Chapter 7 Metabolic Engineering Strategies To Convert CA 2016 Biofuels For

C H A P T E R
7
Metabolic Engineering Strategies to
Convert Carbohydrates to Aviation
Range Hydrocarbons
A. Bergman1,2 and V. Siewers1,2
1
Department of Biology and Biological Engineering, Chalmers University of Technology,
Gothenburg, Sweden, 2Novo Nordisk Foundation Center for Biosustainability,
Chalmers University of Technology, Gothenburg, Sweden
7.1 INTRODUCTION field. In this introductory section we discuss

the advantages of using cell factories for pro-
Metabolic engineering enables the creation duction of biofuels and primary strategies and
of modified biological systems with a precise principles within the field of metabolic
mission the conversion of biomass and engineering.
carbon-containing gases into specific biochem-
icals. This chapter presents recent progress in
the development of cell factories capable of
7.1.1 Why Use Microbes for Fuel
producing aviation range biofuels. It is divided
into two major parts the first covering pro-
Production?
duction routes towards fatty acid-based bio- The use of yeast for ethanol formation and
fuels, such as alkanes, and the second covering molds for production of penicillin are exam-
metabolic pathways and strategies targeting ples of well-known biotechnological industrial
isoprenoid-based fuels, of which there are com- processes today [1,2]. Both are illustrations of
mercial examples available. Each section covers classical uses of cell factories to convert carbo-
natural precursor metabolism, information on hydrate substrates into specific metabolic pro-
central biofuel-forming pathways, metabolic ducts, where the natural substrate-product
engineering examples to generate efficient cell spectrum of the producing organism is
factories for the discussed fuels, and a discus- exploited. However, this limits the range of
sion on challenges and opportunities in the possible products because it is problematic to
Biofuels for Aviation.

DOI: http://dx.doi.org/10.1016/B978-0-12-804568-8.00007-X 151 © 2016 Elsevier Inc. All rights reserved.
152 7. METABOLIC ENGINEERING STRATEGIES TO CONVERT CARBOHYDRATES TO AVIATION RANGE HYDROCARBONS
culture many organisms on a large scale, and substrates, which is questionable both from an
often the compound of interest is present only environmental as well as a food-versus-fuels
in low quantities [3]. Progress in genomics and point-of-view [9]. The microbial community,
genetic engineering during the last few dec- on the other hand, has conquered virtually
ades has allowed us to vastly expand the every environmental niche imaginable, and
microorganism production portfolio. Today, has consequently adapted to metabolize
nearly any metabolic product detected in a life numerous food sources traits that can also
form can be produced in a nonnative host as be transferred between organisms by genetic
long as the metabolic pathway and genetic ele- engineering. Thus, the future holds great
ments within the parent organism are eluci- promise in using current waste products from
dated. This has spurred a great interest in the agriculture and forestry industries to produce
research fields of synthetic biology and next-generation biofuels if microbes can be
metabolic engineering: to design and create constructed to catalyse an efficient conversion,
custom-made organisms with high product-to- for example, yeasts engineered to utilize cellu-
substrate ratios and low by-product formation lose and hemi-cellulose as substrates [10].
[4]. Furthermore, it allows for a sustainable Additionally, a microbial reaction is catalysed
economy, where carbon dioxide-consuming under mild and physiological conditions,
plants and organisms can act as the substrates, which reduces the formation of toxic by-
or even as cell factories, themselves [5]. products.
A major challenge in the field of metabolic
engineering is to develop economically viable
alternatives to petroleum-based products, espe-
7.1.2 Natural Fuel Molecules and
cially within the massive fuel market. Ethanol
is a strong biofuel candidate for markets
Prospective Production Hosts
directed towards regular combustion engines, There are two major cellular metabolic path-
but to be able to power the aviation industry, ways that give rise to precursor hydrocarbons
more energy-dense fuel molecules have to be with relevance for the jet fuel industry fatty
established that can be used in gas-turbine acid and isoprenoid metabolism [11]. Alkanes,
engines [6]. Jet fuels tend to be comprised of a alkenes, fatty alcohols, and fatty acid ethyl
mixture of hydrocarbons most commonly lin- esters (FAEE) are all prospective fuel mole-
ear, branched, and cyclic alkanes that bring cules that can be derived from natural fatty
about different attributes to the fuel, for exam- acid metabolism, which is an essential path-
ple, low-temperature flow properties, high com- way in all organisms in order to produce lipids
bustibility, and high-energy content [7]. (eg, membrane lipids). For example, the major-
Hydrocarbons are naturally present in a variety ity of cyanobacterial species and certain plants
of organisms [8], and there are numerous bio- can naturally produce alkanes and alkenes
logical compounds that are of interest as jet fuel [12,13], and several plants generate biodiesel-
candidates, such as alkanes/alkenes and like molecules such as wax esters [14].
isoprenoid-based fuels. Farnesene and bisabolene are both prospective
A great advantage of using fermentation- aviation range fuels belonging to the group of
based technologies instead of chemical conver- isoprenoid compounds. Isoprenoid production
sion of plant-based material is the broad range is also essential in all living organisms, and
of substrates that can potentially be utilized. these molecules constitute the largest group of
Currently, chemical production of biodiesel chemicals produced in biological systems,
relies to a large extent on vegetable oils as entailing more than 40,000 compounds [15].
II. THE SCIENCE AND TECHNOLOGY OF DEVELOPING BIOFUELS FOR AVIATION

7.1 INTRODUCTION 153
With regards to the different kinds of factories. This section gives a summary of the
microbial cell factories to use, cyanobacteria different strategies adopted to optimize micro-
have been discussed as the gold standard, as bial production of metabolites.
they can convert light energy to chemical The metabolic engineering process is
energy by fixating carbon dioxide (instead of depicted in Fig. 7.1. It is inherently an iterative
converting biomass); many research groups process due to the complexity of biological sys-
focus on optimizing such microorganisms tems and our incomplete understanding of
[16]. However, scaling-up of such processes is them. Commonly, an engineering strategy is
troublesome because cyanobacteria need based on the available knowledge of metabo-
direct access to light [16], and currently yields lism of the chosen organism, such as simpli-
of biofuel-related compounds remain at a fied biochemical reaction networks. The
proof-of-principle stage [17]. A large number strategy is then implemented in the host strain
of the metabolic engineering efforts for bio- through the use of recombinant DNA technol-
fuel production have gone into research and ogy, such as plasmid cloning strategies and
development with the traditional workhorses genome engineering tools, a process referred
of biotechnology that can consume carbohy- to as strain construction. The created strains
drates: the yeast Saccharomyces cerevisiae [18] are then tested in different cultivation settings
and the bacterium Escherichia coli [19]. and samples are taken to quantify production
Naturally, these organisms do not produce capacity and detect physiological changes on
large amounts of fuel precursors, but are different levels. In the concluding step, the
genetically very flexible. Extensive knowledge obtained data are analysed to verify if the ini-
exists about their biology, there are many tial strategy was successful and detect anoma-
tools for modifying them, and they are well lies that might explain unpredicted behaviour.
known in industry. More work is also going The obtained knowledge can then be added to
into utilizing certain yeast species that natu- the previous model, and the cycle can restart
rally accumulate high quantities of lipids to optimize the production of the target com-
oleaginous yeasts, of which Yarrowia lipolytica pound. Potentially, joint advances in systems
has become a model organism [20]. Such biology and metabolic modelling can help cre-
yeasts are promising because they would con- ate improved predictive genome-scale meta-
stitute a good platform for fatty acid-based bolic models constrained by experimental
fuel production, but they are less well charac- data, such as fluxomics, to aid in strain con-
terized compared to S. cerevisiae, from an struction strategies [21].
applied as well as a fundamental point of The re-direction of carbon flux in targeted
view. This chapter mainly focuses on prog- biochemical reaction networks is a central con-
ress made in S. cerevisiae and E. coli, but to cept in metabolic engineering. Increased flux
some extent other organisms as well. through a certain pathway can, for example,
be achieved by increasing the activity of path-
way enzymes occurring naturally in the organ-
ism of choice, or by introducing genes from
7.1.3 General Metabolic Engineering
heterologous (nonnative) sources into the host,
Principles which can provide the cell with new, desired
Genetic engineering has paved the way for functions. The activity of a gene and corre-
the engineering of life forms to make them sponding enzyme of interest can in turn be
optimal for production of a certain biochemical modulated on many different levels. Increased
compound or protein the engineering of cell transcriptional activity (generation of messenger

RNA that later on can be translated into a pro- in prokaryotic species by modulating ribosome
tein) is commonly accomplished either by binding sites to increase the absolute enzyme
increasing gene copy number or utilizing a number [22]. In eukaryotes as well as prokary-
strong promoter (the regulatory DNA otic species, translational efficiency of heterolo-
sequence upstream of a gene that recruits the gous genes can be increased by performing
transcriptional machinery). After transcription, so-called codon-optimization, through which a
the translational efficiency can also be increased gene sequence is adjusted to match the most
FIGURE 7.1 The iterative metabolic engineering cycle used to construct cell factories.

7.2 FATTY ACID-DERIVED AVIATION RANGE BIOFUELS 155
prevalent codon usage in the receiving organ- 7.2 FATTY ACID-DERIVED
ism [23]. The enzymatic efficiency can also be AVIATION RANGE BIOFUELS
modified, for example, by making targeted
engineering efforts to the catalytic site of the The possibility to construct cell factories that
protein or abolishing post-translational regula- produce the prospective aviation fuel molecules
tion of the enzyme [24]. alkanes and alkenes has become a reality due to
A metabolic engineering scheme usually tar- the recent advances in elucidation of the genetic
gets metabolism in an organism at many dif- and biochemical character of the final convert-
ferent points simultaneously. Since it is ing enzymes in these metabolic pathways.
beyond the scope of this chapter to describe Microbial alkane-formation is dependent on
them in detail, a small summary of different enzymatic conversion of fatty acid species
engineering approaches and their purposes is aliphatic carboxylic acids. Therefore, a critical
made in Table 7.1. For a more comprehensive part in the process of engineering a microbial
summary of metabolic engineering strategies, factory for alkane-based fuels is to engineer the
see the review by Lee et al. [25]. endogenous fatty acid metabolism.
TABLE 7.1 Summary of Commonly Utilized Metabolic Engineering Strategies
Strategy Example
Optimize/expand substrate Engineering of cells to co-utilize natural substrates in order to increase productivity, or
utilization installing heterologous genes that help cells utilize substrates that could not be used
naturally.
Reduce/eliminate by- Deletion of genes that enable cells to produce nonessential metabolites in order to
product formation optimize yield of the desired product, or down-regulating genes that are essential but
divert flux from the pathway of interest.
Increase precursor Up-regulating genes responsible for catalysis of reactions upstream of a terminal (eg,
metabolite pool biofuel-forming) enzyme of interest to increase substrate availability and reaction rate of
the terminal enzyme.
Rerouting of metabolic Use of heterologous pathways that can catalyse a certain reaction faster/with fewer
pathways intermediate catalytic steps/from another intracellular metabolite/with reduced energy
consumption, in order to optimize productivity.
Co-factor engineering Modifying a metabolic pathway enzyme requiring a certain co-factor to prefer one that is
more abundant in the biological system, or engineering the cell to increase production of
required co-factors.
Synthetic enzyme scaffolds Utilizing a protein-based structure that can dock multiple enzymes in order to bring
proximity of enzymes in a modular pathway and their respective substrates, thereby
increasing reaction rate, and/or to be able to dock additional copies of a slower enzyme
onto the scaffold in order to balance the carbon flux.
Pathway optimization Optimizing expression of the pathway enzymes or construction of a system where the
metabolic pathway gene expression is regulated by substrate presence, which can generate
flux balance and lead to a more energy efficient system.
Adapted after J.W. Lee, et al., Systems metabolic engineering of microorganisms for natural and non-natural chemicals, Nat. Chem. Biol. 8 (6) (2012)
536546.

