|Ashby, Richard - Rick|
Submitted to: Current Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/3/2005
Publication Date: 6/1/2005
Citation: Solaiman, D., Ashby, R.D. 2005. Genetic characterization of the poly(hydroxyalkanoate) synthases of various pseudomonas oleovorans strains. Current Microbiology. 50(6):329-333.
Interpretive Summary: Many bacteria produce a family of bioplastics called poly(hydroxyalkanoates) (PHAs). Depending on the producing organism, these biopolymers can have the properties of hard plastics, stretchy elastomers, or sticky adhesives. Since these biomaterials are biodegradable and can be produced using agricultural fats, oils, and byproducts as substrates for fermentation, they are useful as environmentally friendly replacements for petroleum-based polymers. To expand the application field of these polymers, however, PHAs with new compositions and improved performance properties are desired. One way to achieve this is through the finding or creating of new genes coding for the enzymes (biological catalysts) responsible for the synthesis of the polymers within the bacterial cells. In this report, we describe the finding and characterization of two new genes from bacteria called Pseudomonas. These genes drive the synthesis of hard plastic-type PHA called PHB. Detailed sequence analyses showed that they are very different from a currently available PHB gene (from a bacterium called Wautersia eutropha). One of the new genes, the phbC14682, contains an extra DNA piece that governs the degradation of the gene product. It is only the second such gene ever reported. The results also show that the two new genes use very similar codes (called codons) to other Pseudomonas genes responsible for the synthesis of adhesive-type PHAs, and may thus facilitate the mixing of these genes through a process called gene shuffling to create new genes for novel PHA production. These new plastic materials should open up additional applications for PHA, leading to the availability of more biobased end-user products that will benefit consumers and the environment.
Technical Abstract: We identified the poly(hydroxyalkanoate) synthase (PHAS) genes of three strains of Pseudomonas oleovorans by using polymerase-chain-reaction (PCR)-based detection methods. P. oleovorans NRRL B-14682 contains Class I PHA synthase gene (phaC), NRRL B-14683 harbors Class II phaC1 and phaC2 genes, and NRRL B-778 contain both the Class I and II PHA synthase genes. Inverse-PCR and chromosomal walking techniques were employed to obtain the complete sequences of the Class I phaC's of NRRL B-778 (phbC778; 1698 bps) and B-14682 (phbC14682; 1899 bps). BLAST search indicated that these genes are new and had not been previously cloned. The gene product of phbC778 (i.e., PhbC778; 566 amino-acid residues) is homologous to the Class I PHA synthases of Pseudomonas sp. HJ-2 and Pseudomonas sp. strain 61-3, and that of phbC14682 (PhbC14682; 632 amino acids) is homologous to PHAS of Delftia acidovorans. The PhbC14682 contains an extra sequence of 33 amino acids in its conserved alpha/beta-hydrolase domain, making it only the second reported Class I PHA synthase to contain this cellular proteolytic sequence. Consistent with their Pseudomonas origin, the codon-usage profiles of PhbC778 and PhbC14682 are similar to those of Pseudomonas Class II PHAS's. These new Pseudomonas Class I phbC genes provide valuable addition to the gene pool for the construction of novel PHAS's through gene shuffling.