Biosynthesis And Property Modificationof Polyhydroxyalkanoates

Polyhydroxyalkanoates (PHAs) are a class of polyesters produced by awide variety of bacteria as intracellular carbon and energy storage materials.Recently, they have attracted increasing attention from scientific and industrialcommunities due to their interesting properties including biodegradability,biocompatibility and piezoelectricity, which promising applications in variousareas.Focused on developing novel PHA materials, two parallel issues wereinvestigated separately in this study. Surface physicochemical properties andbiocompatibility of the blend consisted of poly-3-hydroxybutryrate (PHB) andpoly-(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) was evaluated invitro to drop a new hint for modifying existed PHA materials. On the otherhand, primary genetic engineering studies were also applied to develop novelPHA for a wider application. Following results were obtained:1. PHBHHx was completely miscible with PHB in their blendPHB/PHBHHx. Crystallization behavior of these polyester blends played animportant role in determination of the surface physicochemical propertiesincluding chemical states, surface free energy and polarity. Non-dispersioncomponent governed the total surface free energy in these blends, whichdemonstrated that this kind of polyesters were typical polar semi-crystallinebiomaterials.2. Protein adsorption and cell adhesion were determined by the surfacefree energy of the PHB/PHBHHx blend films. Surface physicochemicalproperties also influenced the cell response to PHB/PHBHHx polyester films,including cell attachment, proliferation, distribution and differentiation. Basedon the above results, it suggested the probability of adjusting materialbiocompatibility via regulating material crystallization behavior.3. (R)-3-hydroxyacyl-acyl carrier protein (ACP)-coenzyme A (CoA)transacylase (PhaG) from Pseudomonas strains was found to convertintermediates of fatty acid de novo biosynthesis pathway tomedium-chain-length (MCL) PHA precursors. In this study, a touch-downpolymerase-chain-reaction (PCR) protocol was developed for the rapid andspecific identification of PhaG structural gene—phaG from various bacteria,including non-Pseudomonas strain Burkholderia caryophylli AS1.2741. Itsuggested that PhaG-mediated pathway was widespread among bacterial flora.4. For the first time, valuable chemical 3-hydroxydecanoic acid (3HD)was directly biosynthesized from carbohydrates, such as glucose and fructose,by heterologous expressing phaG in recombinant Escherichia coli.Fermentation conditions were also optimized in the issued patent.5. Thioesterase II was mobilized in the study of PHA and its monomermetabolism for the first time. It was proven that thioesterase II played animportant role in cleaving 3-hydroxydecanoly-CoA to yield 3HD in thePhaG-mediated pathway. On the other hand, by co-expressing phaG and PHAsynthase gene in thioesterase II negative mutant strain E. coli CH01, whichwas constructed in this study, it was realized the direct bio-production ofMCL-PHA from carbohydrates.6. By employing PhaG-mediated 3-hydroxydecanoic acid biosynthesispathway for high level accumulation of intracellular 3-hydroxydecanoyl-CoA,thioesterse II was found to play a role in preventing accumulation ofintracellular acyl-CoA and thus in maintaining an appropriate acyl-CoA pool invivo. It will be instructive for revealing the physiological role of thioesterase II,which confusing the researches for about 40 years.