Available online at www.sciencedirect.com

ScienceDirect Metabolic engineering for the production of hydrocarbon fuels Sang Yup Lee1,2, Hye Mi Kim1 and Seungwoo Cheon1 Biofuels have been attracting increasing attention to provide a solution to the problems of climate change and our dependence on limited fossil oil. During the last decade, metabolic engineering has been performed to develop superior microorganisms for the production of so called advanced biofuels. Among the advanced biofuels, hydrocarbons possess high-energy content and superior fuel properties to other biofuels, and thus have recently been attracting much research interest. Here we review the recent advances in the microbial production of hydrocarbon fuels together with the metabolic engineering strategies employed to develop their production strains. Strategies employed for the production of long-chain and short-chain hydrocarbons derived from fatty acid metabolism along with the isoprenoid-derived hydrocarbons are reviewed. Also, the current limitations and future prospects in hydrocarbon-based biofuel production are discussed. Addresses 1 Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus Program), BioProcess Engineering Research Center, and Center for Systems and Synthetic Biotechnology, Institute for the BioCentury Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon 305-701, Republic of Korea 2 Bioinformatics Research Center, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 305-701, Republic of Korea Corresponding author: Lee, Sang Yup ([email protected], [email protected])

Current Opinion in Biotechnology 2015, 33:15–22 This review comes from a themed issue on Energy biotechnology Edited by E Terry Papoutsakis and Jack T Pronk

http://dx.doi.org/10.1016/j.copbio.2014.09.008 0958-1669/Published by Elsevier Ltd.

and corn because several microorganisms naturally produce it at high level. Bioethanol can be used as a fuel itself or fuel additive to gasoline at different percentages. However, ethanol has lower energy content than gasoline. Also, it is hygroscopic, which causes the corrosiveness to transportation and storage infrastructures [1,2]. Higher alcohols such as isopropanol, butanol, and isobutanol are considered as better biofuels than ethanol due to their higher energy density and less hygroscopicity [3–5]. Despite of these properties superior to bioethanol, however, current applications of higher alcohols are rather limited to their use as fuel additives [3,6]. In petrochemical refinery, crude oil is fractionated according to the boiling point of the molecules (Figure 1a). Typical liquid transportation fuels, commonly referred to as gasoline, jet fuel (kerosene), and diesel, are hydrocarbons, of which carbon chain lengths are distributed from C5 to C20, possessing energy density around 40 MJ/kg [7]. Long-chain hydrocarbons (C10– C23) are major components of jet fuel and diesel, whereas short-chain hydrocarbons (C5–C12) are used as gasoline [2,7]; it should be noted that the definitions of ‘longchain’ and ‘short-chain’ hydrocarbons can vary from one study to another. Recently, development of bio-based processes for the production of hydrocarbons has attracted great interest with the expectation to replace such petroleum-derived hydrocarbons using renewable biomass or even carbon dioxide as a raw material (Figure 1b). For the bio-based production of hydrocarbons and their derivatives, metabolic engineering strategies have been focused on microbial fatty acid and lipid metabolism and isoprenoid pathways for the production of long-chain (C10–C23) and short-chain (C5–C12) hydrocarbons [2,8–10,11]. In this paper, metabolic engineering strategies employed for the microbial production of hydrocarbon fuels are reviewed, and their limitations together with future prospects are discussed.

Production of diesel and long-chain hydrocarbons Introduction Conventional petroleum-based chemical industry, although economically still thriving, is now facing great socio-political challenges due to the increasing concerns on climate change and limited fossil resources. Thus, there has been much interest in developing biorefineries for the production of fuels and chemicals from renewable resources. Bioethanol has been the most prevalent biofuel produced from carbon sources derived from sugarcane www.sciencedirect.com

As shown in Figure 1b, the feedstocks for biodiesel production can be categorized into edible oils, non-edible oils, and microbial lipids [12]. Biodiesel has been conventionally produced by stepwise processes from feedstock treatment to trans-esterification of the edible and non-edible oil feedstocks [13,14]. However, the use of these feedstocks raised some concerns due to the several problems such as ‘food versus fuel’ issue, fuel quality, or farmland utilization. In order to overcome such concerns, Current Opinion in Biotechnology 2015, 33:15–22

16 Energy biotechnology

Figure 1

(a)

(b)

Bio-based production of hydrocarbon fuels E. coli

in vivo

Fractional distillation of crude oil

Nonane yeast

C5-C9 (Chemicals)

C20-C50 (Lubricating oil) C20-C70 (Ship oil)

Tetradecane

2nd generation feedstocks Jatropha Beef tallow Jojoba oil Tabacco oil Pork lard

3rd generation feedstocks (Oleaginous microorganisms)

Hexadecane

Limonene

microalgae

in vitro

Crude oil

C10-C23 (Diesel and jet fuels)

Tridecane 1st generation feedstocks Soybean Coconut Sunflower Rapeseed Palm oil

C5-C12 (Gasoline)

1-Octene

bacteria

in vivo

C1-C4 (LPG)

Dodecane

Monodus subterraneus Nannochloropsis sp. Schizochytrium sp. 39.3% 31-68% 50-77%

Oil content (% dry wt)

Pinene

yeasts Lipomyces starkeyi 68% Oil content (% dry wt)

Candida curvata 58%

Cryptococcus albidus 58%

Anthrobacter sp. 40%

Rhodococcus opacus 88%

Farnesene

bacteria

FAEEs Oil content (% dry wt)

