Journal of Biotechnology 309 (2020) 1–19

Contents lists available at ScienceDirect

Journal of Biotechnology
journal homepage: www.elsevier.com/locate/jbiotec

Review

Enzymatic reactions and pathway engineering for the production of T

renewable hydrocarbons
Juthamas Jaroensuk1, Pattarawan Intasian1, Watsapon Wattanasuepsin,
Nattanon Akeratchatapan, Chatchai Kesornpun, Narongyot Kittipanukul, Pimchai Chaiyen*
School of Biomolecular Science and Engineering, Vidyasirimedhi Institute of Science and Technology (VISTEC), Wangchan Valley, Rayong 21210, Thailand

A R T I C LE I N FO A B S T R A C T

Keywords: Hydrocarbons such as alkanes and alkenes are extensively used as organic compounds for combustion reactions
Biocatalysis and as building block components for the synthesis of numerous materials. Various synthetic enzymatic cascades
Hydrocarbon and engineered metabolic pathways can be used to produce alkanes and alkenes from bio-based materials. An
Enzyme understanding of the native reactions and pathways used by various organisms to synthesize these compounds
Metabolic engineering
together with novel approaches in biocatalysis and synthetic biology have been instrumental in the development
Scale-up
Fatty acid
of methods to produce alkanes and alkenes with reasonable yield. This article discusses the present state of
knowledge regarding hydrocarbon biosynthetic pathways and discusses current mechanistic understanding of
relevant enzymatic reactions in cyanobacteria, aerobic bacteria, insects, algae, and plants. Recent advancements
in metabolic engineering and process scale up for production of hydrocarbons from fatty acids are also discussed.
This technology is important for sustainability, as it provides a clean and eco-friendly method for the future
production of fuels and industrial materials. Further development towards whole cell biocatalysts that are able to
provide good yield with a low production cost may allow countries without big oil reserves to be capable of
producing precursors for the materials industries in the future.

1. Importance of bio-based hydrocarbon production for
sustainable fuels and materials
Hydrocarbons such as alkanes and alkenes are extensively used as
organic compounds for combustion and as building block components
for the synthesis of various materials. Alkanes are widely used in
heating and motor fuel applications. The particular usage and appli-
cation of a specific hydrocarbon depends on its carbon chain length.
Short-chain alkanes (C2-C4) are gases at atmospheric pressure. They are
mainly used in household cooking and for generating electricity by
incineration. They can be liquified into liquified petroleum gas (LPG)
for use as transportation fuels. Medium-chain alkanes (C5-C17) can be
used as gasoline and diesel fuels for transportation as well as used as
solvents for industrial applications. Very long-chain alkanes (> C18)
are generally used as lubricating oils. Some of these alkanes are solids
and are thus commonly used as waxes for coating, cosmetic and phar-
maceutical applications. Hydrocarbons also serve as important mono-
mers for polymer production (Fig. 1). Alkenes such as ethene, propene
and 1,3-butadiene, are often used in the manufacture of commodity
products such as plastics and detergents. Moreover, these alkenes can
also be used in biomedical applications i.e. dental polymers and wound
dressings (Thome et al., 2015). The numerous applications using hy-
drocarbons have made a significant contribution to industrial and
urban development since the 19th century (Rogner, 1997)
Currently, most of the hydrocarbons used are derived from fossil
fuels. In the US, seventy percent of ethylene monomer production can
be obtained from naphtha steam cracking or ethane thermal cracking
processes (Ghanta et al., 2014). Fossil fuels are not renewable, and
require millions of years to form. Thus, once depleted, they cannot be
replaced in a short period of time. Renewable sources of hydrocarbons
are necessary for global energy security. Moreover, current methods
used for production of hydrocarbons (petroleum refining of crude oil
and thermal cracking process) are harsh and unfriendly to the en-
vironment. Therefore, an alternative method that is both clean for the
environment and has economic viability for large-scale production is
necessary for future socio-economic sustainability.
There have been significant advancements in the development of
alternative energy devices during the past decade, challenging the roles

