Cloning, Expression And Functional Study Of Polyhydroxyalkanoate Synthase Genes

Polyhydroxyalkanoates ( PHA), a family of intracellular polyester synthesized by many bacteria, have received increasing attention due to its excellent biodegradability, biocompatibility, optical activity and piezoelectricity, as well as potential applications in areas of biodegradable packaging, tissue engineering and drug delivery.This thesis focused on the study of PHA synthase gene (phaC), which is a key enzyme for PHA production. The purpose of the thesis is to discover and produce novel PHA through studies on different phaC genes with special functions. Following results were achieved from this thesis.(1) A rapid and easy PCR method was established to clone type II PHA biosynthesis genes. Highly conserved sequences in the coding regions of the phaC genes have been found by gene alignment ofphaC genes cloned from 5 known Pseudomonas spp., and these highly conserved sequences can be used as primers for the PCR screening of phaCl/phaC2-containing microorganisms. Touch-down PCR has been adopted for the cloning of type II PHA synthase genes. This method can be widely applied in type II PHA synthase gene cloning.(2) Totally 7 type II PHA synthase genes have been cloned from 4 different PHA producing microorganisms using the method mentioned above. The 4 PHA producing microorganisms includes 3 Pseudomonas strains and 1 Burkholderia strain, and they were Pseudomonas pseudoalcaligenes YS1, Pseudomonas pseudoalcaligems HBQ06, Psedomonas nitroreducens 0802 and Burkholderia caryophylli YS13. Integrated phaCl and phaC2 were identified through sequence analysis. The analysis of these genes with the known PHA synthases showed over 70% high homologous.(3) A fatty acid degradation pathway (fadE) deleted mutant of Escherichia coliKM32B (fadB::Tef) was constructed by homologous recombination. PCR identification showed the whole region offadB gene was replaced by Tet gene.(4) Expressional plasmids containing the cloned PHA synthase genes were constructed and transformed into wild type E. coli strains and E. coli fadB deleted mutant strain KM32B. In most wild type E. coli strains containing heterologous PHA synthase genes, the amount of PHA accumulation was almost undetectable while in E. coli KM32B the amount of PHA accumulation were clearly visible.(5) A different PHA biosynthesis pathway was found in Burkholderia caryophylli YS13. The shake flask experiments showed that B. caryophylli YS13 could accumulate polyhydroxyalkanoates containing monomers ranging from 3-hydroxybutyrate (3HB) to 3-hydroxydecanoate (3HD) when cultivated on glucose and/or fatty acids. Previous study of Burkholderia strain showed that this strain possessed at least two PHA biosynthesis pathways, one of which is similar with Ralstonia eutropha (type I PHA biosynthesis pathway). The type II PHA biosynthesis genes cloned from B. caryophylli YS13 using the described PCR method demonstrated that PHA biosynthesis in Burkholderia strain has an additional pathway to the normally type I pathway.