This subsection is divided into four parts catalysed by acetyl-CoA carboxylase (Acc1 in
the first discusses key features of fatty acid yeast and AccABCD in E. coli, respectively)
metabolism in yeast and E. coli, the second [27]. The malonyl-moiety is transferred to an
deals with metabolic engineering strategies to acyl-carrier protein (ACP), and then serves as
overproduce fatty acids in these hosts, the the extender unit in FAB, in which the fatty
third summarizes information about the termi- acid chain is elongated by two carbons at a
nal enzymes responsible for natural straight- time.
chain hydrocarbon formation and engineering In eukaryotic organisms such as yeast, the
strategies to produce these molecules in cytosolic fatty acid synthase (FAS) enzyme con-
microbes, and the final section discusses chal- sists of two distinct polypeptides encoded by
lenges and opportunities in the field. FAS1 and FAS2 containing all enzymatic
Fatty alcohols and FAEE are also prospec- domains required for FAB, a system called
tive biofuels derived from fatty acids that do type I FAS [27]. In the bacterial machinery,
not, however, have optimal properties for jet the enzymes involved in FAB are instead
fuel purposes, though research in this field is composed of individual proteins, called the
described in detail in chapter ‘The Suitability type II FAS system [28]. The main yeast FAB-
of Fatty Acid Methyl Esters (FAME) as system is located in the cytosol, but yeast also
Blending Agents in Jet A-1’ [26]. When the contain a mitochondrial system of type II,
general approach to producing them pertains which is essential for respiratory growth due
to the production of alkanes/alkenes, they will to the mitochondrial requirement for lipoic
be brought into the discussion. acid [29].
In yeast, cytosolic FAB is initiated through
the transfer of Ac-CoA to ACP located within
the Fas2-polypeptide, while ACP-bound inter-
7.2.1 Fatty Acid Biosynthesis and
mediates in E. coli are formed in a dissociated
Degradation state (as ACP is expressed as an individual poly-
The overall reaction principles of fatty acid peptide). The first reaction in the FAB cycle is a
biosynthesis (FAB) are evolutionarily con- condensation reaction between the acetyl and
served from bacteria to eukaryotes, but the malonyl unit catalysed by a ketoacyl synthase
enzymatic machinery differs from a struc- (KS subunit or FabH), during which one carbon
tural point of view. In short, the synthesis is is lost in the form of CO2. In yeast, the acetyl
based on a cyclic system, where aliphatic unit is bound to ACP, while in E. coli the acetyl-
thioester-intermediates are extended by two unit remains in CoA form. The chain elongation
carbons at a time through four steps: (1) con- beyond the first cycle is catalysed in E. coli by
densation, (2) reduction, (3) dehydration, and other enzymes than FabH (ie, FabB/FabF) due
(4) reduction. The mechanisms are illustrated to the required change in substrate specificity
in Fig. 7.2. (CoA to ACP), while the KS module per-
The first and committed step of FAB is initi- forms all condensation reactions in yeast. After
ated when the compound acetyl-coenzyme A condensation, the produced ketoacyl-ACP is
(acetyl-CoA, Ac-CoA), a two-carbon metabolic reduced to β-hydroxyacyl-ACP in an NADPH-
intermediate at the junction of catabolic and consuming step by a ketoacyl reductase
anabolic pathways, is converted to malonyl- (KR subunit or FabG). In the next stage, the
CoA (Ma-CoA) by addition of dissolved β-hydroxyacyl-ACP is dehydrated to β-enoylacyl-
CO2 in a biotin- and ATP-dependent reaction ACP by enoyl dehydratase (DH subunit or

FIGURE 7.2 The reaction principles and enzymatic components of the fatty acid biosynthesis system in Saccharomyces
cerevisiae and Escherichia coli, respectively. Fas1/2, fatty acid synthase subunits; Acc1 and AccABCD, acetyl-CoA carboxyl-
ase; ACP, acyl-carrier protein; AT, acetyl transferase; MPT and FabD, malonyl-palmitoyl transacylase; KS and FabB/F/H,
ketoacyl synthase; KR and FabG, ketoacyl reductase; DH and FabA/Z, enoyl dehydratase; ER and FabI, enoyl reductase.
FabZ/FabA), and further reduced to an acyl- end product of FAB in yeast is generated when
ACP molecule of Cn12 while consuming an the Fas2-bound acyl-chain is enzymatically
additional molecule of NADPH through the released as acyl-CoA, while the bacterial end
action of an enoyl reductase (ER subunit or product is acyl-ACP. This difference has impli-
FabI). This newly formed acyl-ACP then under- cations in downstream applications, since
goes subsequent elongation cycles to reach a biofuel-converting enzymes have different
final length of normally 16 or 18 carbons, which specificities towards these intermediates.
constitute the major fatty acid types in both The formed acyl-chains have multiple desti-
E. coli and yeast [27,28]. A distinction is that the nations within cells. A fundamental function is

the formation of membrane lipids. This requires The previously described FAB-system for
that the fatty acids be converted into amphiphi- yeast is valid for both S. cerevisiae and Y. lipoly-
lic phospholipids that can form bilayers. A tica. However, one important distinction
large proportion of the yeast fatty acids formed, between the two is the presence of an ATP cit-
around 7580%, are enzymatically desaturated rate lyase (ACL) in Y. lipolytica, as well as in
by individual enzymes, a feature that helps to other oleaginous yeasts. This enzyme produces
increase membrane fluidity [30]. The desatura- cytosolic acetyl-CoA from citrate, which can be
tion process in E. coli instead takes place during exported out of the mitochondrion, thus
the fatty acid chain synthesis by the activity of increasing the precursor supply for FAB [32].
the enzymatic players FabA and FabB as During lipid accumulation for example, in
opposed to FabF and FabZ, which will produce nitrogen-limited conditions when nucleotides
saturated fatty acid species [31]. and proteins cannot be produced the mito-
Compared to bacteria, the complexity of chondrial isocitrate dehydrogenase is down-
lipid metabolism in yeast increases due to the regulated, increasing the flux through ACL
distinct feature of eukaryotic cells: compart- and towards TAG-synthesis [37]. ACL is there-
mentalization, which is the presence of multi- fore hypothesized to be a critical component
ple types of membrane-enclosed organelles adding to the oleaginous character of this and
with different function and physiology. For other yeasts.
example, in the endoplasmic reticulum, fatty
acid elongation up to C26 is executed by the
7.2.2 Engineering Examples to Increase
proteins Elo1, 2, and 3 [27], in the lipid droplet
triacylglycerol (TAGs) and sterol esters (SEs)
FAB and Lipid Production
are formed and stored [32], and in the peroxi- There have been extensive efforts to opti-
somes β-oxidation (the breakdown of fatty acid mize microbial FAB for oleochemical pro-
chains) takes place. β-Oxidation is a critical duction purposes. This includes increasing
system for cells in order to grow on fatty acid precursor supply as well as FAB, altering regu-
substrates, and essentially consists of the latory mechanisms within the concerned sys-
reversed reactions compared to FAB: cyclic tem, and exploring synthetic systems.
steps of (1) oxidation, (2) hydration, (3) oxida- Representative examples of this are described
tion, and (4) thiolytic cleavage. The mechan- below (see also Fig. 7.4).
isms in E. coli and S. cerevisiae are depicted in
Fig. 7.3. 7.2.2.1 Engineering Acetyl-CoA
FAB demands a lot of energy, and produc- Metabolism
tion of one C18-fatty acid will consume nine The metabolic intermediate acetyl-CoA is
Ac-CoA, eight ATP, and 16 NADPH. Thus, the universal precursor for biosynthesis of
in order to not waste the cell’s energy fatty acids, and also for isoprenoids, in yeast.
resources, the process is heavily regulated, Accordingly, multiple efforts to up-regulate
where transcriptional, translational [27,28], as the precursor pool of acetyl-CoA in cell facto-
well as posttranslational mechanisms are ries with the aim to increase production of
involved [33]. Furthermore, fatty acid synthe- such compounds have been made [38].
sis is subject to product inhibition by acti- Due to compartmentalization, there exist
vated fatty acid species on multiple levels distinct acetyl-CoA pools in yeast, of which
[3436]. This regulation poses great chal- the cytosolic one is responsible for FAB for
lenges to the overproduction of fatty acid- membrane and storage lipid biosynthesis. An
derived compounds. example of cytosolic acetyl-CoA engineering

FIGURE 7.3 The reaction principles and enzymatic components of β-oxidation in Saccharomyces cerevisiae and
Escherichia coli, respectively. Faa1/2/3/4 and FadD, fatty-acyl CoA synthase(s); Fat1, very long chain fatty acyl-CoA
synthase; Pox1 and FadE, acyl-CoA oxidase; Mfe2 and FadB, enoyl-CoA hydratase/hydroxyacyl-CoA dehydrogenase;
Pot1 and FadA, ketoacyl-CoA thiolase.
in yeast was set by Shiba et al., who targeted considered, in part since the native route is
the pyruvate dehydrogenase (PDH) bypass. energy demanding. In addition, the native
By overexpressing the endogenous acetalde- route bypasses the metabolite acetaldehyde,
hyde dehydrogenase Adh6 and a deregulated, which can be efficiently converted to etha-
heterologous version of acetyl-CoA synthase nol, a by-product that severely lowers the
(acs) from Salmonella enterica, acsL641P, flux theoretical product-on-substrate yield for
towards the cytosolic acetyl-CoA node, and fatty acid and isoprenoid-based products. For
the isoprenoid product amorphadiene was example, pathways based on phosphoketo-
increased fourfold at the expense of ethanol lase [39], ACL [4042], pyruvate formate
formation [24]. lyase [43], acetylating acetaldehyde dehydro-
Many heterologous pathway options to pro- genase (A-ALD) [43], and the PDH complex
duce cytosolic acetyl-CoA in yeast have been [44] are all acetyl-CoA-forming pathways

used to channel carbon towards the cytosolic 7.2.2.2.1 OVEREXPRESSION OF ENZYMES

acetyl-CoA node in yeast to increase acetyl- DIRECTLY RELATED TO FAB
CoA-derived product formation. The develop- In order to overproduce fatty acids, the
ment of an efficient alternative route will help strategies described in the previous section to
decrease the amount of carbon lost to ethanol increase acetyl-CoA levels are applicable. The
as well as reduce energy costs. In addition, it subsequent step is to focus on FAB. A straight-
could complement the deletion of the three iso- forward and beneficial principle in yeast
enzymes encoding pyruvate decarboxylase has been to co-overexpress the endogenous
(Pdc1, 5, 6), creating a strain that is completely acetyl-CoA carboxylase and the FAS-enzymes
deficient in ethanol production, and thus has [50]. The sole overexpression of AccABCD in
potential to act as a yeast platform strain with E. coli had no impact on fatty acid yields,
low by-product formation [45]. possibly due to product inhibition of acyl-
In E. coli, several enzymes intersect the ACP, but a small positive effect when a “pull”
acetyl-CoA metabolism for FAB since no com- from the acyl-ACP pool was implemented [51].
partmentalization is present. To keep a high Acc1 in S. cerevisiae is inhibited by posttransla-
acetyl-CoA level, many strategies have aimed tional phosphorylation, and removal of this
to delete metabolic pathways leading to fer- regulation increases its activity in metabolic
mentation products, such as acetate [46,47], engineering strategies [33,52]. The utilization
up-regulating the acetyl-CoA-forming enzymes, of heterologous FAS-systems in S. cerevisiae
such as PDH [48], and fast-channeling the has also been reported, for example, functional
formed acetyl-CoA to downstream pathways, utilization of a modified human FAS-system
for example, by overexpressing the first enzyme [53], or utilization of a bacterial FAS type I
in FAB, acetyl-CoA carboxylase [49]. system which is assumed to bypass native reg-
More details on the subject of engineering ulation [54]. Heterologous expression of a
acetyl-CoA metabolism in microorganisms are FASI-system in E. coli has also been reported
available in a comprehensive review recently in an attempt to overproduce fatty alcohols
published on the topic [38]. [55]. The terminal enzyme for fatty alcohol
production acts upon the intermediate acyl-
CoA, which is directly produced by the FASI
7.2.2.2 Fatty Acid and Lipid
FAB-system, but requires two additional enzy-
Overproduction
matic reactions from acyl-ACP in prokaryotic
Fatty acids and lipids are not only valuable systems. The system was functional in vivo,
as biofuel precursors, but also as food addi- but failed to increase yields compared to the
tives, consumer products, etc. This is why endogenous system.
attempts and strategies to overproduce these
compounds in microorganisms are numerous.
Fatty acid-accumulating strains can be used as 7.2.2.2.2 RELEASE OF FREE FATTY ACIDS AS A
platform strains for specific applications (eg, MEANS TO INCREASE FLUX THROUGH FAB
for alkane synthesis). Due to the sheer quantity Due to the previously mentioned repression
of studies performed to increase yields, and of the FAB-machinery by their products, acyl-
numerous strategic overlaps in these studies, ACP in E. coli and acyl-CoA in yeast, a well-
the principle targets of these strategies are explored method to increase yields of fatty
described, and a few key studies presented in acids in E. coli is to convert these end products
more detail. into free fatty acids (FFA), a reaction catalysed

