Plasmid Construction for Introducing aroY and aroZ Genes into Pseudomonas putida F1 to Produce Muconic Acid from Glucose
| Autor: | Lin,Jian-Jie, 林建傑 |
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| Rok vydání: | 2015 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 103 Adipic acid is an important platform chemical in industry and is primarily used for production of nylon 6,6. Traditional industrial producing adipic acid relies on benzene or its derivative as feedstock with nitrate and metal oxides as catalysts. However, using these processes result in the production of nitrous oxide that is a potent greenhouse gas. On the other hand, adipic acid can also be produced by bio-fermentation after hydrogenation of cis,cis-muconic acid. This method can reduce the production of greenhouse gas. Catechol, one of the intermediates in the metabolic pathway, is toxic to cells. Because Pseudomonas putida is able to withstand a higher concentration of catechol, we chose Pseudomonas putida F1 as a host to synthesize cis,cis-muconic acid from glucose. To endow Pseudomonas putida with a capacity to produce cis,cis-muconic acid from glucose, we transformed aroZ (3-dehydroshikimate dehydratase) and aroY (protocatechuic decarboxylase) into Pseudomonas putida. aroZ and aroY were separately amplified from Klebsiella pneumonia using polymerase chain reaction (PCR). aroZ and aroY, each containing a ribosome binding site, were ligated into pTZ57R/T vector by Exonuclease III. Finally, two 50bp homologous sequences of catBC were flanked to the aroZ-aroY fragment via PCR. The DNA fragment aroZ-aroY was electro-transformed into Pseudomonas putida F1. The chromosomal catBC promoter is used to express aroZ-aroY. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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