Направленное изменениеEscherichia coliMG1655 с целью получения мутантов, продуцирующих гистидин

Abstract

Strain MG 1655+hisGr hisL'-Delta, purR, which produces histidine with a weight yield of approximately 12% from glucose, was constructed through directed chromosomal modifications of the laboratory Escherichia coli strain MG 1655+, which has a known genome sequence. A feedback-resistant ATP-phosphoribosyl transferase encoded by the mutant hisGr (E271 K) was the main determinant of histidine production. A further increase in histidine production was achieved by the expression enhance of a mutant his operon containing hisGr through the deleting attenuator region (hisL'-Delta). An increase in the expression of the wildtype his operon did not result in histidine accumulation. Deletion of the transcriptional regulator gene purR increased the biomass produced and maintained the level of histidine production per cell under the fermentation conditions used.

Specific features of l-histidine production by Escherichia coli concerned with feedback control of AICAR formation and inorganic phosphate/metal transport

“…One of the obtained Cm R -ductants, an MG1655-derivative that had earlier passed through several stages of selection for increased His overproduction, was used in the present study as the donor for P1-duction of the purA -carrier marked cassette. This cassette was P1-duced into the His-overproducing strain KF37 [ 13 ], followed by evaluation of His accumulation by the obtained Cm R -ductants in test tube fermentations. Two groups of P1-ductants were selected according to their ability to overproduce His.…”

“…His overproduction cannot be engineered solely by enabling the resistance of the first biosynthetic enzyme, HisG, to feedback inhibition (see e.g., [ 13 ]). The effective metabolism of AICAR to ATP is also required to prevent the undesirable accumulation of AICAR, which is a HisG inhibitor, and to enhance the supply of ATP, which is an essential component for the synthesis of this amino acid.…”

“…Feedback-resistant HisG-mutant enzymes from S. typhimurium [ 9 ] and C. glutamicum [ 10 , 11 ] have been previously characterized. The HisG E271K , Fbr-mutant from E. coli was obtained by traditional selection methods several decades ago [ 12 ], and its properties were later investigated [ 13 ]. Despite having complete resistance to feedback inhibition by His, this mutant enzyme is still susceptible to competitive inhibition by purine nucleotides, particularly ADP and AMP [ 12 ].…”