by thioesterases. Early attempts to utilize this production and secretion [50,62]. The combi-
principle for engineering of fatty acid metabo- nation of FAA1/FAA4 deletion with expres-
lism showed that overexpression of a native sion of a heterologous thioesterase with
thioesterase, TesA, modified to localize to the broad substrate-range led to the production
cytosol [56], and a thioesterase from of approximately 490 mg/L extracellular FFA,
Umbellularia californica [51], both improved where a significant proportion (B40%) was
FFA-yields significantly in E. coli. Subsequent unsaturated, compared to the WT, where no
studies of a broad range of thioesterases in unsaturated FFAs were detected [62].
E. coli showed a great variety of substrate speci-
ficity (C4 to C16) and activity (up to 2 g/L pro- 7.2.2.2.3 PREVENTING DEGRADATION OF
duced FFA by overexpression of a single FATTY ACIDS BLOCKING β-OXIDATION
thioesterase), suggesting thioesterases as the In E. coli, activation of fatty acids to acyl-
major determinants of fatty acid chain length, CoA constitutes the initiation of β-oxidation. A
thus providing an indispensable tool for engi- functional β-oxidation pathway will diminish
neering of fatty acid metabolism [5760]. The yields when fatty acids are the aim of produc-
thioesterase principle has been shown to work tion, which is why many studies in E. coli
in S. cerevisiae as well. Expression of the trun- involved deletion of the endogenous gene
cated TesA variant from E. coli improved FFA fadD, which encodes an acyl-CoA ligase
levels eightfold [50], and expression of a recom- [49,58]. It should however be noted that this is
binant human FAS-system where the thioester- not a preferable choice if a terminal enzyme
ase domain was replaced with one specific utilizing acyl-CoA is exploited. Therefore, tar-
towards short- and medium-chain acyl-CoAs geting the second enzyme involved in the
enabled the production of 111 mg/L short- oxidation process, fadE, is more favourable
chain fatty acids [53]. In yeast, the system offers [56]. Deletion of β-oxidation in yeast, which
less flexibility due to the fungal FAB system, exclusively takes place in the peroxisomal
where the growing acyl chain is attached to the compartment, has also been targeted, mainly
ACP units of the FAS complex, which is why a through deletion of POX1, the gene encoding
soluble thioesterase only has access to the the enzyme catalysing the first step in the
released C16C18 acyl-CoA species. oxidation process, the conversion of per-
The opposite reaction, the conversion of oxisomal acyl-CoA into trans-2-enoyl-CoA
FFAs into acyl-CoAs, has also been targeted [50,63,64]. A recent study also showed that
with metabolic engineering. In S. cerevisiae, the effect can be enhanced by combining the
the genes FAA1/2/3/4 all encode fatty acid- POX1 deletion with deletion of genes encod-
activating enzymes and are essential for ing Pxa1, which transports acyl-CoA into
growth if fatty acids are used as the sole the peroxisome, and Faa2, an acyl-CoA ligase
carbon source, due to the necessity of acyl- located in the peroxisome, possibly due to
CoA species as substrates for β-oxidation the presence of auxiliary enzymes in the
and incorporation into membrane or storage β-oxidation pathway [64].
lipids. The protein products of FAA1 and
FAA4 have been shown to be responsible 7.2.2.2.4 MANIPULATING STORAGE LIPID
for the vast majority ($98%) of the long- FORMATION IN YEAST FOR INCREASED
chain fatty acid activation, such as C16C18 FREE FATTY ACIDS
[61]. Thus, deletion of these two particular Another option to generate FFAs in yeast by
genes in yeast enables high levels of FFA reducing by-product formation is to modulate

storage lipid and lipid body formation. There fatty acid titres to 5.2 6 0.5 g/L, which is 7.5
are four main genes related to the generation times higher than the control strain with nor-
of the two storage lipid species in yeast: DGA1 mal FadR levels, and corresponds to a yield of
and LRO1 responsible for TAG formation, and 0.26 g FA/g glucose or 74% of the theoretical
ARE1 and ARE2 in charge of SE synthesis. maximum yield [66]. In yeast, a study was
Storage lipid formation is not essential in conducted to investigate if the deletion of neg-
yeast, and deletion of the four genes men- ative regulators of phospholipid synthesis
tioned above will deplete lipid body formation could increase production of fatty alcohols,
and reduce fitness in yeast [65]. A study that which are directly obtained from acyl-CoA
coupled the deletion of storage lipid formation [42]. The logic behind this strategy is that it
with improved production of the fatty acid- would simultaneously increase the flux
derived fuel FAEE in S. cerevisiae reported a through the ACC/FAS system because fatty
1.7-fold increase in yield, and if β-oxidation acids are required as precursors for phospho-
gene POX1 was additionally knocked out, a lipid synthesis. Out of six deletions, the one of
2.9-fold increase in FAEE was seen compared RPD3 helped to increase the fatty alcohol titre
to the control [63]. from 71 to 140 mg/L. A study performed in
A less direct approach is to instead Y. lipolytica also showed that the deletion of
increase the flux through the lipid body by the global regulator Snf1 increased the lipid
overexpressing storage lipid formation genes content in the yeast by 2.6 times [67].
in concert with lipases that are responsible However, such a correlation could not be seen
for degrading TAGs and SEs into FFAs. By between a knockout of Snf1 and fatty alcohol
overexpressing the TAG synthase Dga1 and production in S. cerevisiae, which was unex-
the lipase Tgl3 in combination with eliminat- pected, since Snf1 is known to negatively regu-
ing the fatty acid-activating enzymes Faa1, late Acc1 in this host [42].
Faa4, Fat1, and deleting β-oxidation genes
POX1, FAA2, and PXA1 in S. cerevisiae, an 7.2.2.2.6 INCREASING STORAGE LIPID
extracellular FFA titre of 2.2 g/L could be FORMATION IN YEAST
achieved the highest reported so far [64]. Most strategies described above aim to
A benefit of this strategy in comparison to increase the yield of FFAs or activated fatty
the deletion of storage lipid formation genes acids instead of focusing on increasing the
is that it consumes the acyl-CoA molecules storage lipid content. This is due to the fact
that otherwise would inhibit FAB, enabling a that fatty acids in the form of TAG/SE cannot
faster fatty acid biogenesis. directly be converted enzymatically into fuel
molecules within the cell. However, the option
to produce microbial lipids, which can be
7.2.2.2.5 INFLUENCING FATTY ACID converted catalytically to fuels, is an alterna-
METABOLISM THROUGH REGULATORY tive option. Examples of boosting storage lipid
COMPONENTS formation in S. cerevisiae exist. Optimization
Due to the tight regulation of FAB, another of TAG formation was investigated through
strategy has been to focus on global regulators the expression of a native Dga1 with an
coupled to fatty acid metabolism. For example, N-terminal deletion, DgaΔNp, in yeast back-
a pioneering study made in E. coli shows that grounds where the native DGA1 locus and,
overexpression of the transcriptional regulator in particular, its 30 terminal region had been
FadR, activating unsaturated FAB and deacti- deleted. These led to a lipid content close
vating β-oxidation, could increase the total to 45% of cell dry weight, corresponding to

an oleaginous phenotype. The phenotype is reversed. As opposed to FAB, β-oxidation is a
thought to be connected to the disruption and reversible process, which can function in an
lowered expression of ESA1, encoding a his- opposite direction in the absence of fatty acids.
tone acetyltransferase, which is chromosomally In this mode, the enzymatic steps are identical
adjacent to DGA1 in S. cerevisiae [68]. to the natural FAB except for the initiation,
The collective metabolic engineering efforts which employs acetyl-CoA as an extending
in the oleaginous yeast Y. lipolytica have, on unit. This means that one ATP can be spared
the other hand, primarily been aimed at pro- per elongation cycle. In the absence of fatty
ducing maximal amounts of TAG stored in acids, E. coli will strongly repress the expres-
lipid droplets. In a study from 2014, Y. lipolyti- sion of β-oxidation-related operons in order to
ca engineered to overproduce lipids and cells prevent the reversed reaction. In the study by
accumulated lipids to about 90%, reaching Dellomonaco et al., E. coli was modified in sev-
titres around 25.3 g/L, which represented a eral regulatory aspects, such as exploiting
greater than 60-fold improvement over the mutated versions of FadR and AtoC (negative
parental strain [69]. The result was based on regulators of β-oxidation), allowing for consti-
deletion of genes connected to β-oxidation and tutive expression of the β-oxidation genes even
peroxisome biogenesis, overexpression of a in the absence of fatty acids and in the pres-
gene involved in TAG synthesis, as well as ence of glucose. Furthermore, several fermen-
optimization of cultivation conditions. In 2015, tation pathways were blocked. In combination
a significant achievement was accomplished with expression of enzymes with terminal
when Qiao et al. identified a human gene pres- activity, high titres and yields could be
ent in cellular obese phenotypes encoding a achieved. For example, when the n-butanol-
delta-9-steroyl-CoA desaturase (SCD) generat- forming enzyme FucO was expressed in the
ing mono-unsaturated fatty acids. Expression deregulated strain cultivated in high-glucose
of SCD in Y. lipolytica similarly managed to media, around 14 g/L of n-butanol were pro-
reduce product inhibition (from saturated duced, corresponding to 0.33 g butanol/g
acyl-CoA) in fatty acid synthesis as well as glucose consumed and a productivity of
stimulate TAG-synthesis. Co-expressing SCD 2 g/gCDW/h. In combination with over-
with native ACC1 and DGA1 genes led to a expression of a 3-ketoacyl-CoA thiolase with
strain with a product-on-substrate yield of broad chain-length specificity and a thioester-
84.7%, lipid titres close to 55 g/L, and a maxi- ase with long-chain specificity, and a deletion
mal productivity of about 1 g/L per h [70]. of fadD, efficient production of FFAs was
enabled with titres and yield of 7 g/L and
7.2.2.2.7 FAB THROUGH REVERSED 0.28 g/g [71]. In a following study, instead of
β-OXIDATION utilizing the regulatory approach, several
Production of bulk chemicals, such as bio- possible enzyme candidates for the reversed
fuels, requires very high yields in order to be reactions were characterized in vitro. The
economically viable. The high energy input best candidates were overexpressed with
required in natural FAB means on a practical thioesterases of different chain-length speci-
level that the theoretical yield is fairly low. ficity, and reasonably high titres for shorter-
The energy demand is high due to the use of chain (C4) products were obtained, while
malonyl-CoA/ACP as an extender unit in longer-chain products (up to C12) were
FAB, since its biosynthesis requires one ATP. detected at lower titres [72].
In 2011, a pioneering study was performed Recently, fatty acid synthesis through rever-
where the β-oxidation process in E. coli was sal of β-oxidation was also implemented in

