A perspective: Photosynthetic production of fatty acid-based biofuels in genetically engineered

cyanobacteria

1. Energy resource platforms

Biomass can be transformed into biofuel by chemical or biological conversion or a combination of
both strategies. Yet, each type of biomass presents its own set challenges that must be overcome
if biofuel is to be produced at low fiscal and environmental costs. For example, chemical (acid or
base) or enzyme (lipase) catalysis can be used to liberate the fatty acids from triacyglycerols (TAG)
produced in “oil” production plants or algae for use as the corresponding methyl or ethyl esters as
biofuels. However, the inadequate supply of TAG is the bottleneck for current biodiesel
production. At present, microalgae are considered to be the highest potential TAG resource
because of the high oil content and faster growth compared to plants (Chisti, 2007).

2. Microbial systems
As a candidate for biofuel-producing microbial systems, cyanobacteria are attractive because they
incorporate the favorable characteristics of prokaryotics and plants. In contrast to the generally
utilized biofuel-producing microbes (E. coli, Z. mobilis, S. cerevisiae and others), cyanobacteria are
photosynthetic microbes, which can absorb solar energy and fix carbon dioxide. It is more efficient
to convert solar energy and carbon dioxide into biofuels in one biological system than using plants
to make polysaccharides (cellulose and hemicellulose) from solar energy and carbon dioxide
through photosynthesis, and microbes to make biofuels from glucose produced from the
polysaccharides through fermentation (Angermayr et al., 2009).
Compared to general photosynthetic plants and eukaryotic microalgae, cyanobacteria are more
amenable to genetic manipulation for installing biofuel-producing chemical pathways.
Finally, Angermayr et al. (2009) have outlined a promising approach to biofuel production based
on the redirection of cyanobacterial intermediary metabolism.
Direct conversion of carbon dioxide to biofuels in photosynthetic cyanobacteria can significantly
improve the efficiency of biofuel production. A theoretical calculation shows that the productivity
of ethanol in a photosynthetic organism can reach ca. 5,280 gal/acre/ year (Angermayr et al.,
2009). Algenol Biofuels Inc. has developed an innovative cyanobacteria-based technology that is
reported to produce ethanol at a rate of 6000 gal/acre/year. In contrast, the annual yield of
ethanol from corn is 321 gal/acre/year, from sugar cane 727 gal/acre/year (Brazil Institute Special
Report, 2007), from switchgrass 330–810 gal/acre/year, and from corn stover 290– 580
gal/acre/year (Sanderson, 2006).

3. Biofuel molecules
The exploration and development of novel biofuels, which possess high energy density, are
hydrophobic and are compatible with the existing liquid fuel infrastructure (i.e., fuel engines,
refinery equipment and transportation pipelines) is in great demand. In terms of fuel properties,
the best replacement of petroleum fuels is “Petroleum Fuels”. In other words, ideal biofuels
produced from biological systems should be chemically similar to petroleum, like fatty acid based
molecules including fatty acid esters, fatty alcohols and fatty alkanes.

4. Fatty acid-based biofuels in cyanobacteria

The biosynthesis of fatty acid-based biofuels in cyanobacteria includes two steps, overproduction
of fatty acids and transesterification of fatty acids to form fatty acid ethyl esters or reduction of
fatty acids to form fatty alcohols or fatty alkanes (Fig. 2).
Fatty acid metabolism has been extensively studied. Acetyl-CoA carboxylase catalyzes the first step
of the fatty acid biosynthetic pathway, involving formation of malonyl-CoA from acetyl-CoA and
bicarbonate. Fatty acyl-ACPs (acyl carrier proteins) are synthesized from malonyl-CoA by a multi-
subunit fatty acid synthase. The fatty acyl moiety is eventually incorporated into phospholipids for
cell membrane synthesis. The fatty acid yield ultimately depends on regulation (Magnuson et al.,
1993). Feedback inhibition by fatty acyl- ACPs is considered as the main form of regulation of fatty
acid biosynthesis. Feedback inhibition might be targeting the proposed rate-limiting step of fatty
acid biosynthesis catalyzed by acetyl-CoA carboxylase (Davis et al., 2000)
Enzymatic conversion of fatty acid to fatty alkane can be accomplished in a two step reaction
sequence, which consists of reduction of the fatty acid to the fatty aldehyde followed by
conversion to the fatty alkane. Bioluminescent bacteria, such as Vibrio harveyi and
Photobacterium phosphoreum, have been shown to produce fatty aldehydes using the protein
products of the Lux operon (Meighen, 1998).
The demonstrated success in the overproduction of fatty acids, de novo biosynthesis of FAEE and
fatty alcohols in genetically engineered E. coli, the recently published work of increasing
production and secretion of fatty acids in genetically engineered cyanobacteria, and the
preliminary experimental results from studies directed at producing FAEE and fatty alcohol in
genetically engineered cyanobacteria are evidence that efficient photosynthetic production of
fatty acid-based biofuels in cyanobacteria can be achieved through genetic engineering. The
biosynthesis of fatty acid-based biofuels in cyanobacteria is expected to accelerate the application
of biofuels because (1) the use of solar energy avoids the technical bottlenecks associated with
biomass supply and deconstruction, (2) biofuels are directly made in a single biological system
which eliminates the cost of downstream refining, and (3) fatty acid esters, long chain fatty
alcohols and fatty alkanes chemically similar to fossil fuels and are thus are more compatible with
current engine and transportation systems.
Lu, X. (2010). A perspective: photosynthetic production of fatty acid-based biofuels in genetically
engineered cyanobacteria. Biotechnology advances, 28(6), 742-746.