Gordonia sp. 72%

Bisabolene

in vivo products Current Opinion in Biotechnology

Comparison of petroleum-based and bio-based processes for the production of hydrocarbons. (a) In conventional petroleum distillation process, the types of fuel product are fractionated according to the carbon chain lengths for their appropriate uses. In general, C5–C23 hydrocarbons can be used as transportation fuels: long-chain hydrocarbons as diesel and jet fuel and short-chain hydrocarbons as gasoline. (b) So far, the typical way of producing bio-based hydrocarbon fuels has been in vitro transesterification of natural lipids: the first generation edible oils, the second generation nonedible oils, and the third generation microbial lipids. More recently, metabolic engineering studies have been performed for the in vivo production of such hydrocarbon fuels in engineered microorganisms.

microbial oils have risen as one of the most promising alternative sources for biodiesel production (Figure 1b). A representative third generation biodiesel feedstock is microalgae, which can accumulate lipid to 80% of the dry cell weight [15]. In addition to the high oil content reachable, the capabilities of utilizing carbon dioxide as a primary carbon source [16] and growing on non-potable wastewater [17] make microalgae an eco-friendly biodiesel feedstock. Production of microalgae-based biodiesel has been studied with respect to three main aspects: selection of an appropriate species [18,19], optimization of culture condition for the enhancement of lipid productivity [20–22], and development of an efficient lipid extraction procedure [23]. The main reason for the limited metabolic engineering studies on microalgae for biofuel production is the lack of appropriate genetic manipulation tools. The gene manipulation tools for microalgae are being actively developed for metabolic engineering studies [24,25]. More information on the Current Opinion in Biotechnology 2015, 33:15–22

current status and future aspects of microalgae-based biorefinery processes can be found elsewhere [26,27]. A number of yeast species belonging to the genus Candida, Cryptococcus, Rhodotorula and Yarrowia are known to accumulate significant amount of lipids (up to 70% of the biomass in some yeasts) comprised of C16:0, C18:0, C18:1, and C18:2 fatty acids under suitable nutrient limiting condition in the presence of excessive carbon source [28]. Among these, Yarrowia lypolytica has been the most representative model species for metabolic engineering due to the availability of suitable metabolic engineering tools and its full genome sequence [29]. Therefore, Y. lypolytica has been actively studied for the production of long-chain hydrocarbons although its wild-type strain accumulates lipid to the level less than that other yeast strains do [28]. For example, Tai and Stephanopoulos [30] developed an expression system using the introncontaining translation elongation factor 1-a promoter and www.sciencedirect.com

Microbial production of hydrocarbons Lee, Kim and Cheon 17

used it to overexpress diacylglycerol acyltransferase, which is the final step of the triacylglycerol biosynthesis. This resulted in the accumulation of lipid up to 33.8% of dry cell weight, which was a fourfold higher than that obtained with the wild-type strain. When diacylglycerol acyltransferase and acetyl-CoA carboxylase were overexpressed together, cells were able to accumulate lipid up to 61.7% of dry cell weight with the overall yield and productivity of 0.270 g/g and 0.253 g/L/hour, respectively, in 2-L fermentation [30]. In another study, more thorough systems metabolic engineering was performed to enhance lipid production followed by direct chemical conversion of the lipid to diesel [31]. Optimization of the native lipogenic pathway and central metabolism was performed through combinatorial multiplexing of lipogenesis targets with phenotypic induction by overexpressing five enzymes, AMP deaminase, ATP-citrate lyase,

malic enzyme, and acetyl-CoA:diacylglycerol acyltransferases I and II, in four Y. lypolytica strains having different genetic backgrounds. After optimization of fermentation condition, the best strain was able to produce 25 g/L of lipid with the content of as high as 90% of dry cell weight [31]. In the aforementioned studies, lipids were first produced and then were converted to diesel by in vitro transesterification. Such additional in vitro processes including prelipid extraction treatment and lipid separation steps inevitably reduce the cost-effectiveness and overall production efficiencies. Thus, much effort has been exerted to develop microorganisms that can accumulate lipid and convert it to diesel in vivo (Figure 2). In this aspect, yeasts are good candidates for in vivo diesel production, in particular fatty acid ethyl esters (FAEEs) because they

Figure 2

xyn10B

xsa

(a)

Glucose

Xylose

Hemicellulose

Glycerol gup1

Glucose-6-P

Glucose

Ribulose-5-P

Glycolysis

(b)