S. cerevisiae in a synthetic biology approach metabolic engineering strategies to improve

[73]. In this system, the native Pot1, which their production. The pathways include a
catalyses the last step in β-oxidation, or the reductive two-step pathway from activated fatty
acetoacetyl synthase Erg10, were used as acid species or FFA, a head-to-head condensa-
ketoacyl-CoA synthases (KS). Two mitochon- tion reaction between two fatty acid chains, and
drial β-oxidation enzymes from Y. lipolytica a one-step reduction of free fatty acids to termi-
normally catalysing hydration and the subse- nal alkenes. Of the discussed pathways, the first
quent dehydrogenation of 2-enoyl-CoA were one is by far the most studied and extensively
utilized for their reversed reactions (DH and used in metabolic engineering strategies for
KR activity, respectively). The last selected microbial alka(e)ne production. To visualize and
gene was the yeast native ETR1, whose protein put the pathways into context, a brief summary
product catalyses the last reductive step in of the heterologous pathways and the cellular
mitochondrial FAB. Instead of targeting regu- substrates they would act on in S. cerevisiae
latory components of the system, the genes can be found in Fig. 7.4 (indicated with green
were simply overexpressed from a plasmid. By (dark grey in print versions) arrows/text), and a
co-expressing genes involved in n-butanol, detailed overview of the three reaction types is
FAEE, and fatty alcohol formation, and detect- summarized in Fig. 7.5.
ing the corresponding products, the system
proved to be functional up to medium-chain 7.2.3.1 Two-Step Reduction: Alkane
products (4-cycle reversal). However, obtained Formation Through the Combined Action of
yields were on the milligram scale, which is Fatty Acid Reductases and Fatty Aldehyde
far from what was obtained in E. coli. The low Deformylating Oxygenases
yields were in part attributed to the limitation Schirmer et al. first elucidated the cyanobac-
of acetyl-CoA concentration in the cytosol. terial pathway for straight-chain alkane
Nevertheless, reversal of β-oxidation remains a (C15C17) synthesis in 2010 [75]. By perform-
promising strategy to produce fatty acid-based ing subtractive genome analysis between
products at higher theoretical yield. alkane- and nonalkane-producing species, the
authors found the pathway to be composed of
two enzymes an acyl-ACP reductase (AAR)
7.2.3 Natural Straight-Chain and an aldehyde decarbonylase (AD; later sug-
Hydrocarbon-Forming Pathways and gested to be an aldehyde deformylating oxyge-
nase, ADO, which will be the name used from
Metabolic Engineering Strategies to
here onwards), and their activity was verified
Produce Alka(e)nes by heterologous expression of the correspond-
For many decades it has been known that ing genes and product quantification in E. coli.
both microorganisms and higher organisms The AAR catalyses the reaction from an acti-
naturally have capacities to produce alkanes vated fatty acid to the corresponding alde-
and alkenes (alka(e)nes) [74], but it was not hyde, while the ADO decarbonylates the
until recently that strong efforts were put into aldehyde into an alka(e)ne of chain length
investigating the genetic components of these Cn21. This is why the alkanes found in organ-
pathways, most likely as a response to the isms that almost exclusively produce even-
urgent need of developing renewable biofuels. chain fatty acids consist of uneven carbon
Here, three mechanistically different routes for chain length. Moreover, in vitro studies of the
natural alkane/alkene formation from fatty enzymes also revealed that ADO requires
acid intermediates are presented, alongside reducing equivalents (NADPH) and an

FIGURE 7.4 Overview of fatty acid metabolism in Saccharomyces cerevisiae and examples of heterologous enzymes
capable of converting fatty acid species into alka(e)ne biofuels. Native genes are shown in orange (light grey in print ver-
sions) notation with a blue (black in print versions) arrow while heterologous genes and pathways used in metabolic strat-
egies for alka(e)ne production are marked in green (dark grey in print versions). See main text for more detailed
information.
electron donor system, which in E. coli can be NADPH as co-factor and is activated by K1. The
supplied by ferredoxin (F) and ferredoxin reduc- kcat of S. elongatus upon stearoyl-CoA was
tase (FNR). When Synechococcus elongatus AAR measured to be 0.36 min21, indicating a rather
and Nostoc punctiforme ADO were expressed inefficient enzymatic conversion [82]. However,
in E. coli, the total alkane titre was around initial studies suggest that AAR has a specificity
300 mg/L where pentadecane was found in for acyl-ACP rather than acyl-CoA, supported
majority [75]. The study has generated numer- by in vitro assays as well as an in vivo experi-
ous follow-up reports characterizing the gene ment [75]; thus, the true activity of AAR might
products and their reaction components with be higher. This is also supported by a sub-
the main focus on ADO [7682]. sequent study on N. punctiforme AAR [83].
The reduction of activated fatty acids by AAR The ADO reaction has been proven to
requires divalent metal ions (eg, Mg21) and require both oxygen and NADPH while

FIGURE 7.5 Overview of alka(e)ne biosynthesis pathways based on fatty acid intermediates. Dashed lines within the
molecule structure indicate that the fatty acid intermediate can be unsaturated due to normal cellular metabolism, thus
giving rise to an alkene product. AAR, acyl-ACP reductase; FAR, fatty acyl-CoA reductase; CAR, carboxylic acid reduc-
tase; ADO, aldehyde deformylating oxygenase; OleTJE, P450 fatty acid decarboxylase; OleABCD, head-to-head condensa-
tion enzymes from Micrococcus luteus.
releasing formate, which is why the enzyme increased fivefold in vivo by fusing ADO to
now is referred to as aldehyde-deformylating catalase, an enzyme that converts H2O2 to the
oxygenase [78,79]. The kinetic properties of ADO-substrate O2 [81].
ADO are poor, and the enzyme has been seen A recent study characterizing both AAR
to only achieve 35 catalytic turnovers in and ADO from the cyanobacterial species N.
in vitro experiments [76,78,79]. ADO is sug- punctiforme showed that the two enzymes form
gested to be the rate-limiting step of the paired a tight complex, where transfer of the cytosoli-
enzyme reaction [83]. In vitro studies also cally insoluble fatty-aldehyde intermediate to
revealed that ADO is reversibly inhibited by ADO is facilitated, which increases the cata-
hydrogen peroxide (H2O2), a compound that lytic capacity of ADO as compared to when it
can form when NADPH and oxygen are in is supplied with exogenous fatty aldehydes at
excess. Since both NADPH and oxygen are high concentration [83]. Whether this interac-
vital for ADO function, and are likely to form tion is conserved in AAR/ADO enzyme pairs
H2O2 (also in vivo), ADO activity has been from other species remains to be answered,

but the consequence is that utilization of microbially produced alkanes until now [86].
ADOs with alternative, noncognate fatty acid Accordingly, substrate specificity of CER1
reductases (FARs) might affect the efficiency appears to be relatively broad in vivo, as it is
of the terminal catalytic step. A potential way responsible for production of VLC alkane spe-
to reduce such an effect is to bring the path- cies in its native host. Furthermore, the fact
way enzymes in proximity to each other, either that sole expression of CER1 generated alkanes
by protein fusion or by using protein scaffolds. in E. coli when a heterologous acyl-CoA reduc-
Rahman et al. investigated whether alkane tase was expressed supports the hypothesis
production could be increased in this manner, given by Bernhard et al. that CER3 might cata-
and saw a 4.8-fold increase for an AAR/ADO lyse the acyl-CoA reductase reaction [85], since
fusion construct, and an 8.8-fold increase when individual CER1 expression in yeast failed to
a scaffold that had space for one AAR and produce any alkanes.
three ADO enzymes (both from S. elongatus) The carboxylic acid reductase (CAR) from
compared to the control strain where the Mycobacterium marinum has been observed to
enzymes were expressed separately [84]. This have a broad range activity towards FFAs
indicates a general importance of proximity with chain lengths ranging from C6 to C18,
of the two pathway enzymes for optimal and converts them into fatty aldehydes [87].
productivity. This is in contrast to the pathway previously
The plant Arabidopsis thaliana produces presented from cyanobacteria, where the
very-long chain alkanes as constituents of a AAR relies on an activated form of fatty
protective wax. The gene CER1 has for many acids, primarily fatty acyl-ACPs [75]. As pre-
years been presumed to be involved in long- viously mentioned, fatty acid synthesis is
chain alkane biosynthesis [13], and its protein subjected to product inhibition by activated
product was recently shown to catalyse the fatty acid species on multiple levels [3436].
reaction of VLC (very long chain, BC30) acyl- Thus, utilization of this pathway, in combina-
CoA to VLC alkanes together with CER3 and tion with an enzyme that can release FFAs
CYTB5 in the heterologous host S. cerevisiae from activated ones, can pose a useful way to
(modified to produce VLC fatty acids) [85]. circumvent the problem.
The proposed enzymatic mechanism of action A study showed that a CAR-related enzyme
is that CER1 and CER3 form a heterodimer from Nocardia sp. requires posttranslational
where CER3 produces an aldehyde intermedi- activation by a phosphopantetheine transferase
ate that can be converted to an alkane by (PPTase) for functionality [88]. Akthar et al.
CER1, while CYTB5 acts as an electron donor expressed M. marinum CAR and an activating
to CER1, which contains a di-iron catalytic PPTase from Bacillus subtilis, Spf, in E. coli to
core also found in cyanobacterial ADO [85]. obtain fatty aldehydes. Furthermore, they
Thus, the pathway is presumed to have the overexpressed the endogenous thioesterase
same catalytic reaction setup as the cyanobac- TesA to obtain high intracellular levels of
terial AAR/ADO-system. In an E. coli strain FFA from acyl-ACP, as well as a fatty alde-
engineered to overproduce short- and hyde reductase (AHR) to obtain fatty alcohols
medium-chain fatty acids (C8C14), CER1 from aldehyde intermediates. In a strain over-
from A. thaliana was used in combination with expressing all the above-mentioned enzymes,
a fatty acyl-CoA reductase (Acr) from they managed to produce around 360 mg/L
Clostridium acetobutylicum to produce short- of fatty alcohols [87]. The CAR/Spf system
chain alkanes in a fed-batch fermentation at a was also utilized in a study attempting to pro-
titre of 0.58 g/L the highest reported titre of duce the gaseous alkane propane in E. coli [59].

A heterologous butyryl-ACP-specific thio- synthesis in Jeotgalicoccus sp. ATCC 8456,

esterase was introduced that was capable of which was named OleTJE and is proposed to
intervening in the native fatty acid synthesis, perform a decarboxylation of the fatty acid
thus releasing byturic acid (C4), which sub- while using H2O2 as sole co-factor [90]. The
sequently could be converted to C3 alkane by enzyme has been found to accept and convert
combining it with the activities of CAR/Spf a range of different fatty acid substrates
and a cyanobacterial ADO/FNR-system. By (C12C20) [91].
deleting native AHRs responsible for fatty Hydrogen peroxide causes oxidative damage
alcohol formation and optimization of O2 in cells and is therefore not an optimal co-factor
availability, a final titre of 32 mg/L propane for efficient biosynthesis of alkenes. To investi-
was obtained. The low titres were believed to gate whether OleTJE could function through
be connected to low affinity of ADO towards a monooxygenase mechanism, a research
short-chain aldehydes. group fused OleTJE to a reductase domain from
Proof-of principle production of alkanes in Rhodococcus sp., RhFRED. This fusion con-
E. coli with the ADO-system has been abun- struct enabled H2O2-independent production of
dant, but was first demonstrated in S. cerevisiae alkenes by E. coli in the presence of NADPH
in 2015 [89]. The authors expressed the FAR and O2, with a maximum titre of 97.6 mg/L [92].
and ADO encoded by S. elongatus orf1594 and In a recent report, several homologs to
orf1593, respectively, in yeast, without being OleTJE were expressed in S. cerevisiae to screen
able to detect any alkanes. By co-expressing for an optimal enzyme candidate. The most
the alkane pathway with the redox-system active candidate was a codon-optimized ver-
F/FNR from E. coli, still, only trace amounts of sion of OleTJE, and maximum titres after exten-
heptadecane were produced. By further delet- sive engineering efforts, including precursor
ing a native aldehyde dehydrogenase specu- enhancement and co-factor engineering as well
lated to convert the produced aldehydes to as optimization of gene expression and cultiva-
FFAs, thus competing with an already kineti- tion conditions, reached 3.7 mg/L below
cally challenged ADO, the yield was increased what has been obtained in E. coli [93].
to 22 μg/gCDW. The simultaneous production In 2014, another one-step decarboxylation
of fatty alcohols at a level of 520 μg/gCDW pathway for terminal alkene synthesis was
indicates that ADO is a limiting factor in the discovered. Within the genus Pseudomonas,
system, and that more enzymes (AHRs) need several species produce 1-undecene (C11).
to be potentially eliminated to reduce by- Expressing a genomic library from Pseudomonas
product formation. fluorescens in E. coli and selecting clones posi-
tive for 1-undecene production enabled determi-
7.2.3.2 One-Step Reduction: Terminal nation of the gene undA to be responsible for
Alkene Synthesis alkene formation in the native host. Biochemical
Another biosynthetic route for hydrocar- characterization of the enzyme in vitro revealed
bons is widespread in species of the that UndA is a nonheme protein that catalyses
Jeotgalicoccus genus [90]. These organisms pro- an iron- and oxygen-dependent conversion
duce terminal alkenes through the action of a of medium chain (C10C14) FFAs to terminal
P450 enzyme that uses FFA as substrates, thus alkenes, constituting a previously unidentified
constituting a direct pathway for hydrocarbon enzyme family [94]. Expression of several UndA
synthesis. In 2011, Rude et al. first identified homologs in E. coli resulted in titres ranging
the gene and protein responsible for this between 36 mg/L.