⁎
Corresponding author.
E-mail address: pimchai.chaiyen@vistec.ac.th (P. Chaiyen).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.jbiotec.2019.12.010
Received 3 September 2019; Received in revised form 14 December 2019; Accepted 15 December 2019
Available online 19 December 2019
0168-1656/ © 2019 Elsevier B.V. All rights reserved.
J. Jaroensuk, et al.
Fig. 1. Contribution of bio-based hydrocarbon production as sources of energy and materials. Alkanes and alkenes remain indispensable as power sources (shown in
green) and as chemical building blocks or precursors for materials (shown in orange). Unlike fossil-derived sources which are hazardous for the environment, the bio-
based method offers a clean, renewable and sustainable way for the production of these important chemicals. (For interpretation of the references to colour in this
figure legend, the reader is referred to the web version of this article).
and importance of fossil fuels as energy sources for mobile vehicles.
Currently, the high efficiency of electric vehicle batteries (EVBs) allows
EVBs to serve as power supplies for electric vehicles. In the future, a
large part of ground transportation is predicted to be diverted from
petroleum-reliant engines to electric engines (Dhameja, 2002;
Tsiropoulos et al., 2018). However, electric vehicles still require energy
input from charging stations. While further innovations will likely lead
to more efficient methods for harnessing power from solar, wind and
hydro sources, the use of hydrocarbons as energy sources for power
stations and for industrial operation will still be indispensable for many
decades to come (Fig. 1). Thus, the development of methods for hy-
drocarbon production from renewable sources together with energy
conversion and improved storage technology will contribute to greater
sustainability and security of global energy resources (Evans et al.,
2009).
Bio-based methods are alternative, renewable and sustainable ways
for producing alkanes and alkenes. In addition to the benefits of bio-
based production of hydrocarbons for energy, this technology would
also be highly beneficial to the production of alkanes and alkenes as
monomers or precursors for material industries. Therefore, this review
article highlights recent findings regarding the enzymatic reactions and
metabolic pathways which convert fatty acids to alkanes and alkenes.
Here we discuss the natural biosynthetic pathways and enzymatic re-
actions found in various organisms and artificial enzymatic cascades
that can be used for hydrocarbon production. The scope of this review
does not include a well-known and industrial used process of biodiesel
production as this topic has been covered in great detail in previous
references (Sivaramakrishnan and Incharoensakdi, 2018; Thangaraj
et al., 2018). The report also highlights the recent developments in
metabolic and process engineering as related to the production of al-
kanes and alkenes by whole cells in large-scale systems. Future per-
spectives on the technologies required for sustainable production of
alkanes and alkenes are also discussed.
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Journal of Biotechnology 309 (2020) 1–19
2. Natural pathways for hydrocarbon production
2.1. Biosynthesis of hydrocarbons in cyanobacteria
Previous studies revealed that many cyanobacteria can accumulate
hydrocarbon molecules by storing them inside their cells or storing
them in membrane lipid form to help cyanobacteria respond to a
variety of environmental changes (Wada and Murata, 1998; Wang
et al., 2013). Cyanobacteria can convert a variety of carbon feedstocks
such as CO2, glucose or fatty acids to alkane or alkene products using
their unique metabolic pathways. Two different pathways for alkane or
alkene biosynthesis exist in cyanobacteria. The first pathway uses acyl-
[acyl carrier protein (ACP)] reductase (AAR) to generate fatty aldehyde
from fatty acyl-ACP and aldehyde-deformylating oxygenase (ADO) to
decarbonylate fatty aldehyde to yield alkanes or alkenes (Fig. 2a). De-
pending on the substrate captured by AAR, this pathway can yield sa-
turated alkanes, branched alkanes and alkenes containing internal
double bonds as products. The second pathway utilizes a modular
multi-domain protein called olefin synthase (OLS) to synthesize alkenes
from fatty acids. OLS is comprised of three modules (Coates et al.,
2014b; Mendez-Perez et al., 2011; Zhu et al., 2018). The first module is
the activation module which generates fatty acyl-ACP, the second
module is an extension module that can extend the fatty acyl-ACP via a
malonyl-coenzyme A (malonyl-CoA) unit, and the last module is the
alkene formation module which generates the terminal alkene (Fig. 2b)
(see more discussion in Section 3.3.1).
2.2. Biosynthesis of hydrocarbons in bacteria
During the past few years, several hydrocarbon biosynthesis path-
ways in bacteria have been explored. In bacteria other than cyano-
bacteria, the synthesis of hydrocarbons mainly leads to formation of
alkenes. Two different pathways for alkene biosynthesis exist in these
bacteria. The first pathway used is internal alkene biosynthesis via
head-to-head condensation of fatty acids. In some bacteria such as
J. Jaroensuk, et al.
Micrococcus luteus, Xanthomonas campestris and Shewanella oneidensis,
the OleABCD protein complex catalyzes the formation of internal al-
kenes using an initial reaction of OleA to convert β-ketoacyl-CoA (β-
oxidation intermediate) into an intermediate which is then condensed
with another fatty acyl-CoA via non-decarboxylative Claisen
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Journal of Biotechnology 309 (2020) 1–19
Fig. 2. Overview of enzymatic reaction for hydrocarbon
biosynthesis. (a) Reaction catalyzed by AAR and ADO in
alkane synthesis. AAR, acyl-[acyl carrier protein (ACP)]
reductase; ADO, aldehyde deformylating oxygenase. (b)
Reaction catalyzed by OLS in terminal alkene synthesis.
FAAL, fatty acid-ACP ligase; ACP1, acyl carrier protein 1;
KS, ketosynthase; AT, acyltransferase; KR, ketoreductase;
ACP2, acyl carrier protein 2; ST, sulfotransferase; TE,
thioesterase. (c) Reaction catalyzed by OleABCD in an
internal alkene synthesis via head-to-head condensation.
(d) Reaction catalyzed by fatty acid decarboxylase OleT,
UndA and UndB in terminal alkene synthesis. Pd,
Pseudomonas putida ferredoxin; PNR, Pseudomonas putida
ferredoxin reductase. (e) Reaction catalyzed by acyl-ester
reductase/CYP4Gs and FAR/CYP4Gs in hydrocarbon bio-
synthesis in insects. (f) Photodecarboxylation of fatty acid
by CvFAP. (g) Reaction catalyzed by CER in conversion of
very long-chain fatty acyl-CoA to alkanes and carbon
monoxide in plants.
condensation to yield a β-keto acid and the release of 2CoASH. The β-
keto acid is subsequently converted into a β-hydroxy acid by OleD
through an NADPH-dependent reduction. Formation of the alkene
products is catalyzed by OleC using adenosine triphosphate (ATP)
(Amelia et al., 2019; Davis et al., 1987; Frias et al., 2010; Sukovich
J. Jaroensuk, et al.
et al., 2010) (Fig. 2c). The function of OleB remains unclear. Based on
sequence homology analysis, OleB belongs to the α/ß hydrolase en-
zyme family (Frias et al., 2011; Kang and Nielsen, 2017). The second
pathway for alkene biosynthesis is the formation of terminal alkenes.
Bacteria from the genus Jeotgalicoccus have been reported to synthesize
1-alkenes through the activity of a fatty acid decarboxylase (OleTJE)
which is a cytochrome P450 enzyme (CYP450) (Fig. 2d) (see more
discussion in Section 3.3.2 and 4.4). Another route for 1-alkene
synthesis is via the reaction of nonheme iron oxidase UndA or mem-
brane-bound desaturase-like enzyme UndB from Pseudomonas. UndA
and UndB are nonheme mononuclear iron (FeII) oxidases. UndA uses
medium-chain fatty acids (C10-C14) and while UndB uses also short-
chain fatty acids (C6-C16) as substrates. These enzymes convert these
fatty acid substrates into 1-alkenes through an oxidative decarboxyla-
tion (Fig. 2d) (see more discussion in Section 3.3.3 and 4.4). Both en-
zymes were first discovered by Prof. Zhang laboratory in UC Berkeley
and their sequences show no homology (Rui et al., 2015, 2014).
2.3. Biosynthesis of hydrocarbons in insects
Long-chain hydrocarbons are abundant components found in cuti-
cular lipids located on the surface of all insects. The hydrophobic sur-
face of the insect cuticle plays many important roles in the lives of the
insects (Mpuru et al., 1996). The cuticle protects insects from various
conditions such as preventing penetration of inorganic chemicals and
acting as a barrier against microorganisms. Insects also use hydro-
carbons as chemosensory compounds for chemical communication, and
most importantly, for regulating the uptake and loss of water (Herman
and Zhang, 2016). The cuticular lipids are found widely among various
insect species. The cuticles of the majority of insects possess branched
and unsaturated hydrocarbons that all have the same basic structures,
generally consisting of 19–35 carbon atoms with different ratios of in-
dividual hydrocarbons in the cuticular lipids varying from 3 % to 95 %
among different species (Lockey, 1988).
Based on the experiments from Reed et al. (1994) which used mi-
crosomal preparations from the house fly, Musca domestica, fatty acyl-
CoAs can be reduced to aldehydes by the acyl-ester reductase and then
converted to hydrocarbons by the enzyme aldehyde decarbonylase. The
carbonyl carbon from the aldehyde is released as CO2 in the microsome
during hydrocarbon formation. The data also demonstrate that the
conversion of aldehyde into hydrocarbon in house fly microsomes re-
quires NADPH and O2 for the activity of the enzyme aldehyde dec-
arbonylase. The data indicate that the enzyme mechanism may be si-
milar to those of other CYP450 type reactions (Reed et al., 1994). Qiu
et al. (2012) identified that the insect aldehyde decarbonylase is a
membrane-bound CYP450 type enzyme. The results from in vitro ana-
lyses using purified microsomes expressing the recombinant fusion
protein of CYP450 reductase (CPR) with cytochrome P450 4G1
(CYP4G1) from Drosophila melanogaster and cytochrome P450 4G2
(CYP4G2) from house fly showed that the insect CYP4Gs function as
oxidative decarbonylases which require O2 as a substrate to generate
alka(e)ne from aldehydes (Fig. 2e).
Recently, MacLean et al. (2018) reported another pathway in which
insect cuticular hydrocarbons are formed by fatty acyl-CoA reductase
(FAR), which converts fatty acyl-CoAs to alcohols. The alcohol moieties
are then oxidized to aldehydes and to alka(e)nes by CYP4Gs dec-
arbonylases (Fig. 2e). As the oxidation of primary alcohols can be
catalyzed by many types of cytochrome P450 s (MacLean et al., 2018),
the ability of CYP4Gs to oxidize primary alcohols to aldehydes and then
decarbonylate the aldehydes to alkanes can be expected.
2.4. Biosynthesis of hydrocarbons in algae
Algae and microalgae have been proposed as potential organisms
for use in the synthesis of hydrocarbons from fatty acids because of
their ability to produce plenty of biomass and fatty acids (Beer et al.,
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Journal of Biotechnology 309 (2020) 1–19
2009; Harwood and Guschina, 2009; Hu et al., 2008; León-Bañares
et al., 2004; Liu and Benning, 2013; Malcata, 2011; Sorigué et al., 2016;
Wijffels et al., 2013). Several algal and microalgal such as Chlamydo-
monas, Chlorella, Nannochloropsis, Ostreococcus, and Phaeodactylum
species were found to be capable of synthesizing long-chain alka(e)nes
by light-dependent pathways (Sorigué et al., 2016).
Despite their valuable ability to produce hydrocarbons, some of the
key enzymes responsible for alkane biosynthesis in algae were only just
identified in 2017. The algal photoenzyme in Chlorella variabilis NC64A
designated as fatty acid photodecarboxylase (CvFAP) uses blue light
(450 nm) and its cofactor, flavin adenine dinucleotide (FAD) bound at
the enzyme active site, to convert fatty acid substrates into alka(e)nes
and CO2 by decarboxylation (Fig. 2f) (Sorigue et al., 2017). The type of
alkane or alkene products from CvFAP usually depends upon the
properties of the starting substrates (i.e. saturated vs. unsaturated fatty
acid). Besides fatty acids, CvFAP can also use short-chain carboxylic
acids to produce short-chain alkanes (Zhang et al., 2019). The enzyme
can convert acetic acid to methane (Zhang et al., 2019). Therefore, this
reaction is useful and has great versatility in terms of biofuel produc-
tion.
2.5. Biosynthesis of hydrocarbons in plants
The ability of plants to produce very long-chain alkanes is well
known. Long-chain alkanes are major compounds in the cuticle located
on the primary aerial surface of most terrestrial plants. These com-
pounds play a key role in minimizing non-stomatal transpiration and
also serve as barriers to protect plants against ultraviolet radiation,
pathogens and insects, and provide heat insulation and transpiration
control (Bourdenx et al., 2011). Most of the cuticle in plants is located
on the outer side of epidermal cell walls and consists of a cutin matrix
as well as cuticular waxes which are abundant in very long-chain fatty
acids, alcohols, aldehydes, alkanes, ketones, and esters, as well as small
amounts of triterpenoids, flavonoids, and sterols (Samuels et al., 2008).
Types and amounts of cuticular wax compounds vary widely among
plant compartments and species. Their contents in several genus such as
Cactaceae, Cupressaceae or Poaceae have been identified (Bi et al., 2005;
Bugalho et al., 2009; Buschhaus et al., 2008; Busta et al., 2016; Dodd
et al., 1998; Dominguez et al., 2011; Dragota and Riederer, 2007;
Diefendorf et al., 2011; Gao and Huang, 2013; Guhling et al., 2005;
Hegebarth et al., 2017; Hegebarth and Jetter, 2017; Jetter and
Riederer, 2011; Bird and Gray, 2003; Mihailova et al., 2015; Nadiminti
et al., 2015; Titah et al., 2013; Zhang et al., 2004; Zheng et al., 2005).
Interestingly, the data suggest that very long cuticular waxes con-
stituents are limited to only certain hydrocarbons mostly alkanes. Other
compounds such as alcohols or aldehydes chain lengths between C24
and C34 tend to be found in cuticular waxes (Bugalho et al., 2009).
Although their involvement in cuticle formation is known, the plant
alkane biosynthesis pathway is still not thoroughly understood. The
biosynthesis pathway of Arabidopsis thaliana (A. thaliana) is the most
investigated pathway regarding this aspect. This plant can produce very
long-chain (VLC) alkanes dominated by nonacosane (C29 alkane),
which accounts for up to 70 % of the total cuticular wax content in the
leaf (Jetter and Kunst, 2008). A. thaliana was used as a model organism
for investigating the mechanisms of alkane biosynthesis in plants. It is
currently believed that long-chain fatty acyl-CoAs are modified by the
enzymes in the alkane formation pathway. CER3 reduces long-chain
fatty acyl-CoAs into long-chain fatty aldehydes with predominantly
even carbon numbers and CER1 enzymes consecutively decarbonylates
the aldehyde to generate an alkane production carbon shorter than the
substrate (Fig. 2g) (Hegebarth and Jetter, 2017).
J. Jaroensuk, et al.
Fig. 3. Reaction mechanism of CAR catalysis of the reduction of a carboxylic acid to its corresponding aldehyde (Finnigan et al., 2017; Gahloth et al., 2017; Horvat
et al., 2019; Kramer et al., 2018; Qu et al., 2019, 2018; Stolterfoht et al., 2018; Winkler, 2018). Reaction steps consist of activation of the carboxylic acid through
adenylation, thioesterification of the acyl group, and transfer of the acyl thioester from the A-domain to the R-domain.
3. Enzymes used in biocatalysis for production of hydrocarbons
from fatty acids
3.1. Enzymes involved in the reduction of fatty acid or fatty acid derivatives
to fatty aldehydes
3.1.1. Carboxylic acid reductase (CAR)
Carboxylic acid reductase (CAR) is an important enzyme that has
been employed in several synthetic pathways for the production of al-
dehydes, key intermediates for the production of hydrocarbons and
other fine chemicals (Finnigan et al., 2017; Kramer et al., 2018; Qu
et al., 2018; Schwendenwein et al., 2016, 2019; Winkler, 2018; Wood
et al., 2017). CAR is a large (MW ∼ 130 kDa) multidomain protein
consisting of an N-terminal adenylation domain (A-domain), a thiola-
tion domain (T-domain), and a C-terminal reductase domain (R-do-
main) (Finnigan et al., 2017; Kramer et al., 2018; Qu et al., 2018;
Stolterfoht et al., 2017, 2018; Winkler, 2018).
The mechanism of CAR catalysis of the reduction of a carboxylic
acid to its corresponding aldehyde has been proposed to contain 4 re-
action steps (Fig. 3) (Finnigan et al., 2017; Gahloth et al., 2017; Horvat
et al., 2019; Kramer et al., 2018; Qu et al., 2019, 2018; Stolterfoht et al.,
2018; Winkler, 2018). The first step is the activation of the carboxylic
acid through adenylation. At this step, the carboxyl group of the acid
reacts with the α-phosphate of ATP to form an acyl-AMP intermediate
with release of pyrophosphate (PPi). The X-ray crystal structures of
CARs in complex with AMP reveal that the universal conserved lysine
residue (K629 in Segniliparus rugosus CAR (SrCAR)), located in the
mobile region of the A-domain (residues 527–654 in SrCAR), promotes
adenylation in the catalytic region of the A-domain by stabilizing and
bringing together ATP and carboxylic acid to facilitate the reaction
(Finnigan et al., 2017; Gahloth et al., 2017; Horvat et al., 2019; Kramer
et al., 2018; Qu et al., 2019, 2018; Stolterfoht et al., 2018; Winkler,
2018). The crystal structure of SrCAR during the adenylation step and
the interaction between the AMP-bound domain and the mobile region
of the A-domain is shown in Fig. 4a (Gahloth et al., 2017). The second
step is thioesterification, in which the thiol group of the phospho-
pantetheine arm attached to the conserved serine residue (S702 in
SrCAR) on the T-domain forms a thioester with an acyl group of the
acyl-AMP intermediate with concomitant release of AMP. The phos-
phopantetheine arm on the T-domain is post-translationally attached by
a phosphopantetheinyl transferase (PPT). In the thioesterification state,
structural transformation of the mobile region of the A-domain and the
T-domain is required to orient the T-domain to be close to the catalytic
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Journal of Biotechnology 309 (2020) 1–19
site of the A-domain, thus promoting the thioesterification (Finnigan
et al., 2017; Gahloth et al., 2017; Horvat et al., 2019; Kramer et al.,
2018; Qu et al., 2019, 2018; Stolterfoht et al., 2018; Winkler, 2018).
The crystal structure of SrCAR during the thioesterification step is
shown in Fig. 4b (Gahloth et al., 2017). The third step is the transfer of
the acyl thioester from the T-domain to the R-domain. This reaction
step is executed by the swing of the phosphopantetheine arm on the T-
domain. The final step is the reduction of the acyl thioester inter-
mediate by NADPH and the release of aldehyde and NADP+ products. It
has been speculated that the aspartate D998 in SrCAR located near the
NADPH binding site plays a crucial role in regulating the reduction of
carboxylic acid to aldehyde as a final product. The D998 G variant of
SrCAR showed that the rate of benzoic acid reduction to form benzyl
alcohol was lowered than the wildtype enzyme (Gahloth et al., 2017).
Recently, Stolterfoht et al. (2018) identified a set of amino acid
residues that are important for CAR activity. Mutagenesis of two re-
sidues in the A-domain (H237A and G433A in Neurospora crassa CAR
(NcCAR)), one residue in the T-domain (S595A in NcCAR, equivalent to
S702 in SrCAR), or two residues in the R-domain (Y844A and K848A in
NcCAR) abolished CAR activity, indicating these residues are essential
for carboxylic acid reduction (Stolterfoht et al., 2018). Moreover, mu-
tation of P234A, P285A and E441A in NcCAR increased the specific
activity of NcCAR towards pentanoic acid, hexanoic acid, heptanoic
acid, octanoic acid substrates, as compared to the wild type NcCAR,
suggesting these residues play a key role in substrate binding and
substrate preference (Stolterfoht et al., 2018).
CAR is widely distributed in numerous fungi and actinobacteria.
Purified CARs from these organisms have been reported to be active
against a broad range of substrates such as C4-C18 fatty acids, hydro-
xyacids, dicarboxylic acids, and (hetero)aromatic acids (Finnigan et al.,
2017; Horvat et al., 2019; Kramer et al., 2018; Schwendenwein et al.,
2016; Stolterfoht et al., 2017). To date, all of the reported CARs require
NADPH and ATP as co-substrates to complete the reduction of car-
boxylic acid to aldehyde (Finnigan et al., 2017; Horvat et al., 2019;
Kramer et al., 2018; Schwendenwein et al., 2016; Stolterfoht et al.,
2017). The Km values for NADPH and ATP have been reported in a
range of 24–36 μM and 64–84 μM, respectively (Finnigan et al., 2017).
Biochemical characterizations of purified CARs from Mycobacterium
phlei (MpCAR), Mycobacterium smegmatis (MsCAR), Nocardia iowensis
(NiCAR), Nocardia otitidiscaviarum (NoCAR), and Tsukamurella paur-
ometabola (TpCAR) showed that CARs from these organisms prefer to
use C8-C12 fatty acids as substrates more than benzoic acid and its
derivatives as judged by the kcat/Km value (Finnigan et al., 2017). When
J. Jaroensuk, et al. Journal of Biotechnology 309 (2020) 1–19

