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).