7.2.3.3 Head-to-Head Condensation modest titres of 25 mg/L alka(e)nes were
In 2010, several orthologous bacterial path- reached. To investigate whether the utilized
ways for long-chain alkene synthesis were FAR system was limiting alkane production,
also genetically elucidated and characterized. fatty aldehyde and fatty alcohol titres were also
This type of alkene formation relies on a con- quantified. The result showed that fatty alcohol
densation reaction of two molecules of acyl- titres were high compared to fatty aldehydes,
CoA to bring about alkenes of chain length thus the low titres most likely depend on com-
C23C33 [9598]. The reactions were found to peting enzymes consuming the formed alde-
be catalysed by an enzyme group referred to hyde intermediate before ADO converts them
as OleABCD, where OleA performs the first into alkanes [100].
step through a Claisen condensation analo- Another strategy included expression of an
gous to the one executed in FAB by ketoacyl- alternative enzyme to initiate FAB in E. coli
ACP synthase III, to which OleA shares [101]. The native enzyme, FabH, has high spec-
sequence homology [96]. Subsequent in-depth ificity for acetyl-CoA, which results in a profile
studies of these biosynthetic enzymes sug- of even-chain fatty acids. To alter the profile,
gested that OleA generates a β-ketoacid that an alternative enzyme, FabH2 from B. subtilis
can be converted in the presence of OleC was utilized, which has a higher activity
and OleD into the corresponding alkene, toward longer-chain acyl-CoAs. When the
while the function of OleB remained elusive AAR/ADO pathway from S. elongatus was
[99]. OleD is speculated to be a NADPH- expressed in combination with FabH2, alkane
dependent reductase [96]. For aviation fuel titres were doubled, however, only a low per-
purposes, however, long-chain alkenes are not centage of the alkanes present were even-
optimal as increased chain length increases chained. When propanoate was fed to the cells,
the melting point. the even-chained alkenes increased from 6% to
27% of the total amount, indicating that the
precursor propionyl-CoA was limiting.
7.2.3.4 Attempts to Diversify Alkane Most recently, Cao et al. attempted to widen
Production the product scope by utilizing an
Since fatty acid species in organisms are α-dioxygenase from rice (α-DOX) whose activ-
even-chained due to the FAB mechanism, the ity forms “C-1” aldehyde intermediates from
alkane products obtained are odd-chained fatty acids, in contrast to the previously
while fatty alcohols remain even-chained. described fatty acid-based reductases. By com-
There have been some efforts to expand this bining α-DOX with a thioesterase and either
product profile to respond to the wide range an AHR or ADO, odd-chained alcohols, as
of alkane species present in current petroleum- well as even-chained alkanes, could be formed.
based fuels. By optimizing enzyme expression and cultiva-
The use of an alternative FAR system from tion conditions, the highest fatty alcohol titre
Photorhabdus luminescens consisting of three thus far could be reached: 1.95 g/L [102].
genes (luxC, luxD, and luxE) also enabled
reduction of FFAs in E. coli. A thioesterase from
Cinnamomum camphora was used to create a free
fatty acid of altered chain-length, enabling both
7.2.4 Limitations and Future Directions
even- and odd-chained alkane production. Yet, There is a huge gap between current micro-
in combination with N. punctiforme ADO, only bial alkane yields and titres, currently in the

mg/L range, and what is required for commer- short-chain alkane sensors as well as medium-
cial production. As described previously, long-chain alkane sensors have been generated
endogenous fatty acid metabolism and cultiva- [108,109], and in 2015, a sensor based on tran-
tion conditions have successfully been targeted scriptional regulators from the hydrocarbon-
to optimize production yields of lipids in pro- utilizing organisms Acinetobacter baylyi and
spective cell factories, but the main bottleneck Pseudomonas oleovorans was also used to detect
of the alkane pathway is the poor enzymatic medium- and long-chain alkanes produced by
activity of AAR/ADO. Natural alkane require- the AAR/ADO-system [110]. These, and deri-
ments in hosts such as plants, cyanobacteria, vatives thereof, will likely become instrumen-
and insects are low, which is why it is plausible tal in the development of more efficient
that evolutionary pressure to increase activity alkane-producing enzymes.
of these enzymes has remained low. Therefore, Metabolic sensors also show great promise
to make microbial alkane production more effi- in the optimization of metabolic pathway
cient, much work has to be put into engineering expression systems. The most common
FAR, AAR, CAR, and ADO for higher efficacy. approach to reach high production yields in
To make rational enzyme modifications, consid- cell factories has for a long time been to simply
erable knowledge of the structurefunction overexpress the essential enzymes in the bio-
relationship of the enzyme is required. synthetic pathway. However, this drains con-
Moreover, each individual mutant/enzyme var- siderable amounts of energy towards protein
iant has to be evaluated through product quan- synthesis and production of metabolic inter-
tification. The overall process of a rational mediates that might not be optimally utilized
design cycle is therefore labor-intensive. due to low substrate levels and inefficient
In order to create a high-throughput screen- downstream enzymes, respectively. Thus, the
ing approach, one alternative is to construct creation of dynamic systems able to respond to
libraries of randomly mutagenized enzymes environmental stimuli, for example, to over-
and combine these with a metabolite sensor express a pathway enzyme only if there is
[103]. Such sensors can rely on transcription abundant substrate present for it to catalyti-
factor-based systems coupled to a reporter cally convert, is highly sought after. Present
gene, for example, yielding a fluorescent out- examples are a dynamic sensor-regulator sys-
put if the metabolite is detected. Thus, tem developed to regulate expression of
enzymes with improved activity could easily enzymes related to FAEE production based
be selected by fluorescence-activated cell sort- on the presence of the substrate acyl-CoA
ing (FACS). Sensors should optimally have a [107], and the use of a malonyl-CoA sensor to
large dynamic range rather than an on-off optimize fatty acid production [111]. A sensor
signal meaning that they should produce an activated by fatty acid species could in a
increase in output with increasing stimulus similar fashion be applied for the alkane bio-
to be able to single out high producers from synthetic pathway.
intermediate ones. Sensors that can detect If alkane titres can be improved, another
intermediates in alkane synthesis have been issue will inevitably rise product toxicity.
developed; for example, malonyl-CoA sensors Alkanes are toxic to many microorganisms,
functional in several different organisms and due to their hydrophobic nature, they
[104106], and also a sensor system responsive are likely to cause problems with the mem-
to fatty acyl-CoA [107]. To increase activity of brane integrity of the cells producing them. To
AAR/ADO-enzymes, metabolic sensors specif- find a solution, the toxicity problem can be
ically sensing alkanes must be utilized. Both tackled by different means. By analysing

7.3 ISOPRENOIDS AS AVIATION FUELS 171
transcriptional data of cells exposed to the of isoprenoids, however, function as nones-
stressor [112,113] or performing adaptive labo- sential secondary metabolites, especially in
ratory evolution in increasing concentration of plants, where they serve, for example, as
stressor [114,115], changes in transcriptome insect attractants or defense molecules against
and genome, respectively, can be detected that herbivores or pathogens [119]. Due to their
potentially could be beneficial for the cell versatile function, many of these secondary
when exposed to the stressor. A more direct metabolites have gained industrial interest as
approach is to take advantage of the fact that fragrances, flavours, pesticides, or pharma-
cells tend to expel unwanted metabolites from ceuticals [15,119,120].
the cell by so-called efflux pumps, and try to All isoprenoids are derived from the C5 iso-
identify pumps specific to the product of inter- prene unit, and more precisely, from its two
est [116]. For example, Chen et al. expressed isomeric activated forms, isopentenyl diphos-
ABC-transporters from Y. lipolytica in S. cerevi- phate (IPP) and dimethylallyl diphosphate
siae, resulting in increased tolerance (about 80- (DMAPP). Depending on the number of incor-
fold) to exogenously added alkanes mediated porated isoprene units, they are classified as
by an ability to keep intracellular alkane levels hemiterpenes (C5), monoterpenes (C10), sesqui-
low [117]. A transcriptional data analysis study terpenes (C15), diterpenes (C20), etc. Mono- and
of S. cerevisiae exposed to medium-chain sesquiterpene hydrocarbons have been pro-
alkanes enabled identification of two endoge- posed as alternative aviation fuels or fuel addi-
nous efflux pumps, Snq2 and Pdr5, that were tives because they have properties (carbon
shown to reduce intracellular levels of alkanes chain length, density, freezing point, and heat
and increase tolerance [113]. Transporter engi- of combustion) similar to conventional,
neering in cells can also increase product for- petroleum-derived jet fuel [15,121]. Examples
mation, as a concentration gradient is formed include the monoterpenes pinene, sabinene,
as the products exit the cells, and potential limonene, phellandrene, and terpinene, as well
substrate inhibition can be reduced. An as the sesquiterpenes farnesene, bisabolene,
exported product is also beneficial for product and valencene (Fig. 7.6).
recovery at the commercial scale. A major disadvantage of these molecules is,
however, that they are unsaturated and there-
fore contain highly reactive CQC bonds.
7.3 ISOPRENOIDS AS AVIATION Reduction of these bonds can be achieved by
FUELS hydrogenation in the presence of a catalyst, as
has been demonstrated for limonene and myr-
Besides fatty acid-derived alka(e)nes, iso- cene, for example, [122]. Another method of
prenoids constitute the second class of com- chemical modification is the dimerization of
pounds derived from cellular metabolism cyclic monoterpenes, which can increase the
with promising prospects as biofuels. With density and heat of combustion to values simi-
more than 40,000 identified structures, iso- lar to high-density tactical fuels [123].
prenoids represent the largest group of Production of naturally occurring mono- [124]
chemical products. They occur universally in and sesquiterpenes by engineered micro-
all classes of organisms, where they fulfil organisms can therefore be combined with
essential functions as, for example, mem- further chemical conversions to produce
brane components, vitamins, hormones, pig- suitable aviation fuels with desired properties.
ments, membrane anchors, and electron This chapter covers the two major biosyn-
transport molecules [118]. The major group thetic pathways for isoprenoid biosynthesis,

FIGURE 7.6 Examples of monoterpene and sesquiterpene hydrocarbons.
describes general engineering strategies to mevalonate by HMG-CoA reductase (HMGR)

increase isoprenoid formation in different host consuming two NADPH for each mevalonate
organisms (mainly S. cerevisiae and E. coli), and formed. S. cerevisiae contains two HMGR
then gives specific examples on the production isoenzymes, Hmg1 and Hmg2 [130], which
of mono- and sesquiterpene hydrocarbons are differentially regulated [131,132] and inte-
suitable as aviation fuel precursors. grated into the membrane of the endoplasmic
reticulum via an N-terminal noncatalytic
domain involved in regulating protein degra-
7.3.1 Biosynthesis dation [133]. Mevalonate is phosphorylated
The isoprenoid precursors IPP and DMAPP to mevalonate 5-diphosphate by mevalonate
are produced via two different biosynthetic kinase (MK) and phosphomevalonate kinase
pathways: (1) the mevalonate (MVA) pathway (PMK). Finally, decarboxylation of mevalo-
in eukaryotes, in archaea [125], and in some nate 5-diphosphate, catalysed by mevalonate
eubacteria [124]; and (2) the nonmevalonate diphosphate decarboxylase (MDD), leads to
route, also called the methylerythritol (MEP) formation of IPP. In some archaea, conver-
or deoxyxylulose phosphate (DXP) pathway, sion of mevalonate to IPP follows an alter-
found in most eubacteria [126], plastids of native route via isopentenyl phosphate
plants [127], and in apicomplexa [128]. [125,134]. IPP can be converted into its
The MVA pathway uses acetyl-CoA as isomer, DMAPP, and vice versa by IPP
precursor (Fig. 7.7) [129]. Three molecules of isomerase (IDI).
acetyl-CoA are combined in two consecutive In contrast to the MVA pathway, the MEP
condensation reactions to form one molecule pathway proceeds from glyceraldehyde 3-
of 3-hydroxy-3-methylglutaryl-CoA (HMG- phosphate and pyruvate as direct precursors
CoA). The first condensation is catalysed by (Fig. 7.7) [135,136]. Both molecules are con-
acetyl-CoA acetyltransferase (ACAT), while densed to DXP by DXP synthase (DXS) under
the second one is catalysed by HMG-CoA release of CO2. DXP reductoisomerase (DXR)
synthase (HMGS). In a two-step reduction, then converts DXP into MEP, a branched
which is considered the rate-limiting reaction aldose. Coupling of MEP to cytidyl mono-
of the pathway, HMG-CoA is converted to phosphate (CMP) catalysed by MEP