Fig. 4. (a) The crystal structure of SrCAR during the adenylation step (PDB ID code 5MSW) and (b) the thioesterification step (PDB ID code 5MSS) (Gahloth et al.,
2017). During the adenylation step, the T-domain is located away from the A-domain and the conserved lysine (K629 in SrCAR) rotates into the catalytic site. K629
makes a contact with ATP and catalyzes the adenylation by bringing together ATP and carboxylic acid to achieve formation of the acyl-AMP intermediate. After the
adenylation step, the mobile regions of the A-domain and the T-domain rotate into the form that enables the thioesterification (K629 moves away from the catalytic
site of the A-domain, and S702 moves to be closer to the phosphate of the bound AMP). The residues S702, G532, K528, and, G532 exhibit large movements when the
enzyme switches from the adenylation step to the thioesterification step.

the reactions of benzoic acid substrates were compared, the data
showed that substituted benzoic acids with electron-withdrawing
groups at the para- and meta-positions had lower values of Km/kcat
compared to that of benzoic acid without any substituents while ben-
zoic acids with ortho-substituents could not be used as substrates for
CAR. For benzoic acids with electron-donating groups at the ortho-po-
sition, the absence of activity was observed for MpCAR, MsCAR, NiCAR,
and NoCAR, while a decrease in activity was observed for TpCARs. The
steric hindrance of ortho- substituents is thought to be a major cause of
the activity lowering effect in CARs (Finnigan et al., 2017). For benzoic
acids with electron-donating groups at the para- and meta-position,
activities of CARs were only found to be slightly decreased (Finnigan
et al., 2017).
Several hydroxyacids (C2-C6) and dicarboxylic acids (C2-C6) have
also been shown to be substrates for CARs, in which the reactions of
hydroxyacids are more favored than those of dicarboxylic acids

