| Summary: | 碩士 === 國立中興大學 === 化學工程學系所 === 103 === Caldimonas manganoxidans, a thermophilic bacterial strains of β-Proteobacteria isolated from the hot spring in Japan, was attractive for its ability to accumulate and degrade PHB. PHA biosynthesis genes of Caldimonas manganoxidans consisting of three genes encoding β-ketothiolase (phaACm gene, 1179 bp), acetoacetyl-CoA reductase (phaBCm gene, 738 bp) and PHA synthase, (phaCCm gene, 1694 bp) were identified in this study. Sequence alignment of PHA synthases revealed that phaCCm showed at least 60% identity with those of class I strains Alcaligenes latus, Cupriavidus taiwanensis, Comamonas sp. EB 172, and Cupriavidus necator. However, the phaCCm showed low identities of 41%, 22%, and 23% with those of Pseudomonas aeruginosa (class II), Allochromatium vinosum (class III), and Bacillus megaterium (class IV). The PHA biosynthesis genes were successfully cloned in Escherichia coli. E. coli BL21 (DE3) harboring pT7pha1A2B and pBADphaC grown on LB agar plate containing Nile Blue A emitted fluorescence under UV light irradiation (312 nm). PHB granule could clearly be seen in TEM. These results revealed that recombinant E. coli could accumulate PHB. LB medium containing 20 g/L glucose was also used to confirm PHB production in this study. The result of OD600 showed that the control group of wild type E. coli BL21 (DE3) is only 7-8 whereas E. coli BL21 (DE3) harboring pT7pha1A2B and pBADphaC is as high as 39 (data not shown). The PHB content and the PHB concentration of 52.2±2.0% and 6.38±0.15 g/L can be reached in recombinant E. coli.
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