| In this work, the productions of human epidermal growth factor (hEGF) as well as penicillin acylase (PGA) by recombinant microorganisms were selected as examples for solving several key problems in the cultivation, such as plasmid stability, inhibition by metabolic by-products and oxygen supply in high density cultivation. The purpose is to enhance the product expression level with various new engineering aspects.1. In-situ adsorption of acetic acid via macro-pore ion-exchange resin during recombinant E. coli JM101 cultivation for hEGF productionAcetic acid is one of the major by-products during E. coli recombinants cultivation, which will inhibit the cell growth and product expression. The effects of acetic acid and sodium acetate additions on the E. coli JM101 growth and hEGF expression were evaluated. It was found that, with the addition of sodium acetate, the lag-phase of cell growth was lengthened. Increasing the sodium acetate concentration would cause the stronger inhibitory effect for cell growth. When sodium acetate concentration reached 30g-L-1, the growth of E. coli JM101 was completely inhibited, whereas, only 20g-L-1 was high enough for the complete inhibition of hEGF expression.Based on the physical and chemical properties, one anion-exchange resin and one cation-exchange resin, with high ion-exchange capability of acetic acid, were screened from various resins. The results showed that the addition of l.Og resin per 30ml medium was insignificant for the cell growth, however, could improve the hEGF expression.The addition of ion-exchange resin into the culture medium was able to reduce the acetic acid concentration in the medium because of in-situ separation by resin, which was favorable for the cell growth and hEGF expression. The experimental data of batch cultivation in a B. Braun 2.5 L fermenter, coupled with in-situ separation of acetic acid via ion exchange, showed that the cultivation period was shortened by 2 hours and the expression level of hEGF reached as high as 225mg-L-1.2. Three-phase fluidized-bed bioreactor for the PUF immobilized E.coli JM101 (pWKW2) cultivation and hEGF productionThe immobilization of genetic microorganisms was preferred for higher plasmid stability as well as cell density. A new immobilization material, polyurethane foam (PUF), which is sterizable, easy for cell immobilization, cheap in price and low in mass transfer resistance, was adopted to immobilize recombinant E.coli JM101. The experimentalresults from shake-flask cultivation showed that both cell density and hEGF expression were improved greatly. A new type three-phase fluidized-bed bioreactor for PUF immobilized cells was developed. The effects of aeration rate and PUF particle amount were examined, and the results indicated that, for a 0.8L reactor with 500ml medium, the highest hEGF was observed with aeration rate of 3vvm and total PUF particles of 5.0 g. A mathematical model considering plasmid stability was proposed to describe the growth of both free- and immobilized-cells and the expression of hEGF during batch cultivation in the three-phase fluidized-bed bioreactor. The agreement between the experimental data and the calculated results indicated that the model was able to describe the bioprocesses of hEGF culture.A new two-stage cultivation strategy was applied to maintain the plasmid stability and to increase the hEGF expression level. In the first stage, thee three-phase fluidized-bed bioreactor was applied, and high antibiotics selective pressure was maintained to keep rapid growth of plasmid-carrying cells. The cell immobilization with PUF matrix also increased the plasmid stability. The free-cells produced in the first stage was pumped into the second stage, which is a bubble column for hEGF expression induced by IPTG. The effect of dilution rate on the two-stage process was evaluated. The results indicated that, with the increase in dilution rate, the free cell density was reduced, but plasmid stability and substrate concentration increased, in the first reactor. In t...
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