Dihydroxyacetone

gcy1

Glycerol

Xylulose-5-P dak1

Glyceraldehyde3-P dxr

Deoxyxylulose phosphate

dxps pdc

Glycerol 3phosphate

adhB

Acetaldehyde

Pyruvate

fps1

Glycerol

ack

Acetate

Acetyl-P

Ethanol Dimethylallyl diphosphate

idi

Isopentenyl diphosphate

Acetyl-CoA

gpps

Limonene Geranyl pyrophosphate

ms

wax-dgaT ws2

(c) Fatty acyl-ACP

Pinene

Oleic acid

pta

atoB

AcetoacetylCoA

Mevalonate

Oleoyl-CoA

Glycerol Oleic acid

2-C-Methylerythritol-4phosphate

Dihydroxyacetone phosphate

orf1594

Fatty aldehyde orf1593

‘tesA

C12-C23 hydrocarbons

fpps

Farnesene

fadD

Free fatty acid

Fatty acyl-CoA

fadD

C10-C15 hydrocarbons

ss

Farnesyl pyrophosphate

Bisabolene

Fatty acyl-CoA

acr

Fatty aldehyde

CER1

C5-C12 hydrocarbons Current Opinion in Biotechnology

Metabolic pathways for the microbial production of hydrocarbon fuels. Metabolic pathways for the biosynthesis of long-chain and short-chain hydrocarbons have been established in various microorganisms. A solid line arrow indicates a single enzyme reaction, while a dotted line arrow indicates a set of multiple enzyme reactions. (a) Fatty acid ethyl ester (FAEE) biosynthesis is catalyzed by a wax ester synthase (wax-dgaT, ws2), which condenses fatty acyl-CoA and ethanol. Besides glucoses, alternative carbon sources such as oleic acid and glycerol can also be used for FAEE production. (b) Cyclic or branched hydrocarbons are produced from isoprenoids generated through the mevalonate (MEV) pathway or deoxyxylulose phosphate (DXP) pathway. (c) Production of short-chain hydrocarbons (gasoline) has successfully been demonstrated via engineering of fatty acid metabolism in E. coli and establishing a heterologous pathway toward hydrocarbon biosynthesis. Gene abbreviations are: ack, acetate kinase; acr, acyl-CoA reductase; adhB, alcohol dehydrogenase B; atoB, acetyl-CoA acetyltransferase; CER1, fatty aldehyde decarbonylase I; dxps, deoxyxylulose phosphate synthase; dak1, dihydroxyacetone kinase; dxr, deoxyxylulose phosphate reductoisomerase; fadD, acyl-CoA synthetase; fpps, farnesyl pyrophosphate synthase; gcy1, glycerol dehydrogenase; gpps, geranyl pyrophosphate synthase; gup1, glycerol uptake protein; idi, isopentenyl pyrophosphate isomerase; ms, monoterpene synthases; orf1593, fatty aldehyde decarbonylase; orf1594, fatty acyl-ACP reductase; pdc, pyruvate decarbonylase; pta, phosphotransacetylase; ss, sesquiterpene synthases; ‘tesA, Acyl-Acyl carrier protein thioesterase I; wax-dgaT, wax ester synthase/acyl-coenzyme A:diacylglycerol acyltransferase; ws2, wax ester synthase 2; xsa, xylanase; xyn10B, endoxylanase catalytic domain. www.sciencedirect.com

Current Opinion in Biotechnology 2015, 33:15–22

18 Energy biotechnology

naturally produce ethanol. By introducing wax ester synthase (ws), FAEEs can be biosynthesized by esterifying fatty acyl-CoAs with ethanol, both produced in vivo. Five different wax ester synthases isolated from Acinetobacter baylyi ADP1, Marinobacter hydrocarbonoclasticus DSM 8798, Rhodococcus opacus PD630, Mus musculus C57BL/6, and Psychrobacter arcticus 273-4 have mainly been studied. These enzymes were evaluated for FAEE production in Saccharomyces cerevisiae. Among the engineered S. cerevisiae strains examined, the one overexpressing the M. hydrocarbonoclasticus wax ester synthase produced the highest level of FAEEs (6.3 mg/L) from glucose. Moreover, up-regulation of acetyl-CoA carboxylase expression further improved the FAEE production by 30% [32]. In their continued study, two strategies for increasing the NADPH and acetyl-CoA pools required for acyl-CoA synthesis were employed for the enhanced production of FAEEs [33]. First, the carbon flow toward acetyl-CoA was re-channeled by employing the ethanol degradation pathway through the overexpression of alcohol dehydrogenase (ADH2), acetaldehyde dehydrogenase (ALD6), a heterologous acetyl-CoA synthase (acsSEL641P), and M. hydrocarbonoclasticus wax ester synthase. Second, the phosphoketolase pathway was established in S. cerevisiae by expressing the Aspergillus nidulans xpkA and ack genes. When the M. hydrocarbonoclasticus wax ester synthase was co-expressed, the FAEE production was successfully improved by 1.7-fold in the engineered strain compared to the control [33]. In a different study, the use of glycerol, a byproduct of biodiesel industry, was also examined for the production of FAEEs by S. cerevisiae. When the bi-functional wax ester synthase/acyl-coenzyme A:diacylglycerol acyltransferase (wax-dgaT) from A. baylyi ADP1 and the glycerol utilizing genes were introduced into S. cerevisiae, the engineered strain produced 0.24 g/L of FAEEs from glycerol and externally fed oleic acid. When the glycerol exporter was deleted in this strain, production of 0.52 g/L of FAEEs was achieved [34]. As can be seen from the examples above, the titers of FAEEs produced by engineered S. cerevisiae strains have been rather low, suggesting that the studies so far conducted have been rather proofof-concept demonstration that yeasts can be engineered to produce FAEEs. Escherichia coli has been widely employed for the production of desired bioproducts through systems metabolic engineering because the genetic engineering tools are well established compared to other microbial species, and its metabolism and physiology are relatively well understood. Such advantages led to the development of engineered E. coli strains capable of hydrocarbon production. The first report on the production of FAEEs by engineered E. coli appeared in 2009 [35]. In this study, a novel Current Opinion in Biotechnology 2015, 33:15–22

