|Ashby, Richard - Rick
Submitted to: Federation of European Microbiological Societies Microbiology Letters
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/22/2002
Publication Date: N/A
Interpretive Summary: Many bacteria can produce a type of polymer called poly(hydroxyalkanoates) (PHAs). These biopolymers have the properties of household plastics, elastomers or adhesives depending on their chemical compositions. Because they are biodegradable, PHAs are attractive, environmentally friendly substitutes for petroleum-based polymers in many applications, such as wrappings, coatings, and the manufacturing of fibers and containers. To expand their potential markets into more areas of end-use applications, there is a need to produce PHAs with new materials properties. Modifying the genes responsible for PHA synthesis in bacteria provides one way to change the chemical compositions and hence the properties of the biopolymers produced by the organisms. In this paper, we report the development of a method to mix portions of different PHA synthesis genes to create new genes. When portions of two different genes, each capable of directing the synthesis of elastomeric/adhesive- (e/a-) PHAs, were combined, the new chimeric genes were capable of also synthesizing similar types of PHA. Several trials of mixing genes that separately were responsible for the synthesis of e/a-PHAs and hard plastic-type PHAs produced inactive chimeric gene products. With the success achieved with the e/a-PHA genes, however, further study using variations of this approach should yield chimeric PHA-synthesis genes capable of producing biopolymers with new properties. The results should greatly expand the application potential of this class of biodegradable polymers, making them more available to the consumers in the form of environmentally friendly end-use products.
Technical Abstract: Pseudomonas resinovorans phaC1(Pre) and phaC2(Pre) genes coding for poly(hydroxyalkanoate) (PHA) synthases were cloned by polymerase-chain- reaction and expressed in E. coli LS1298 (fadB). Repeat-unit composition analysis showed that beta-hydroxydecanoate (67-75 mol%) and beta- hydroxyoctanoate (25-33 mol%) are the major monomers of the PHA produced in ncells grown on octanoate. Sequence analysis showed that the gene products of phaC1(Pre) and phaC2(Pre) had 61% identical (75% positive) amino-acid sequence matches, and both sequences contained a conserved alpha/beta- hydrolase fold in the carboxy-terminal portion of the proteins. Switching the alpha/beta-hydrolase folds of phaC1(Pre) and phaC2(Pre) yielded chimeric pha7 and pha8 genes that afforded PHA synthesis in E. coli LS1298. The repeat-unit compositions of PHA in cells containing pha7 and pha8 were similar to those found in transformants containing the parental genes. Deletion mutants of phaC1(Pre) and phaC2(Pre) that resulted in potential translational fusions also supported PHA synthesis with similar repeat-unit compositions. Chimeric genes obtained from the switching of fragments containing the alpha/beta-hydrolase folds of phaC1(Pre) and Ralstonia eutropha phbC did not direct the synthesis of PHA in transformed cells.