L-tryptophan production by regulatory mutants of escherichia coli

Abstract

For the purpose of high production of L-tryptophan using Escherchia coli, various mutant strains were developed and optimal fermentation conditions were investigated. Recombinant microorganism, E. coli W3110tnaΔtrpEA[pCRT185], accumulated about 0.03 g/l of L-tryptophan after cultivation for 38 hr in a jar fermentor. Since only small amount of L-tryptophan was produced by this recombinant cell may be due to the complex regulatory function in L-tryptophan biosynthesis, analogue resistant mutants were derived from the prototype E. coliW3110. Among various analogues tested 5-methyltryptophan, 5-fluorotryptophan, β-thienylalanine were found to be very effective and these analogues were used to release the feedback inhibition and the feedback repression of tryptophan biosynthesis by the end products of aromatic amino acid pathway. Mutant strain SB 1007 resistant to these analogues was isolated and strain SB 1007 produced 0.76 g/l of L-tryptophan in the test tube culture. Furthermore in order to increase the metabolic flux of tryptophan specific pathway from chorismate, double auxotrophic mutant SE 2756 strain was derived from the strain SB 1007 by blocking the side-pathway to phenylalanine and tyrosine. SB2756 strain accumulated 2.6 g/l of tryptophan in the test tube culture. The effects of the precursors on L-tryptophan production were examined using mutant strain SB 1007. Both anthranilate and indole inhibited the cell growth and production of L-tryptophan reduced. Therefore various surfactants have been used as reservoirs of precursors. Among the nonionic surfactants tested, Triton X-100 was found to be very effective for L-tryptophan production. When the strain SB 1007 was cultivated in shake-flask in the fermentation medium containing 1.0 g/l of indole and 2% (volume%) of Triton X-100, L-tryptophan production was increased about 2.5 times compared with control value. The effect of oxygen supply and of the temperature on L-tryptophan production were investigated ...