Biomolecular Engineering Solutions for Renewable Specialty Chemicals. Группа авторов
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of metabolic pathways for synthesis of secondary metabolites and full genome sequencing made fabrication of microbial cell factories easier (Keasling, 2012). Fabrication generally involves introduction of the gene of interest in the host organism so the heterologous pathway begins in it. It also includes exclusion of native pathways that are not crucial for the growth of the host organism (Tyo et al., 2007). Apart from the development of genetically modified strains, optimum conditions according to the product of interest and growth of strains is to be provided in a contained environment (bioreactor). This is done to observe the full potential of the strain (Kiss and Stephanopoulos, 1992).
1.3.1 Biopolymers
Any substance that is made up of repeating monomeric units is called as polymer. The polymers that are naturally synthesized by the living organisms are called biopolymers such as proteins, polysaccharides, polyphenols, lipids, polyamides, and polyhydroxyalkanoate (PHA) (spanning from liquid solutions to bioplastics). They are biodegradable hence eco‐friendly. Naturally occurring polymers form the basis for life. Polynucleotides (DNA and RNA) and proteins (amino acids) are present in every living form. Starch is the reserve form of carbohydrate in which plants store food and is the main carbohydrate in the human diet. Lipids store energy and its bilayer acts as a barrier in the living cells. Whereas cellulose is the primary component in the cell wall of plant kingdom providing structural rigidity. Microbial synthesis of biopolymers is known from old age. Louis Pasteur and Van Tieghem, in the mid nineteenth century, discovered that Leuconostoc mesenteriodes synthesize dextran. 40 years after, and PHA reserves were found in B. megaterium, which serves as a basis for bioplastics to date. As stated earlier, understanding in the molecular pathways for synthesis of biopolymers leads to the engineering of microorganism for the production of custom made, altered biopolymers (Rehm, 2010).
Biopolymers have a wide range of application in biomedical, food industry, packaging to cosmetics, electronics, etc. Its biomedical applications are in drug delivery, tissue engineering, wound healing, and medical and dental devices. Porous gelatin, electrospun poly (lactic‐co‐glycolic acid), etc. are used as a scaffold for this purpose (Van Vlierberghe et al., 2007; Zhao et al., 2016). Each biopolymer has a specific characteristic to be used for particular conditions. Poly (D,L‐lactides) scaffolds are used for bone engineering, whereas poly (trimethylene carbonate) scaffolds are used for soft tissue engineering. Poly(L‐lactide‐co‐glycolide) scaffolds provides good adhesion and proliferation (Ulery et al., 2011). Some of the commercially available scaffolds are BioFiber™, an orthopedic scaffold made of a leno weave of P4HB monofilaments (Tornier) for tendon repair, BioFiber®‐CM, an orthopedic scaffold made of P4HB coated with bovine collagen (Tornier) for tendon repair, etc. Use of plastic in packaging industry is growing day by day leading to their disposal problem. Plastic films production takes one‐third portion of the total plastic production. Cellulose and starch are widely used for this purpose after some modifications. The material to be used for packaging should have properties similar to polyethylene terephthalate (PET) i.e. barrier, sealing and thermal properties. Standard cosmetics have nonbiodegradable polymers in them such as polyethylene microparticles in face and body scrubs. These microparticles eventually enter in the ecosystem through waste water stream. Biodegradable poly lactic acid (PLA) powder can be used in exfoliating agents. Chitin absorbs UV light and effectively used in sunscreen lotions. Also, PLA mixed with titanium dioxide and zinc oxide powder shows photocatalytic activity.
Some of the biopolymers having industrial application are highlighted in the section below with their structures shown in Figure 1.2.
