Читать книгу Biomolecular Engineering Solutions for Renewable Specialty Chemicals - Группа авторов - Страница 46

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).