Microbiology & Immunology - Theses

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Controlled production of tryptophan by genetically-manipulated strains of Escherichia coli

Cowan, Peter J. ( 1992)

The tryptophan productivity of the genetically-manipulated strain JP4153 was increased 2.5-fold by introducing pMU78, a medium copy-number plasmid carrying a feedback-resistant trp operon. JP4153(pMU78) produced 23.5 g/l of tryptophan at a rate of 0.7 g/l/h when grown at 37 degrees C in a defined glucose and ammonium salts medium in a bench-scale fermentor. During prolonged cultivation in the presence of antibiotic, the recombinant strain generated faster-growing, production-defective variants, which harboure mutated derivatives of pMU78. Insertion sequences were responsible for the two predominant types of mutation. The plasmid element ISI02 mediated deletions extending into the promoter-proximal region of the plasmid-borne trp operon. ISI0-Right, a chromosomal element, inserted into the promoter/trpE region of the plasmid. Three methods were employed to increase the structural stability of JP4153(pMU78) during the course of the production process. First, the growth of seed cultures was carried out at 30 degrees C, the permissive temperature for the trpS378 mutation carried by the host strain. Second, the seed culture medium was modified by the addition of yeast extract, which appeared to reduce the selective disadvantage conferred by the plasmid. Third, ISI02was deleted from pMU78 to create pMU88. (For complete abstract open document)