1.3.1.1 Cellulose
Cellulose is the major biopolymer found in nature and commercially extracted from wood (Klemm et al., 2005). It is homopolysaccharide of glucose linked by β‐1,4 glycosidic bonds (Cannon and Anderson, 1991). In 2015, the value of cellulose market calculated was US$20.61 billion and is estimated to reach US$48.37 billion by 2025 (Cellulose Fiber Market Size and Share Industry Report, 2014–2025 market [Accessed 20 December 2017]). To meet such high demands overcutting of trees are being done leading to imbalance in nature and global warming. Bacterial cellulose (BC) is the alternative and green approach toward it which is biodegradable, nontoxic, and biocompatible. Apart from this, BC can be used directly in its native form as it is free from contaminants such as lignin, pectin, hemicellulose, and other constituents of lignocellulosic materials (Rahman and Netravali, 2016). There are several bacterial strains that synthesize BC specially the acetic acid bacteria group as they are generally recognized as safe (GRAS) such as Komagataeibacter xylinus, Komagataeibacter hansenii, Komagataeibacter medellinensis, Komagataeibacter nataicola, Komagataeibacter oboediens (Škraban et al., 2018; Castro et al., 2012,2013). Bacterial cellulose synthase (Bcs) is the primary enzyme for cellulose synthesis which adds glucose monomers to the growing chain. There are two subunits BcsA and BcsB necessary for the formation of polysaccharide chain (Römling and Galperin, 2015). Commercial usage of BC and low yields from the strains producing BC lead to the development of microbial cell factories. These cell factories overexpress genes essential for BC like cmc (carboxymethylcellulose), ccp (cellulose complementing factor protein), cesAB, cesC, cesD, bgl, bcsABCD (BC synthase operon), etc. into the host organism to increase the yields and crystallinity of BC.
Figure 1.2 Representative chemical structures of biopolymers.
Gluconacetobacter xylinus six genes cmc–ccp–cesAB–cesC–cesD–bgl were overexpressed in Synechococcus sp. PCC 7002 and resulted in very high‐yield production of extracellular type‐I cellulose (Zhao et al., 2015). BC synthase operon (bcsABCD) from Gluconacetobacter hansenii was engineered in E. coli with its upstream operon (cmcax and ccpAx) giving BC of 1000–3000 μm and a diameter of 10–20 μm (Buldum et al., 2018). Cellulose synthase D subunit (bcsD) increases the crystallinity structure of BC, but not the yield. G. xylinus bcsD when engineered in E. coli synthesizes BC with high crystallinity. FTIR results showed crystallinity index of 0.84, which was 17% more than the wild‐type strain (Sajadi et al., 2017). Oxygen plays an important role in the synthesis of BC. G. xylinus was engineered with Vitreoscilla hemoglobin (VHb)‐encoding gene vgb. vgb help cells to grow in hypoxic conditions. The mutant strains showed significant increase in BC in oxygen tension also (Liu et al., 2018). Recently, Komagataeibacter sp. nov. CGMCC 17276 genome was completely sequenced. BC operon genes were aligned with other BC producers to find sequence similarity. Apart from this growth rate, substrate utilization, BC production, etc. were evaluated. This gives future opportunity to engineer this strain for better BC synthesis (Jang et al., 2019).
1.3.1.2 Poly‐ϒ‐glutamic Acid
Poly‐ϒ‐glutamic acid is a naturally occurring, abundant poly amino acid which is water‐soluble, anionic, biodegradable, and edible biopolymer produced primarily by Bacillus subtilis. Glutamic acid can be L‐ and D‐ in nature linked by ϒ‐amide bond (Cao et al., 2018). Due to the presence of ϒ‐amide linkage, it is resistant to cleavage by proteases (Candela and Fouet, 2006). It is gaining importance because of being the potential candidate for drug delivery, cryoprotectant, thickening agent, biopolymer flocculant, bioabsorption of heavy metals, etc. (Bhattacharyya et al., 1998; Shih and Van, 2001). It is also widely used in skin serums in combination with vitamin C to increase skin elasticity and making it smooth (Tanimoto, 2010; BEN‐ZUR and GOLDMAN, 2007). Predominantly, Bacillus sp., such as Bacillus licheniformis, Bacillus subtilis, B. megaterium, Bacillus pumilis, Bacillus mojavensis, and Bacillus amyloliquefaciens, are producer of PGA. It is synthesized in ribosome independent manner. Glutamate is the precursor of PGA, which is derived from glutamic acid biosynthesis or glutamate transportation. In turn, α‐keto glutaric acid serves as a precursor for glutamate in tricarboxylic acid cycle (TCA) also known as Krebs cycle. The reaction is catalyzed by