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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 (leesy@kaist.ac.kr,
mbelmbel99@gmail.com)
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 [35].
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 (C5C12) 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 (C10C23) and short-chain (C5C12)
hydrocarbons [2,810,11]. 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.
16 Energy biotechnology
Figure 1
(a)
(b)
in vivo
Fractional distillation
of crude oil
Nonane
yeast
C5-C9
(Chemicals)
C20-C50
(Lubricating oil)
C20-C70
(Ship oil)
Tetradecane
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
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, C5C23 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.
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
2-C-Methylerythritol-4phosphate
Dihydroxyacetone phosphate
orf1594
Fatty aldehyde
orf1593
tesA
C12-C23
hydrocarbons
fpps
Farnesene
fadD
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.
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18 Energy biotechnology
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
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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, 33:1522
20 Energy biotechnology
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
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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.
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