Study On Functions Of Isozymes Related To PHA Synthesis Pathway By Using Mutations Of Pseudomonas Putida And Biosynthesis Of Novel Medium-Chain-Length-PHA Materials

Polyhydroxyalkanoates (PHA) are produced by various bacteria as intracellular carbon and energy storage materials under nutrient-limitation conditions in the presence of excess carbon source. They have attracted increasing attention from scientific and industrial communities due to their interesting properties including biodegradability, biocompatibility and piezoelectricity. However, the physical characters of PHA which had been developed as commercial materials could not fit their utilization completely,it was hard to separate typical medium-chain-length PHA hydrolyzed product because of its various of monomers component. Therefore, researchers all over the world are focusing on mcl-PHA with better physical characters or mcl-PHA homopolymers. This study mainly focused on using genetic engineering technology to construct mutant recombinants to biosynthesis better physical character material with high content of long-chain-length monomer.P.putida KTOY08 was used as the parent to construct mutant recombinants by knocking out ten genes which related to PHA synthesis orβ-oxidation. The functions of the genes which knocked out in this study were analyzed. Lauric acid was used as the single substrate to culture the mutant strains. By analysis the PHA and its monomer content detected by Gas chromatography, five genes were found important to PHA synthesis andβ-oxidation. Deletion of gene PP3280 and PP4636, which were encoding isozymes of Acetyl-CoA acetyl-transferase (FadA), lead a high-yield of PHA about 90wt% of CDW. The lack of other three genes PP2051 (encoding Acetyl-CoA acetyl-transferase, FadA), PP2047 (encoding 3-hydroxyactyl- CoA dehydrogenase, FadB) and PP2048 (encoding Actyl-CoA dehydrogenase, FadE) would weakenβ-oxidation strongly.Meanwhile, one high-yielding strain KTQQ12 and one long-chain-length homopolymer and novel medium-chain-length PHA production strain KTQQ20 were obtained. When KTQQ12 were cultured by single substrate lauric acid, the product of PHA would be 40wt% higher than the parent strain. The long-chain-length homopolymer polydecanoate (PHD) which firstly obtained in vivo could be biosynthesized by KTQQ20 under the single substrate decanoate. In addition, the mutant KTQQ20 could also produce P(16mol% 3HD-co-84mol% 3HDD) and P(21 mol% 3HDD-co-79mol% 3HTD) by using dodecanoate and tetradecanoate as single substrate respectively.At last, molecular weight, physical properties of PHD, P(16mol% 3HD-co-84 mol% 3HDD) and P(21mol% 3HDD-co-79mol% 3HTD) were characterized. P(16 mol% 3HD-co-84mol% 3HDD) has the highest melting temperature of 78oC among mcl-PHA been reported. The tensile strength, strength at break and elongation at break were 12 MPa, 12 MPa, 313%, which were obviously higher than typical mcl-PHA. Therefore, there would be a wider application of novel mcl-PHA: P(16 mol% 3HD-co-84mol% 3HDD).