Читать книгу Biomolecular Engineering Solutions for Renewable Specialty Chemicals - Группа авторов - Страница 47

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 et al., 1988). PHB synthase gene from R. eutropha was cloned in E. coli. The engineered E. coli is able to synthesize PHB but lack the ability to accumulate it.

Increasing the number of PHA synthase gene copies in an organism only increases the PHA levels occasionally that too when the cell dry weight increases. This indicates the importance of cell dry weight over additional copies of PHA synthase gene and shown by many researchers (Tombolini et al., 1995; Huisman et al., 1992; Huisman et al., 1991). E. coli binary fission pattern is changed to multiple fission, by deleting the genes minC and minD (related to binary fission) and overexpressing the genes ftsQ, ftsL, ftsW, ftsN, and ftsZ (related to cell division) (Wu et al., 2016a). This results in more dry weight and more PHB accumulation. Apart from the cell dry weight, cell shape also matters. Along with the deletion of minC and minD gene, deleting gene envC and nlpD which degrades peptidoglycan layer leads to filamentous shape and accumulated 70% PHB (Wu et al., 2016b). All carbon source getting converted to biomass and no product is not beneficial industrially. Therefore, a glucose‐sensing toggle switch is created that automatically senses glucose limitation and activates product formation leading to shorter growth phases and good titers of PH (Bothfeld et al., 2017).

There is no doubt that genetic engineering increases the production of PHAs in many aspects. But the substrate used in PHA production is costly, and unusual substrates (whey, lipid, molasses, glycerol, etc.) are not directly converted to PHA. Whey contains 4.5% of lactose, engineering Cupriavidus necator which is the natural producer of PHA so that it can grow on lactose. For this purpose, lac operon consisting of lac I, lac Z, lac O from E. coli is inserted into C. necator (Povolo et al., 2010). The lac operon is inserted within the depolymerase gene (phaZ1) which reduces the depolymerization of the polymer and increase in its accumulation. E. coli which naturally utilizes lactose but do not have PHA synthase gene is engineered with PHA synthase gene from C. necator. The recombinant strain was grown in cell recycle fed‐batch culture giving 87% PHB content (Ahn et al., 2001). Molasses, which is the predominant waste produced by sugar industry, contains almost 50% sucrose. Microorganisms should be engineered with sucrose utilization genes in order to use molasses as the carbon source for PHA production. One such gene is sucrose permease (csc) of E. coli which has been engineered in C. necator to produce PHBHHx using sucrose as a sole carbon source (Loo et al., 2005). Glycerol is the major waste of soap industry by fat hydrolysis and is now also being generated from biodiesel manufacturing. Aerobic metabolism gene from E. coli, glycerol kinase, aquaglyceroporin, FAD‐dependent glycerol 3‐phosphate dehydrogenase are transformed in C. necator to obtain PHB accumulation (Fukui et al., 2014).