MCB-9514100 Dennis Polyhydroxyalkanoates (PHAs) are energy-reserve polymers that are accumulated by many bacterial species in times of nutritional stress. Coincidentally, these polymers may also be utilized as biodegradable plastic. The physical properties of the plastic are dictated by the length of the carbon chain in the monomer unit with C4 homopolymers being relatively brittle and C8 homopolymers being elastomeric. Copolymers tend to have physical properties intermediate between their monomer units. The enzymes that mediate the polymerization, PHA synthases, are grouped into three distinct categories based on their substrate specificity and their molecular structure. Type I PHA synthases have a single polypeptide chain and form predominately C4 (3-hydroxybutyrate; 3HB) monomer units. Type II PHA synthases also have a single polypeptide chain, but they form C4 to C12 (3-hydroxydodecanoate; 3HDD) monomer units, with Cg (3-hydroxyoctanoate; 3HO) and C10 (3-hydroxydecanoate; 3HD) being the preponderant units. Type III PHA synthase are composed of 2 polypeptide chains and form largely 3HB. However, some members of this class are able to incorporate mostly 3HV into P(3HB-co-3HV) copolymer. The PHA synthase from N. corallina (phaCNc), a bacteria which makes a substantial amount of 3-HV from unrelated carbon sources such as glucose and fructose has been cloned. Preliminary data suggests that this PHA synthase does not completely meet the specifications for any of the three classes of synthases. It has a single polypeptide chain and is genetically most similar to Type II PHA synthases, yet its substrate specificity appears to be largely in the 3HV and 3HH range, though its total substrate range is from 3HB to 3HHp (3-hydroxyheptanoate). This is quite exciting for several reasons. First, phaCNc represents a potentially new class of PHA synthase. Second, it is a PHA synthase which makes new types of polyhydroxyalkanoates that have been little-characterized. Third, it is well-expressed in Pseudomona s putida, reasonably expressed in Alcaligenes eutrophus and Klebsiella aerogenes, and poorly expressed in Escherichia coli. Fourth, it is a PHA synthase that may have the capacity to be utilized in plant constructs because it accesses long- and medium-chain fatty acid metabolism, yet only forms copolyesters of 3HB and 3HHx (3-hydroxyhexanoate) or 3HV (3-hydroxyvalerate) and 3HHp. This research continues the analysis of phaCNc in two specific areas. In the first, expression of phaCNc will be analyzed in the four different recombinant hosts named above. Analyses will be conducted for enzymatic activity, quantitation of phaCNc transcripts, and quantitation of PhaCNc levels. This data will be correlated with the levels of expression of polymer in each recombinant host. In addition, the effect of rare codon usage in each host will be analyzed by selecting specific codons, genetically altering them to more prevalent codons, and then examining the result of this alteration on enzymatic activity, phaCNc transcript levels, protein levels, and polymer levels. Finally, the effect of placing phaCNc under the control of a strong, regulatable promoter will be determined. The expected result of this phase of the project is an understanding of the factors which limit or facilitate phaCNc expression. An additional result is the construction of phaCNc genes which are well-expressed in recombinant hosts. In the second phase of the project studies will be initiated that focus on identifying regions of phaCNc which mediate substrate specificity. The first strategy used will be to randomly mutagenize phaCNc and screen for altered substrate specificity of PhaCNc. This will done enzymatically utilizing a novel screening assay developed in our laboratory. phaCNc genes which exhibit altered substrate specificity will be analyzed as to the specific change in their DNA sequence. The second strategy used to to identify regions that regulate substrate specificity will be to construct chimeras from different PHA synthase g enes and analyze the construct for enzymatic activity and polymer composition. Though many (approximately 19) different PHA synthases have been cloned, only one has been rigorously studied, the Alcaligenes eutrophus PHA synthase, and these studies did not extend to the level proposed here for phaCNc- phaCNc has advantages over most other PHA synthase genes in that its substrate specificity falls into a range that is more likely to be of commercial interest. Studies proposed here should be applicable not only to phaCNc, but to other PHA synthase genes as well. %%% This research has application to the inexpensive production of biodegradable plastics. In addition, it accomplishes the training of undergraduate students in the techniques of molecular biology and their applications to problems of both basic and applied interests. ***