FIGURE 7.7 Biosynthesis of mono- and ses-
quiterpenes via the MVA and MEP pathway.
Metabolites: HMG-CoA, 3-hydroxy-3-methylglu-
taryl-CoA; DXP, 1-deoxy-D-xylulose 5-phosphate;
MEP, 2-C-methyl-D-erythritol 4-phosphate; CDP-
ME, 4-diphosphocytidyl-2-C-methyl-D-erythritol;
CDP-MEP, 4-diphosphocytidyl-2-C-methyl-D-
erythritol 2-phosphate; MEcPP, 2-C-methyl-
D-erythritol 2,4-cyclodiphosphate; HMBPP, (E)-4-
hydroxy-3-methyl-but-2-enyl diphosphate; IPP,
isopentenyl diphosphate; DMAPP, dimethylallyl
diphosphate; GPP, geranyl diphosphate; FPP, far-
nesyl diphosphate. Enzymes: ACAT, acetyl-CoA
C-acetyltransferase; HMGS, 3-hydroxy-3-methyl-
glutaryl-CoA synthase; HMGR, 3-hydroxy-3-
methylglutaryl-CoA reductase; MK, mevalonate
kinase; PMK, phosphomevalonate kinase; MDD,
mevalonate diphosphate decarboxylase; DXS,
DXP synthase; DXR, DXP reductoisomerase;
MEPCT, MEP cytidylyltransferase; CMK, CDP-
ME kinase; MCS, MEcPP synthase; HDS, HMBPP
synthase; HDR, HMBPP reductase; IDI, isopente-
nyl diphosphate isomerase; GPPS, GPP synthase;
FPPS, FPP synthase.

cytidyltransferase (MEPCT) and subsequent production of various mono- and sesquiter-

phosphorylation of the resulting intermediate penes in S. cerevisiae, E. coli, and other micro-
4-diphosphocytidyl-2C-methyl-D-erythritol bial hosts. Although not all of these strategies
(CDP-ME) catalysed by CDP-ME kinase were specifically applied to potential aviation
(CMK) leads to formation of CDP-ME 2-phos- fuel precursors, they can still easily be
phate (CDP-MEP). In an intramolecular adapted for this purpose and will also be
transphosphorylation, 2C-methyl-D-erythritol discussed here.
2,4-cyclodiphosphate (MEcPP) is formed under
release of CMP. The last two enzymes of the 7.3.2.1 Saccharomyces cerevisiae
pathway 1-hydroxy-2-methyl-2-(E)-butenyl- As S. cerevisiae contains the MVA pathway
4-diphosphate (HMBPP) synthase (HMS) and starting from acetyl-CoA for isoprenoid pro-
HMBPP reductase (HMR) both contain an duction, approaches similar to the ones
iron-sulphur cluster and are highly sensitive to described above for increasing the production
oxygen [137,138]. HMR produces a mixture of of fatty acid-derived products have also been
both IPP and DMAPP at a ratio of about 5:1 in employed to increase isoprenoid synthesis in
E. coli [139]. In addition, most organisms that yeast. Engineering of the PDH bypass by
use the MEP pathway for IPP and DMAPP expression of a deregulated acetyl-CoA synthe-
synthesis also possess an IDI to balance the tase from S. enterica and overexpression of
ratio between the two metabolites [140]. aldehyde dehydrogenase Ald6 either alone
When comparing the overall stoichiometry or in combination with overexpression of alco-
of both pathways for IPP production from hol dehydrogenase Adh2 and ACAT (Erg10)
glucose, the MVA pathway consumes 1.5 mol increased sesquiterpene production by up to
of glucose and 3 mol of ATP per mol IPP, 75% [24,144]. Additional inhibition of acetyl-
while 1 mol of glucose and 2 mol of ATP are CoA consumption in the glyoxylate cycle was
required for production of 1 mol of IPP via able to further double sesquiterpene produc-
the MEP pathway [141,142]. This renders the tion [144]. Further strategies to increase the
MEP pathway superior in terms of maximum cytosolic acetyl-CoA supply in yeast are men-
isoprenoid yields that can theoretically be tioned in section 7.2.2.1.
achieved. Engineering of the MVA pathway in yeast
After synthesis of IPP and DMAPP, prenyl- has mainly focused on modulation of key
transferases catalyse the successive head-to-tail enzymatic reactions, one of them being HMGR
condensation of IPP to an allylic diphosphate (see above). The most common strategy is the
(DMAPP in the first cycle) to produce the overexpression of a truncated version of
direct precursors of monoterpenes (geranyl Hmg that is, a version only consisting of the
diphosphate, GPP), sesquiterpenes (farnesyl C-terminal catalytic domain and devoid of the
diphosphate, FPP), and longer-chain isopre- N-terminal membrane anchor [145147].
noids (Fig. 7.7) [143]. However, overexpression of full-length Hmg1
[148], a stabilized variant of Hmg2, Hmg2
(K6R) [149,150], or expression of a heterolo-
gous HMGR from Staphylococcus aureus [151],
7.3.2 Metabolic Engineering to Increase
were also proven to be successful in increasing
Isoprenoid Production in Microbial Hosts
isoprenoid production. Moreover, additional
Numerous studies have investigated possi- genetic perturbations stabilizing HMGR pro-
bilities to produce and enhance the tein levels and thus leading to improved

sesquiterpene production in yeast were identi- squalene synthase (Erg9), and then further to
fied [152]. ergosterol, transcription of the ERG9 gene has
Also, balancing the ratio between IPP and been modulated in order to reduce the flux
DMAPP appears to be a crucial factor for iso- towards ergosterol and increase the availability
prenoid biosynthesis. Overexpression of IDI of FPP for sesquiterpene production. In several
alone in yeast was able to boost monoterpene cases, the ERG9 promoter was exchanged with
production by up to fivefold [150,153]. the MET3 promoter that is repressed by methi-
When producing either mono- or sesquiter- onine [163,164]. This, however, requires the
penes, it is important to consider that a single constant addition of methionine to the medium
enzyme, FPP synthase Erg20, is responsible in because methionine is metabolized by the cells.
yeast for the two enzymatic steps leading to Other studies therefore used the copper-
production of both GPP, the precursor of repressible CTR3 promoter [165], or a more
monoterpenes, and FPP, the sesquiterpene pre- dynamic approach where ERG9 was controlled
cursor. The wild-type enzyme, however, either by the glucose-regulated HXT1 pro-
releases only small amounts of the intermedi- moter, which repressed ergosterol formation
ate GPP [154,155]. Overexpression of Erg20 has when glucose was consumed after the expo-
therefore mainly been used to increase the pro- nential growth phase [166], or by promoters
duction of sesquiterpenes [145,150,156]. To repressed by ergosterol itself as feedback inhi-
enhance the formation of monoterpenes, on bition [167].
the other hand, mutant variants of the enzyme, Another sink for FPP is its dephosphoryla-
such as Erg20(K197E) or Erg20(F96W-N127W), tion to farnesol. It is believed that two lipid
that favour GPP over FPP production, were phosphatases, Lpp1 and Dpp1, are involved in
employed [153,155,157159]. Alternatively, this process [168]. In one study, deletion of
specific heterologous GPP synthases derived DPP1 in an lpp1Δ background strain was able
from plants, for instance, can be used [160]. to both reduce farnesol levels and increase
Instead of concentrating on selected enzymatic sesquiterpene production [166], whereas other
reactions of the MVA pathway, other studies studies saw no, or even a negative, effect of
took a more global approach, such as expres- deletion of DPP1 either alone or in combination
sion of a mutated version of a transcription with LPP1 on sesquiterpene titres [155,169,170].
factor (Upc2-1) that led to enhanced transcrip- While FPP represents the major branch point
tion of several sterol pathway genes but in the isoprenoid biosynthesis, DMAPP is also
alone only had a minor effect on sesquiterpene consumed by other metabolic pathways, namely
production [145,156,161] or by simply over- the prenylation of tRNA. Liu and co-workers
expressing all genes of the MVA pathway, therefore overexpressed MAF1 encoding a nega-
which led to a significant increase in sesquiter- tive regulator of RNA polymerase III, which
pene formation [162]. doubled monoterpene production [153].
Apart from increasing the flux through the Deletion or down-regulation of competing
MVA pathway towards FPP or GPP, it is also pathways represents one way of reducing the
crucial to limit the consumption of FPP by drain of precursors or intermediates. Another
other metabolic pathways. Most of these path- possibility is spatial control, for example, by
ways, such as the formation of ergosterol or exploiting compartmentalization of a eukary-
dolichols, are, however, essential, and can otic cell. Targeting of sesquiterpene synthases
therefore not be entirely disrupted. Since the to the mitochondria drastically increased prod-
majority of FPP is converted to squalene by uct formation, which was further enhanced by

targeting a heterologous FPP synthase to the isoprenoid synthesis is dependent on the

same compartment [170]. However, it remains background strain [179181].
speculative whether shielding from compet- As in S. cerevisiae, overexpression of IDI to
ing reactions or other possible explanations balance the ratio between the two precursor
such as a more favourable environment for isomers is a useful tool to increase isoprenoid
enzyme activity are the actual cause for the accumulation [174,176,182,183]. Also like in
observed effect. yeast, overexpression of FPP synthase (IspA)
A more direct method of metabolite was used to increase the production of sesqui-
channeling by spatial control is the fusion of terpenes and longer terpenes [176,182,184],
consecutive pathway enzymes. Fusion of while either mutated versions of IspA or
Erg20 to a sesquiterpene synthase as well as heterologous GPP synthases were employed
fusion of a GPP producing Erg20 mutant to a to establish production of monoterpenes
monoterpene synthase both successfully [185188].
increased product formation [155,171]. It While heterologous expression of the MEP
should be noted, however, that orientation of pathway in yeast seems to be challenging (see
the enzymes within the fusion construct and above), a functional MVA pathway has been
the choice of a potential linker peptide may be successfully established in E. coli, and often, its
of importance. introduction had a greater effect than engi-
With regard to the fact that the MEP path- neering the endogenous pathway, despite its
way would be the more efficient isoprenoid theoretically lower yields [178,189191]. This
pathway in terms of carbon preservation and may be due to the fact that enzymes of the
energy consumption, it was also attempted to MEP pathway are subject to feedback regula-
heterologously express this pathway in yeast, tion [192] and expression of a heterologous
albeit without success [142,172]. It was pathway can therefore benefit from circum-
assumed that this was due to the nonfunction- venting regulatory mechanisms. However,
ality of the two iron-sulphur cluster-containing accumulation of certain MVA pathway inter-
pathway enzymes in the yeast cytosol. mediates such as HMG-CoA and prenyl
diphosphates (IPP, DMAPP, FPP) seems to be
7.3.2.2 Escherichia coli toxic to E. coli and efficient downstream
Similar to increasing the flux through enzymes and careful pathway balancing are
the MVA pathway in yeast, engineering of necessary to avoid growth retardation of the
the MEP pathway in E. coli mainly focuses engineered strains [189]. Pathway balancing
on overexpression of several key enzymes, was performed by adding additional gene cop-
usually in combination. One crucial factor ies of postulated rate-limiting enzymes
seems to be the early pathway steps because [178,193,194], recruitment of enzymes from
overexpression of DXS and/or DXR often other sources for improved activity, reduced
resulted in increased isoprenoid levels (eg, feedback inhibition or co-factor preference
[173175]). However, in some cases this [195197], or by modulating the strength of
strategy even had a negative effect on terpe- ribosome binding sites determining translation
noid production, which suggests that careful efficiency of the different pathway genes [198].
fine-tuning of the expression levels and A fourth approach to increase the flux through
the genetic context are important [176178]. the MVA pathway in E. coli was the assembly
Similarly, the effect of overexpressing other of pathway enzymes to a protein scaffold
pathway genes, such as ispG and ispH encod- using proteinprotein interaction domains, a
ing HMS and HMR, respectively, on concept similar to enzyme fusion described

