PHB Biosynthesis

PHB synthesis from glucose using Azotobacter beijerinkii revealed substantial amounts of polymer accumulation under oxygen limitation conditions. The key feature of control in A. beijerinckii is the pool size of acetyl-CoA, which may either be oxidized via the tricarboxylic acid (TCA) cycle or can serve as a substrate for PHB synthesis the diversion depends on environmental conditions, especially oxygen limitation, when the NADH/NAD ratio increases. Citrate synthase and isocitrate dehydrogenase are inhibited by NADH, and as a consequence, acetyl-CoA no longer enters the TCA cycle at the same rate. Instead acetyl-CoA is converted to acetoacetyl-CoA by p-ketothiolase, the first 

There are four dilferent pathways for the synthesis of PHAs found to date. 

eutrophus, p-ketothiolase carries out the condensation of two molecules of acetyl-CoA to acetoacetyl-CoA. An NADPH-dependent acetoacetyl-CoA reductase then carries out its conversion to 3-hydro-xybutyryl-CoA. The third and the final step is the polymerization reaction catalysed by PHB synthase.  

In Rhodopsuedomonas rubrum, the pathway differs after the second step where the acetoacetyl-CoA formed by p-ketothiolase is reduced by a NADH-dependent reductase to L-(+)-3-hydroxybutyryl-CoA which is then converted to D-( )-3-hydroxybutyryl-CoA by two enoyl-CoA hydratases. 

A third type of PH A biosynthetic pathway is found in most Psuedomonas species belonging to rRNA homology group I. P. oleovorans and other Psuedomonas species accumulate PHA consisting of 3-hydroxyalkanoic acid of medium-chain length (MCL) if cells are cultivated on alkanes, alkanols or alkanoic acids.  

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In combination of this polymerase with purified propionyl-CoA transferase of Clostridium propionicum, a two-enzyme in vitro PHB biosynthesis system was established which allowed the PHB synthesis from (R)-hydroxybutyric acid as substrate [119]. In this way, the PHB synthesis was independent of the consumption of the expensive CoA, and hence PHA could be readily produced in a semipreparative-scale... 

Peoples, O. P. Sinskey, A.J. (1989). Poly-beta-hydroxybutryate (PHB) biosynthesis in Alcaligenes eutrophus H16. Identification and characterization of the PHB polymerase gene (phbC). Journal of Biological Chemistry, 264, 15 298-303-... 

Masamune, S., Walsh, C. T., Sinskey, A. J., Peoples, O. P. Poly-(R)-3-hydroxybutyrate (PHB) biosynthesis mechanistic studies on the biological Claisen condensation catalyzed by -ketoacyl thiolase. PureAppl. Chem. 1989, 61,303-312. 

Because only about 17% of the total sugar produced is diverted to PHB biosynthesis, the polymer production will not affect sugar stocks and will not cause a... 

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-... 

For large-scale recombinant production of bacterial polymers, non-polymer producing bacteria were exposed to biosynthesis pathways. Polymers such as PHA, CGP (cyanophycin granule peptide), HA (hyaluronic acid), and PGA [poly-y-glutamate] were produced by these methods [89, 85-96]. For example, recombinant E.coli [89] was fermented for the lai e-scale production of PHA [89]. In addition the PHB biosynthesis genes of Ralstonia eutropa were harbored in E.coli to produce poljmers such as PHA composed of (R)-S-hydroxybutyrate and (R)-3-hydroxyvalerate and/or (R)-3-hydroxyhexanoate which showed preferable properties for use in industrial applications [97-99, 85-96]. 

Polyhydroxyalkanoates biosynthesis is regulated, on one hand, by the activity of 3-ketothiolase (EC 2.3.1.16), and on the other hand of acetoacetyl-CoA reductase (EC 1.1.1.36) intracellular PHA breakdown is dependent on the activity of 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30). Besides these three enzymes, the following compounds can be pointed out as major factors responsible of the activities of the key enzymes acetyl-CoA, free CoA, NAD(P) + (or NAD(P)H2, respectively) and, to a lower extent, ATP, pyruvate and oxalacetate. In any case, acetyl-CoA can be considered as the central metabolite both for biomass formation and PHB biosynthesis. This compound stems from the catabolic break down of carbon substrates like sugars (mainly catabolized by the 2-Keto-3-desoxy-6-phosphogluconate pathway) or fatty acids (converted by 6-oxidation). 

Figure 7.3 illustrates the basic metabolic route from carbon substrates to PHB biosynthesis and its subsequent remobilization. 

Ultra-high-molecular-weight P(3HB) samples (UHMW-P(3HB), M = 1.3-16 X 10 ) were produced by E. coli XLl-Blue (pSYL105)" containing a stable plasmid harboring the R. eutropha H16 (ATCC 17699) PHB biosynthesis genes operon phbCAB. Two-step cultivation of the recombinant E. coli... 

Escherichia coli Succinate and polyhydroxybutyr-ate (PHB) A/B Since PHA/PHB is an intracellular compound and succinate is an extracellular compound, they can be easily separated, either by centrifugation or filtration. The NADPH consumed during PHB biosynthesis can be regenerated by pushing the metabolic pathway toward the TCA cycle. 32,33... 

E. coli Tissue plasminogen activator (tPA) and PHB A/C In vivo PHB biosynthesis provides the cytoplasm as a suitable environment that can be further oxidized for the formation of multidisulfide bonds in tPA. tPA is a value-added drug compound for thrombolytic therapy. 35... 

Mahishi LH.Tripathi G, Rawal SK. Poly (3-hydroxybutyrate) (PHB) synthesis by recombinant Escherichia coli harbouring Streptomyces ameofaciens PHB biosynthesis genes effect of various carbon and nitrogen sources. Microbiol Res 2003 158(2003) 19-27. 

The biochemical pathways of PHB biosynthesis are reasonably well understood and start with the conversion to acetate of an appropriate carbon substrate, such as low alcohols and acids, sugars or even gaseous mixtures of hydrogen and carbon dioxide. " The bacteria put a handle on the acetate molecules in the form of an enzyme cofactor linked by a thioester bond. Two molecules of this acetyl coenzyme-A can then be condensed to give acetoacetyl coenzyme-A which is subsequently reduced to 3-hydroxybutyryl coenzyme-A. The final polymerase enzyme joins these units together to form PHB, and recycles the cofactor as shown in Fig. 2. 

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. 

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