Biosynthesis of PHB

The phbA, phbB, and phbC genes from Alcaligenes eutrophus (Ralstonia eutrophus) encoding the biosynthetic enzymes (3-ketothiolase, acetoacetyl-CoA reductase (NADPH-dependent), and PHB synthase, respectively, have been cloned into E. coli (Scheme 19.42).339-342 The use of in vitro evolution using error-prone polymerase chain reaction has led to enhanced accumulation of PHA in a resultant recombinant strain.343 Additional studies to enhance the biosynthesis of PHB through the use of metabolic engineering have been discussed.344... 

Summarizing, the biosynthesis of PHB can be understood in terms of the kinetic processes of initiation, propagation and chain transfer and the affect of latex particle size on these. Rirther, the colloidal aspects of the formation of PHB latex particles can be explained by the homogeneous nucleation mechanism, well known in conventional emulsion polymerization processes. 

LabuZek S, Radecka I (2001) Biosynthesis of PHB tercopolymer by Bacillus cereus UW85. J Appl Microbiol 90 353-357... 

Lee KH, Park JH, Kim TY, Kim HU, Lee SY (2007) Systems metabolic engineering of Escherichia coli for L-threonine production. Mol Syst Biol 3 1-8 Lim SJ, Jung YM, Shin HD, Lee YH (2002) Amplification of the NADPH-related genes zfvf and gnd for the oddball biosynthesis of PHB in an E. coli transformant harboring a cloned phbCAB Operon. J Biosci Bioeng 93 543-549... 

In Ralstonia eutropha, two p-ketothiolases, enzyme A and enzyme B, have been discovered to take part in biosynthesis of PHB. Enzyme A is a homo-tetramer of 44-kDa subunits and enzyme B, a homo-tetramer of 46 kDa subunits. (Madison and Huisman, 1999). Slater et al. (1998) have shown that enzyme B is the primary catalyzer for the P(3HB-3HV) formation. The enzymatic mechanism of P-ketothiolase consists of biological condensation of two acetyl-Co A-moieties withformation of carbon-carbon bond. 

Expression of PHB biosynthesis genes in animal cells has been achieved in the cells of insect Spodoptera frugiperda and Trichopulsiani. In Spodoptera, an alternative pathway for the biosynthesis of PHB is created. The dehydratase domain mutant rat fatty acid synthase cDNA and phaC were expressed simultaneously which resulted into PHB synthesis (Wilhams et al., 1996). AhSiCv o vm, Autographa califomica nuclear polyhedrosis vims system has been also used to express PhaCj in Trichopulsiani cells. These cells accmnttlated PHB synthase in 50% of the total cell protein. 

Patients with Lesch-Nyhan syndrome have hyperuricemia, indicating an increased biosynthesis of purine nucleotides, and markedly decreased levels of hypoxanthine phbs-phoribosyl transferase (HPRT). The hyperuricemia can be explained on the basis of a decrease in which regulator of purine biosynthesis ... 

As part of on going efforts of Williams and his coworkers [46] to elucidate the biosynthesis of the core bicyclo[2.2.2] ring system of the related alkaloids the brevianamides [51], they have applied methodology originally developed for the stereocontrolled total synthesis of (-)-brevianamide B [52] to complete the first stereocontrolled total synthesis of (+)-PHB. 

After biosynthesis of the polyester and separation of the bacterial biomass from the supernatant, the required recovery process (typically a solid-liquid extractiOTi procedure) can constitute another not negligible cost factor, especially in large-scale production. Here extraction solvents that can easily be recycled will be of interest [53]. In order not to leave the patterns of sustainability in biopolymer production, it will be indispensable to concentrate the development of new extraction processes on such recyclable solvents that are also of environmentally sound nature [54], Typical harmful chlorinated solvents like chloroform must be avoided. A PHB production process embedded in an ethanol production plant has the advantage to utilise the medium chain length alcohol fraction (fusel alcohols) from the distillery step, consisting mainly of iso-pentanol. The application of the fusel alcohols as extracting solvents unites two important points On the one hand, this liquid normally constitutes a surplus product that has little market value. When used as an extraction solvent the costs for alternative solvents are saved. Furthermore, this extraction solvent is less harmful to handle than the classical extraction solvent chloroform [27],... 

A dramatic rise of the cost for liquid and gaseous hydrocarbons stimulates development of novel biopolymers which production is not depended on fossil fuels. Fermentative biosynthesis of poly(3-hydroxybutyrate) [PHB] and its homologues - poly(3-hydroxyalkonoates) [PHAs] bases on using renewable organic substrates. Hydrocarbons wastes of food- and wine/juice industries (sugars, melissa, starch et al.) present the basic structural material for bacterial PHB (and PHA). Utilization of hydrocarbons dining biosynthesis of PHA is favorable to eco-efficiency. 

The PhaC is soluble in the cytoplasm when the cells do not accumulate PHB. The enzyme becomes particle bound upon the onset of PHB biosynthesis [18]. According to the micelle model of PHA granule formation and according to the mechanism of PHA formation (see below) the enzyme is covalently bound to the growing polyester chain. Therefore, the enzyme is found in most PHA-... 

The cell as a biosynthesis machine can use cheap carbon sources (waste products) as precursor substrates to produce bacterial polymers. However, the in vitro synthesis of biopolymers requires costly purified key enzymes and precursor molecules such as ATP, coal, coal bolsters, and nucleotide sugars or sugar acids to synthesize polymers such as PHA, cellulose, alginate, and PGA. Consequently, these polymers have limited commercial applicability due to their very high production costs. It is estimated the production of PHB by in vitro synthesis would amount to a cost of around US 286,000 per gram of PHB whereas, bacterial production of PHB was estimated to cost about 0.0025 per gram of PHB, and this is still 5-10 times as expensive to produce as the respective petroleum-based polymers. 

Poly-(R)-3-hydroxyalkanoates (PHAs) are linear biopolyestos produced by a wide variety of bacteria as a reserve of carbon and energy [39]. Recently De Koning and Maxwell [40] drew an analogy between the conventional emulsion polymerization process and the biosynthesis of PHAs. Based on this Kuija et al. [41,42] made a more quantitative description of the accumulation of poly-(R)-3-hydroxybutyrate (PHB) in Alcaligenes eutrophus. 

Lutke-Eversloh T, Bergander K, Luftmann H, Steinbuchel A (2001) Biosynthesis of poly (3-hydroxybutyrate-co-3-mercaptobutyrate) as a sulfur analogue to poly(3-hydroxybutyrate) (PHB). Biomacromolecules 2 1061-1065... 

Other approaches to improve the properties of PHB is the production of derivatives based on PHB via the biosynthesis of copolyesters containing PHB units with other 3-hydroxyalkanoates units [19], such as poly [3-hydroxybutyrate-co-hydroxyvalerate] [PHBV] [20] or poly [3-hydroxybutyrate-co-3-hydroxyhexanoate] [21], with different molar ratios of hydroxycarboxylic acids. This approach has been investigated extensively [22] because it can... 

Labuzek, S., Radecka, I. Biosynthesis of copolymers of PHB tercopolymer by Bacillus cereus UW85 strain. J. Appl. Microbiol. 90, 353-357 (2001)... 

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