Recombinant E.coli for Rhodobacter sphaeroides 5-aminolevulinic acid synthase(ALAS) expression and 5-aminolevulinic acid production
| Autor: | 游志平 |
|---|---|
| Rok vydání: | 2004 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 92 5-aminolevulinic acid(ALA) is a five carbons amino acid and already has many application in the field of agriculture and medicine for many years. In recent years, it has been developed as one of the second generation of photosensitive reagent for Photodynamic Therapy(PDT). ALA is produced by many organisms as a precursor for the bio-synthesis of heme. One of the ALA biosynthesis routes is through the ALA synthetase(ALAS) reaction with glycine and succinyl-CoA. Therefore, in this thesis, ALAS gene(hemA) of R.sphaeroides was expressed in E.coli BL21(DE3) to study the ALA production from E.coli. Expression vector pHmA based on pET30b(+) was constructed. In order to stabilize this pHmA vector, sopABC gene was co-expressed in the construct pPhmA. Without IPTG induction, BL21(DE3) harboring pPhmA could express a large amount of ALAS. As a consequence, ALA of 3.1 g/L could be obtained at the end of 24h fermentation using LB medium supplemented with 30mM glycine、90mM succinate. A very stable single copy number expressing vector pBhmA derived from mini-F plasmid pBeloBAC11 was also constructed. With 0.1mM IPTG induction, ALA of 1.88 g/L could be obtained at the end of 25h fermentation. Probably due to its single copy number, the ALA produced by the E.coli harboring this vector was much lower than that of pPhmA. High cell density fermentation had been carried out, high cell conc.(OD600=80) was obtained but did not enhance the ALA production. As a consequence, ALA of 2.64 g/L was obtained. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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