Cyanobacterial biofuel production

Several emerging technologies are being implemented in order to overcome the problem of
greenhouse gas (GHG) pollution and replace fossil fuels by promoting viable production of liquid
fuels such as fatty acid esters (biodiesel) (Kalscheuer et al., 2006), alkanes (Schirmer et al., 2010),
and higher alcohols from renewable sources (Atsumi et al., 2008a,b; Bond-Watts et al., 2011;
Shen et al., 2011). However, most of these bioprocesses have a major drawback: they rely on
microorganisms that metabolize carbohydrate sources from land-based feedstocks.

The direct conversion of solar energy into liquid fuel using photosynthetic microorganisms is an
attractive alternative to fossil fuels. There are several advantages to using organisms such as
microalgae and cyanobacteria: their readily available genetic tools and sequenced genomes; their
higher growth rate compared to plants; and their ability to thrive in areas that cannot support
agriculture.

2.1. Isobutanol production from S. elongatus using a keto acid pathway

Isobutanol is a higher alcohol used as a solvent in the manufacture of pesticides, flavor and
fragrances, and also used to produce corrosion inhibitor additives for lubricant oils. Recently,
isobutanol has been designated as a promising gasoline substitute (Atsumi et al., 2008b).
Compared to ethanol, isobutanol is a better candidate for gasoline replacement due to its low
hygroscopicity, higher energy density and high compatibility with current infrastructure.
Isobutanol and 1-butanol share similar physicochemical properties, except that isobutanol has a
higher octane rating (RON = 113, MON = 94 for isobutanol compared to RON = 96, MON = 78 for 1-
butanol; Wallner et al., 2009). However, no organism can naturally synthesize isobutanol at high
yield and productivity. Recently, a 2-ketoacid-based pathway was developed by Atsumi et al.
(2008b) (Fig. 1). 2-Ketoacids are intermediates in amino acid biosynthesis pathways and can be
converted to alcohols by 2-ketoacid decarboxylase and alcohol dehydrogenase (Fig. 1)
(Atsumi et al., 2008b). An l-valine precursor, 2-ketoisovalerate, was utilized to produce isobutanol.
Several synthetic approaches to produce isobutanol from carbohydrate compounds using
metabolic engineering have been reported in different organisms including Corynebacterium
glutamicum (Smith et al., 2010), Clostridium cellulolyticum (Higashide et al., 2011), and
Saccharomyces cerevisiae (Chen et al., 2011). To test whether cyanobacteria are capable of direct
synthesis of isobutanol, three alcohol dehydrogenases were tested to convert isobutyraldehyde to
isobutanol. YqhD from E. coli (Sulzenbacher et al., 2004) showed the best performance (Atsumi et
al., 2009). The strain expressing the l-valine pathway (ALS-IlvCD) and alcohol pathway (Kivd and
YqhD) produced 0.450 g/L isobutanol in 6 days (Table 1 and Fig. 1).
2.2. 1-Butanol production from S. elongatus using a modified CoA-dependent pathway
Another higher-chain alcohol of interest as a biofuel and chemical feedstock is 1-butanol. This is a
primary alcohol produced industrially from the petrochemical feedstock propylene and used
as an intermediate in the production of butyl esters. It is also used as a solvent for the extraction
of essential oils, a solvent for paints, natural and synthetic resins and gums. 1-Butanol has been
proposed as a substitute for diesel fuel and gasoline because of its low hygroscopicity and energy
content (27 MJ/L), which is similar to gasoline (32 MJ/L). 1-Butanol can also be produced as a
byproduct of microbial fermentation processes. At the beginning of the last century, industrial
butanol production was carried out by fermentation of Clostridium acetobutylicum (Jones and
Woods, 1986).
This process produced mainly acetone, 1-butanol, and ethanol (referred to as the ABE
fermentation) at a total concentration of solvents produced ranging from 12 to 20 g/L in batch
fermentation starting from 55 to 60 g/L of sugar substrate, it results in solvent yields
approximately 0.35 g/g sugar (Green, 2011) with solvent ratio of 6:3:1 (butanol–acetone–ethanol)
(Jones and Woods, 1986; Lee et al., 2008). In Clostridium, 1-butanol is produced by a pathway
branched from the butyrate pathway (Jones and Woods, 1986), referred to as the CoA-dependent
pathway. Pyruvate, resulting from glycolysis, is cleaved by pyruvate ferredoxin oxidoreductase in
the presence of coenzyme A (CoA) to yield carbon dioxide, acetyl-CoA, and reduced ferredoxin.
The metabolic pathway from acetyl-CoA to 1-butanol requires the following enzymes: acetyl-CoA
acetyltransferase (also called thiolase, THL); -hydroxybutyryl-CoA
dehydrogenase (HBD); 3-hydroxybutyryl- CoA dehydratase (crotonase, CRT), butyryl-CoA
dehydrogenase (BCD); electron transferring protein A and B (ETF-A, ETF-B) and bifunctional
butyraldehyde dehydrogenase (BYDH)/butanol dehydrogenase (BDH) (Jones and Woods, 1986; Li
et al., 2008). Interest
in production of biobutanol from biomass has shone new light on 1-butanol biosynthesis research,
redirecting it toward production by non-native and user-friendly hosts that are facultative
anaerobes with fast growth rates, have readily available tools for genetic manipulation, and have
well characterized physiological regulation. Butanol pathways have been re-constructed in
different non-native hosts including E. coli (Atsumi et al., 2008a; Inui et al., 2008), S. cerevisiae
(Steen et al., 2008), Lactobacillus brevis (Berezina et al., 2010), B. subtilis and Pseudomonas putida
(Nielsen et al., 2009).

