In vitro PHA biosynthesis

Intermediates of the metabolism have so far not been identified as inhibitors or activators of PHA synthases. The only exception is coenzyme A which inhibits the PHA synthases of R. eutropha, C. vinosum, and R aeruginosa at relatively low concentrations [73,74]. It is not known whether this inhibition is physiologically relevant. The inhibition by coenzyme A has, however, to be taken into account during the design of in vitro PHA biosynthesis processes, if PHA is being prepared on a preparative scale recycling of coenzyme is then recommended not only to reduce the costs but also to improve the kinetics of PHA formation. 

Expression in E. coli will make PHA synthase proteins more readily accessible for purification and therefore available in large quantities for further studies such as detailed biochemical analysis, generation of antibodies and in vitro PHA biosynthesis. 

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... 

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... 

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. 

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