Prominent fuel molecules produced by modified
cyanobacteria
In recent years, cyanobacteria has been a suitable candidate for
metabolic engineering for the production of potential fuels to overcome
concerns related to energy crises and greenhouse gas emission and could
be a great alternative of sustainable and renewable energy (Melis, 2009
and Woo, 2017). Cyanobacteria harvest solar energy through
photosynthesis and synthesize simple sugars and a variety of metabolite
intermediates which functions as precursors of biofuels (Knoot et al.,
2018). Till now many fuel molecules have been efficiently produced by
metabolically engineered cyanobacteria in good yields which are shown in
Table 6. Schematic biosynthetic pathway of various molecules having fuel
properties are demonstrated in figure 2.
<Table 6 >
<Figure 2 >
4.1 Isoprene
Isoprene (2-methyl-1,3-butadiene), a volatile hydrocarbon molecule which
is naturally synthesized in the leaves of deciduous and perennial plants
like oak, kudzu and eucalyptus and emitted in the environment at higher
temperature (Melis, 2012; Chaves, 2018). Naturally microorganisms like
algae bacteria and cyanobacteria do not synthesize isoprene. However
isoprene synthase gene from the higher plants can be isolated and
transferred in microbes for microbial production of isoprene. Nowadays
cyanobacteria have attracted researcher’s attention for their
capabilities of fast growth rate and simple genetic composition which
qualifies them as great photosynthetic chassis for biofuel production.
Cyanobacteria don’t possess isoprene synthase gene which catalyses the
conversion of di-methylallyl diphosphate (DMAPP) to isoprene, the final
step of isoprene synthesis. However they are equipped with methyl
erythritol phosphate (MEP) pathway (figure 2) for the synthesis of a
variety of terpenoid molecules (Lichtenthaler, 2000). The first step of
MEP isoprenoid biosynthetic pathway is catalysed by deoxy xylulose
synthase (DXS) enzyme which utilises glyceraldehyde 3 phosphate (G3P)
and pyruvate as initial substrate and converts into deoxy-xylulose
phosphate (DXP). Which is further converted to di-methylallyl
diphosphate (DMAPP) and isopentenyl diphosphate (IPP) through a series
of enzyme catalysed reactions. Cyanobacteria also have been reported to
contain an IPP isomerase that catalyses the inter-conversion of IPP and
DMAPP (Barkley et al., 2004). Heterologous expression of isoprene
synthase gene from plant has been the strategy of many researchers to
produce isoprene in cyanobacterial system. First cyanobacterial
production of isoprene was reported by Lindberg et al. (2010). They
introduced plant’s (Pueraria montana ) isoprene synthase gene
(IspS) into Synechocystis PCC 6803 under the light regulated
PsbA2 promoter. The yield of isoprene was 50 μgg-1DCW. Another research group used intermittent addition of
CO2 using isoprene synthase (IspS) gene engineeredSynechocystis sp . PCC 6803 and observed over 192 hour for
isoprene production in a closed system. 120 μgg-1 DCW
yield was found (Bentley et al., 2012). When, isoprene synthase gene is
expressed in combination with the MVA (Mevalonic acid) pathway enzymes,
2.5 fold isoprene yield was enhanced, (Bentley et al., 2014). In another
study Synechococcus elongatus PCC 7942 was engineered for the
production of isoprene by over expressing isopentenyl pyrophosphate
isomerase (idi) in combination with isoprene synthase which resulted
1.26g/l isoprene production (Gao et al, 2016).
4.2 Limonene
Limonene, a 10-carbon isoprenoid molecule is mainly synthesized in
plants. Limonene is commonly found in the peel of citrus fruits and
smells like orange. Limonene has been recognized as a substitute fuel
for diesel and jet fuels (Tracy et al., 2009; Chucks et al., 2014). In
cyanobacteria, isoprenoids are synthesized by MEP (methyl erythritol 4
phosphate) pathway. The end products of MEP pathway are IPP and DMAPP
which acts as precursors of limonene and can be converted to limonene by
limonene synthase enzyme. Although cyanobacteria do not possess limonene
synthase gene, researchers utilize limonene synthase gene from the
plants and transfer into cyanobacteria. In a research, limonene synthase
gene from the plant Schizonepeta tenuifolia was introduced intoSynechocystis sp. PCC 6803 under the control of a strong
promoter. They also cloned three genes that are involved in the
synthesis of precursors of limonene, dimethyl allyl pyrophosphate
(DMAPP) and isopentenyl pyrophosphate (IPP) via methyl erythritol 4
phosphate (MEP) pathway (Kiyota et al., 2014). In another studySynechococcuselongatus 7942 was genetically modified with limonene
synthase gene from the spearmint (Mentha spicata ) under the
control of isopropyl β-D galactopyrenoside (IPTG) inducible promoter
Ptrc (Wang et al 2016). Mentha spicata andCitrus limon origin limonene synthase gene were transferred in
cyanobacterial strain Synechocystis6803 to enhance the limonene
production. Two-fold higher limonene was produced by limonene synthase
from M . spicata compared to C . limon (Lin et
al., 2017).
