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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)

Current Opinion in Biotechnology 2015, 33:1522

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 [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.

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
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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, 33:1522

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, 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.

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 [2022], 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, 33:1522

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
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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.
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Current Opinion in Biotechnology 2015, 33:1522

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, 33:1522

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
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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
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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

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, 33:1522

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.

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