6
J. Jaroensuk, et al.
(Kramer et al., 2018). Steady-state kinetic analyses using the purified
CARs from Mycobacterium avium (MavCAR), Mycobacterium ar-
omaticivorans (MarCAR), and Kutzneria albida (KaCAR) showed that the
catalytic efficiency of these purified CARs with hydroxyacids and di-
carboxylic acids increases with longer chain length, but lower than that
with benzoic acid (Kramer et al., 2018). Overall, the results indicate
that CARs prefer electron-rich acid substrates in which a carboxyl group
is the only charged group (Finnigan et al., 2017; Kramer et al., 2018).
Additionally, CARs have a higher tolerance towards para- and meta-
substituents than to ortho-substituents (Finnigan et al., 2017). Recently,
Schwendenwein et al. (2019) reported identification of a hot spot re-
sidue at the active site of NiCAR which increased its activity towards
meta-and ortho-substituents. Mutagenesis of Q283 to P283 increases
NiCAR affinity to 2‐methoxybenzoic acid, about 9-fold compared to
wild type NiCAR. In addition, the catalytic performance of Q283 P
NiCAR appeared to improve about 3-fold and 4-fold towards 2‐sub-
stituted (2‐Cl, 2‐Br), and 3‐ and 4‐substituted benzoic acid (3-OMe and
4-OMe), respectively.
NADP+, AMP, and PPi have been shown to inhibit the activity of
CARs in vitro. Finnigan et al. (Finnigan et al., 2017) demonstrated that
NADP+ and AMP inhibit CAR by competing against NADPH (Ki =
143 ± 8 μM for NADP+) and ATP (Ki = 8200 ± 900 μM for AMP).
While PPi exhibits mixed type inhibition against ATP (Ki = 220 ± 50
μM), it showed competitive inhibition against the 4-methylbenzoic acid
substrate (Ki = 340 ± 40 μM).
3.1.2. Acyl-[acyl carrier protein (ACP)] reductase (AAR)
Acyl-[acyl carrier protein (ACP)] reductase (AAR) is a key enzyme
for alkane biosynthesis in cyanobacteria. It catalyzes NAD(P)H-depen-
dent reduction by reducing fatty acyl-ACP/CoA substrate to its corre-
sponding fatty aldehyde and releasing a free ACP or CoASH (Fig. 2a).
To date, the AAR crystal structure has not yet been reported.
Steady-state kinetic analysis of the purified AAR from Synechococcus
elongatus PCC7942 (SeAAR) revealed that SeAAR catalyzes the reduc-
tion of fatty acyl-ACP substrate to aldehyde via formation of an acyl-
enzyme thioester intermediate with a cysteine catalytic residue of
SeAAR before the intermediate is reduced to form aldehyde by NADPH.
The SeAAR enzyme is exclusively specific for NADPH
(Km = 35.6 ± 4.9 μM) because NADH cannot be used to generate the
aldehyde product in vitro (Lin et al., 2013). Based on the investigations
of AAR from Nostoc punctiforme PCC73102 (NpAAR) by Warui et al.
(2015), the intermediate was reported to form through the thiol group
of C294 in the active site of NpAAR.
Substrate-specificity analysis demonstrated that AARs can utilize
C12-C20 fatty acyl-CoA and C15-C18 fatty acyl-ACP as substrates (Lin
et al., 2013; Schirmer et al., 2010; Warui et al., 2015). When tested with
C8-C20 fatty acyl-CoA, the purified SeAAR showed the highest activities
towards C18:0-CoA (kcat = 0.36 ± 0.023 min−1, and
Km = 31.9 ± 4.2 μM), followed by C20:0-CoA, and C18:1-CoA/C16:0-
CoA/C12:0-CoA. There was no enzyme activity detected with C8:0-CoA
(Lin et al., 2013). Similar substrate preference with respect to acyl
chain length was observed when the purified NpAAR was tested with
C18-C12 fatty acyl-ACP. NpAAR exhibits a maximum activity towards
C18:0 fatty acyl-ACP (kcat = 0.17 min−1, and Km = 24 ± 6 μM), fol-
lowed by C16:0-ACP, C15:0-ACP and C18:1-CoA. There was no activity
observed when substrates with acyl chain lengths shorter than C14
were used (Warui et al., 2015). It is worth mentioning that a mono-
valent cation (K+) and divalent cations (Mg2+, Mn2+, and Ca2+) are
required for enhancing the in vitro activity and stability of the purified
SeAARs and NpAAR (Lin et al., 2013; Warui et al., 2015).
Kudo et al. (2016) revealed that AARs derived from marine cya-
nobacteria and from freshwater cyanobacteria have different substrate
specificities. Co-overexpression of AARs derived from marine cyano-
bacteria such as Prochlorococcus marinus MIT9313 (PmAAR) or Sy-
nechococcus sp. PCC7336 (7336AAR) with Nostoc punctiforme
PCC73102 ADO (NpADO) in E. coli resulted in formation of
7
Journal of Biotechnology 309 (2020) 1–19
pentadecane (C15 alkane) as a major product (glucose was used as sole
carbon source), indicating that PmAAR and 7336AAR have higher ac-
tivity toward C16-ACP than other substrates. On the contrary, hepta-
decane (C17 alkane) is a primary product produced in the E. coli co-
expressing NpADO and AARs derived from freshwater cyanobacteria
such as SeAAR, Microcystis aeruginosa (MaAAR), or Thermo-
synechococcus elongatus BP-1 (BP-1 AAR). The data imply that C18-ACP
may be the most preferred substrate for AAR derived from fresh water
marine bacteria. However, the type and amount of alkanes produced in
hosts cannot be used as absolute indication about substrate preference
because the data also depend on the type and amount of substrates that
can be produced by hosts.
3.1.3. Fatty acyl-CoA reductase (ACR)
Fatty acyl-CoA reductase (ACR) is a key enzyme involved in wax
ester biosynthesis in Acinetobacter species (Reiser and Somerville,
1997). This enzyme can be utilized for generating aldehyde inter-
mediates in the same manner as CAR. Although ACR can be expressed
well in E. coli, it could not be purified to homogeneity. Therefore, no
ACR crystal structure is available to date.
An enzyme activity assay using ACR in crude lysate from
Acinetobacter calcoaceticus revealed that ACR catalyzes the formation of
fatty aldehyde by using NADPH for reduction of fatty acyl-CoA (Reiser
and Somerville, 1997). Substrate specificity assays indicated that ACR
can utilize a broad range of fatty acyl-CoA as substrates (C14 to C22).
However, ACR has the highest activity towards C16:0-CoA, followed by
C18:0-CoA, C14:0-CoA, C20:0-CoA, and C22:0-CoA respectively. There
was no activity observed when the enzyme was tested with substrates of
acyl chain lengths shorter than C12 (Reiser and Somerville, 1997). ACR
can also be overexpressed in conjunction with ADO to produce alkanes
in engineered metabolic pathways (Jaroensuk et al., 2019), see more
discussion in Section 4.3.
3.2. Enzymes involved in decarbonylation of fatty aldehyde
3.2.1. Aldehyde deformylating oxygenase (ADO)
Aldehyde deformylating oxygenase (ADO) is a non-heme di-iron
enzyme that catalyzes the last step in the biosynthesis of long-chain
hydrocarbons. It catalyzes the decarbonylation of fatty aldehydes to
form Cn-1 alka(e)nes through a redox oxygenation process which re-
quires O2 for activity to provide one oxygen atom which is incorporated
into formate and also requires a reducing system consisting of NADPH,
ferredoxin (Fd), and ferredoxin reductase (FNR) to provide four elec-
trons per turnover (Buer et al., 2014; Das et al., 2014; Li et al., 2012,
2011; Paul et al., 2013; Rajakovich et al., 2015; Zhang et al., 2013).
The mechanisms of ADO underlying catalysis of decarbonylation of
fatty aldehydes to Cn-1 alka(e)nes have been proposed (Fig. 5) (Buer
et al., 2014; Das et al., 2014; Li et al., 2012, 2011; Paul et al., 2013;
Rajakovich et al., 2015; Zhang et al., 2013). The reaction of ADO begins
with (I) the aldehyde substrate binds to the di-iron center (Fe2II/II) and
forms the intermediate 1 (Fe2II/II-aldehyde). Based on investigations of
substrate binding to ADO using NMR, Buer et al. (2014) showed that
the non-hydrated aldehyde is in the aldehyde form when bound to the
enzyme. In addition, the X-ray crystal structure of metal bound ADO by
Buer et al. (2014) also revealed that the aldehyde oxygen is ligated to
Fe2 of the di-iron center, while the water molecule is ligated to Fe1. (II)
Molecular O2 binds to the intermediate 1 at the di-iron center (Fe2II/II)
and generates the di-iron peroxo species (Fe2III/III) which then nucleo-
philically attacks the carbonyl group of the aldehyde. (III-IV) The OeO
bond of the intermediate breaks and receives one electron to generate a
hemiacetal radical, followed by scission of the C1-C2 bond to yield an
alkyl radical. (V) The alkyl radical abstracts one proton and one elec-
tron from the iron-bound water molecule, resulting in formation of an
alkane and regeneration of the Fe1IV/Fe2III cluster (Buer et al., 2014;
Waugh and Marsh, 2014). Buer et al. (2014) reported that the water
molecule enters the enzyme (PmADO) through the channel formed
J. Jaroensuk, et al.
Fig. 5. The mechanism proposed for the decarbonylation of aldehydes to form Cn-1 alka(e)nes catalyzed by ADO (Buer et al., 2014; Das et al., 2014; Li et al., 2012,
2011; Paul et al., 2013; Rajakovich et al., 2015; Zhang et al., 2013). The left and right iron atoms are referred to as Fe1 and Fe2, respectively.
between helices 1 and 3. (VI) The reaction center receives one proton
and one electron from the reducing system and the alkane product is
released. (VII) Formate is released as a final product while the enzyme
accepts two electrons from the reducing system to convert di-ferric iron
(Fe2III/III) to be the active di-ferrous iron (Fe2II/II) center for the sub-
sequent turnover. The key reductant interacting with ADO during the
catalytic turnover is reduced Fd which is generated by FNR using
NADPH as the ultimate reductant. Recently, Jaroensuk et al. showed
that NADH can also be used by FNR as a reductant to generate reduced
Fd, which in turn can be used to support the activity of ADO. In vitro
analysis using a C14 aldehyde as a substrate indicates that the
Fd–NADH–FNR–ADO reconstituted system can generate tridecane as
effectively as the Fd–NADPH–FNR–ADO system (Jaroensuk et al.,
2019).
To identify residues important for the activity of ADO, site directed
mutagenesis of residues identified by X-ray crystal structures were
carried out (Bao et al., 2016; Jia et al., 2015; Khara et al., 2013; Wang
et al., 2017). Bao et al. (2016) demonstrated that mutation of residues
which are located close to the hydrophobic tail of the aldehyde sub-
strate (Fig. 6) resulted in variants (Y21R and C70 F in SeADO) that have
lower activities with medium- and long-chain aldehydes than the wild-
type enzyme: (I) Y21R exhibits low activities with C10:0 and C12:0
aldehydes; (II) Y21R and C70 F exhibit low activities with C14:0-C18:0
aldehydes; (III) Y21R and C70 F exhibit comparable activities as the
wild-type enzyme when using short-chain aldehydes as substrates. The
data clearly indicate that Y21 and C70 are important for medium- to
long-chain substrate selectivity. Wang et al. (2017) reported that mu-
tation of residues close to the di-iron center and active site of the en-
zyme (Y122F and R62A SeADO, Fig. 6) resulted in a significant decrease
in SeADO activity, with the apparent kcat dropping by 70 % for Y122 F
and 92 % for R62A. This may due to the disruption of the hydrogen
bonding network around the di-iron cofactor, impairing protein stabi-
lity and activity. In addition, mutation of residues at the protein surface
such as W178R in SeADO Fig. 6 caused the apparent kcat of W178R
SeADO to increase 3-fold compared to that of the wild type SeADO. The
author suggested that this improvement was due to the increase of
protein solubility and removal of the side chain that blocks substrate
entry (Wang et al., 2017).
Investigation of ADO substrate profiling revealed that ADOs can use
short-, medium-, and long-chain fatty aldehydes (C4–C18) as substrates
(Andre et al., 2013; Bao et al., 2016; Khara et al., 2013). This indicates
8
Journal of Biotechnology 309 (2020) 1–19
that ADO has broad substrate specificity with respect to acyl chain
length. Nevertheless, redesigning the enzyme by site-directed muta-
genesis of the amino acids involved in substrate channeling and the
specificity of ADO resulted in the enzyme with significantly improved
catalytic properties (Bao et al., 2016; Khara et al., 2013; Wang et al.,
2017). For example, the catalytic efficiency (kcat/Km) of the variant
A121 F of SeADO towards the C6 aldehyde was improved 3.8-fold
compared to the wild-type enzyme (Bao et al., 2016).
3.3. Enzymes involved in the decarboxylation of fatty acids
3.3.1. Olefin synthase (OLS)
Olefin synthase (OLS) is a modular multi-domain enzyme consisting
of three modules (Fig. 2b) (Chatterjee et al., 2018; Coates et al., 2014a;
Gu et al., 2009; Mendez-Perez et al., 2011; Moss et al., 2016; Zhu et al.,
2018). The first module is the substrate (fatty acid) activation module
located at the N-terminus of OLS, comprised of a fatty acyl-ACP ligase
(FAAL) and ACP1. FAAL uses ATP to activate fatty acids to yield fatty
acyl-AMP, which is then attacked by the SH group of the phospho-
pantetheine moiety on ACP1 to generate a fatty acyl-ACP1 intermediate
(Fig. 2b, Activation module). This intermediate is later transferred to
the extension module. On this module, the acyl chain of the substrate is
elongated by the activities of ketosynthase (KS), acyltransferase (AT),
ketoreductase (KR), and ACP2. The KS and AT add two carbons from
malonyl-CoA to fatty acyl-ACP2 and the β-carbonyl group is reduced to
form a hydroxy group by the KR domain. The resulting fatty acyl-ACP2
is converted into a terminal alkene by the activities of enzymes in the
terminal alkene formation module, which are sulfotransferase (ST) and
thioesterase (TE). In this step, the ST domain adds a sulfonate group to
the β-hydroxy group via the sulfonate donor, phosphoadenosinepho-
sphosulfate (PAPS), followed by decarboxylation and hydrolysis of the
β-sulfate to form the terminal alkene as catalyzed by the TE domain
(Chatterjee et al., 2018; Coates et al., 2014a; Gu et al., 2009; Mendez-
Perez et al., 2011; Moss et al., 2016).
Almost all filamentous cyanobacteria that contain the OLS pathway
have the FAAL-ACP1 and KS-AT-KR-ACP2-ST-TE systems encoded in
two open reading frames except Leptolyngbya sp. PCC 7376 (unicellular
and baeocystis cyanobacteria), in which all enzyme genes are encoded
in only one reading frame.
J. Jaroensuk, et al. Journal of Biotechnology 309 (2020) 1–19

Fig. 6. The crystal structure of SeADO (PDB ID code 4RC5) (Jia et al., 2015). Residues close to the di-iron center (Y39, R62, Y122, D143) are shown, as well as a key
residue (W178) located on the protein surface and close to the active site, and residues located close to the hydrophobic tail of the aldehyde substrate (Y21 and C70).

