Biomolecular Engineering Solutions for Renewable Specialty Chemicals. Группа авторов

of endogenous alcohol dehydrogenase and hampering the biosynthetic pathway of poly‐β‐hydroxybutyrate, PCC6803 gives an ethanol efficiency of 5.50 g/l (Gao et al., 2012). Synechococcus sp. PCC7002 is more tolerant to higher temperature as compared with PCC6803. Glycogen synthesis was blocked in PCC7002 by introducing two glycogen synthase genes. This lead to hamper the growth of the cyanobacteria. This was accommodated by incorporating double copies of ethanolgenic pathways giving 2.2 g/l ethanol in 10 days (Wang et al., 2020). Pdc and Adh with E. coli lac and CI‐PL temperature inducible promoter increase ethanol productivity in Synechococcus sp. PCC 7942 (Dexter et al., 2015). It is said that increased NADPH increases biomass and ethanol yield. This was confirmed by endogenous expression of glucose‐6‐phosphate dehydrogenase (zwf) gene in PCC6803. zwf gene of pentose phosphate pathway increases NADPH production and hence biomass and ethanol production (Choi and Park, 2016).

      1.4.4 Terpenoids

Terpenoids are also called as isoprenoids and are a large class of plant specialized secondary metabolites, derived from 5 carbon (C5) isoprenoid unit. These precursors are produced by two biosynthetic pathways, the methylerythritol phosphate pathway (MEP) in the chloroplast and the classical mevalonate pathway (MVA). MEP pathway is found in prokaryotes whereas the MVA pathway in eukaryotes. Terpene synthases (TPSs) in plants have been classified into seven subfamilies, designated as TPS‐a, TPS‐b, TPS‐c, TPS‐d, TPS‐e/f, TPS‐g, and TPS‐h, according to similarities in their amino acid sequences (Chen et al., 2011). Table 1.1 shows the genome‐wide analysis of TPSs from different plants. Farnesene, isoprene, etc. are some of the terpenoids which can be used as biofuel.

Farnesene belongs to the Sesquiterpenes class of terpenes. Farnesene (3, 7, 11‐trimethyldodeca‐1, 3E, 6E, 10‐tetraene) is the simplest sesquiterpenes produced by plants. It is a 15‐carbon compound (C₁₅H₂₄). It was first discovered in apple peels and was found to play a role in plant defence. It can be used as a promising replacement for diesel. It has a major role in cosmetics industry as an antiaging and moisturizing lotions. Being less hygroscopic in nature and having high‐energy density (cetane numbers of 58), it forms the precursor for jet biofuel (Yang et al., 2016). Its market size was reported to be 8.51‐kilotons in 2015 and is expected to reach 180.9 kilotons and up to $485 million by 2023. Amyris Biotechnologies, USA, dominates the global farnesene market (Biofene). Intrexon and chromatin, USA, is in emerging state to reach production level. Farnesene is basically synthesized by plants. So, adding this machinery to the prokaryotes will increase its production. E. coli, S. cerevisiae, and Yarrowia lypolytica are heterotrophic organisms that were engineered by adding farnesene synthase gene to produce farnesene (Wang et al., 2011; Tippmann et al., 2016; Yang et al., 2016). The heterotrophic organisms require continuous supply of carbon sources. Therefore, using cyanobacteria, i.e. autotrophic organisms can be beneficial as they utilize carbon dioxide as the carbon source and also helps in CO₂ sequestration. Table 1.2 shows the list of photosynthetic organisms engineered to produce farnesene. Much research is needed to be done in the field of farnesene synthesis and is a booming biofuel.

Table 1.1 Genome wide analysis of terpene synthase genes.

Plant species	Description	References
Jatropha curcas	59 putative TPS genes were identified.Among them 26 belongs to TPS‐a family.	Xiong et al. (2016)
Ananas comosus	21 putative TPS genes were identified.Divided into five sub families.	Chen et al. (2017)
Citrus sinensis	55 putative TPS genes identified out of which 28 are TPS‐A, 18 are TPS‐b, and 5 are TPS‐g.Only two of them are TPS‐e/f each.	Alquezar et al. (2017)
Ocimum sanctum	81 putative genes identified.Further only 47 putative genes were found to be functional.	Kumar et al. (2018)

Table 1.2 Photoautotrophic production of farnesene.

Microorganism	Promoter used	Description	Max. farnesene concentration (mg/l)	References
Anabaena sp. PCC 7120	Ptrc	Codon optimized farnesene synthase gene from Norway spruce is taken.Expression plasmid was used for the production.	0.0691	Halfmann et al., (2014)
Synechococcus elongatus PCC 7942	Ptrc	Codon optimized farnesene synthase gene from Malus domestica and Picea abies was taken.In addition to the above‐mentioned genes dxs, idi and ispA gene were also incorporated int the genome. Thus, optimizing Methylerithritol phosphate (MEP) pathway.	4.6 ± 0.4	Lee et al., (2017)

      dxs, deoxy xylulose synthase; idi, isopentynyl pyrophosphate isomerase; ispA, farnesyl diphosphate synthase.

Isoprene (C₅H₈) is another industrially important chemical. It is a volatile hydrocarbon produced by plants under stress condition. One million tons of it is produced from petrochemicals to reach the global need (Morais et al., 2015). Isolating such huge amount of isoprene from plants is not feasible. Therefore, microbial production of isoprene is gaining interest recently. Cyanobacteria are genetically modified for isoprene synthase (Isps) gene, which is usually absent in them. Isoprene synthase gene catalyzes the conversion of di‐methylallyl diphosphate to isoprene. Lindberg et al., (2010) reported first isoprene synthesis from cyanobacteria. Isps gene from Pueraria montana was introduced in PCC6803 under the light‐regulated PsbA2 promoter giving yield of isoprene 50 μg/g DCW. Engineering isoprene synthase gene with other MVA pathway genes improves yield of isoprene by 2.5‐folds (Bentley et al., 2014). Apart from introduction of isoprene synthase gene, overexpression of IPP isomerase (IDI) gene leads to 40% photosynthetically fixed carbon toward isoprene (Gao et al., 2016b).

1.5 Conclusion

      Biocommodities are now being produced by microbial cell factories. These microorganisms are engineered to increase their robustness. Increasing knowledge of the omics technologies as now we have full access to whole genome makes easy to manipulate any organism. Engineering of microbial cell factories depends on the required end product. Number of genetic tools are now available that makes biocommodity engineering easy. Despite of the plethora of literature available on genetic engineering of microorganisms, very few of them are able to perform well industrially. Therefore, focus is to be made on scale up of the existing cell factories, and new engineered strains should also be taken to industrial level for better production.

References

      1 Ahn, W. S., Park, S. J., & Lee, S. Y. (2001). Production of poly (3‐hydroxybutyrate) from whey by cell recycle fed‐batch culture of recombinant Escherichia coli. Biotechnology Letters, 23(3), 235–240.

      2 Alquézar, B., Rodríguez, A., de la Peña, M.,