vector, p(Microdiesel), containing the Zymomonas mobilis pyruvate decarboxylase ( pdc) alcohol dehydrogenase B (adhB) genes, and the A. baylyi wax-dgaT was constructed. The engineered E. coli strain harboring p(Microdiesel) was able to produce 1.28 g/L of FAEEs from glucose and exogenous oleic acid (Figure 2a) [35]. In their follow-up study, pilot-scale fermentation of E. coli p(Microdiesel) strain was performed using glycerol as a carbon source for biomass production, and then glucose and oleic acid were added for the production of FAEEs; this allowed production of 19 g/L of FAEEs with a productivity of 0.29 g/ L/hour in 16-L fermentation [36]. In a different study, Steen et al. [37] first demonstrated in vivo production of FAEEs solely from plant-derived sugars without the supplementation of fatty acids. To achieve this, the fatty acid biosynthetic pathway in E. coli was engineered to enrich free fatty acid production; a leaderless variant of E. coli thioesterase (‘tesA) was overexpressed, while the fadE gene was deleted to prevent degradation of fatty acids via b-oxidation. Introducing the pdc, adhB and wax-dgaT genes into the above strain resulted in the production of 37 mg/L of FAEEs from glucose. It was found that the conversion of free fatty acids into acyl-CoAs was a bottleneck, which could be solved by introducing the mutant fadD gene (M335I); this resulted in sixfold increase in the FAEE titer to 233 mg/ L. By further increasing the expression level of the waxdgaT gene and preventing the evaporation of FAEEs, the maximum titer of 647 mg/L of FAEEs was achieved. Finally, a proof-of-concept experiment on the production of FAEEs directly from hemicellulose was performed as follows. To enable E. coli to utilize xylan, the Clostridium stercoainum xyn10B and Bacteoides ovatus xsa genes fused to the C-terminus of the E. coli osmY gene were introduced for the excretion of the encoded enzymes. This engineered strain was able to produce 11.6 mg/L of FAEE from 2% (w/v) xylan [37]. In their continued study, a dynamic sensor-regulator system (DSRS) was developed for further improvement of the strain performance. First of all, fatty acid/fatty-acyl CoA (FA/acyl-CoA) biosensors were established by regulating the DNA-binding activity of FadR and were used to screen fatty acid overproducing strains. The FAEE production pathway was divided into three parts: module A related to fatty acids production, module B related to ethanol production, and module C containing fadD and wax-dgaT. By applying the DSRS to the three modules of the strain, the FAEE titer could be further increased to1.5 g/L [38]. Beyond the production of FAEEs, production of alka(e)nes using metabolically engineered E. coli has also been reported. Through the genome analysis of 10 different hydrocarbon producing strains, the orf1593 and orf1594 from Synechococcus elogatus PCC7942 were found to encode an aldehyde decarbonylase (Adc) and an acylACP reductase (Aar), respectively. When these two www.sciencedirect.com

Microbial production of hydrocarbons Lee, Kim and Cheon 19

proteins were introduced into E. coli, 0.3 g/L of alka(e)ne mixture consisting of C13, C15, and C17 was successfully produced [9]. In a recent study, the native E. coli FabH involved in the first step of fatty acid elongation was replaced with Bacillus subtilis FabH2, which has been reported to have higher activity than that of E. coli on acylCoAs. This allowed extension of the profile of the hydrocarbons produced in engineered E. coli expressing Adc and Aar [10]; an alkane mixture composed of C13, C15 and C17, and additionally, C14 and C16 alkanes was produced. Furthermore, the proportions of the produced alkanes could be altered by the addition of alternative acyl-CoA substrates such as propanoate during the cultivation of the above strain, resulting in fourfold increase of C16 (3.7 to 14.3 mg/L) and ten fold increase of C14 (1.2 to 11.9 mg/L) [10].

Production of isoprenoid-derived hydrocarbons Isoprenoids are naturally produced metabolites having diverse structures and functions that are of great interest to healthcare and food industries as medicines, fragrances, and flavors. Two major pathways for the biosynthesis of isoprenoids, namely the D-xylulose-5-phosphate (DXP) pathway and mevalonate (MEV) pathway (Figure 2b), have been identified [39]. Two isomeric five-carbon monomers, dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP), are generated via DXP or MEV pathway. DMAPP and IPP are then condensed by prenyltransferases to form isoprenoid precursors such as geranyl pyrophosphate (GPP, C10) and farnesyl pyrophosphate (FPP, C15) [6]. There has recently been much interest in developing microorganisms capable of producing isoprenoid-derived hydrocarbons, mostly belonging to monoterpenes (C10) or sesquiterpenes (C15), as candidates for jet fuel and biodiesel, because of the desirable properties such as low freezing temperature and high ignition stability resulting from their branched or cyclic structure [2]. The carbon lengths of the isoprenoidderived hydrocarbons are between those of short-chain and long-chain hydrocarbons, but can be considered as long-chain hydrocarbons since their fuel properties typically resemble those of diesel and jet fuel. Monoterpenes are C10 isoprenoids derived from GPP (Figure 2b). GPP is synthesized by the GPP synthase which condenses two units of five-carbon building blocks, IPP and DMAPP, and is subsequently converted to diverse monoterpenes by monoterpene synthases. Although monoterpenes have been typically used as flavors and fragrances, their possible applications as alternative fuel source have recently been pursued by a number of researchers. In one study, 2,6-dimethyloctane and 1-isopropyl-4-methylcyclohexane, the fully saturated forms of myrcene and limonene, respectively, were found to be good for blending with conventional diesel fuel [40]. Other studies have also suggested that the chemically www.sciencedirect.com

