Biomolecular Engineering Solutions for Renewable Specialty Chemicals. Группа авторов

units are joined together to produce PGA through racemization, polymerization, and anchoring or releasing. This can be studied in detail in review by Najar and Das (2015). PGA synthetase (Pgs), a membrane associated enzyme polymerizes glutamate to PGA. Genes encoding Pgs are different in different Bacillus species. Like Pgs is encoded by four genes (pgsB, C, A, and E) in B. licheniformis and B. amyloliquefaciens, capB, C, A, and E, in B. anthraci, while in B. subtilis they are ywsC, ywtABC. Role of pgsBCA operon was shown in B. subtilis for PGA production. Mutants with disrupted pgsBCA did not showed PGA synthesis. While the xylose‐induced pgsBCA operon started PGA synthesis (Ashiuchi et al., 2006). Sometimes the overexpression of pgsBCA leads to decrease in PGA production as seen in mutants of B. amyloliquefaciens (Feng et al., 2015).

      Under nutrient deprivation conditions PGA is sometimes hydrolyzed by PGA hydrolases. Deletion of PGA hydrolases can increase the yield of PGA. Depending on where the PGA hydrolase will cleave PGA, it can be (i) PgdS (the γ‐DL‐glutamyl hydrolase) cleaves the γ‐glutamyl bond between D‐ and L‐glutamic acids residues of γ‐PGA; (ii) the D/L‐endopeptidase hydrolyzes the peptide bond between the glutamic acid residues; and (iii) the γ‐glutamyltransferase (GGT) possesses a powerful exo‐γ‐glutamyl hydrolase activity. The GGT is different from PgdS and D/L‐endopeptidase as it hydrolyzes the γ‐PGA from the N‐terminal to release both D‐ and L‐glutamic acids (Cao et al., 2018).

      1.3.1.3 Hyaluronic Acid

Hyaluronic acid (HA) also called as hyaluronan is a structural biopolymer produced by extracellular matrix of animal and human epithelial, neural, and connective tissues. HA is copolymer disaccharide between N‐acetyl‐o‐glucosamine and o‐glucuronic acid linked by β1 → 3 and β1 → 4 glycosidic bonds. Due to the water retention properties of HA, it is widely used in cosmetics as it binds water to collagen and makes skin look plumper and more hydrated. Hyaluronic acid synthase (HAS‐a) is the integral enzyme present in the plasma membrane of mammalian cells and bacteria and synthesizes HA (Chen, 2002). It has several isoforms depending on the organism in which it is forming. Traditionally, it was extracted from animals that pose threat of infection from the animal source. Its bacterial production started with using Streptococcus zooepidemicus with a production titer value of 5–10 g/l in batch fermentation. Later, S. zooepidemicus was considered to be a human pathogen and was not GRAS. HA being viscous in nature pose problems in fermentation leading to lower yields. This leads to optimization of fermentation conditions and improvement in the strains genetically (Chong et al., 2005).

      ^{Hyaluronidase enzyme activity in the strains is not beneficial as it depolymerizes HA. Kim et al. (1996a) selected Streptococcus equi chemically derived mutant strain (after nitroglycerine treatment) which is nonhemolytic, hyaluronidase‐negative in nature, thus producing high molecular weight HA. Controlled expression of hyaluronidase is beneficial too as it reduces the molecular weight and viscosity of HA. Expressing tuaD, gtaB, glmU, glmM, and glmS genes plus glycolytic pathway genes in B. subtilis with controlled expression of hyaluronidase by N‐terminal engineering increased production from 5.96 to 19.38 g/l (Jin et al., 2016). HA produced from this strategy was low in molecular weight in the range of 2.20 × 103 to 1.42×106 Da. One of the reasons Streptococcus sp. were not considered as GRAS is that they produce streptolysin which causes haemolysis. Streptococcus equisimilis CVCC55116 mutated by ultraviolet ray combined with 60C_o‐γ ray treatment produces 174.76 mg/L HA without producing streptolysin (Chen et al., 2012).}

