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Previously, we established an easy and quick construction method for obtaining a stable and highly productive gene-amplified recombinant Chinese hamster ovary (CHO) cell line. With a gradual increase in methotrexate (MTX) concentration, gene-amplified cell pools had high and stable specific growth and production rates. Moreover, the phenotype of gene-amplified cells seemed to be affected by the location of the amplified gene in chromosomal DNA. We suspected that various kinds of gene-amplified cells might appear during the long-term selection to construct gene-amplified cell pools. To clarify the behavior of gene-amplified cell pools during a stepwise increase of MTX concentration, we isolated gene-amplified clones derived from gene-amplified cell pools. We compared the characteristics of isolated clones, such as the productivity of recombinant protein, stability of amplified genes, and the location of amplified genes. As a result, telomere-type clones, in which the amplified gene was located near the telomeric region, were found to be more stable and productive than other types of clones. Telomere-type clones had over 100 copies of amplified genes in the chromosomal DNA. In contrast, a large number of other types of clones had less than 10 copies of amplified genes. During long-term cultivation in the absence of MTX, in other types of clones, amplified genes rapidly decreased in the chromosomal DNA.

During fed-batch cultivation of a recombinant Escherichia coli AT2471 harboring plasmid pSY130-14 for phenylalanine production, a large amount of acetic acid was excreted by the cells and accumulated in the culture medium. Acetic acid concentration reached 30-35 g/L at the end of a process conducted without special precautions for the reduction of this excretion. Cell growth stopped when acetic acid concentration was about 15 g/L, resulting in poor growth, 16 g/L cell concentration, and poor production - 8 g/L phenylalanine. A novel control strategy, called a balanced DO-stat. was developed to prevent acetic acid excretion. It represents a model-independent two-loop control structure, which is simple, reliable, and convenient for computer application. Using the balanced DO-stat, implemented in a computer control system, acetic acid concentration was kept at zero during the entire cultivation period. As a result, the cell concentration increased to 36 g/L and phenylalanine concentration reached 24 g/L. Aside from the phenylalanine fermentation, the proposed control approach might be applied to cultivation of other bacterial and yeast strains which have similar mechanism of the excretion of fermentative by-products.

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