2.4. Fatty acid production in cyanobacteria

The gradual exhaustion of fossil-based energy resources is driving chemical industries to search for
alternatives to products of the petroleum industry. One of the energy alternatives for
petroleumbased diesel fuel is biodiesel. Biodiesel is a clean-burning fuel made by
transesterification of triacylglycerides (TAGs) purified from plant oils, yielding fatty acid methyl
esters (FAMEs) and fatty acid ethyl esters (FAEEs). Production is dependent on the availability
of the specific vegetable oil feedstock and the plant oil must undergo transesterification with
short-chain alcohols, usually methanol or ethanol followed by purification. These downstream
processes and supply factors can affect the final price and quality of biodiesel (Kalscheuer et al.,
2006). Fatty acids are energy-rich molecules composed of long alkyl chains used by cells for energy
storage and chemical production. Fatty acid metabolism in bacteria has been extensively
investigated (Cronan and Thomas, 2009).
E. coli has been engineered to be an efficient producer of fatty acids (2.5 g/L) by blocking fatty acid
degradation, increasing the supplement of malonyl-CoA (an intermediate in fatty acid
biosynthesis), and releasing feedback inhibition (Lu et al., 2008). Oil-producing algae are a more
prominent biological approach to producing biodiesel because of their capacity to sequester and
fix CO2. However, there are still technological barriers and economic issues that hamper the
industrial development of high-yield oil-producing algal strains (Robertson et al., 2011; Sheehan et
al., 1998)
The FFAs produced from CO2 by cyanobacteria can be converted into biodiesel by esterification,
and to alkanes and alkenes, through a two-step pathway recently identified in cyanobacteria.
Schirmer et al., identified two enzyme families related to alkane biosynthesis, an acyl-acyl-carrier
protein reductase and an aldehyde decarbonylase (Schirmer et al., 2010). Both enzymes were
coexpressed in E. coli, which allowed for production of alkanes. Their model alkane biosynthesis
pathway in cyanobacteria consisted of reduction and decarboxylation, with formation of carbon
monoxide as a coproduct. When the model was tested, formate was formed rather than CO
during conversion of fatty aldehydes to alkanes (Li et al., 2011; Warui et al., 2011).