catalyzed dimers of cyclic monoterpenes such as camphene, pinene, and limonene are indeed good fuels having volumetric heating values of up to 39.5 MJ/L [41,42]. Recently, Amyris Inc. successfully conducted a flight demonstration using their bio-jet fuel product called AMJ-700, more than a half of which corresponds to monoterpene derivatives (URL: http://www.amyris.com) [43]. On the basis of such good potentials of monoterpenederived biofuels, many studies have been carried out to establish platforms for the microbial production of monoterpenes using S. cerevisiae and E. coli. Although the de novo monoterpene synthesis platform was established in S. cerevisiae a few years ago [44], current status of monoterpene production is still in the developmental stage. To increase monoterpene biosynthesis, the yeast sterol biosynthetic pathway genes, HMG2, ERG20, and IDI1, were incorporated into the genome of S. cerevisiae. With the expression of two terpene synthases from Salvia fruticosa or Salvia pomifera, the engineered S. cerevisiae could produce monoterpene up to 1 g/L [45]. Bacterial monoterpene synthesis has been first established by the expression of S. cerevisiae mevalonate pathway genes in E. coli [46]. Recently, a new potential monoterpene-based biofuel precursor called sabinene was produced by engineered E. coli [47]. In this study, the methylerythritol 4-phosphate (MEP) or the MEV pathway was established in E. coli. With the expression of sabinene synthase derived from S. pomifera, 82.18 mg/ L of sabinene could be produced under optimal culture condition. Fermentation of this strain resulted in the production of 2.65 g/L of sabinene using glycerol as a carbon source. Sesquiterpene (C15 isoprenoid) is another representative isoprenoid-derived fuel source which has already been commercialized, or at least near commercialization [43,48]. For example, Amyris Inc. has recently announced the formation of Total-Amyris fuels partnership for the development of renewable diesel and jet fuel using farnesene produced by metabolically engineered S. cerevisiae (URL: http://www.amyris.com). In another study, bisabolane, the fully reduced form of bisabolene, has been identified as an excellent D2 diesel alternative in terms of physical and chemical properties. For bisabolene production in E. coli and S. cerevisiae, six different bisabolene synthases isolated from Arabidopsis thaliana, Picea abies, Pseudotsuga menziesii, or Abies grandis were examined. Using the best enzyme identified, the engineered E. coli and S. cerevisiae strains produced more than 0.9 g/L of bisabolene [49]. S. cerevisiae and E. coli are not the only microorganisms employed for the production of such sesquiterpenes. More recently, Streptomyces venezuelae has been employed as a model actinobacterium species for bisabolene production. Through the engineering of the native secondary Current Opinion in Biotechnology 2015, 33:15–22

20 Energy biotechnology

metabolism (DXP pathway) and overexpressing the A. grandis bisabolene synthase, the recombinant S. venezuelae strain was able to produce bisabolene up to 10.5 mg/L [50].

Production of short-chain hydrocarbons As described above, there have been a number of studies on microbial production of long-chain hydrocarbons. However, it had been rather difficult to produce shortchain (C4-C12) hydrocarbons suitable for substituting gasoline. Biological production of short-chain hydrocarbons is inherently difficult because the natural fatty acids synthesized in microorganisms mainly comprise C14-C18 for their use in making cell membranes and other cellular components. To produce short-chain alkanes, it is necessary to generate short-chain fatty acids and their derivatives in the cell. Recently, there has been an excellent study on the production of short-chain fatty acids of all even and odd chain lengths from 4 to 13 carbons in E. coli [51]; the authors named them as medium-chain fatty acids. Long-chain acyl-ACP elongation was selectively inhibited by engineering the ketoacyl synthases, which resulted in the production of short-chain fatty acids up to several hundreds of mg/L depending on the fatty acids. Also, short odd-chain fatty acids could be produced by making propionyl-CoA available in vivo either by supplementing propionic acid or by introducing the propionylCoA biosynthetic pathway. Another recent study reports the production of shortchain fatty acids (C6-C10) by employing a human fatty acid synthase (hFAS) in S. cerevisiae. In this study, the native C-terminal thioesterase (TE) domain of hFAS, which cleaves fatty acid from fatty acyl carrier protein (ACP), was replaced with heterologous short-chain TEs: one from Cuphea palustris (CpFatB1) and the other from Rattus norvegicus (TEII). When the hFAS mutants and the short-chain TEs, either linked or plasmid-based, were overexpressed, caprylic acid and total short-chain fatty acids were produced to 63 and 68 mg/L, respectively. When the phosphopantetheine transferase was overexpressed with the hFAS mutant, C8 fatty acid and total short-chain fatty acid concentrations increased to 82 and 111 mg/L, respectively [52]. Recently, Choi and Lee [11] have reported the development of a platform E. coli strain that is able to produce 580.8 mg/L of short-chain hydrocarbons, mainly consisting of C9 and C12 (Figure 2c). In a fadE-deleted E. coli strain, the activity of 3-oxoacyl-ACP synthase (FabH) was increased to enhance the initiation of fatty acid biosynthesis by the deletion of the fadR gene. The deletion of the fadR gene prevents upregulation of the fabA and fabB genes responsible for the biosynthesis of unsaturated fatty acids, which inhibit FabH. A modified fatty acyl-ACP thioesterase was employed for the conversion of short-chain fatty acyl-ACPs to short-chain free Current Opinion in Biotechnology 2015, 33:15–22

fatty acids. Then, a synthetic pathway composed of E. coli fatty acyl-CoA synthetase, Clostridium acetobutylicum fatty acyl-CoA reductase (acr) and A. thaliana fatty aldehyde decarbonylase (CER1) was designed and introduced for the conversion of short-chain fatty acids to their corresponding alkanes. Using this E. coli strain, various shortchain fatty esters and fatty alcohols can also be produced by introducing responsible enzymes such as wax ester synthase and alcohol dehydrogenase [11]. This was the first report on the production of short-chain hydrocarbons by metabolic engineering of E. coli, the strategies of which can be applied for the production of other fatty acid-derived products in other microbial species. As can be seen from above, production of short-chain fatty acids and hydrocarbons is still far from commercial applications. In vivo generation of sufficient amount of shortchain fatty acids or their derivatives is a big challenge. In this aspect, the work of Dellomonaco et al. [53] might present an excellent alternative way for hydrocarbon fuel production. The b-oxidation cycle was functionally reversed to synthesize alcohols and carboxylic acids having desired chain lengths. Highly efficient production of these products is possible by directly using acetyl-CoA for acyl-chain elongation without the need to use ATP-dependent activation of malonyl-CoA. When this platform is coupled with hydrocarbon biosynthetic pathway, it might be possible to more efficiently produce short-chain hydrocarbons having desired chain lengths.

