Biomolecular Engineering Solutions for Renewable Specialty Chemicals. Группа авторов

growing and can fix CO₂ (Dragone et al., 2010). Fourth‐generation biofuels use genetically modified organisms such as algae and cyanobacteria and metabolic engineering to produce biofuels (Dutta et al., 2014). Bioethanol, biodiesel, biogas, biohydrogen, etc. come under biofuel.

      1.4.1 Biohydrogen

      Biohydrogen is the clean fuel as it burns leaving only water. It is produced biologically and therefore is a promising future fuel (Hosseinpour et al., 2017). Hydrogenase or nitrogenase are the crucial enzyme for biohydrogen regulation in both prokaryotes and eukaryotes. Autotrophic organisms such as microalgae, green algae, cyanobacteria, etc. are most efficient for biohydrogen production. The main principle behind the hydrogen production is the electrons generated during metabolism inside the cells by the action of hydrogenase enzyme are made to form hydrogen. Genetic manipulation and metabolic engineering of autotrophic organisms for production of biohydrogen has received much attention.

      In cyanobacteria three alternative pathways (i) photolysis of water using photosystems (PS); (ii) fermentative pathway; and (iii) photofermentative pathway for the production of biohydrogen. In the first pathway, PSI and PSII through light‐dependent reaction transfer electron from water to ferredoxin producing NADPH leading to biohydrogen formation (Carrieri et al., 2011). In second, the source of NADPH is degraded polysaccharides or lipids, and the electrons are transferred to plastoquinone pool for hydrogen formation (Ghirardi et al., 2000). Third pathway is the combination of first two. Cyanobacteria having heterocyst use it as the sites for nitrogen fixation. A vegetative cell originates NADPH and transport electrons to the plastoquinone pool inside the heterocyst. Inside this hydrogenase gets inactivated and nitrogenase reaction take place (Kufryk, 2013). Before the hydrogen production by cyanobacteria can become industrially viable improvements in strains are to be done. There are two types of hydrogenase: uptake hydrogenase (Hup) and bidirectional hydrogenase (Hox). Anabaena sp. PCC 7120 contains both Hup and Hox (Masukawa et al., 2002). The species were tested for both Hup and Hox inactivation. Hup inactivated strain gave four to seven times increase in H₂ production. In another study, Nostoc sp. PCC 7422 was chosen having highest nitrogenase activity and was subjected to Hup gene disruption (Yoshino et al., 2007). The generated mutant was able to produce hydrogen at a rate of threefold than wild type. Electrons play an important role in hydrogen production. Electrons generated by ferredoxin and NADPH are sometimes transferred to other competing pathways (e.g. nitrate assimilation pathway). This can be engineered to redirect electron flow to, for example, Hox (Baebprasert et al., 2011). Disrupting nitrate assimilating pathway in Synechocystis sp. strain PCC 6803 results in higher hydrogen production as electron gets redirected to Hox. LDH mutants of Synechococcus 7002 lacking have exhibited 5 times greater hydrogen production compared with the wild type (Park et al., 2008). In a different study, Cyanothece sp. ATCC 51142, a unicellular diazotrophic cyanobacterium demonstrated the ability to generate high levels of hydrogen (465 μmol H₂/mg of chlorophyll h) using glycerol as a substrate under aerobic conditions (Bandyopadhyay et al., 2010).

Green algae have two light‐dependent and one light‐independent pathway mediated by [Fe] or [FeFe] hydrogenase for hydrogen synthesis (Meyer, 2007). In it [Fe] or [FeFe] hydrogenase is the key catalyst and ferredoxin is electron donor. In the first pathway, water is used as the electron sink and biophotolysis of water takes place. Here water splitting PSII and ferredoxin‐reducing PSI act together. The second pathway is PSII independent where electrons from metabolic pathways like glycolysis or citric acid are transferred to electron transport chain. The third pathway that is light independent or dark fermentation of decarboxylated pyruvate. Chlorella vulgaris strain when overexpressed with hydrogenase gene can produce hydrogen even in atmospheric conditions (Hwang et al., 2014). These strains were able to show maximum production at low intensities of light much less than that is received on Earth by Sun. This can be overcome by decreasing the number of light harvesting antenna as done in Chlamydomonas reinhardtii tla1 strain (Kosourov et al., 2011). In total, 50% truncated photosynthetic light harvesting antenna increases hydrogen production. Flavodiiron (Flv3B) proteins have crucial and specific roles in photoprotection of photosystems I and II in cyanobacteria. Absence of Flv3B leads to impaired growth of cyanobacteria in the presence of oxygen. Overexpression of Flv3B in Nostoc PCC7120 significantly increases the hydrogen production (Roumezi et al., 2020).

