production of advanced biofuels in engineered E. coli, Curr Opin Chem Biol (2013), http://dx.doi.org/10.1016/j.cbpa.2013.03.034

COCHBI-1053; NO. OF PAGES 8

Production of advanced biofuels in engineered E. coli Wen, Bond-Watts and Chang 7

10. Rude MA, Schirmer A: New microbial fuels: a biotech perspective. Curr Opin Microbiol 2009, 12:274-281. 11. Peralta-Yahya PP, Zhang F, del Cardayre SB, Keasling JD: Microbial engineering for the production of advanced biofuel. Nature 2012, 488:320-328. 12. Lennen RM, Pfleger BF: Engineering Escherichia coli to synthesize free fatty acids. Trends Biotechnol 2012, 30:660-667. 13. Lee SK, Chou H, Ham TS, Lee TS, Keasling JD: Metabolic engineering of microorganisms for biofuels production: from bugs to synthetic biology to fuels. Curr Opin Biotechnol 2008, 19:556-563. 14. Atsumi S, Liao JC: Metabolic engineering for advanced biofuels production from Escherichia coli. Curr Opin Biotechnol 2008, 19:414-419. 15. Nielsen DR, Leonard E, Yoon SH, Tseng HC, Yuan C, Prather KL: Engineering alternative butanol production platforms in heterologous bacteria. Metab Eng 2009, 11:262-273. 16. Liu T, Khosla C: Genetic engineering of Escherichia coli for biofuel production. Annu Rev Genet 2010, 44:53-69. 17. Mehta RN, Chakraborty M, Mahanta P, Parikh PA: Evaluation of fuel properties of butanol-biodiesel-diesel blends and their impact on engine performance and emissions. Ind Eng Chem Res 2010, 49:7660-7665. 18. Szwaja S, Naber JD: Combustion of n-butanol in a sparkignition IC engine. Fuel 2010, 89:1573-1582. 19. Dickinson JR, Harrison SJ, Hewlins MJ: An investigation of the metabolism of valine to isobutyl alcohol in Saccharomyces cerevisiae. J Biol Chem 1998, 273:25751-25756. 20. Dickinson JR, Harrison SJ, Dickinson JA, Hewlins MJ: An investigation of the metabolism of isoleucine to active amyl alcohol in Saccharomyces cerevisiae. J Biol Chem 2000, 275:10937-10942. 21. Atsumi S, Hanai T, Liao JC: Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels. Nature 2008, 451:86-89. 22. Bramucci MG, Flint D, Miller ES, Nagarajan V, Sedkova N, Singh M, Van Dyk TK: Method for the production of isobutanol. US Patent 2008, 20080274526 23. Shen CR, Liao JC: Metabolic engineering of Escherichia coli for 1-butanol and 1-propanol production via the keto-acid pathways. Metab Eng 2008, 10:312-320. 24. Atsumi S, Higashide W, Liao JC: Direct photosynthetic recycling of carbon dioxide to isobutyraldehyde. Nat Biotechnol 2009, 27:1177-1180. 25. Li H, Opgenorth PH, Wernick DG, Rogers S, Wu TY, Higashide W, Malati P, Huo YX, Cho KM, Liao JC: Integrated electromicrobial conversion of CO2 to higher alcohols. Science 2012, 335:1596. 26. Zhang K, Sawaya MR, Eisenberg DS, Liao JC: Expanding metabolism for biosynthesis of nonnatural alcohols. Proc Natl Acad Sci U S A 2008, 105:20653-20658. 27. Marcheschi RJ, Li H, Zhang K, Noey EL, Kim S, Chaubey A, Houk KN, Liao JC: A synthetic recursive ‘‘+1’’ pathway for carbon chain elongation. ACS Chem Biol 2012, 7:689-697. 28. Huo Y-X, Cho KM, Rivera JGL, Monte E, Shen CR, Yan Y, Liao JC:  Conversion of proteins into biofuels by engineering nitrogen flux. Nat Biotechnol 2011, 29:346-351. To tap into carbon sources trapped in nitrogen containing compounds, three transamination and deamination cycles are introduced into host’s metabolism. A thermodynamic driving force is created that allows utilization of protein hydrosylate as carbon source by blocking ammonia assimilation pathways. 29. Jones DT, Woods DR: Acetone–butanol fermentation revisited. Microbiol Rev 1986, 50:484-524. 30. Inui M, Suda M, Kimura S, Yasuda K, Suzuki H, Toda H, Yamamoto S, Okino S, Suzuki N, Yukawa H: Expression of Clostridium acetobutylicum butanol synthetic genes in Escherichia coli. Appl Microbiol Biotechnol 2008, 77:1305-1316. www.sciencedirect.com