above [199]. However, in this case the scaffold combined with either overexpression of the
allows for modulating the amount of each endogenous FPP/GPP synthase or expres-
enzyme per complex, thus compensating for sion of a GPP synthase derived from Abies
different enzyme activities. grandis, whereupon the latter drastically
improved pinene production. α-Pinene titres
7.3.2.3 Other Organisms were further enhanced by expression of a
Besides S. cerevisiae and E. coli, other micro- hybrid MVA pathway based on genes from
organisms have also been engineered for Enterococcus faecalis and S. cerevisiae, res-
increased production of isoprenoid precursors, pectively, and reached up to 5.4 mg/L in
for example, overexpression of endogenous shake flask cultivations and 970 mg/L in a
MEP pathway genes or establishment of the fed-batch fermentation.
heterologous MVA pathway in cyanobacteria To establish a more sustainable process for
[200202]. Another photosynthetic bacterium the production of biofuels, it is important to
that has been engineered through introduc- develop strains that are able to grow on
tion of a heterologous MVA pathway, in this more complex carbon sources such as cellu-
case encoded by an operon derived from losic biomass. Bokinsky et al. [215] therefore
Paracoccus zeaxanthinifaciens, is Rhodobacter engineered two E. coli strains with the ability
sphaeroides, which increased sesquiterpene to grow on cellulose and xylan, respectively.
formation sixfold [203]. Nonphotosynthetic As described above, a heterologous MVA
bacteria, in which either endogenous or pathway in this case entirely from S. cerevi-
heterologous isoprenoid pathways have siae GPP synthase from A. grandis, and
been improved, comprise, among others, pinene synthase from P. taeda were employed
Lactococcus lactis [204], Corynebacterium gluta- to confer pinene synthesis in both strains.
micum [205,206], Streptomyces venezuelae [207], Since the two plant enzymes are normally
Pseudomonas putida [208], and B. subtilis targeted to the chloroplast, their targeting
[209]. With regard to eukaryotic microorgan- signals were removed and the genes codon
isms, the endogenous MVA pathway was optimized for E. coli. A co-culture of both
modulated in several non-Saccharomyces spe- strains was able to produce 1.7 mg/L of
cies, including Y. lipolytica and Pichia pas- pinene from switchgrass pre-treated with
toris, which in most cases focused mainly on ionic liquids. While Yang et al. [214] mea-
overexpression of either full-length or trun- sured pinene in the gaseous phase of the
cated HMGR [210213]. fermentation vessel, Bokinsky et al. [215]
used an organic solvent, dodecane, to capture
pinene in the liquid phase.
7.3.3 Production of Monoterpene Efficient secretion of the produced biofuel
Hydrocarbons precursor is important for two reasons. On the
one hand, it facilitates product recovery, and
7.3.3.1 Pinene on the other hand it may alleviate the toxic
Pinene, which occurs as an α- and effect that many terpenes, especially monoter-
β-isomer, is a bicyclic monoterpene produced penes, have on the producing microbes. To
by pine trees and other plants. Pinene synthe- address this issue, a gene encoding an inner
sis from GPP in a heterologous organism is membrane transporter of E. coli was randomly
established by expression of a single enzyme, mutagenized and strains expressing this gene
pinene synthase. Yang et al. [214] expressed library selected for improved tolerance
a Pinus taeda α-pinene synthase in E. coli towards (ie, presumably improved secretion

of) n-octane or 1-octanol [216]. Remarkably, Optimization of a two-phase fed-batch fer-

the mutated transporter not only improved the mentation process using a similar MVA
export of n-octane, but also of α-pinene, up to pathway-containing strain allowed accu-
fourfold. mulation of up to 2.7 g/L of limonene (in
Because apart from pinene the precursor the organic phase) from glycerol with a pro-
GPP may also be toxic to E. coli, Sarria et al. ductivity of 22.6 mg/L per h [187].
[186] tested combinations of three different In an experimental setup similar to the one
GPP synthases with three different pinene described above for transporter optimization, a
synthases as well as fused versions of both library of E. coli strains expressing 43 different
enzymes in an MVA pathway-expressing efflux pumps was used to select for enrich-
strain yielding up to 32 mg/L of pinene. They ments under cultivation in the presence of lim-
also showed that the enzyme combination onene and other terpenes [116]. A pump
influenced the ratio between α- and β-pinene conferring higher resistance to limonene
produced. The best strain derived from this derived from Alcanivorax borkumensis also
study expressing a fusion protein of GPP increased limonene formation in a producer
synthase and α-/β-pinene synthase (both strain by about 50%.
from A. grandis) was also used to produce To enable the production of limonene
pinene from macroalgal hydrolysate at up to directly from CO2, limonene synthases from
21 mg/L [217]. different sources were introduced into cyano-
As an alternative to E. coli as a host, pinene bacterial species, including the two unicellular
production was also established in model organisms Synechococcus sp. [220] and
Corynebacterium glutamicum using two different Synechocystis sp. [202], and the filamentous
GPP synthases and two pinene synthases in N2-fixing Anabaena sp. [221]. The produced
connection with overexpression of DXS and limonene was either collected in a dodecane
IDI of the endogenous MEP pathway, resulting overlay [220] or captured from the gas phase.
in up to 176 μg/L of pinene [205]. While (over)expression of endogenous or
heterologous DXS, IDI, and GPP synthase to
7.3.3.2 Limonene improve precursor supply by the endogenous
The monocyclic hydrocarbon limonene MEP pathway had a positive effect on limo-
was first produced in E. coli as an intermedi- nene formation [150,202], inhibiting formation
ate of the pathway leading to carvone synthe- of the storage carbohydrate and by-product
sis [218]. Expression of the homodimeric GPP glycogen did not [220]. In all cases, produc-
synthase of A. grandis together with limonene tion levels have remained rather low, in the
synthase from Mentha spicata omitting their μg/L range.
targeting peptides led to a final limonene The same is true for limonene production in
titre of 5 mg/L. Expression of IDI from S. cerevisiae so far [159]. In one study, a
Mentha x piperita only slightly increased (1)-limonene synthase from Citrus limon and
limonene formation. Expression of both genes a (2)-limonene synthase from Perilla frutescens
in a strain containing the yeast MVA path- were expressed in a yeast strain expressing
way together with optimization of certain Erg20 (K197G) (see above) both as full-length
steps such as replacement of HMGS and versions and without their respective plastid-
HMGR with the respective enzymes from targeting signal, where the truncated versions
Staphylococcus aureus and optimization of of the enzymes proved to be superior. When
gene expression levels drastically enhanced comparing different methods for product
limonene production to 435 mg/L [219]. recovery, using a dodecane overlay or

continuous headspace trapping were most effi- diploid background strain overexpressing
cient. While many of the above-mentioned Hmg2(K6R) and carrying heterozygous dele-
engineering strategies will be useful in further tions in ERG20 and ERG9 to further reduce
increasing limonene production in yeast, the FPP formation as well as FPP flux towards
screening of yeast strain libraries producing ergosterol, leading to production of 17.5 mg/L
limonene and other monoterpenes may also be of sabinene.
supported by a recently developed colouri- Proof-of-principle studies also showed the
metric assay based on 2,2-diphenyl-1-picrylhy- production of monocyclic phellandrene using
drazyl [222]. a terpene synthase from Lavandula angustifolia
Apart from low production levels, mono- [227] and acyclic myrcene using myrcene
terpene toxicity is a major issue in yeast. synthase from Quercus ilex [228] in E. coli. As
Among five different monoterpenes tested, in the examples mentioned above, engineer-
limonene had the strongest inhibitory effect ing strategies included the expression of a
on the yeast growth rate [223] and mainly truncated plant GPP synthase, the mevalonate
seems to affect its cell wall [224]. While the pathway, and balancing of gene expression
use of organic solvents in two-phase fer- levels. In addition, phellandrene was also
mentation set-ups was able to reduce limo- produced in Synechocystis sp. for evaluating
nene toxicity [223], the overexpression of different transcriptional and translational reg-
endogenous ATP-binding cassette transpor- ulatory elements for optimal expression of the
ters had no effect [225]. Analysis of strains terpene synthase gene after chromosomal
evolved for increased limonene tolerance integration [229].
revealed that a single frame shift mutation in
TCB3 encoding a protein of the ER could con-
fer monoterpene tolerance, potentially by 7.3.4 Production of Sesquiterpene
affecting cell wall integrity [226]. Hydrocarbons
7.3.3.3 Sabinene, Phellandrene, and 7.3.4.1 Valencene
Myrcene Like many other mono- and sesquiterpene
The bicyclic sabinene was produced in both hydrocarbons, the bicyclic valencene does not
E. coli [185] and S. cerevisiae [155] employing a only attract interest as a potential fuel, but
sabinene synthase from Salvia pomifera. In also as an aroma compound. While valencene
E. coli, expression of the terpene synthase was represents an aroma substance itself, it also
combined with expression of the A. grandis serves as a precursor for the high-value
GPP synthase and the hybrid Enterococcus fae- nootkatone. Valencene, among other sesqui-
calis/S. cerevisiae MVA pathway similar to the terpenes, was first produced in yeast to study
strategy described for pinene above [214]. the influence of regulating ERG9 expression
Optimization of the fermentation medium and with the repressible MET3 promoter [163].
gene induction levels finally resulted in pro- Here, a valencene synthase gene from Citrus
duction of 2.65 g/L of sabinene from glycerol x paradisi was expressed under control of
in a fed-batch approach. The study in S. cerevi- the inducible GAL1 promoter leading pro-
siae mainly concentrated on engineering Erg20 duction of up to 3 mg/L. Farhi et al. [170]
to reduce FPP formation and increase GPP for- targeted valencene synthesis to the mitochon-
mation. The resulting dominant negative dria with the rational that FPP-consuming
mutant enzyme Erg20(F96W-N127W) was then enzymes are located in both the cytosol and
fused to sabinene synthase and expressed in a mitochondria that is, a precursor pool

should be present in different compartments. codon-optimized for E. coli. In both heterolo-