Conclusions Recently, there have been a number of studies on the development of microbial strains for the production of long-chain and short-chain hydrocarbons as fuel substitutes. There have been some successful developments on the production of isoprenoid-derived biofuels in largescale. However, by contrast to bioethanol that has already been produced in large-scale, most of the studies on hydrocarbon biofuel have rather been of proof-of-concept. One of the main reasons for the relatively low titers of hydrocarbon products is low flux toward hydrocarbon synthesis due to the low activities of the enzymes involved. The use of computational and high-throughput approaches will allow development of better enzymes for hydrocarbon production. Also, instead of employing the best studied strains such as S. cerevisiae and E. coli, superior oleaginous microorganisms capable of accumulating lipids to greater amounts can be employed as host strains for more efficient hydrocarbon production [54]. After establishing a more efficient host strain equipped with better pathway enzymes, systems metabolic engineering can be performed for optimizing the cellular performance under industrially relevant condition [55] toward the efficient production of hydrocarbons to high titers with high productivities and yields. With advances in engineering the fatty acid metabolism allowing increased metabolic fluxes, it is expected that more www.sciencedirect.com

Microbial production of hydrocarbons Lee, Kim and Cheon 21

efficient bioprocess for the production of hydrocarbon biofuels from renewable carbon substrates will be developed in the near future.

Acknowledgements This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries from the Ministry of Science, ICT and Future Planning (MSIP) through the National Research Foundation (NRF) of Korea (NRF2012M1A2A2026556). Further support from the Advanced Biomass R&D Center of Korea (NRF-2010-0029799) through the Global Frontier Research Program of the MSIP is appreciated.

References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as:  of special interest  of outstanding interest

14. Rincon LE, Jaramillo JJ, Cardona CA: Comparison of feedstocks and technologies for biodiesel production: an environmental and techno-economic evaluation. Renew Energy 2014, 69:479487. 15. Chisti Y: Biodiesel from microalgae. Biotechnol Adv 2007, 25:294-306. 16. Schenk PM, Thomas-Hall SR, Stephens E, Marx UC, Mussgnug JH, Posten C, Kruse O, Hankamer B: Second generation biofuels: high-efficiency microalgae for biodiesel production. Bioenergy Res 2008, 1:20-43. 17. Delanoue J, Laliberte G, Proulx D: Algae and waste-water. J Appl Phycol 1992, 4:247-254. 18. Yoo C, Jun S-Y, Lee J-Y, Ahn C-Y, Oh H-M: Selection of microalgae for lipid production under high levels carbon dioxide. Bioresour Technol 2010 http://dx.doi.org/10.1016/ j.biortech.2009.03.030. 19. Griffiths MJ, Harrison STL: Lipid productivity as a key characteristic for choosing algal species for biodiesel production. J Appl Phycol 2009, 21:493-507. 20. Zhu L, Hiltunen E, Shu Q, Zhou W, Li Z, Wang Z: Biodiesel production from algae cultivated in winter with artificial wastewater through pH regulation by acetic acid. Appl Energy 2014, 128:103-110.

1.

Peralta-Yahya PP, Keasling JD: Advanced biofuel production in microbes. Biotechnol J 2010, 5:147-162.

2.

Peralta-Yahya PP, Zhang FZ, del Cardayre SB, Keasling JD: Microbial engineering for the production of advanced biofuels. Nature 2012, 488:320-328.

3.

Harvey BG, Meylemans HA: The role of butanol in the development of sustainable fuel technologies. J Chem Technol Biotechnol 2011, 86:2-9.

4.

Connor MR, Cann AF, Liao JC: 3-Methyl-1-butanol production in Escherichia coli: random mutagenesis and two-phase fermentation. Appl Microbiol Biotechnol 2010, 86:1155-1164.

23. Reddy HK, Muppaneni T, Sun Y, Li Y, Ponnusamy S, Patil PD, Dailey P, Schaub T, Holguin FO, Dungan B et al.: Subcritical water extraction of lipids from wet algae for biodiesel production. Fuel 2014, 133:73-81.

5.

Zheng Y, Liu Q, Li L, Qin W, Yang J, Zhang H, Jiang X, Cheng T, Liu W, Xu X et al.: Metabolic engineering of Escherichia coli for high-specificity production of isoprenol and prenol as next generation of biofuels. Biotechnol Biofuels 2013 http:// dx.doi.org/10.1186/1754-6834-6-57.

24. Trentacoste EM, Shrestha RP, Smith SR, Gle´ C, Hartmann AC, Hildebrand M, Gerwick WH: Metabolic engineering of lipid catabolism increases microalgal lipid accumulation without compromising growth. Proc Natl Acad Sci U S A 2013, 110:19748-19753.

6.

Zhang F, Rodriguez S, Keasling JD: Metabolic engineering of microbial pathways for advanced biofuels production. Curr Opin Biotechnol 2011, 22:775-783.