3.3.2. Olefin-forming fatty acid decarboxylase (OleT) utilizing H2O2 (peroxide shunt pathway) to generate a ferric
Olefin-forming fatty acid decarboxylase (OleT) is an attractive en- (FeIII)‐hydroperoxo (compound 0) which is then protonated and dehy-
zyme for various applications and has drawn great interest due to its drated to yield a high energy ferryl (FeIV)-oxo species (compound I).
ability to transform free fatty acids to 1-alkenes in a single-step reac- Compound I was observed in single turnover reactions of OleTJE using
tion. OleT found in Jeotgalicoccus sp, Bacillus subtilis, Corynebacterium stopped‐flow absorption spectroscopy (Grant et al., 2015) ; (V) Com-
efficiens, Kocuria rhizophila, and Methylobacterium populi belong to the pound I abstracts a hydrogen atom from the β-carbon position of the
cytochrome P450 CYP152 family and are known to catalyze the dec- fatty acid, generating a substrate radical and the ferryl (FeIV)-hydroxo
arboxylation of medium- to long-chain (C12-C20) fatty acids into 1- species (compound II). (VI) Compound II in OleTJE undergoes two
alkenes (C11-C19) using H2O2 as an oxidant and electron donor possible pathways which are decarboxylation of the fatty acid to form
(Belcher et al., 2014; Matthews et al., 2017; Munro et al., 2018; Rude 1-alkene (g–h) or hydroxylation the substrate at the α‐ or β‐position
et al., 2011; Zachos et al., 2015). Moreover, OleT also catalyzes α- and (i–j).
ß- hydroxylation of fatty acids as a side reaction (Lee et al., 2003;
Matsunaga et al., 2000; Rude et al., 2011; Shoji et al., 2007).
3.3.3. Nonheme mononuclear iron oxidase UndA and UndB
Besides direct addition of H2O2 to the reaction to support OleTJE
UndA and UndB are two major enzymes responsible for the bio-
production of 1-alkenes from fatty acids, the use of light or an H2O2-
synthesis of 1-alkenes in the Pseudomonas species (Rui et al., 2015,
forming enzyme to generate H2O2 for OleTJE has also been explored
2014). Biochemical analysis of UndA demonstrated that UndA requires
(Matthews et al., 2017; Zachos et al., 2015). Fusion of an H2O2-gen-
FeII as a cofactor and O2 as an activator for catalyzing the decarbox-
erating enzyme, alditol oxidase (AldO) from Streptomyces coelicolor,
ylation of medium-chain fatty acids (C10-C14) to generate 1-alkene
with OleTJE has been employed to continuously supply H2O2 for the
with release of CO2 (Rui et al., 2014). Biochemical characterization of
oxidative conversion of fatty acids into 1-alkenes (Matthews et al.,
UndA showed that the stoichiometry of O2 binding to UndA is
2017). This approach was used successfully for the production of 1-
equivalent to the formation of 1-alkene. The results of an in vitro assay
alkenes with a 98 % conversion yield of myristic acid (C14:0) which is
using lauric acid (C12:0) as a substrate also showed that UndA can only
higher than that obtained by direct addition of H2O2 at the start of the
catalyze one turnover of lauric acid (C12:0) to form 1-undecene. Ad-
reaction (87 % conversion yield).
dition of reductants including ascorbate, PMS/NADH, and PQQ/cy-
Recently, Fang et al. (2017) also showed that in addition to H2O2,
steine, could improve the turnover number of UndA in vitro to about 4
OleTJE can also use other redox partner proteins/NAD(P)H-utilizing
turnovers per hour (Rui et al., 2014).
systems to generate 1-alkenes from fatty acids with a 94.4 % yield. The
The results of substrate specificity analyses using lauric acid (LA)
most efficient redox partner protein for 1-alkene production reported in
derivatives which are 2-hydroxydodecanoic acid (AHDA), [α,α-D2]LA,
this study was Fd from Corynebacterium glutamicum and FNR from S.
[D23]LA, 3-hydroxydodecanoic acid (BHDA), and 2,3-dodecanoic acid
elongatus.
(DEA), showed that (I) UndA could convert α-hydroxy fatty acids into
The mechanism of OleT catalysis of the decarboxylation of fatty
1-alkenes but could not use ß-hydroxy fatty acids as a substrate, (II) 1-
acids into 1-alkenes or α- or ß-OH fatty acids has been proposed (Fig. 7)
undecene produced from [α,α-D2]LA retained both deuterium atoms,
(Belcher et al., 2014; Lee et al., 2003; Munro et al., 2018; Rude et al.,
and (III) the UndA assay with [D23]LA resulted in the formation of
2011). The reaction of OleT consists of six steps: (I) Fatty acids bind to
[D22]1-undecene (Rui et al., 2014). Based on this biochemical analysis,
the iron center by displacing the axial water molecule and converting
Rui et al. (2014) proposed a mechanism to describe UndA catalysis of
the heme iron from a low spin to a high spin state (Sligar and Gunsalus,
the decarboxylation of fatty acids into 1-alkenes. The reaction of UndA
1979); (II-IV) OleT bypasses the O2-redox partner system (a–d) by
consists of three steps: (I) fatty acid binds to the mononuclear iron (FeII)

9
J. Jaroensuk, et al.
Fig. 7. Proposed mechanism of OleT catalysis of the decarboxylation of fatty acids into 1-alkenes or α- or ß−OH fatty acids (Belcher et al., 2014; Lee et al., 2003;
Munro et al., 2018; Rude et al., 2011).
Fig. 8. Proposed mechanism of UndA synthesis of an alkene with a terminal double bond (Rui et al., 2014).
center at the active site of UndA and forms intermediate 1 (FeII-fatty
acid), (II-III) the Fe(III)-superoxide complex formed at the active site of
UndA following O2 binding abstracts a hydrogen from the ß-carbon of
the fatty acid, followed by one electron transfer and scission of the
carbon-carbon bond, resulting in formation of 1-undecene, CO2 and
H2O (Fig. 8) (Rui et al., 2014). Structural analysis of UndA in complex
with 3-hydroxydodecanoic acid (BHDA) also supports the mechanism
proposed above because the position of the ß-hydrogen of the fatty acid
is ideal for abstraction, as the distance between the ß-hydroxyl oxygen
of BHDA and the distal oxygen atom is only 2.45°A (Rui et al., 2014).
UndB, a membrane-bound desaturase-like enzyme, catalyzes the
formation of 1-alkene from short- to medium-chain fatty acid (C6-C16)
in the same manner as UndA. UndB is only distributed in a few species
of Acinetobacter and Pseudomonas, such as P. fluorescens and P. mendo-
cina. Overexpression in E. coli of UndB with UcFatB2, a thioesterase
which specifically hydrolyzes the lauryl chain from ACP resulted in a
100-fold improvement in 1-undecene production as compared to a
strain in which UndB was overexpressed alone. The results indicate that
free fatty acids are the preferred substrates of UndB. Additionally, co-
expression of electron transfer systems from E. coli (e.g. ferredoxin-
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Journal of Biotechnology 309 (2020) 1–19
ferredoxin reductase (EcFd-EcFNR), flavodoxin-ferredoxin reductase
(EcFld-EcFNR)), and from Pseudomonas putida (e.g. putidaredoxin-puti-
daredoxin reductase (Pd-PNR)) with UndB also increased the produc-
tion of 1-alkenes in engineered E. coli by approximately 1.5-, 1.75-, and
2.25-fold as compared to the engineered strain harboring only UndB,
respectively (Zhou et al., 2018a).
3.3.4. Fatty acid photodecarboxylase (FAP)
FAP is a flavoenzyme found in Chlorella green algae that possesses
the ability to produce hydrocarbons through the photodecarboxylation
of fatty acids (Sorigue et al., 2017; Yu et al., 2019). This enzyme needs
photons from blue light (400−520 nm) to activate its FAD cofactor for
catalysis as shown in Fig. 9 (Huijbers et al., 2018; Sorigue et al., 2017;
Sorigué et al., 2016; Zhang et al., 2019). Several studies have shown
that FAP exhibits turnover numbers (TON) in the range of thousands
(Huijbers et al., 2018; Zhang et al., 2019), suggesting that use of FAP
for large-scale production of alka(e)nes from fatty acids may be feasible
(Hoeven et al., 2019; Scrutton, 2017).
The mechanism of FAP catalysis of the decarboxylation of fatty
acids to Cn-1 alka(e)nes has been proposed (Fig. 9) (Huijbers et al.,
J. Jaroensuk, et al. Journal of Biotechnology 309 (2020) 1–19

Fig. 9. The reaction mechanism of FAP conversion of a fatty acid into alka(e)ne starts by a base abstraction of a fatty acid to result in a carboxylate anion and FAD
(RCOO−-FAD). The di-radical complex (RCOO●…●-FAD) is produced after an excited singlet FAD* abstracts one electron from the carboxylate substrate. Finally, the
reduction of the alkyl radical results in release of the alka(e)ne and CO2 (Huijbers et al., 2018; Sorigue et al., 2017; Zhang et al., 2019).