      In an attempt to find the GRAS producers of HA, B. subtilis, Lactococcus lactis, E. coli, Pichia pastoris, Corynebacterium glutamicum, etc. were engineered to express HAS‐a, b (UDP‐glucose 6‐dehygrogenase), c (glucose‐1‐P uridyltransferase), d and e gene, which using precursors N‐acetyl‐o‐glucosamine and o‐glucuronic acid forms HA (Jia et al., 2013; Kaur and Jayaraman, 2016; Jeong et al., 2014). Lactococcus lactis is not the natural producer of HA due to absence of HAS gene. The attempt to express HAS gene in L. lactis was performed by number of research groups by using non‐integrative plasmids pRKN, pNZ8148, etc. (Prasad et al., 2010; Chien and Lee, 2007; Prasad et al., 2012). Strains expressing only HAS‐a and b genes give a production of 0.097 g/l, while the one expressing HAS‐c gene also gives the maximum production of 0.234 g/l (Prasad et al., 2010). This indicates the importance of HAS‐c gene expression for HA production. This is due to the fact that HAS‐c diverts the flux of glucose‐1‐phosphate toward UDP‐glucose synthesis, which is critical for HA synthesis. Genome integrative approach is also carried in Lactobacillus sp. giving twofold increase in the production and molecular weight (MW) of HA in comparison to plasmid bearing strains (Hmar et al., 2014). It was explained by the precursors (N‐acetyl‐o‐glucosamine/o‐glucuronic acid) and HAS‐a/HAS‐b mRNA ratio. In genome‐integrated strains HAS‐b expression was high compared to HAS‐a, which gives greater availability of precursors to bind to HA synthase leading to high MW HA. E. coli the most common microbe to be engineered has a wide variety of genetic tools available to be modified. It was the first microorganism in which human HAS was successfully expressed (Hoshi et al., 2004).

      1.3.1.4 Polyhydroxyalkoate

      Poly‐3‐hydroxyalkanoates (PHA) are polyesters that have gained economic importance due to their biodegradability and tendency to replace petroleum synthetic polymers. PHAs are produced in the form of intracellular inclusion bodies under nutrient deprivation and carbon source excess conditions. These inclusion bodies or PHA granules serve as carbon reserves for microorganisms during stress conditions (Sudesh et al., 2000). The inclusion bodies have PHA hydrophobic core surrounded by PHA synthase involved in synthesis of PHA (Grage et al., 2009). Depending on the number of monomeric units PHA can be divided into short chain length (SCL), medium chain length (MCL), and combination of both (SCL/MCL). SCL PHA have three to five carbons in the monomeric unit, is thermoplastic nature, brittle, and lacks toughness (3‐hydroxypropionate). MCL PHA have monomers consisting of six to fourteen carbons and have elastomeric property (3‐hydroxytetradecanoate). SCL/MCL PHA are derived from both short‐chain‐length and long‐chain‐length PHA having three to fourteen carbons. It has a wide range of physical and thermal properties. The generalized structure of PHA is shown in Figure 1.2. Till date 150 different PHAs composition have been identified according to the modifications made in existing PHA and genetically engineered organisms to produce PHA (Loos, 2011; Zinn and Hany, 2005). Apart from its application to produce bioplastics PHA has application in the field of tissue engineering, drug carrier, biomedical, etc. The widespread use of PHA is still restricted due to the fact that the cost of production of PHA is 5–10 times the cost of petrochemical‐derived plastic (Chen, 2009).

PHA synthase (PHAc) is the primary enzymes which polymerizes R‐3‐hydroxyacyl‐CoA precursors. For this all the carbon metabolism is shifted to form R‐3‐hydroxyacyl‐CoA thioester. PHA synthase is broadly classified into four classes. Class I consists of one type of PHAc, while class II contains two type of synthases PHAc1 and PHAc2. Classes II and IV consist of PHAc‐PHAe and PHAc‐PHAr units, respectively (Pötter and Steinbüchel, 2005). Classes I, III, IV favor SCL‐PHA, while Class II favors MCL‐PHA synthesis. Ralstonia eutropha is a model organism for PHA production as it accumulates polymer in nutrient‐deficient condition. Its wild type only synthesizes SCL‐PHA and has to be modified for tailor‐made PHAs (Pohlmann et al., 2006). R. eutropha is engineered to produce copolymer poly(3‐hydroxybutyrate‐co‐3‐hydroxyhexanoate) (PHBHHx) from palm oil. Engineered strain contains PHAc from Rhodococcus aetherivorans, enoyl‐CoA hydratase gene (PHAj) from Pseudomonas aeruginosa, and acetoacetyl‐CoA reducatase gene (PHAb). PHAj accumulates PHA, while PHAb alters the level of hydroxyhexanoate (Budde et al., 2011). Since R. eutropha grows slowly and difficult to lyse engineering E. coli is better option. E. coli is not a natural producer of PHAs, as it does not have PHA synthase gene. The first heterologous production of polyhydroxybutyrate (PHB) in E. coli was done in 1988 (Schubert