7.

Petrus L, Noordermeer MA: Biomass to biofuels, a chemical perspective. Green Chem 2006, 8:861-867.

25. Yu WL, Ansari W, Schoepp NG, Hannon MJ, Mayfield SP, Burkart MD: Modifications of the metabolic pathways of lipid and triacylglycerol production in microalgae. Microb Cell Fact 2011 http://dx.doi.org/10.1186/1475-2859-10-91.

8.

Lennen RM, Braden DJ, West RM, Dumesic JA, Pfleger BF: A process for microbial hydrocarbon synthesis: overproduction of fatty acids in Escherichia coli and catalytic conversion to alkanes. Biotechnol Bioeng 2010, 106:193-202.

9.

Schirmer A, Rude MA, Li X, Popova E, del Cardayre SB: Microbial biosynthesis of alkanes. Science 2010, 329:559-562.

10. Harger M, Zheng L, Moon A, Ager C, An JH, Choe C, Lai Y-L, Mo B, Zong D, Smith MD et al.: Expanding the product profile of a microbial alkane biosynthetic pathway. ACS Synth Biol 2013, 2:59-62. 11. Choi YJ, Lee SY: Microbial production of short-chain alkanes.  Nature 2013, 502:571-574. This paper reports microbial production of short-chain alkane for the first time. Short-chain fatty acid pathway was first established by manipulating several fatty acid biosynthesis and degradation genes, and introducing a modified thioesterase. A short-chain alkane biosynthetic pathway was then established in E. coli by introducing fatty acyl-CoA reducatase gene from Clostridium acetobutylicum and the decarbonylase gene from Arabidopsis. This platform strain can be further modified for the production of short-chain fatty esters and fatty alcohols. 12. Ahmad AL, Yasin NHM, Derek CJC, Lim JK: Microalgae as a sustainable energy source for biodiesel production: a review. Renew Sust Energy Rev 2011, 15:584-593. 13. Adewale P, Dumont M-J, Ngadi M: Rheological, thermal, and physicochemical characterization of animal fat wastes for use in biodiesel production. Energy Technol 2014, 2:634-642. www.sciencedirect.com

21. Lv JM, Cheng LH, Xu XH, Zhang L, Chen HL: Enhanced lipid production of Chlorella vulgaris by adjustment of cultivation conditions. Bioresour Technol 2010, 101:6797-6804. 22. Liang K, Zhang Q, Gu M, Cong W: Effect of phosphorus on lipid accumulation in freshwater microalga Chlorella sp.. J Appl Phycol 2013, 25:311-318.

26. Lam MK, Lee KT: Microalgae biofuels: a critical review of issues, problems and the way forward. Biotechnol Adv 2012, 30:673-690. 27. Pragya N, Pandey KK, Sahoo PK: A review on harvesting, oil extraction and biofuels production technologies from microalgae. Renew Sust Energy Rev 2013, 24:159-171. 28. Beopoulos A, Cescut J, Haddouche R, Uribelarrea JL, MolinaJouve C, Nicaud JM: Yarrowia lipolytica as a model for bio-oil production. Prog Lipid Res 2009, 48:375-387. 29. Barth G, Gaillardin C: Physiology and genetics of the dimorphic fungus Yarrowia lipolytica. FEMS Microbiol Rev 1997, 19:219237. 30. Tai M, Stephanopoulos G: Engineering the push and pull of lipid  biosynthesis in oleaginous yeast Yarrowia lipolytica for biofuel production. Metab Eng 2013, 15:1-9. For the enhanced production of lipid in oleaginous yeast Yarrowia lipolytica, the importance of metabolite generation (push) and consumption (pull) was suggested. This ‘push-and-pull’ strategy was successfully employed for the improvement on lipid production in Y. lipolytica through the enhancement of diacylglycerol acyltransferase expression (pull; TAG biosynthesis) employing the intron-containing translation elongation factor 1-a promoter, while acetyl-CoA carboxylase was simultaneously overexpressed (push; fatty acid biosynthesis). 31. Blazeck J, Hill A, Liu L, Knight R, Miller J, Pan A, Otoupal P, Alper HS: Harnessing Yarrowia lipolytica lipogenesis to create a platform for lipid and biofuel production. Nat Commun 2014 http://dx.doi.org/10.1038/ncomms4131. Current Opinion in Biotechnology 2015, 33:15–22

