Site‐saturation engineering of proline 474 in pyruvate carboxylase from R. oryzae was done to improve fumaric acid production in S. cerevisiae (Xu et al., 2012). Ethanol yield was lower down by deleting fumarate hydratase gene. The generated strain gives a yield of 314.5 ± 3.8 mg/l.
Similarly, E. coli can also be engineered by overexpressing and deleting the abovementioned genes. The iclR gene encoding a repressor of glyoxylate shunt operon was deleted to direct carbon flux to glyoxylate shunt (Song et al., 2013). All three fumarase genes were also deleted enhancing fumaric acid production to 1.45 g/l. in a strain with all three fumarase genes deleted with the overexpression of phosphoenolpyruvate carboxylase gene gives yield of 3.65 g/l fumaric acid (Li et al., 2014).
1.3.3 Therapeutic Proteins
Proteins are the building block of our body and are essential for proper functioning of body. Any defect in a particular protein can lead to a disease such as hemophilia (lack of various blood‐clotting factors), dwarfism (lack of growth hormone), and diabetes (less or no insulin production) (Vajo et al., 2001; Takeda et al., 2010). These diseases caused by the deficiencies of the proteins can be treated by regular administration of them. These are synthesized by the other organisms, isolated, purified, and then given to patients. Proteins synthesis for human use is completely different form the enzymes used for chemical industry as for human’s quality constraints are strict and detailed clinical trials are required. More therapeutic proteins are under clinical trial than in the market.
Out of the many recombinant therapeutic proteins human growth hormones (hGH) is at the top. Its market was estimated to be $2850.70 million in 2018 and is anticipated to reach $5653.60 million by 2026 observing a CAGR of 8.2% over the estimated period. Two of the most common hGh currently in the market are Accretropin™ and Sermorelin (Reh and Geffner, 2010). Accretropin™ (recombinant human growth hormone (r‐hGH); somatropin) is a protein, which is manufactured by rDT. The production process includes fermentation of E. coli that gives a protein product containing 192 amino acids. Later process includes removal of the N‐terminal amino acid, methionine that yields a product which is physio‐chemically and chemically similar to pituitary‐derived hGH, comprising of 191 amino acids. These are arranged in a single polypeptide chain. In general terms, Accretropin vitalize linear growth in those patients whose body lacks sufficient production of the endogenous growth hormones. Other than that, it also shows substantial effect in process such as tissue growth, metabolism of protein, carbohydrate, and lipids along with minerals. The drug got approved by FDA on 23 January 2018. It is manufactured by Cangene Corporation, Canada.
Similarly, Sermorelin or growth hormone releasing factor 1‐29 NH2‐acetate is classified as a member of the growth hormone‐releasing hormone analogue class drug. It is made up of 29 amino acids out of the 44 that originate in its natural state (Walker, 2006). It can notably increase the release of the growth hormone in the body. It also aims to enhance growth hormone serum concentrations and insulin‐like growth factor 1 (IGF‐1). The bodies of those children, which does not produce enough growth hormone, can be provided with Sermorelin to assist with the increase in amount of growth hormone processed by the pituitary gland. Other than that, it is also commonly prescribed for the treatment of adult growth hormone deficiency. It is also getting popular with those doctors who use them partly in their medical weight loss plans. The drug was first developed during early 1980s and was approved by the FDA by 1997 for sale via prescription.
Another fine example is IGF‐1 (Laron, 2001). They can be related to the family of insulin‐related peptides, which comprise of relaxin and various other peptides, which are extracted from lower invertebrates. Being a small peptide and comprising of 70 amino acids and with a molecular weight of 7469 Da, it binds to the insulin receptor with a relatively low affinity. Along with IGF‐2, they were first found in 1957 by Salmon and Daughday. The IGF‐1 plays major role in proliferation and function of just about every cell, tissue, and organ in the body. Their action mechanism conciliates through IGF/IGFR binding, the activation of kinase, and signaling via AKT pathway.
1.4 Photosynthetic Production of Biofuels
The increase in energy demand globally is affecting environment due to global warming and depletion of nonrenewable energy sources. Conventional fossil fuel sources such as petrol, diesel, natural gas, and coal, which were considered to be the primary sources earlier are getting exhausted due to extensive uses (Kumar et al., 2017). A 37% surge in fuel demand by 2040 has been estimated due to the rapid increase in demand (Joshi et al., 2017). Besides this the petroleum‐based fuel upon burning releases greenhouse gases. This requires a renewable substitute of petroleum‐based fuels. Biofuels are the renewable energy source produced by photosynthetic organisms utilizing Earth’s biggest fuel source, the Sun as the carbon source. Harvesting solar energy via photosynthesis is one of nature’s noteworthy achievements that could also be a solution for the future worldwide economy. Earlier biofuels were produced from plants known as the first generation of biofuels. They are mainly generated from wheat, barley, corn, oilseed, sunflower, etc. the plants, which are rich in carbohydrates and oils. Biofuels produced by them compete with agriculture croplands leading to crisis in food production for human beings (Surriya et al., 2015). Additionally, harvesting of plants take full season and then processing the complex sugars from plants to simpler sugars that can be used by microorganisms limits the production of biofuel (Voloshin et al., 2019). Second generation of biofuels solves the problem of utilizing croplands as they are based on agricultural waste, forest dregs, waste wood residues, and organic waste materials. Biofuels from algae are considered to be third‐generation biofuels. Microalgae and cellulolytic bacteria are fast 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
