Genetically Engineered Bacteria Synthesis Of Poly Hydroxybutyric Acid Hydroxyl Esters

Polyhydroxyalkanoates (PHAs) are polyesters produced by a wide variety of bacteria ascarbon and energy storage material under unbalanced growth conditions. Aeromonas punctataFA440 (formerly named Aeromonas caviae FA440) and Aeromonas hydrophila 4AK4 couldsynthesize poly(3-hydroxy--butyrate-co-3-hydroxyhexanoate) (PHBHHx) from lauric acid(dodeconoate), one of the member of polyhydroxyalkanoate (PHA). And PHBHHx has morewider applied prospects than other PHA due to its superior material properties. PHBHHx withdifferent 3HHx fraction has different properties and applications. Howerver, the constant 15mol% 3HHx content of PHBHHx produced in A. hydrophila 4AK4 limited its wide use,furthermore; PHBHHx were only produced from expensive related carbon sources such asfatty acids or oils,which enhanced the cost of PHBHHx production. Therefore, the aim of thestudy was to overcome the above difficulties to some extent.In order to clarify the factors affecting the monomer composition and PHBHHx content,genes encoding phasin (phaP), PHA synthase (phaC) and (R)-specific enoyl-CoA hydratase(phaJ) from A. punctata were introduced individually or jointly into A. hydrophila 4AK4,dueto the high homology of the corresponding genes in the two strains. PhaC increased 3HHxfraction more significantly than phaP, while phaJ had little effect. Expression of phaC aloneincreased the 3HHx fraction from 14 mol% to 22 mol%. When phaC was co-expressed withphaP and phaJ, the 3HHx fraction increased from 14 mol% to 34 mol%. Expression of phaPor phaC alone or with another gene enhanced PHBHHx content up to 64%, cell dry weight(CDW) as much as 4.4 g L-1 and PHBHHx concentration to 2.7 g L-1 after 48 h in shake flaskculture. The results suggest that much higher PHA synthase activity could lead to a higher3HHx fraction and PHBHHx content. Co-expression of phaJ with phaC or phaP would favorPHA accumulation, although over-expression of phaJ did not affect PHA synthesis much. Inaddition, after phaB gene encoding acytoacetyl-CoA from Wautesia eutropha H16 wasintroduced into wild type A. hydrophila 4AK4, PhaB could supply more 3HB-CoA precursorfor accumulating PHBHHx of lower 3HHx content in the recombinant strain than in wild typestrain. And PHBHHx with variable 3HHx content could be synthesized from the aboveconstructed recombinant strains, which would widen the use of PHBHHx in manyapplications, especially in tissue engineering. And the effect of acrylate, which was aninhibitor of β-oxidation, on PHA synthesis in A. hydrophila 4AK4 was also studied. Theaddition of acrylate in the culture of A. hydrophila 4AK4 enhanced PHBHHx content.However, no monomers longer than 3HHx were detected. It could be concluded that thesubstrate specificity of PhaC in A. hydrophila 4AK4 was restricted to monomers of 3HB-CoA...