Abstract

Azotobacter vinelandii is a bacterium able to produce only short chain length PHAs. On carbohydrates it produces the polyhydroxybutyrate homopolymer, whereas on valerate, heptanoate or nonanoate it is able to accumulate the copolymer polyhydorxybutyrate-co-hydroxyvalerate. Some of the closely related Pseudomonas, like P. putida, are able to synthesize medium chain length PHAs from carbohydrates. The precursors for the synthesis of PHAs in this case are produced through the fatty acids biosynthetic pathway. The link between fatty acids synthesis and the synthesis of PHAs is provided by a β-hydroxyacyl ACP:CoA acyltransferase encoded by phaG. The aim of this study was to construct a recombinant strain of A. vinelandii capable of expressing a β-hydroxyacyl-ACP:CoA acyltrasferase in order to explore the possibility to produce mcl-PHAs from carbohydrates and to learn about the biosynthetic capabilities of the type I PHA synthase of A. vinelandii. To express the enzyme ACP:CoA acyltransferase, the phaG gene of P. putida was cloned and integrated in the A. vinelandii chromosome. When growing on carbohydrates and under non-inducing conditions, the recombinant strain produced a polymer composed only of hydroxybutyrate units. When the phaG gene expression was induced, the monomer composition of the polymers produced changed, obtaining a copolymer composed of hydroxybutyrate and hydroxyhexanoate (~90 and 10% respectively). This result shows that the phaG gene product is able to confer A. vinelandii the ability to synthesize a polymer not previously produced by this bacterium. Although the enzyme introduced has been shown to be able to generate precursors of longer chain length in other bacteria, no other monomers were detected in the polymer of the recombinant A. vinelandii. These results suggest that the PHA synthase of A. vinelandii has no capacity to polymerize hydroxyalkanoates containing more than 6 carbons. This limitation could also be affecting other characteristics of the copolymer produced. When the molecular weight of the hydroxybutyrate-co-hydroxyhexanoate PHA was estimated, it showed a considerably diminished molecular size when compared to that of the hydroxybutyrate homopolymer produced under non-inducing conditions. A possible explanation is that although the A. vinelandii PHA synthase incorporated the six carbon monomers, its ability to polymerize them is probably limited, leading to the production of a polymer of shorter chain length.