4.3 α-Farnesene
α-Farnesene (3,7, 11-trimethyldodeca-1,3E,6E,10-tetraene) plays a role
in plant defence and was found first in apple peel. It is one of the
simplest acyclic sesquiterpenes. Naturally, it helps in pollination,
seed dispersion, etc. and acts as a chemical signalling agent (Köllner
et al., 2009; Pechous and Whitaker, 2004). Being less hygroscopic in
nature and having high energy density (cetane numbers of 58) it forms
the precursor for jet biofuel (Peralta and Keasling, 2010; Yang et al.,
2016, Renninger and Mcphee, 2008). It has a cloud point of −78 °C
compared with D2 diesel’s cloud point of −3 °C. It also forms the
precursor for solvents, polymers (Yoo et al., 2017), emollients, and
vitamins. Amyris Biotechnologies, headquartered in Emeryville CA, a
renewable products company engineered Saccharomyces cerevisiae to
produce farnesene from sugarcane sucrose. Cyanobacteria have MEP pathway
by which precursors of all sesquiterpenes are formed. Several efforts
have been made to produce α-farnesene through genetic modification and
metabolic engineering such as Escherichia coli (0.38 mg/g of
glycerol) (Wang et al., 2011), Saccharomyces cerevisiae (0.57
mg/g of glucose) (Tippmann et al., 2017) and Yarrowia lipolytica(6.5 mg/g of glucose and fructose) (Yang et al., 2016). The stated
organisms are heterotrophic in nature and require a carbon source for
their growth. Recently researchers have moved their focus to
cyanobacteria, which can utilize carbon dioxide and light to produce
farnesene. Anabaena sp. PCC 7129 (filamentous cyanobacteria)
yielded 305.4 μg/L farnesene in 15 days (Halfmann et al., 2014). The
strain started producing farnesene by directly incorporating plasmid
having farnesene synthase gene (from Norway spruce ). Similarly,Synechococcus elongatus PCC 7942 (naturally competent
cyanobacteria) was engineered to express heterologous farnesene synthase
gene (Lee et al., 2017). The production of α-Farnesene from carbon
dioxide was found to be 4.6 ± 0.4 mg/L in 7 days.
4.4 Alkanes
Alkanes are one of the major constituents of petroleum. They include
gasoline, diesel oil propane, lubricants and many more fuel molecules.
Industrial scale refining of petroleum requires a high energy input and
huge manpower and also many toxic by-products are generated which cause
environmental pollution. Alternatively, alkane can be produced by
cyanobacterial cell factories. There are mainly two alkane biosynthetic
pathways have been identified in cyanobacteria till now. In one pathway
fatty acyl-ACP is converted into fatty aldehydeby the enzyme fatty acyl
ACP reductase (FAR). Fatty aldehyde is further converted into alkanes by
aldehyde deformylating oxigenase (ADO). In second pathway mainly alkenes
are synthesized via a polyketide synthase enzyme. Wang and coworkers
constructed a series of Synechocystis PCC 6803 mutant strains by
over expressing both acyl-acyl carrier protein reductase and
aldehyde-deformylating oxygenase, the maximum yield was found to be
1.3% of DCW (Wang et al., 2013). Alkanes can also be produced by some
cyanobacterial strains in salt stress conditions. When Anabaena
sp. 7120 was grown in salt stress (nitrogen deficiency) condition,
alkane yield was found 1200 μgg-1 DCW (Kageyama et
al., 2015). Another research group overexpressed seven copies of FAR,
ADO and a lipase in Nostoc punctiforme PCC 73102 which
corresponded to 12.9 % alkane of DCW (Peramuna et al, 2015).
4.5 Squalene
Squalene is a 30-carbon isoprenoid molecule, naturally synthesized by
plants, animals and microorganisms via MEP and MVA pathways (Xu et al.,
2016). Apart from many uses like cosmetics, food and medicine, squalene
can be used as fuel instead of petroleum (Englund et al., 2014).
Squalene is synthesized from farnesyl di-phosphate (FPP) in a two-step
reaction catalyzed by squalene synthase. In first step, condensation
reaction occurs between two FPP molecules and presqualene diphosphate
(PSPP) is formed, which is further converted into squalene, utilizing a
molecule of NADPH (Englund et al., 2014). Photosynthetic generation of
squalene from CO2 is a great alternative solution of
higher industrial production cost and minimization of pollutant
emission. A research group predicted that Synechocystis PCC 6803
possess slr 2083 gene which encodes squalene hopene cyclase (shc) enzyme
which catalyzes squalene conversion into hopene. Inactivation of slr
2083 gene resulted into 0.67 mg L-1squalene, seventy
time higher than wild strain (Englund et al., 2014). Squalene production
has also been done in model cyanobacterium Synechococcus elongatus
7942 in which squalene synthase gene was joined to either a key enzyme
FPP of the MEP pathway or the β-subunit of phycocyanin. Engineered
strain resulted squalene production 11.98 mgL-1 (Choi
et. al., 2017).