2018; Sorigue et al., 2017; Zhang et al., 2019). The mechanism consists 4. Metabolic Engineering or cascade reactions for the synthesis of
of three steps: (I) a proton from the fatty acid is abstracted by an un- hydrocarbons
known base (B−) to generate the carboxylate anion that will then form
an intermediate complex with FAD (RCOO−-FAD). Theoretical studies The overall diagram describing the engineered metabolic pathways
from density functional theory (DFT) and time dependent-DFT (TD- for conversion of fatty acids to hydrocarbons is in Fig. 10.
DFT) revealed that coordination between the carboxylate group and the
N5 site of FAD is required to initiate the activity of FAP (Zhang et al.,
4.1. Carboxylic acid reductase-Aldehyde deformylating oxygenase (CAR-
2019). In steps II-III, an excited singlet state, FAD*, generated from the
ADO)
photo excitation, abstracts one electron from the carboxylate substrate
to yield a di-radical complex (RCOO●…●-FAD) which then rapidly
The combined use of CAR and ADO has been employed in various
converts to an alky radical, and later an alka(e)ne. The reduction of an
engineered pathways for the in vivo production of aliphatic hydro-
alkyl radical to an alka(e)ne (step III) has been speculated to occur via
carbons. This system was first reported in 2013 by Akhtar et al. (2013)
two mechanisms (Sorigue et al., 2017): (a) the alkyl radical receives one
in which the CAR enzyme from Mycobacterium marinum (MmCAR) and
electron from FAD●- and one proton from a proton donor via a proton-
the cyanobacterial ADO from Prochlorococcus marinus (PmADO) were
coupled electron transfer mechanism to yield B− and release of alka(e)
co-expressed in E. coli along with two additional enzymes, namely E.
ne; (b) the alkyl radical receives one hydrogen from BH, followed by
coli thioesterase A (TesA) which catalyzes conversion of fatty acyl-ACP/
one electron transfer from FAD●- to B●, generating B− and releasing
CoA to free fatty acids and Bacillus subtilis Sfp as a PPT to activate CAR
the alka(e)ne (Sorigue et al., 2017).
(Beld et al., 2014). In the presence of exogenous fatty acid, the culture
The substrate specificity of FAP has been investigated (Huijbers
of CAR-ADO containing cells produced a mixture of medium-chain
et al., 2018; Zhang et al., 2019). FAP from Chlorella variabilis NC64A
hydrocarbons at an approximate concentration of 2 mg/L. It was,
(CvFAP) exhibits the ability to utilize C1-C20 fatty acids as substrates.
however, suggested that such poor yield was likely the result of very
However, CvFAP showed greater activities towards long-chain fatty
low turnover rates of ADO and accumulation of cellular toxicants
acids (C > 14) than short- and medium-chain fatty acids. It has been
(Andre et al., 2013). This was likely due to the fact that aldehyde re-
reported that a 90–96 % conversion of C16:0, C17:0, C18:0, and C20
ductases and dehydrogenases can also compete with ADO for fatty al-
substrates could be obtained after performing the photoenzymatic re-
dehyde intermediates (Akhtar et al., 2013; Kallio et al., 2014).
action (blue light intensity = 13.7 μEL−1s−1) for 14 h in 100 mM Tris-
The MmCAR-PmADO pathway has also been utilized for the pro-
HCl (pH 8.5) containing 30 mM fatty acid substrate, 30 % DMSO, and
duction of short-chain alkanes such as propane in E. coli (Kallio et al.,
6.0 μM of crude extract CvFAP (Huijbers et al., 2018). Moreover,
2014; Menon et al., 2015). The principal concept for biosynthesis of
Huijbers et al. (2018) also observed that the crude extract CvFAP is
propane by the MmCAR-PmADO pathway is similar to that of the
much more active and stable than the purified CvFAP.
medium-chain alkanes previously mentioned (Beld et al., 2014). Im-
provements of the system were done through (I) the substitution of E.

11
J. Jaroensuk, et al.
Fig. 10. Overview of engineered metabolic pathways for hydrocarbon biosynthesis from fatty acids. The pink panel represents fatty acid biosynthesis, the central
metabolism for the biosynthesis of hydrocarbon using endogenous fatty acid. The gray panel shows the pathway related to alkane synthesis. The yellow panel shows
the pathway involved in alkene synthesis. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
coli TesA with Bacteroides fragilis Tes4 which was reported to have
higher specificity towards C4 butyryl-ACP (Kallio et al., 2014); (II) the
substitution of wild-type PmADO with A134 F PmADO that has a high
affinity and activity towards butyraldehyde (Khara et al., 2013;
Sheppard et al., 2016); (III) the addition of redox partner proteins
(Synechocystis sp. PCC6803 Fd (SynPCC608Fd) and E. coli FNR (EcFNR))
and E. coli catalase (KatE); (IV) deletion of ahr and yqhD which encode
the enzymes that can competitively consume butyraldehyde, the im-
mediate precursor of propane synthesis. This strategy led to a remark-
able improvement in propane production (32 mg/L) after 19-h culti-
vation (Kallio et al., 2014).
Due to the fact that the microbial platforms for propane biosynthesis
rely on the flux of butyryl-ACP (precursor) from fatty acid biosynthesis,
alternative pathways to redirect the precursors towards formation of
propane have been explored. Menon et al. (2015) designed a set of
alternative CoA-dependent butyraldehyde synthetic routes which uti-
lize enzymes acetyl-CoA acetyltransferase (AtoB) and acetoacetyl-CoA
synthase (NphT7) encoded by the genes atoB and nphT7, respectively,
to be overexpressed in E. coli BL21 (DE3). Coupling of MmCAR-A134 F
PmADO-SynPCC608Fd-EcFNR with AtoB and NphT7 was able to pro-
duce propane with a titer of 3.40 ± 0.19 mg/L over 12 h of cultivation
(Menon et al., 2015).
More recently, the CAR-ADO pathway has been investigated in
order to identify the best combination of enzymes for the production of
alkanes from exogenous fatty acids in E. coli. ADOs from various strains
of cyanobacteria were combined with MmCAR, SynPCC608Fd and
EcFNR for C3-C7 alkane biosynthesis (Patrikainen et al., 2017). The
highest yield was obtained with the engineered system bearing NpADO,
giving a heptane yield of around 35 % from exogenous fatty acid. It can
thus be inferred that selection of an efficient ADO is one of the crucial
factors to achieving successful production yields of alkane because the
12
Journal of Biotechnology 309 (2020) 1–19
reaction is widely believed to be the rate-limiting step for bio-alkane
production (Patrikainen et al., 2017; Warui et al., 2015).
Another feasible cell factory explored for hydrocarbon formation
was Saccharomyces cerevisiae. According to Zhou et al. (2016b), the
yeast strain was first engineered to be capable of accumulating high
concentrations of free fatty acids (FFAs) prior to equipping the cell with
the alkane-producing system including E. coli Fd (EcFd), EcFNR,
MmCAR, and NpADO. This engineered yeast strain was found to give
alkane of up to 0.82 mg/L, which is still very low. The yield improve-
ment in yeast could be achieved by peroxisomal compartmentalization
of the biosynthetic routes in order to minimize the cytosolic toxicity of
H2O2 and spatially sequester the synthetic enzymes of interest away
from competing reactions (Zhou et al., 2016a; Zhu et al., 2017). This
strategy resulted in production of alkanes at a level of about 4 mg/L by
the engineered yeast strain with the expression of S. elongatus Fd (SeFd),
S. elongatus FNR (SeFNR), MmCAR, and SeADO in peroxisomes (Zhou
et al., 2016a).
Altogether, it is clear that further improvement is needed for en-
hancing the production efficiency of hydrocarbon biosynthesis while
circumventing the potential bottlenecks, in either yeast or bacteria, for
production of alkanes by the CAR-ADO systems. More investigation of
the CAR-ADO platform in both prokaryotic and eukaryotic hosts as well
as a better understanding of ADO catalysis can improve the perfor-
mance of these engineered cells for hydrocarbon production.
4.2. Acyl-[acyl carrier protein (ACP)] reductase-Aldehyde deformylating
oxygenase (AAR-ADO)
The AAR-ADO system has been investigated in several studies for
the production of long-chain alkanes. With heterologous expression of
SeAAR and NpADO in E. coli, the alkane titer (tridecane, pentadecene,
J. Jaroensuk, et al.
pentadecane and heptadecane) was able to reach 300 mg/L in which
more than 80 % of the product is secreted outside the cell (Schirmer
et al., 2010). The possibility of introducing multiple copies of aar-ado
into the genome of cyanobacterial host strain was also explored. Wang
et al. (2013) succeeded in obtaining the engineered Synechocystis sp.
PCC6803 strain with extra copies of aar-ado in both slr0168 (unknown
function) and slr1556 (2-hydroxyacid dehydrogenase gene, ddh) loci.
The engineered Synechocystis sp. PCC6803 resulted in production of
alka(e)ne with titer of 26 mg/L from a 50 ml culture in a photo-
bioreactor. This level of alkane production is approximately eight-fold
higher than the production levels attained with the wild-type strain,
suggesting that this approach may be used for future hydrocarbon
production by genetically engineered hosts.
Other plausible genetic manipulations, particularly those which
supply other additional associated genes responsible for driving the
metabolic flux towards alka(e)ne biosynthesis, have often been per-
formed following the establishment of AAR-ADO in these cells. For
example, Coursolle et al. (2015) revealed that the co-expression of type-
I fatty acid synthase (FAS) from Corynebacterium ammoniagenes with
SeAAR-SeADO in the BL21(DE3) enhanced the total titer of mixed long-
chain alkane/alcohol to roughly 100 mg/L, in which the pentadecane
content was 57 mg/L. This yield is higher than when only SeAAR-
SeADO (∼8.15 mg/L) was expressed (Coursolle et al., 2015) (Table 1).
One of the most notable improvements for long-chain alkane bio-
synthesis in E. coli via the AAR-ADO pathway was recently reported by
Fatma et al. (2018) In this study, the flux towards fatty acid synthesis
routes in E. coli was enhanced by upregulating the gene encoding for
glucose-6-phosphate dehydrogenase (zwf) to boost the accumulation of
intracellular NADPH. Meanwhile, six sets of genes including the Entner-
Doudoroff pathway (phosphogluconate dehydratase, edd), gluconeo-
genesis pathway (phosphoenolpyruvate synthase, ppsA), fermentative
pathway (D-lactate dehydrogenase, ldhA; pyruvate oxidase, poxB),
glyoxylate pathway (isocitrate lyase, aceA) and phospholipid pathway
(acyl-ACP:phosphate transacylase) were deleted to remove the path-
ways competing for glucose. The engineered cells resulted in an alkane
titer of 425 mg/L. The production was elevated to 2.54 g/L after 72-h
fed-batch cultivation in a 5 L fermenter using glucose as substrate
(500 g/L). The alkane titer from this work has surpassed the previously
highest titer (1.31 g/L) obtained from a 2.5 L fed-batch culture of an
engineered strain with AAR-ADO overexpression and an electron
transfer system from S. elongatus (Cao et al., 2016).
Combination of AAR-ADO expression in yeast resulted in much
lower efficiency of hydrocarbon generation than the CAR-ADO pathway
(Zhou et al., 2016a, b). However, alkane synthesis by strains bearing
either AAR or CAR with various ADO and electron-donating modules
was improved with the compartmentalization approach (Zhou et al.,
2016a, b). Nevertheless, it could be seen that the use of yeast cells for
overexpression of AAR-ADO for hydrocarbon production is not as effi-
cient as the use of bacterial hosts.
4.3. Fatty acyl-CoA reductase-Aldehyde deformylating oxygenase (ACR-
ADO)
Unlike the previous two systems mentioned, overexpression of ACR
and ADO to generate engineered metabolic cells for production of al-
kanes has only been recently investigated (Akhtar et al., 2013;
Jaroensuk et al., 2019). In the past, overexpression of ACR has been
used for production of other fatty acid-derived chemicals such as fatty
alcohol and wax esters (Lehtinen et al., 2018; Santala et al., 2011,
2014). Recently, our team has successfully developed an artificial me-
tabolic pathway comprised of ACR, ADO, Fd, FNR, and XaFDH for the
synthesis of hydrocarbons from exogenous fatty acid. A key invention of
our system is the improvement ACR-ADO mediated alkane production
by addition of formate dehydrogenase (FDH) activity. FDH from Xan-
thobacter sp. 91 (XaFDH was added into the metabolic pathway so that
it can convert formate, an unused co-product of ADO reaction, into
13
Journal of Biotechnology 309 (2020) 1–19
NAD(P)H, an important reductant of alkane biosynthesis (Fig. 10)
(Jaroensuk et al., 2019). Based on this strategy, we managed to produce
alkane with a 50 % yield from exogenous fatty acid which is the best
yield to date. Therefore, this approach should be useful for future
production of fatty acid-derived chemicals.
4.4. Terminal alkene synthesis
OleTJE, UndA, and UndB have been utilized for producing terminal
alkenes in many engineered cell factories such as E. coli and S. cerevisiae
(Zhou et al., 2016a, a). In E. coli, Liu et al. (2014b) engineered E. coli by
deletion of the acyl-CoA synthetase gene (fadD) and overexpression of
acyl-CoA carboxylase (ACC), thioesterase A (TesA) and OleTJE or OleTJE
fused with a CYP450 reductase domain from Rhodococcus sp (RhFRED),
and used the engineered cells for production of 1-alkene. Interestingly,
the engineered E. coli harboring OleTJE produced total 1-alkene
(96.7 mg/mL) more than that of the engineered E. coli harboring OleTJE
fused with RhFRED (6.0 mg/mL). The author suggested that high pro-
duction of alkene in E. coli harboring OleTJE was due to the ability of
OleTJE to use electron transfer protein in E. coli (i.e. Fd & FNR) to drive
catalysis efficiently.
In yeast, Zhou et al. (2018a) demonstrated the overexpression of
OleTJE, P. putida UndA (PpUndA), P. mendocina UndB (PmUndB), and P.
fluorescens (PfUndB) in a fatty acid-overproducing yeast strain (S. cer-
evisiae YJZ06) for production of 1-alkene. All the engineered yeast
strains were able to produce 1-alkene, however with a very low titer
(∼1−6 mg/L). Among the four engineered yeast strains, the strain
harboring PfUndB produced the highest titer of 1-alkenes. To improve
the production, various electron transfer systems were co-expressed
with PfUndB in S. cerevisiae to enhance the reduction of FeIII to FeII, an
important cofactor of UndB. The Pd-PNR showed the highest im-
provement of PfUndB activity. The 1-alkene production was improved
by 50 % compared to the strain expressing only PfUndB. In addition, a
long-chain fatty acid transporter (FATP1) from Homo sapiens was en-
gineered into S. cerevisiae. All efforts led to the production of 1-alkene
with a titer of 35 mg/L, of which, 80 % was secreted into the medium
(Zhou et al., 2018a).
Terminal alkene synthesis by an in vitro enzymatic cascade has also
been explored as an alternative strategy. Dennig et al. (2015) employed
OleTJE and Pd-PNR, an effective redox partner of OleTJE for production
of 1-alkene. By this system, they acquired 0.93 g/L of C17 alkene from
octadecanoic acid as a starting substrate and turnover numbers of more
than 2000 per 8 h. Yan et al. (2015) demonstrated a one-pot reaction
for the synthesis of 1-alkene from triacylglycerols (TAGs) using lipase
from Thermomyces lanuginosus (Tllipase) and OleTJE. Although 1-al-
kenes were produced from the TAGs, the yield and reaction rate were
very low. Recently, Li et al. (2019) developed a new one-pot process for
production of 1-alkenes from TAGs by immobilizing the dockerin-taq
enzymes (Tllipase-D1 and P450 OleTJE-D2) on the cohesion-cellulose
binding module. The results showed that the co-immobilization of
OleTJE with Tllipase on the cellulose matrix enhanced the reaction rate
and conversion of TAGs to 1-alkenes. The reaction rate and yield in-
creased by 9-fold and 2.8-fold respectively, when compared to the re-
action using a mixture of free enzymes. The most efficient cascade for 1-
alkene production was comprised of a 1:2 ratio of Tllipase to OleTJE. In
addition, the stability and recyclability of the immobilized enzyme
complexes were better than those of the free enzyme mixtures. The
activities of the immobilized enzyme complexes remained around 89 %
after reuse for 10 times.
4.5. Fatty acid photodecarboxylase (FAP)
Overexpression of FAP to generate the metabolically engineered
cells for production of alkane has only been recently investigated
(Bruder et al., 2019; Hoeven et al., 2019). Hoeven et al. (2019) de-
monstrated the utilization of a variant CvFAP G462 V for production of
 J. Jaroensuk, et al.