      1.4.2 Biodiesel

      Biodiesel is a renewable liquid transportation fuel consisting of alkyl esters of fatty acids and produced from triacylglycerides (TAGs). It is synthesized by a process called transesterification of fats in the presence of catalyst (alcohol) to form fatty acid methyl esters (Subramani and Gangwal, 2008). Therefore, properties of biodiesel are dependent on the fatty acid from which it was made (Knothe, 2005). Majority of biodiesel currently is being formed by oilseed crops competing with food and cultivable land. Alternative feedstock for the oil includes oleaginous or grease microorganisms, such as microalgae, cyanobacteria, yeast, and bacteria (Hu et al., 2008). Out of these microalgae and cyanobacteria are of importance as they are autotrophic in nature. Some microalgae accumulate 20–50% TAG, which are same as found in oilcrops such as canola and sunflower (Gaurav et al., 2017). Like plants only they utilize sunlight as energy source and CO₂ as carbon source but can be cultivated on little barren land using wastewater (Pittman et al., 2011). Attempts are being made to improve lipid content of microalgae by metabolic engineering and genetic engineering. C. reinhardtii is the model organism for this purpose as its genome is well known.

TAG is derived from acylation or diacylglycerol through acyl‐CoA dependent and acyl CoA‐independent pathway. Rate‐limiting steps of these two pathways can be altered to increase or decrease the TAG production. Inactivation of ADP‐glucose pyrophosphorylase in Chlamydomonas resulted in 10‐fold increase in TAGs (Miller et al., 2010). In another study, Chlamydomonas mutant lacked subunit of ADP‐glucose pyrophosphorylase and accumulated 46.5% total lipids out of which 32.5% were neutral lipids (Li et al., 2010).

      Knockout and overexpression of enzymes important for fatty acid synthesis like acetyl CoA carboxylase (ACCase) and type‐II fatty acid synthase (FAS) are well known to increase lipid content in the cell (Majidian et al., 2018). ACCase catalyzes the rate‐limiting step of fatty acid synthesis and therefore C. reinhardtii was overexpressed with ACCase and showed increase in fatty acid synthesis to 56.15% as compared to wild type (48.39%) (Chen et al., 2019).

      In diatom like Thalassiosira pseudonana, deletion of multifunctional lipase/phospholipase/acyltransferase does not hamper growth and increases (up to threefold) lipid content (Trentacoste et al., 2013). Heterologous expression of two thioesterases in microalga Phaeodactylum tricornutum accumulates shorter chain fatty acids; lauric and myristic acid (Radakovits et al., 2011). These shorter fatty acids are not secreted and gets incorporated in TAG.

      1.4.3 Bioethanol

      Bioethanol (C₂H₅OH) is the most common liquid biofuel. It is used as an additive to gasoline as 5–10% blend. It burns clean and help in reduction of greenhouse gases and is being commercially produced in Brazil, Canada, and United States for transportation (Sirajunnisa and Surendhiran, 2016). Initially, bioethanol was produced from biorefinery of sugar‐rich agricultural biomass (Rastogi et al., 2018). This bioethanol production was limited due to issues with food competition. Photosynthetic biomass production and getting the biomass converted into ethanol is of great interest and is done easily by cyanobacteria. Some strains can naturally convert carbon source to ethanol and some have to be engineered with key enzymes for ethanol production. Simply ethanol production requires two key enzymes pyruvate decarboxylase which converts pyruvate to acetaldehyde and alcohol dehydrogenase converting acetaldehyde to ethanol.

Pyruvate decarboxylase (pdc) enzyme from Zymomonas mobilis is well characterized and used to engineer several cyanobacterial strains such as Synechocystis sp. PCC6803. Pdc and alcohol dehydrogenase from Z. mobilis were integrated into the genome of PCC6803 using double homologous recombination. The genes were under the control of strong light inducible psbAII promoter (Dexter