4.6 Isobutanol
Isobutanol, a branched-chain alcohol consisting of four carbon
molecules, has great importance as gasoline additive for fuel purpose
due to its higher energy value (Lu et al., 2012). It can be used as
substitute (drop in fuel) for a variety of petroleum hydrocarbons
without any modification of engine (Peralta et al., 2012). A research
group introduced CoA (Co-enzyme A) dependent 1- butanol production
pathway into Synechococcus elongatus PCC 7942. In this pathway,
treponemadenticola- coA reductase (ter) works as proton donor and
reduces crotonyl- coA to butyryl-coA. Trans-enoyl-coA activity was
enhanced in the presence of poly histidine tag. 13.6 mg/L 1- butanol was
produced (Lan et al., 2011). In another study, Synechocystis PCC
6803 was engineered for the expression of two heterologous genes from
the Ehrlich pathway, which can synthesize isobutanol in autotrophic and
mixotrophic conditions. Isobutanol was separated from the production
medium by oleyl alcohol as a solvent. 298 mg/L of isobutanol was
produced under mixotrophic condition (Varman et al., 2013). The
biological synthesis of isobutanol can be done by 2- keto acid pathway.
Mainly branched chain amino acids are synthesized by this pathway.Escherichia coli (E. coli ) (Atsumi et al., 2009)Saccharomycescerevisiae (Yuan et al., 2017), and cyanobacteria
(Atsumi et al., 2009, Miao et al., 2017, Miao et al., 2018) has been
metabolically engineered with isobutanol biosynthesis pathway. In a
study cyanobacterium Synechocystis PCC 6803 washeterologously
expressed with an α-ketoisovalerate decarboxylase (Kivd) gene fromLactococcus lactis (L. lactis ) which resulted in an
isobutanol and 3-methyl-1-butanol (3M1B) producing strain (Miao et al.,
2018).
4.7 Fatty acids
Fatty acids are one of the prominent fuel molecules consisting of long
alkyl chains, a great petroleum substitute for energy requirements
(Pandey et al., 2019). Triacylglycerides (TAGs) are converted to fatty
acid methyl esters (FAMEs) and fatty acid ethyl esters (FAEEs) by
transesterification reaction. A prominent biological approach for
biodiesel production is the transesterification of cyanobacterial fatty
acids due to their capacity to capture and fix environmental
CO2. Although cyanobacteria possess lipid biosynthesis
pathway, but they do not accumulate neutral lipids in normal
environmental conditions (Wada et al., 1990). In cyanobacteria lipid
production in nutrient stress conditions has been reported by many
researchers. In a study, supply of nitrogen and phosphorus (important
nutrients) were limited to observe its effect on lipid productivity in
selected cyanobacteria. Oscillatoria sp., Anabaena sp.,
Microcoleus sp., and Nostoc sp. varied in their ability to accumulate
lipids which ranged from a lowest of 0.13% in Anabaena sp . to
the maximum of 7.24% in Microcoleus sp . (Kumar et al., 2017).
Apart from natural lipid synthesis and applying stress condition, the
cyanobacterium can also be genetically modified for the enhanced lipid
synthesis. Liu and co-workers genetically engineered synechocystis
PCC6803 with codon optimized acyl-acyl carrier protein thioesterase
gene. The fatty acid secretion yield was increased up to 197 ±14
mgL-1 (Liu et al., 2011). Synechococcos
elongatus PCC 7942 was engineered for the production of free fatty
acids by knocking out acyl-ACP synthetase encoding gene and thioesterase
encoding gene was over expressed for secretion of free fatty acids which
resulted very low yield (Ruffing and Jones, 2012). A research group
engineered Synechocystis PCC 6803 for enhanced fatty acid
synthesis using a novel strategy. They targeted genes encoding
acetyl-coA carboxylase (fatty acids synthesis), lipase A (phospholipid
hydrolysis) and acyl-acyl carrier protein synthetase (recycling of free
fatty acids). Maximum lipid production was observed up to 34.5%
w/DCW corresponding 41.4 mg/l/d in the strain which was engineered with
acyl-acyl carrier protein synthetase encoding gene (Eungrasamee et al.,
2019). In another study, rbc LXS and glpD genes of calvin- Benson-
basham (CBB) and acyl-ACP synthetase encoding genes were engineered inSynechocystis PCC 6803. Modified strain was reported to produce
35.9% DCW intracellular lipid and 9.6% extracellular free fatty acids
(Eungrasamee et al., 2020).