Table 1
Scale-up production of hydrocarbon compounds by engineered microbial strains.
Ref. Fermentation mode (bioreactor Time (h) Culture media Carbon Feeding solution Microorganism Product titer (g/L) Productivity (g/L/h)
size in liter) source

(Kim et al., Fed-batch mode (5 or 6.6 L); 101.3 Minimal medium 40 g/L 800 g/L glucose, 5 g/L Rhodococcus opacus ROPA2 5.20 (C12, C14-C19, C21, C24, 0.051
2019) initial working volume (2 L); glucose MgSO4•7H2O (100 mL C27 alkanes)
final volume (2.48 L) feeding)
(Fatma et al., Fed-batch mode (5 L); initial 84 Modified M9 medium 20 g/L 200 g/L yeast extract, Escherichia coli strain ZFM8 2.50 (C13, C15, C17 alkanes; 0.031
2018) working volume (3 L) glucose 500 g/L glucose, 12.47 g/L C15, C17 alkenes)
MgSO4
(100 mL feeding)
(Wang et al., Fed-batch mode (5 L); initial 48 Modified M9 medium 30 g/L 2−3 g/L/h glucose E. coli BL21 (DE3) with 1.009 (C15, C17 alkanes; C17 0.021
2018) working volume (n.a.) glucose overexpression of fadR-ΔyqhD alkene)

