Construction of a Novel
Engineering Strain E.coli G830,which
is Adoptable to High-cell-density Fermentation
YANG Yun-Gui,
ZHANG Hui-Tang, TONG Qin, YANG Yan-Hua, WANG You, YANG Sheng-Li, GONG Yi*
( Shanghai Research Center of Biotechnology, Institute for Biological
Science, the Chinese Academy of Sciences, Shanghai 200233, China
)
Abstract A novel
engineered strain G830 adoptable to high�–ell density fermentation by
integrating bacterial hemoglobin vhb (Vitreoscilla hemoglobin
gene) into thr operon in the chromosome of PA1 blocking Pta-Ack
metabolic pathway through the homologous recombination between the homologous fragments
of integrated plasmid and that of chromosome. The engineered strain G830 was
characterized by phenotype observation, PCR, thr mutant, acetate acid
detection, Western blotting and VHb activity assays. In high dentity
fermentation, the cellular respiration, energy metabolism, highest bacterial
density and dry bacteria weight of the G830 strain were markedly better than
control strains PA1 and BL21. The expression of recombinant prolyl
endopeptidase (PEP) in G830 and PA1 under the above condition was high and
stable. Their growth situation and fermentation parameters were similar with
their parental strains without plasmids, and resided plasmids maintained stably
in those strains. It revealed that the integral VHb and acetate metabolism
pathway (Pta-Ack) block improved the growth of host strain under
low-dissolved-oxygen conditions, enhanced the recombinant proteins production
and reduced the accumulation of acetate harmful to bacterial growth. In
conclusion, the novel engineering strain G830 was adoptable to high cell
density fermentation.
Key words high-cell-density fermentation； Vitreoscilla haemoglobin； thronine operon； acetate metabolism pathway； prolyl endopeptidase
*Corresponding author： Tel, 86-21-64700892-371； Fax, 86-21-64700244； e-mail, [email protected]