31. Atsumi S, Cann AF, Connor MR, Shen CR, Smith KM, Brynildsen MP, Chou KJY, Hanai T, Liao JC: Metabolic engineering of Escherichia coli for 1-butanol production. Metab Eng 2008, 10:305-311. 32. Steen EJ, Chan R, Prasad N, Myers S, Petzold CJ, Redding A, Ouellet M, Keasling JD: Metabolic engineering of Saccharomyces cerevisiae for the production of n-butanol. Microb Cell Fact 2008, 7:36. 33. Bond-Watts BB, Bellerose RJ, Chang MCY: Enzyme mechanism  as a kinetic control element for designing synthetic biofuel pathways. Nat Chem Biol 2011, 7:222-227. Production of n-butanol was increased through the introduction of a kinetic trap at the enoyl-CoA reduction step, trapping carbon in the synthetic pathway. 34. Shen CR, Lan EI, Dekishima Y, Baez A, Cho KM, Liao JC: Driving  forces enable high-titer anaerobic 1-butanol synthesis in Escherichia coli. Appl Environ Microbiol 2011, 77:2905-2915. Modifying the fermentation pathways of E. coli, the native reactions to use acetyl-CoA and oxidize NADH were eliminated. These metabolic modifications allowed for the build-up of a driving force that was used to increase flux through the n-butanol producing pathways. 35. 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. Engineering of E. coli native metabolism was utilized to produce nbutanol. The transcriptional regulators of b-oxidation in E. coli were mutated to decouple the pathway from environmental signals. In addition several native enzymes of the b-oxidation pathway were overexpressed to allow for high flux through the pathway in the reverse direction. 36. Hill J, Nelson E, Tilman D, Polasky S, Tiffany D: Environmental, economic, and energetic costs and benefits of biodiesel and ethanol biofuels. Proc Natl Acad Sci U S A 2006, 103:1120611210. 37. Magnuson K, Jackowski S, Rock CO, Cronan JE Jr: Regulation of fatty acid biosynthesis in Escherichia coli. Microbiol Rev 1993, 57:522-542. 38. Lu X, Vora H, Khosla C: Overproduction of free fatty acids in E. coli: implications for biodiesel production. Metab Eng 2008, 10:333-339. 39. Steen EJ, Kang Y, Bokinsky G, Hu Z, Schirmer A, McClure A, del Cardayre SB, Keasling JD: Microbial production of fatty-acidderived fuels and chemicals from plant biomass. Nature 2010, 463:559-562. 40. Liu T, Vora H, Khosla C: Quantitative analysis and engineering of fatty acid biosynthesis in E. coli. Metab Eng 2010, 12:378386. 41. Yu X, Liu T, Zhu F, Khosla C: In vitro reconstitution and steadystate analysis of the fatty acid synthase from Escherichia coli. Proc Natl Acad Sci U S A 2011, 108:18643-18648. 42. Kalscheuer R, Steinbuchel A: A novel bifunctional wax ester synthase/acyl-CoA:diacylglycerol acyltransferase mediates wax ester and triacylglycerol biosynthesis in Acinetobacter calcoaceticus ADP1. J Biol Chem 2003, 278:8075-8082. 43. Stoveken T, Kalscheuer R, Malkus U, Reichelt R, Steinbuchel A: The wax ester synthase/acyl coenzyme A:diacylglycerol acyltransferase from Acinetobacter sp. strain ADP1: characterization of a novel type of acyltransferase. J Bacteriol 2005, 187:1369-1376. 44. Zhang F, Carothers JM, Keasling JD: Design of a dynamic  sensor-regulator system for production of chemicals and fuels derived from fatty acids. Nat Biotechnol 2012, 30:354-359. Using an E. coli strain capable of producing FAEEs, the authors introduced a dynamic sensor-regulator system to control the production of ethanol and fatty acids on the basis of the availability of acyl-CoA, the limiting component in FAEE synthesis. Using this regulation system the authors were able to improve strain stability and increase FAEE production. 45. Bokinsky G, Peralta-Yahya PP, George A, Holmes BM, Steen EJ, Dietrich J, Soon Lee T, Tullman-Ercek D, Voigt CA, Simmons BA et al.: Synthesis of three advanced biofuels from ionic liquidpretreated switchgrass using engineered Escherichia coli. Proc Natl Acad Sci U S A 2011, 108:19949-19954. Current Opinion in Chemical Biology 2013, 17:1–8

Please cite this article in press as: Wen M, et al.: Production of advanced biofuels in engineered E. coli, Curr Opin Chem Biol (2013), http://dx.doi.org/10.1016/j.cbpa.2013.03.034

COCHBI-1053; NO. OF PAGES 8

8 Energy

46. Schirmer A, Rude MA, Li X, Popova E, del Cardayre SB: Microbial biosynthesis of alkanes. Science 2010, 329: 559-562. 47. Mendez-Perez D, Begemann MB, Pfleger BF: Modular synthaseencoding gene involved in a-olefin biosynthesis in Synechococcus sp. strain PCC 7002. Appl Environ Microbiol 2011, 77:4264-4267. 48. Martin VJJ, Pitera DJ, Withers ST, Newman JD, Keasling JD: Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nat Biotechnol 2003, 21: 796-802.

Current Opinion in Chemical Biology 2013, 17:1–8

49. Peralta-Yahya PP, Ouellet M, Chan R, Mukhopadhyay A, Keasling JD, Lee TS: Identification and microbial production of a terpene-based advanced biofuel. Nat Commun 2011, 2:483. 50. Anbarasan P, Baer ZC, Sreekumar S, Gross E, Binder JB, Blanch HW, Clark DS, Toste FD: Integration of chemical catalysis with extractive fermentation to produce fuels. Nature 2012, 491:235-239. 51. Guillena G, Ramo´n DJ, Yus M: Alcohols as electrophiles in C–C bond-forming reactions: the hydrogen autotransfer process. Angew Chem Int Ed 2007, 46:2358-2364.

www.sciencedirect.com

Please cite this article in press as: Wen M, et al.: Production of advanced biofuels in engineered E. coli, Curr Opin Chem Biol (2013), http://dx.doi.org/10.1016/j.cbpa.2013.03.034