Their best producer strain was based on an gous hosts, the codon-optimized version of
industrial yeast strain expressing both a cyto- E-α-bisabolene synthase from A. grandis
solic and a mitochondrial version of valen- (AgBIS) exhibited by far the highest activity.
cene synthase from Citrus x sinensis, a Production levels in E. coli carrying an
heterologous FPP synthase from Arabidopsis optimized MVA pathway reached up to
also targeted to the mitochondria and cyto- 912 mg/L, whereas a S. cerevisiae strain, in
solic tHmg1 yielding 1.5 mg/L, which repre- which tHMGR, Erg20 and Upc2-1 were over-
sents a sevenfold improvement over the expressed and ERG9 was down-regulated,
strain only expressing the terpene synthase produced 994 mg/L of bisabolene. Later, the
in the cytosol. protein structure of AgBIS was determined
Because the low valencene titres obtained in and a catalytic mechanism for bisabolene syn-
this study also in comparison with other ses- thesis was proposed, which opens up oppor-
quiterpenes indicated low activity of the tunities for protein engineering approaches
synthases, Beekwilder et al. [203] isolated and [232]. The fusion of AgBIS to Erg20 increased
characterized valencene synthase from both enzyme expression levels and bisabolene
Callitropsis nootkatensis, which had kinetic production in yeast [233].
properties similar to the one from Citrus x To identify nonobvious targets for increas-
paradisi, but showed a better performance ing bisabolene production, the authors also
when expressed in a nonoptimized yeast strain screened a yeast deletion library expressing a
or in Rhodobacter sphaeroides. When combined carotenoid biosynthetic pathway for enhanced
with expression of the MVA pathway operon colour formation. Gene deletions that were
from Paracoccus zeaxanthinifaciens, valencene able to increase both carotenoid and bisabolene
production increased to 350 mg/L. formation were identified. A strain carrying,
In addition, two other microbial hosts were among other modifications, deletions in two
engineered for production of valencene using genes of unknown function was tested in a
the Callitropsis nootkatensis enzyme: a fed-batch cultivation leading to a production
Corynebacterium glutamicum strain deleted in of 5.2 g/L of bisabolene.
two prenyltransferase genes and expressing Instead of a screening system, Kirby et al.
FPP synthase from either E. coli or S. cerevisiae [234] developed a method that allows for selec-
[206] and, rather unusually, the basidiomycete tion of strains with enhanced production of
Schizophyllum commune [230]. Maximum titres bisabolene (and potentially also other ter-
were 2.4 and 16 mg/L, respectively. In all penes). This method is based on the fact that
studies, dodecane was used to capture valen- cells producing bisabolene are more resistant
cene in situ. to nonionic surfactants than nonproducers,
presumably due to changes in membrane flu-
7.3.4.2 Bisabolene idity. This principle was on the one hand used
The activity of the terpene synthase often to identify AgBIS gene variants leading to
represents a limiting factor in isoprenoid pro- higher bisabolene production, and on the other
duction. To establish bisabolene production in hand to increase production stability in long-
E. coli and in S. cerevisiae, Peralta-Yahya et al. term cultures.
[231] screened bisabolene synthases from Apart from E. coli and Saccharomyces cerevi-
five different sources: two angiosperm and siae, bisabolene production was also
three gymnosperm synthases differing in introduced in Synechococcus sp. [220] and
their domain structure. Most of them were Streptomyces venezuelae [207].

7.3.4.3 Farnesene Ald6 and Acs2. Since many of these genes are
Both farnesene isomers (α- and β-) occur in a regulated by the GAL promoters, galactose reg-
wide range of plant and animal species. Both ulation was modified in a way that enabled
α- and β-farnesene synthases from different activity of these promoters in glucose medium.
plant sources have been heterologously Crucial for the further improvement of far-
expressed in Anabaena sp. [235], E. coli [236], nesene production strains, however, was the
and S. cerevisiae [237]. While titres in Anabaena development of an automation platform for
sp. remained in the μg/L range, engineering an high-throughput strain construction and
E. coli strain through expression of a hybrid screening. This enabled the construction of
MVA pathway and a fusion enzyme of FPP the current production strain, which carries
synthase and Malus x domestica α-farnesene about 80,000 new bp and has 40,000 bp
synthase led to production of up to 380 mg/L of deleted (http://www.biofuelsdigest.com/
α-farnesene when glycerol was fed as an addi- bdigest/2015/08/27/amyris-and-the-velocity-
tional carbon source [238]. Zhu et al. [194] recon- of-renewables/). Production of farnesene
structed the MVA pathway including farnesene from sugarcane is implemented at the Brotas
synthesis in vitro in order to study its kinetics plant in São Paulo, Brazil, which has a capac-
and derive engineering strategies from these ity of 50 M L per year. The applicability of
results. By increasing expression of selected farnesene-derived fuels as a blending compo-
MVA pathway genes in E. coli, they were able to nent in jet fuel has been demonstrated in
enhance the α-farnesene titre to 1.1 g/L. several test flights.
To date, β-farnesene is by far the most
advanced example of isoprenoid hydrocarbon
production. It is produced on an industrial
7.3.5 Conclusion and Perspectives
scale by a heavily engineered S. cerevisiae strain The above examples have shown that
developed by Amyris. One of their intermedi- although mono- and sesquiterpene yields and
ate strains was described in a study where the titres of primary engineered strains are usually
CoA precursor pantothenate was used as a quite low, it is possible to reach industrially
metabolic switch to limit farnesene production relevant production levels. This, however,
during the growth phase and thereby requires extensive strain engineering efforts, as
increased the genetic stability of the popula- seen in the farnesene example. One major limi-
tion [239]. This strain contained, among others, tation is limited availability of GPP and FPP in
the following features: (1) expression of a the microbial hosts. Current and future strate-
mutated version of a β-farnesene synthase gies to establish the production of novel com-
from Artemisia annua with improved catalytic pounds will therefore instead rely on platform
efficiency, the gene being integrated in several strains already optimized, amongst other fea-
copies into the genome; (2) overexpression of tures, for the provision of sufficient terpene
the entire endogenous MVA pathway using precursors using either endogenous, heterolo-
integration of one or two additional copies per gous, or hybrid pathways.
gene as well as expression of an HMGS from Another challenge is the selection or engi-
Brassica juncea and an ACAT from C. acetobuty- neering of efficient terpene synthases. Since
licum; (3) down-regulation of ERG9 with help terpenes are often only required at low
of the MET3 promoter; and (4) overexpression amounts in their native hosts, the evolutionary
of the PDH bypass using a pyruvate decarbox- pressure to select biosynthetic enzymes of high
ylase from Zymomonas mobilis and endogenous catalytic efficiency has been low similar to

the enzymes of the alka(e)ne pathways dis- may not necessarily be a disadvantage. When
cussed above. Although increasing enzyme screening a mutant library of a bisabolene
expression levels through enhancing the gene synthase Kirby et al. [234] identified enzyme
copy number, selection of efficient promoters variants that were able to produce a mixture
and codon optimization may be beneficial, its of both α-bisabolene and β-farnesene at
effect is often limited [229]. As an initial step, different ratios.
it may therefore be useful to screen terpene All in all, it has been shown that the
synthases from different organisms since their microbial production of aviation fuels or their
activities can vary substantially in the heterolo- precursors at industrially relevant levels is
gous host [231]. Furthermore, the increasing possible. The future will show whether exten-
availability of genome sequences will further sive efforts in high-throughput strain engi-
enhance the search space for potential gene/ neering can boost alka(e)ne production to
enzyme candidates. Rational engineering of levels similar to the ones obtained for farne-
terpene synthases for enhanced activity has sene. So far, production of farnesene is based
proven to be difficult, and the screening of on sugarcane. The next challenge will there-
enzyme libraries through direct analysis of fore be to turn first-generation jet fuel pro-
product formation by GC-MS is only feasible duction into second-generation production
at low throughput. based on lignocellulosic biomass analogous
Therefore, indirect screening or selection to second-generation bioethanol plants to
methods for high-throughput analysis of achieve a truly sustainable process.
large terpene synthase libraries generated by
random mutagenesis have been developed.
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C H A P T E R

7.1 INTRODUCTION field. In this introductory section we discuss

Biofuels for Aviation.

II. THE SCIENCE AND TECHNOLOGY OF DEVELOPING BIOFUELS FOR AVIATION

II. THE SCIENCE AND TECHNOLOGY OF DEVELOPING BIOFUELS FOR AVIATION

II. THE SCIENCE AND TECHNOLOGY OF DEVELOPING BIOFUELS FOR AVIATION

TABLE 7.1 Summary of Commonly Utilized Metabolic Engineering Strategies

II. THE SCIENCE AND TECHNOLOGY OF DEVELOPING BIOFUELS FOR AVIATION

II. THE SCIENCE AND TECHNOLOGY OF DEVELOPING BIOFUELS FOR AVIATION

II. THE SCIENCE AND TECHNOLOGY OF DEVELOPING BIOFUELS FOR AVIATION

II. THE SCIENCE AND TECHNOLOGY OF DEVELOPING BIOFUELS FOR AVIATION

II. THE SCIENCE AND TECHNOLOGY OF DEVELOPING BIOFUELS FOR AVIATION

used to channel carbon towards the cytosolic 7.2.2.2.1 OVEREXPRESSION OF ENZYMES

II. THE SCIENCE AND TECHNOLOGY OF DEVELOPING BIOFUELS FOR AVIATION

II. THE SCIENCE AND TECHNOLOGY OF DEVELOPING BIOFUELS FOR AVIATION

II. THE SCIENCE AND TECHNOLOGY OF DEVELOPING BIOFUELS FOR AVIATION

II. THE SCIENCE AND TECHNOLOGY OF DEVELOPING BIOFUELS FOR AVIATION

S. cerevisiae in a synthetic biology approach metabolic engineering strategies to improve

II. THE SCIENCE AND TECHNOLOGY OF DEVELOPING BIOFUELS FOR AVIATION

II. THE SCIENCE AND TECHNOLOGY OF DEVELOPING BIOFUELS FOR AVIATION

II. THE SCIENCE AND TECHNOLOGY OF DEVELOPING BIOFUELS FOR AVIATION

II. THE SCIENCE AND TECHNOLOGY OF DEVELOPING BIOFUELS FOR AVIATION

A heterologous butyryl-ACP-specific thio- synthesis in Jeotgalicoccus sp. ATCC 8456,

II. THE SCIENCE AND TECHNOLOGY OF DEVELOPING BIOFUELS FOR AVIATION

II. THE SCIENCE AND TECHNOLOGY OF DEVELOPING BIOFUELS FOR AVIATION

II. THE SCIENCE AND TECHNOLOGY OF DEVELOPING BIOFUELS FOR AVIATION

II. THE SCIENCE AND TECHNOLOGY OF DEVELOPING BIOFUELS FOR AVIATION

FIGURE 7.6 Examples of monoterpene and sesquiterpene hydrocarbons.

describes general engineering strategies to mevalonate by HMG-CoA reductase (HMGR)

II. THE SCIENCE AND TECHNOLOGY OF DEVELOPING BIOFUELS FOR AVIATION

II. THE SCIENCE AND TECHNOLOGY OF DEVELOPING BIOFUELS FOR AVIATION

cytidyltransferase (MEPCT) and subsequent production of various mono- and sesquiter-

II. THE SCIENCE AND TECHNOLOGY OF DEVELOPING BIOFUELS FOR AVIATION

II. THE SCIENCE AND TECHNOLOGY OF DEVELOPING BIOFUELS FOR AVIATION

targeting a heterologous FPP synthase to the isoprenoid synthesis is dependent on the

II. THE SCIENCE AND TECHNOLOGY OF DEVELOPING BIOFUELS FOR AVIATION

II. THE SCIENCE AND TECHNOLOGY OF DEVELOPING BIOFUELS FOR AVIATION

of) n-octane or 1-octanol [216]. Remarkably, Optimization of a two-phase fed-batch fer-

II. THE SCIENCE AND TECHNOLOGY OF DEVELOPING BIOFUELS FOR AVIATION

II. THE SCIENCE AND TECHNOLOGY OF DEVELOPING BIOFUELS FOR AVIATION

should be present in different compartments. codon-optimized for E. coli. In both heterolo-

II. THE SCIENCE AND TECHNOLOGY OF DEVELOPING BIOFUELS FOR AVIATION

II. THE SCIENCE AND TECHNOLOGY OF DEVELOPING BIOFUELS FOR AVIATION

II. THE SCIENCE AND TECHNOLOGY OF DEVELOPING BIOFUELS FOR AVIATION

II. THE SCIENCE AND TECHNOLOGY OF DEVELOPING BIOFUELS FOR AVIATION

II. THE SCIENCE AND TECHNOLOGY OF DEVELOPING BIOFUELS FOR AVIATION

II. THE SCIENCE AND TECHNOLOGY OF DEVELOPING BIOFUELS FOR AVIATION

II. THE SCIENCE AND TECHNOLOGY OF DEVELOPING BIOFUELS FOR AVIATION

II. THE SCIENCE AND TECHNOLOGY OF DEVELOPING BIOFUELS FOR AVIATION

II. THE SCIENCE AND TECHNOLOGY OF DEVELOPING BIOFUELS FOR AVIATION

II. THE SCIENCE AND TECHNOLOGY OF DEVELOPING BIOFUELS FOR AVIATION

II. THE SCIENCE AND TECHNOLOGY OF DEVELOPING BIOFUELS FOR AVIATION

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