Screening polyhydroxyalkanoate producer with unique composition and analyzing substrate specificity of the PHA synthase

• Main Authors: Yan-Jia Huang, 黃筵嘉
• Other Authors: 李佳音
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/92631143180947412232

碩士 === 國立臺灣大學 === 農業化學研究所 === 93 === We screened the poly(3-hydroxyalkanoates) (PHAs) producers by cultivated in MSM containing 0.5 µg/ml Nile red from environment. Nine PHA producers had been isolated and verified for their PHA accumulation ability by flask fermentation and gas chromatography analysis. Among the wild type strains, TO7, PO6, PO21, TS18, and TF1-1 possess high PHA accumulation ability. The monomer compositions of PHAs produced by PO6 and PO21 possessed short-chain-length (scl) and medium- chain-length (mcl) monomers. The mcl monomer mol% of PHA produced by PO6 and PO21 could be regulated by the C/N ratio in PHA accumulation medium. The PHAs produced by PO6 and PO21 were further identified as scl-mcl blend PHA not the scl-mcl hybrid PHA by hot-acetone fractionation and GC analysis. The monomer composition of PHA accumulated by strains TS18 and TF1-1 was only 3-hydroxybutyrate (3-HB). Strains TS18 and TF1-1 could use cheaper carbon source such as sucrose to produce PHA. The PHA yield of TS18 could reach up to 75.7% of cell dry weight while fructose was used as the sole carbon source. Strains TS18 and TF1-1 both could incorporate 3-hydroxyvalerate (3-HV) monomer into PHB polymer when propionic acid was supplemented in the medium. 3-HV monomer composition of PHA produced by TF1-1 could also be regulated from 17 mol% to 42 mol% by supplementing various concentration of propionic acid with constant fructose as carbon sources, concentration ratio of fructose to propionic acid from 60 to 15. Strain TO7 was the mcl-PHA producer and 3-hydroxyoctanoate (3-HO) monomer composition of mcl-PHA was 93.7% of PHA content from octanoate as sole carbon sources. Strains TO7, PO6 and PO21 were considered to belong to Pseudomonas mosselii, Pseudomonas nitroreducens and Pseudomonas citronellolis. TS18 and TF1-1 were the genus Duganella zoogloeoides by 16S rDNA analysis. PHA synthase of Pseudomonas mosselii, TO7, was analyzed by recombinant P. putida GPp104 harboring PHA synthase gene. The 3-HO monomer was the major composition of PHA when P. putida GPp104 PHA- harboring phaC1 and phaC2 accumulated PHA.