High Cell-density Cultivation Of Recombinant Escherichia Coli

In present paper, the fermentation culture medium and condition for recombinant Escherichia coli(pRIL+pET28-pfa) have been optimized. The optimum medium was composed as follows (in g/L): glucose 10.0, tryptone19.5, yeast extract 11.5, NaCl 0.5, (NH4)2SO4 4.0, K2HPO4·3H2O 18.0, KH2PO4 3.0, MgSO4 1.2, trace element solution 1.0mL. The optimized operational conditions were temperature 37℃, working volume 35mL in a 250mL flask, initial pH7.1, inoculating age 8h and inoculums quantity with 1.0%. When the absorptance A600 of broth given by the recombinant stain up to 7.0, lactose was added so as to the final concentration to 1 g/L, and induction was conducted for 8h at 37℃, theα-amylase activity reached the maximum to 210U/mL and the cell density (at A600) did 12.8, which is almost 3.1 times and 14.9 times of those under the initial fermentation conditions, respectively.Fed-batch process for E. coli(pRIL+pET28-pfa) was carried out in a 15L automatic fermentor. Several feeding strategies, i.e., pH-stat feeding, exponential feeding and the combination, were comparatively investigated. The results indicated that the combined strategy was adoptable. The higher cell density and enzyme activity were obtained with 62.7g/L(by dry cell weight, DCW) and 700 U/mLα-amylase.The potential effect of anaerobic metabolism pathway naturally appeared in E. coli on high cell-density cultivation was investigated. Three gene clusters (pflA/B, pflC/D, pflE/F) encoding pyruvate-formate lyase were disrupted and relative mutants (pflA/B?, pflC/D? and pflE/F?) were developed. By using these mutants as a host cell, the recombinant enzyme activity was significantly increased. On the other hand, comparison to the parent, mutants formed less acetate and pflA/B? mutant did only 30%. However, at the same time, lactate was accumulated with elevated level.