Identification and Analysis of Polyhydroxyalkanoate Biosynthesis Gene Clusters in the Extreme Halophilic Archaeon Haloterrigena sp. H13

• Main Authors: Chih-Chien Lin, 林志鍵
• Other Authors: Mei-Chin Lai
• Format: Others
• Language: zh-TW
• Online Access: http://ndltd.ncl.edu.tw/handle/47857954339349370370

碩士 === 國立中興大學 === 生命科學系所 === 96 === Polyhydroxyalkanoates (PHAs) are a class of biodegradable polyesters of (R)-hydroxyalkanoates. These biopolymers are accumulated by a wide variety of bacteria and haloarchaea when the carbon source is available in excess but other nutrients are growth limiting. PHAs can be used as biodegradable thermoplastics for a wide range of industrial and medical applications. The extremely halophilic Haloterrigena sp. H13 is capable of accumulating large amounts of hPHA under conditions of nitrogen limitation and abundant carbon source. The monomer of hPHA as -[-O-CH(C≡CH)-CH(C2H5)-CHO-]n- is a novel PHA with C≡C bonding. In this study, Southern blot was performed and a 4.6-kb NotI restriction fragment contained PHA biosynthetic gene cluster was cloned from genomic DNA of Haloterrigena sp. H13. This PHA biosynthetic gene cluster included six open reading frames encoding enoyl-CoA hydratase (MaoCH13), AbrB protein (PhaRH13), transducer protein (PhaTH13), polyhydroxyalkanoate synthase subunit (PhaDH13), polyhydroxyalkanoate synthase (PhaCH13), NADPH-dependent acetoacetyl coenzyme A reductase (PhaBH13) and two putative promoter regions. PhaC H13 and PhaD H13 were composed of 538 and 182 amino acid residues respectively and showed low amino acid identity with other class III type PHA synthases. There were 4 bp overlapping of phaDH13 and phaCH13 and a putative promoter regions located upstream of phaDH13. Result of Northern blot hybridization also demonstrated that phaCH13 and phaDH13 were co-transcribed. The amino acids of (Cys-151)-(Asp-306)-(His-335) were proposed as the catalytic nucleoplile for PHA polymerization at PhaCH13. The PHA biosynthetic gene cluster we identified from Haloterrigena sp. H13 should broaden our knowledge in archaeal PHA biosynthesis.