Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1997-06-30
1998-11-17
Prouty, Rebecca E.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
4351721, 43525233, 4352528, 435849, 935 73, C12P 2106, C12N 1500, C12N 120
Patent
active
058374949
DESCRIPTION:
BRIEF SUMMARY
This application is the National Stage Application of PCT/0186, filed Oct. 29, 1996, published as WO97/16530 May 9, 1997, which claims priority from application 1995-3, filed on Oct. 30, 1995 in the Republic of Korea.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is related to a novel E. coli mutant strain with suppressed production of fermentative organic acids (especially acetate) during aerobic growth in glucose-supplemented medium. Reduction of the organic acids production of this mutant strain allows it to be a good host strain for recombinant protein production using high-cell-density aerobic fermentation.
2. Description of the Prior Arts
In order to produce useful recombinant proteins, E. coli is widely used as a host strain for high-cell-density aerobic fermentation. A substantial amount of glucose is added into the growth medium for the high-density growth of the host cells as well as for good expression of the recombinant gene, since it is an inexpensive and readily utilizable carbon and energy source. The major problem in the high-cell-density aerobic fermentation is production of fermentative acidic by-products of which acetate is the most predominant. Production of the acidic by-products, especially acetate, is a major factor in the limitation of high-cell-density growth and thereby the production of recombinant protein production (Han et al., Biotechnol. Bioeng., 39, 663 (1992); Luli et al., Appl. Environ. Microbiol., 56, 1004 (1990)).
In order to solve these problems, fed-batch culture technologies (Fieschko et al., Chem. Eng. Commun., 45, 2875 (1986); Ohta et al., J. Ferment. Bioeng., 75, 155 (1993); Yang, J. Biotechnol., 23, 271 (1992)), methods of removing organic acids produced from the culture (Landwall et al., J. Gen. Microbiol., 103, 345 (1977); Meyer et al., Proceedings of the 3rd European Congress on Biotechnology, (1984); MacDonald, Appl. Environ. Microbiol., 56, 640 (1990)), or changing media compositions (Holmes, Curr. Topics Cell. Regul. 28, 69 (1986); Han et al., Biotechnol. Bioeng., 41, 316 (1993); Reihng, J. Biotechnol. 2, 191 (1985); Mor et al., J. Ferment. Technol., 50, 519 (1972)) has been used. However, these methods also have limitations such that the slow growth rates and metabolically less active cultures resulting by these culture conditions often produce lower recombinant protein yields, that the control of complicated nutrient feeding is cumbersome and prone to errors (Chou et al., Biotechnol. Bioeng., 44, 952 (1994)), and that the salts added to the culture medium for pH control often cause inhibition of host growth and/or product production (Jensen et al., Biotechnol. Bioeng., 36, 1 (1990)).
For the glucose metabolism in E. coli strains under aerobic conditions, the carbon flow exceeding the capacity of TCA cycle, is converted to acetic acid which is excreted outside the cell (Majewski & Domach, Biotechnol. Bioeng., 35, 732 (1990)). The excreted acetic acid inhibits the growth of the host strain and the production of the desired recombinant protein. Acetate is formed from acetyl coenzyme A by the consecutive action of phosphotransacetylase (pia) and acetate kinase (ack). It was shown that mutational inactivation (by deletion of both pts and ack genes) of the acetate-forming enzymes produces a reduced, but still a significant amount of acetate. The mutation also caused lactate and pyruvate to accumulate(Diaz-Ricci et al., Biotechnol. Bioeng., 38, 1318 (1991)) to the level higher than its parental strain. These results suggest that there is an alternative pathway for acetate production and that inactivation of known acetate biosynthetic pathways redirects the carbon flow toward other organic acid.
Therefore, it had been needed a continued research to develop a new host strain deficient in production of organic acids overall and thereby increasing the yield of desired recombinant proteins.
SUMMARY OF THE INVENTION
Thus, an object of the present invention is to provide a new strain of E. coli, of which organic acid production is sup
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Choi Han
Jung Il Lae
Lee Jong Ho
Na Doe Sun
Park Young Min
Hobbs Lisa J.
Korea Green Cross Corporation
Prouty Rebecca E.
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