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Modification of Phosphoenolpyruvate: Carbohydrate Phosphotransferase System of Escherichia Coli by Genetic Engineering and Characterization of Mutants

Author: ZhouJunZhi
Tutor: CaiYaFei
School: Anhui Normal University
Course: Cell Biology
Keywords: Red homologous recombination PTS system ptsHIcrr operon E. coli
CLC: Q78
Type: Master's thesis
Year: 2010
Downloads: 151
Quote: 0
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Abstract


Phosphoenolpyruvate - sugar phosphotransferase system (PTS system for short) play an important role in glucose phosphorylation and translocation process, the use of the I-Sce Ⅰ Red-mediated homologous recombination technology knocked manipulation in addition to E. coli ptsG gene and ptsHIcrr son visits deficient strain of growth performance and its possible applications. To be knockout by overlapping PCR construct containing gene downstream homology arm and both ends containing 18bp targeting specific sequence fragment, digested connect high copy targeting plasmid targeting plasmid and helper plasmid was transformed to host bacteria in vivo, in the L auxiliary the plasmid expression homing endonuclease and Red recombinase - arabinose induction, targeting fragment homologous region on chromosome reorganization knockout. In addition to Escherichia coli DH5α, JM109, HB101, BL21 (DE3), the ptsG gene and DH5α the ptsHIcrr operon step incognito knock; in LB medium containing 1% glucose, DH5α was △ ptsG, △ ptsG JM109 was HB101 was △ ptsG Bl21 (DE3) △ ptsG highest cell density followed by parents 2.99,3.49,1.58,1.70 times; the highest bacteria DH5α △ ptsHIcrr density is 1.51 times parents; E.coli K12 strain DH5α △ ptsG JM109 the △ ptsG production of acetic acid ability decreased gene chip revealed the sterilization of ptsG knock knock sterilization metabolism have a wider impact, especially glycolysis cycle weakened, while the citric acid cycle to strengthen, making tends to be a balance between the two cycles. In LB medium, the DH5α the △ ptsHIcrr the growth behavior DH5α and DH5α △ ptsG significantly different, the highest bacteria density is nearly 2 times DH5α and DH5α △ ptsG. DH5α △ ptsG growth behavior and DH5α no significant difference. In LB containing 1% glucose, DH5α △ ptsHIcrr DH5α △ ptsG showed a growth advantage, the highest bacteria density in turn is DH5α 2.8 times and 2 times; final acetic acid content in the culture medium were DH5α 12.2%, 47 %. M9 modified medium, DH5α △ ptsHIcrr specific growth rate (h -1 ) and specific glucose consumption rate (g · g -1 · h -1 ) was significantly lower than DH5α, and slightly less than DH5α △ ptsG. Results the description, ptsHIcrr operon knock sterilization changed the rate of glucose metabolism, and presents a different metabolic characteristics and the ptsG gene knock sterilization. ptsG-deficient strain is expected to be used to the high-density fermentation; In In addition, DH5α △ ptsHIcrr than glucose consumption rate for the follow-up to further lay the foundation to build the production of shikimic acid and other aromatic amino acids engineered strains.

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