14
(Choi and Lee, Fed-batch mode (6.6 L); initial n.a. MR medium 20 g/L 400 g/L glucose, 14 g/L E. coli w3110 strain GAS3 with 0.580 (C9, C12, C13, C14 n.a.
2013) working volume (2 L) glucose MgSO4•7H2O (100 mL tesA(L109 P) alkanes)
feeding) E. coli w3110 with tesA 0.804 (C13, C15 alkanes) n.a.
(Xin et al., 2017) Batch mode (10 L); initial 120 Heavy oil producing 160 g/L – Aureobasidium melanogenum 9-1 43.0 (89.8 % alkane; 10.19 % 0.358 (product);
working volume (7 L) medium inulin transformant 88 (inulinase C16, C18 fatty acids) 0.354 (alkane)
overexpression)
A. melanogenum 9-1 38.24 (87.12 % C20-C34 0.319 (product);
alkanes; 12.88 % C16, C18 0.278 (alkane)
fatty acids)
(Cao et al., Batch mode (5 L); initial working 50.5 (40.5 h after Mineral medium 15 g/L 500 g/L glycerol, 200 g/L E. coli BL21 (DE3) with plasmid 1.31 (C15 alkane; C15, C17 0.032
2016) volume (2.5 L) induction) glycerol yeast extract, and 2.47 g/L YX10+SeFd/Fnr alkenes)
MgSO4
(Chen et al., Fed-batch mode (5 L); initial 144 YPD medium + G418 30 g/L 200 g/L glucose (150 mL S. cerevisiae strain BY22 0.0037 (C11, C13, C15, C17, 0.000026
2015) working volume (1 L) medium glucose feeding) C19 alkenes)
(Liu et al., Batch mode (10 L); initial 168 Heavy oil producing 120 g/L – A. melanogenum (native strain) 32.5 g/L heavy oil (66.15 % 0.128
2014a) working volume (6 L) medium glucose C13, C15, C24, C26, C27, C28,
C44 alkanes; fatty acids 26.38
%)
Journal of Biotechnology 309 (2020) 1–19
J. Jaroensuk, et al.
propane in E. coli, Halomonas sp, and Synechocystis sp. The engineered
E. coli harboring CvFAP G462 V was found to produce propane
(48.31 + 2.66 mg/L) from 10 mM butyric acid within 18 h. The titer of
propane was further improved by adding ethyl acetoacetate into the
medium to activate a butyrate uptake-transporter, atoE. The titer of
propane obtained by this approach was 97.1 + 10.3 mg/L, the highest
titer reported to date for biopropane production using engineered E. coli
(Hoeven et al., 2019; Kallio et al., 2014; Menon et al., 2015).
The significant improvement of propane production was obtained
when CvFAP G462 V was overexpressed in Halomonas sp. (Hoeven
et al., 2019). Under non-sterile conditions, the engineered Halomonas
can produce propane at a level of 157 +/- 17 mg/L from 80 mM butyric
acid within 48 h, the highest titer reported to date for biopropane
production using an engineered microbe (Hoeven et al., 2019; Kallio
et al., 2014; Menon et al., 2015). To expand the use of CvFAP for
production of propane from CO2, CvFAP was expressed in Synechocystis
sp. The titer of propane produced from this engineered cyanobacterium
was reported at 12.2 +/- 2.6 mg propane/g cells/day (Hoeven et al.,
2019).
Hydrocarbons synthesis by in vitro enzymatic cascade has also been
explored. A full length CvFAP could not be overexpressed. Only the
CvFAP variant lacking a chloroplast-targeting sequence could be over-
expressed in soluble form (Huijbers et al., 2018; Zhang et al., 2019). A
two-step reaction for the synthesis of alkanes from triglycerides using
lipase from Candida rugosa (Crlipase) and the CvFAP variant could be
used to synthesize heptadec-8-ene from triolein with more than 80 %
yield and turnover number (TON = molproduct × molFAP −1) of 8280
(reaction time used of 20 h) (Huijbers et al., 2018). In another recent
study on hydrocarbons synthesis, a decoy molecule was used to fill up
part of the substrate tunnel of CvFAP, resulting in expansion of sub-
strate scope to include carboxylic acids with straight-chain, branched,
and unsaturated structures and to increase the rate of decarboxylation
(Zhang et al., 2019). In addition to hydrocarbon synthesis, CvFAP was
also used to synthesize chiral α-hydroxy/amino carboxylic acids (Xu
et al., 2019). The results demonstrated that a single residue substitution
(G426Y) in the substrate channel of CvFAP can increase stereo-
selectivity of this reaction significantly (ee up to 99 %).
5. Large scale production
Despite there being many types of native microorganisms that are
capable of synthesizing hydrocarbons, their production yields are all
low and impossible to employ in industrial processes. Therefore, most
studies of scaling up fermentation processes have only been focused on
the metabolically engineered or genetically modified (GM) microbial
hosts, e.g. E. coli, Rhodococcus opacus, S. cerevisiae and Yarrowia lipoly-
tica (Jimenez-Diaz et al., 2017; Schirmer et al., 2010, 2014) Redirecting
the host system to produce a higher titer of products via metabolic
engineering is generally perceived as one of the major approaches to
achieve successful production of alka(e)nes for industrial use. In 2013,
the engineered E. coli W3310 was reported to produce alkane up to
0.804 g/L from glucose using fed-batch fermentation (Choi and Lee,
2013). Using the same mode of cultivation, the titers of alkane pro-
duced by the engineered E. coli strain BL21 (DE3) with overexpression
of aar-ado-fadR and deletion of yqhD and the E. coli strain DH5α with
overexpression of aar-ado-zwf and deletion of edd, ppsA, ldhA, aceA,
poxB and plsX reached 1.009 and 2.50 g/L, with productivity rates of
0.021 and 0.031 g/L/h respectively (Fatma et al., 2018; Wang et al.,
2018). The low production titers are thought to be due to the fact that
the glucose substrate fed into the culture is predominantly catabolized
and used in other pathways to generate energy, biomass and other
metabolites besides hydrocarbon (Kim et al., 2019). Further investiga-
tion and evaluation of large-scale microbial fermentation for an im-
proved and sustainable hydrocarbon-producing system are required to
make the systems into economically viable modes of production of
hydrocarbons.
15
Journal of Biotechnology 309 (2020) 1–19
In addition to E. coli, other microbial hosts were also explored to
produce hydrocarbon on a larger fermentation scale. For example, an
engineered S. cerevisiae strain BY22 was reported to produce about
3.7 mg/L of alkene, with a relatively low productivity rate of
0.000026 g/L/h, even after 144 h of fed-batch cultivation using glucose
as carbon source (Chen et al., 2015). Investigations using exogenous
fatty acid as a sole carbon source for alkane biosynthesis by engineered
S. cerevisiae were later explored (Foo et al., 2017). The fermentation of
an engineered S. cerevisiae strain (Δfaa1Δfaa4) with overexpressed
SeADO and the enzyme which can produce Cn-1 aldehydes from fatty
acids (α-dioxygenase (α-DOX)) from Oryza sativa in a medium con-
taining 200 mg/L exogenous fatty acid (C12:0-C18:0) as the sole carbon
source was reported to produce 28.7, 304.0, and 5.1 mg/L of dodecane,
tetradecane, and hexadecane at pH 7.0, after 48 h, respectively (Foo
et al., 2017). Nevertheless, the alkane titers were still low. This has led
to exploration on other microorganisms for biosynthesis and conversion
of carbon sources to hydrocarbons. The bacterium R. opacus has been
used as a host candidate for hydrocarbon synthesis. The engineered R.
opacus strain (ΔfadEΔalk-1) with overexpressed of SeADO and enzymes
for the production of free fatty acids (TAG lipase (LPD05381) from R.
opacus and lipase chaperon (LSF) from Burkholderia cepacian) and en-
zymes for the production of fatty aldehydes (acyl-CoA reductase (ACR)
from Clostridium kluyveri) was able to produce 5.20 g/L of alkane with
the productivity rate of 0.051 g/L/h when glucose was used as a sole
carbon source under a fed-batch process (Kim et al., 2019).
While significant efforts have been devoted to engineer microbial
strains for greater superiority in forming hydrocarbon products, the
culture of some native strains, including Aureobasidium melanogenum
strain P5, an oleaginous fungal endophyte found to be associated with a
mangrove species, has already been observed to give process yields and
productivities acceptable for larger scale production. The strain
P5 grown in a 6 L initial working volume of batch culture yielded a
mixture of n-alkanes and fatty acids (32.5 g/L), with C13, C15, C24,
C26, C28, and C44 alkanes adding up to a total yield of 66 % (21.5 g/L)
with a productivity rate of 0.128 g/L/h, which was higher than those of
the engineered hosts in several other studies (Liu et al., 2014a). How-
ever, the highest production performance to date has been found in a
different strain of A. melanogenum 9-1 with overexpressed inulinase,
giving a yield of 43.0 g/L of heavy oil containing 89.8 % alkane at a
productivity rate of 0.354 g/L/h in a batch process with 7 L starting
volume fed with inulin as the sole carbon source (Xin et al., 2017).
For a scale-up cultivation for hydrocarbon production, it can be seen
that a wide range of carbon sources can be chosen as well as different
fermentation modes. Possible feeding substrates include simple sugars
(e.g. glucose, fructose, galactose and lactose), fatty acids or even bio-
mass such as lignocellulosic materials (Grim et al., 2019; Hoeven et al.,
2019; Kohli et al., 2019; Luo et al., 2019; Ng et al., 2019; Toogood and
Scrutton, 2018; Zhou et al., 2018b). Although direct supplementation of
exogenous fatty acids has not been reported on such scale, some studies
attempted to construct the engineered strains with upregulation of
enzymes such as 3-oxoacyl-ACP synthase (FabH), which is responsible
for the production of endogenous fatty acid to be subsequently con-
verted to hydrocarbon in vivo (Choi and Lee, 2013). As an alternative
feedstock, glycerol, a by-product derived from saponification or trans-
esterification of triglycerides, was also explored as another source of
carbon. Culture of the engineered E. coli BL21 (DE3) with alkane bio-
synthetic genes using a 2.5 L fed-batch process in glycerol feed for
50.5 h yielded a mixture of C15 alkane and C15 and C17 alkenes with a
yield of 1.31 g/L and a productivity rate of 0.032 g/L/h (Cao et al.,
2016). In terms of fermentation mode, either batch or fed-batch process
is commonly implemented to grow producer strains when scaling up.
However, continuous operation for manufacturing hydrocarbon com-
pounds has not been reported. One underlying reason could be due in
part to the low yield of hydrocarbon production. More optimization of
yield improvement is needed so this technology can reach the level
which is needed for economy of scale.
J. Jaroensuk, et al.
6. Future perspectives
Based on the current technologies available (Sections 2–5), it can be
seen that various types of enzymatic cascades and metabolic engineered
cells are capable of enabling de novo synthesis of alkanes and alkenes
from bio-based feedstocks such as sugars and fatty acids (Fig. 10).
As the current and future trends of industries will require all pro-
duction to meet UN 17 sustainable development goals and any in-
dustries with high emission of greenhouse gas will be required to pay
carbon tax (Ramseur and Leggett, 2019), enzymatic and cell-based
methods serve as beautiful alternative technologies for hydrocarbon
production. These methods only involve bioconversion or fermentable
processes in which the whole technology generates very low CO2
emission and does not produce toxic compounds. In addition to being
green and clean for the environment, the source for hydrocarbon pro-
duction by this method is also renewable. The engineered enzymes and
cells have the potential to convert abundant biomass from agriculture,
agro-industry and food waste from household for large scale production
of alkanes and alkenes.
Robust and efficient enzymatic cascades and metabolically en-
gineered cells for producing hydrocarbons also have the potential to be
revolutionary tools for sustainable and also for developing new business
models for alkane and alkene production. Rather than relying on
countries with big oil reserves to provide crude oil and gas for hydro-
carbon production, any country with considerable biomass feedstocks
from agriculture and agro-industries can develop a new business model
for alkane and alkene production to be done locally. The technology
can even be coupled with a waste management program and can po-
tentially solve local waste and environmental problems. For example,
our research group has engaged in a project in which we propose to use
enzymatic cascades and metabolically engineered cells to increase the
value of household food waste from the community where compre-
hensive waste sorting and segregation is implemented (C-ROS, 2019).
We are developing technology to convert food waste into hydrocarbons
to be used as fuels for farming engines. Such value addition to food
waste will support the ZeroWaste program because it will encourage the
community to segregate waste so that food waste can be used for the
production of hydrocarbons. We believe that the bio-based method may
be relevant for industrial hydrocarbon production in the long run be-
cause the process is sustainable, creates no environmental damage and
can be used as an important tool for waste management.
In conclusion, enzymatic cascades and metabolically engineered
cells that can produce good yields of alkanes and alkenes are important,
clean, and sustainable technologies for the future production of fuels
and materials. This technology will allow countries without big oil re-
serves in their homeland to be capable of producing precursors for
material industries. However, more development of and research on
new enzymes, new pathways and new types of engineered cells are
needed in order to obtain the whole cell biocatalysts which can provide
greater yield with lower production costs to meet the techno-economic
feasibility requirement.
Declaration of Competing Interest
The authors declare that there is no conflict of interest.
Acknowledgements
This research was financially supported by The Thailand Research
Fund through grants RTA5980001(to P Chaiyen), and a grant from
Vidyasirimedhi Institute of Science and Technology (VISTEC) (to JJ, PI,
WW, NA, CK, NK and PC). All figures generated with ChemBioDraw
Ultra (Version 12.0), bioRENDER and The PyMOL Molecular graphic
System (Version 2.3.1).
16
Journal of Biotechnology 309 (2020) 1–19
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