22 Energy biotechnology

32. Shi S, Valle-Rodriguez JO, Khoomrung S, Siewers V, Nielsen J:  Functional expression and characterization of five wax ester synthases in Saccharomyces cerevisiae and their utility for biodiesel production. Biotechnol Biofuels 2012 http://dx.doi.org/ 10.1186/1754-6834-5-7. FAEEs (biodiesel) are produced thorough the condensation of fatty acylCoA and ethanol by wax ester synthase. Characterization of various wax ester synthases is thus crucial for enhanced microbial biodiesel production. In this study, five different wax ester syntheses isolated from five different organisms were functionally expressed in S. cerevisiae and characterized with respect to FAEEs production. 33. Jong BW, Shi S, Siewers V, Nielsen J: Improved production of fatty acid ethyl esters in Saccharomyces cerevisiae through up-regulation of the ethanol degradation pathway and expression of the heterologous phosphoketolase pathway. Microb Cell Fact 2014 http://dx.doi.org/10.1186/1475-2859-1339. 34. Yu KO, Jung J, Kim SW, Park CH, Han SO: Synthesis of FAEEs from glycerol in engineered Saccharomyces cerevisiae using endogenously produced ethanol by heterologous expression of an unspecific bacterial acyltransferase. Biotechnol Bioeng 2012, 109:110-115. 35. Kalscheuer R, Stoelting T, Steinbuechel A: Microdiesel Escherichia coli engineered for fuel production. Microbiology 2006, 152:2529-2536. 36. Elbahloul Y, Steinbuechel A: Pilot-scale production of fatty acid ethyl esters by an engineered Escherichia coli strain harboring the p(Microdiesel) plasmid. Appl Environ Microbiol 2010, 76:4560-4565. 37. 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. 38. 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. In this work, a dynamic sensor-regulator system was developed by regulating the DNA-binding activity of FadR, and was used to screen fatty acid overproducing strains. By optimizing the modules related to fatty acids production, ethanol production, and the wax ester synthase, 1.5 g/L of FAEEs could be produced. 39. Kuzuyama T: Mevalonate and nonmevalonate pathways for the biosynthesis of isoprene units. Biosci Biotechnol Biochem 2002, 66:1619-1627. 40. Tracy NI, Chen D, Crunkleton DW, Price GL: Hydrogenated monoterpenes as diesel fuel additives. Fuel 2009, 88:22382240. 41. Harvey BG, Wright ME, Quintana RL: High-density renewable fuels based on the selective dimerization of pinenes. Energy Fuels 2010, 24:267-273. 42. Meylemans HA, Quintana RL, Harvey BG: Efficient conversion of pure and mixed terpene feedstocks to high density fuels. Fuel 2012, 97:560-568. 43. Renninger NS, Ryder JA, Fisher KJ. Jet fuel compositions and methods of making and using same. US 11/986,485; 2011. 44. Carrau FM, Medina K, Boido E, Farina L, Gaggero C, Dellacassa E, Versini G, Henschke PA: De novo synthesis of monoterpenes by Saccharomyces cerevisiae wine yeasts. FEMS Microbiol Lett 2005, 243:107-115.

Current Opinion in Biotechnology 2015, 33:15–22

45. Ignea C, Cvetkovic I, Loupassaki S, Kefalas P, Johnson CB, Kampranis SC, Makris AM: Improving yeast strains using recyclable integration cassettes, for the production of plant terpenoids. Microb Cell Fact 2011 http://dx.doi.org/10.1186/ 1475-2859-10-4. 46. Martin VJ, 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. 47. Zhang HB, Liu Q, Cao YJ, Feng XJ, Zheng YN, Zou HB, Liu H, Yang JM, Xian M: Microbial production of sabinene — a new terpene-based precursor of advanced biofuel. Microb Cell Fact 2014 http://dx.doi.org/10.1186/1475-2859-13-20. 48. Renninger NS, McPhee DJ. Fuel compositions comprising farnesane and farnesane derivatives and method of making and using same. PCT/US2007/021890; 2008. 49. Peralta-Yahya PP, Oullet M, Chan R, Mukhopadhyay A, Keasling JD, Lee KS: Identification and microbial production of a terpene-based advanced biofuel. Nat Commun 2011 http:// dx.doi.org/10.1038/ncomms1494. 50. Phelan RM, Sekurova ON, Keasling JD, Zotchev SB: Engineering terpene biosynthesis in streptomyces for production of the  advanced biofuel precursor bisabolene. ACS Synth Biol 2014 http://dx.doi.org/10.1021/sb5002517. Most metabolic engineering studies on the production of isoprenoidderived hydrocarbons employed S. cerevisiae or E. coli as a host strain. This study presents a new platform host for the production of bisabolene, a sesquiterpene, in Streptomyces venezuelae, representing a successful demonstration that other hosts can be considered for the production of isoprenoid-derived biofuels. 51. Torella JP, Ford TJ, Kim SN, Chen AM, Way JC, Silver PA: Tailored  fatty acid synthesis via dynamic control of fatty acid elongation. Proc Natl Acad Sci U S A 2013, 110:11290-11295. E. coli was engineered to produce short-chain fatty acids of all even and odd chain lengths from 4 to 13 carbons. Long-chain acyl-ACP elongation was selectively inhibited by engineering the ketoacyl synthases, which resulted in the production of short-chain fatty acids up to several hundreds of mg/L depending on the fatty acids. Feeding propionic acid or generating in vivo propionyl-CoA allowed production of short odd-chain fatty acids. 52. Leber C, Da Silva NA: Engineering of Saccharomyces cerevisiae for the synthesis of short chain fatty acids. Biotechnol Bioeng 2014, 111:347-358. 53. 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. The b-oxidation cycle was functionally reversed to synthesize alcohols (C4 and higher alcohols) and long chain fatty acids (C > 10) having desired chain lengths. Acetyl-CoA was directly used for chain elongation without the need to use ATP-dependent activation of malonyl-CoA. 54. Kurosawa K, Boccazzi P, de Almeida NM, Sinskey AJ: High-celldensity batch fermentation of Rhodococcus opacus PD630 using a high glucose concentration for triacylglycerol production. J Biotechnol 2010, 147:212-218. 55. Park SH, Kim HU, Kim Y, Park JS, Kim SS, Lee S: Metabolic engineering of Corynebacterium glutamicum for L-arginine production. Nat Commun 2014 http://dx.doi.org/10.1038/ ncomms5618.

www.sciencedirect.com

Metabolic engineering for the production of hydrocarbon fuels.

Biofuels have been attracting increasing attention to provide a solution to the problems of climate change and our dependence on limited fossil oil. D...
886KB Sizes 0 Downloads 8 Views