A pool of the mutant genes carB has been obtained by a partial randomization of 15-nucleotide fragment of the gene that encodes a segment of the CPSase protein mol. from Leu947 to Glu951.The fbr-variants of the enzyme with different levels of specific activity (up to the level exceeding that of the wild-type CPSase) were selected as a result of screening the recombinant clones.It was established that the amino acid sequence from 947-951 residues determines the intensity of the enzyme inhibition by UMP, effects catalytic activity, and to some extent influences the specific activity of CPSase toward glutamine or ammonium in the mutant strain.In Escherichia coli strains producing orotic acid and arginine, the expression of mutant CPSase gene insensitive to retroinhibition leads to an increased synthesis of the products of pyrimidine and arginine metabolic pathways.

Biotekhnologiya

Insertion of the symmetrical O_lac-ideal between the "-35" and "-10" regions of the hybrid P_trc/O_lac promoter leads to a significant increase in the efficiency of the LacI-driven repression

Author: Skorokhodova, A. Yu. ; Biryukova, I. V. ; Mashko, S. V. ; Gulevich, A. yu. ; Zimenkov, D. V. ; Minaeva, N. I.

The present work is the continuation of the earlier published investigations concerning modification of the well-known E. coli hybrid promoters, P_trp-P_lacUVS/O_lacs, due to the insertion of the perfectly sym. artificial O_lac-ideal operator in the promoters.That time the work was aimed at increasing in LacI-driven repression efficiency of the new regulatory elements, with the simultaneous retaining their high promoter strength.As previously, the transcription efficiency of the new promoters under various conditions was evaluated using the lacZ as a reporter gene.The expression efficiency of lacZ reporter gene was estimated in E. coli-Δlac strain (the maximum induction of the O_lac-bearing promoters) as well as in the cells of LacI-superproducing strain (lacl^Q) grown in the absence (the repression conditions) or in the presence (partial induction) of IPTG in a culture medium.P_trc/O_lac and earlier obtained on its basis O_lac-ideal-P_trc/O_lac were used as precursors for the new hybrid promoters.The latter were obtained by the insertion of the ideally sym. operator in the (-92)-(-73) region upstream of the transcription start so that the distance between the centers of O_lac-ideal and O_lac was 92.5 bp.The initial promoters were modified in order to obtain 19 from 20 bp between the conventional ≪-35≫ and ≪-10≫ regions of O_lac-ideal.Another two initial lactose operators were saved (O_lac-ideal-P_trc/O_lac-ideal/O_lac) or deleted (O_lac-ideal-P_trc/O_lac-ideal or P_trc/O_lac-ideal/O_lac).Three new hybrid promoters were as strong as the initial P_trc/O_lac under the conditions of the derepression, and they were repressed by LacI much more efficiently, with the coefficient of repression even exceeding that of the native O₃-P_lacUV5/O_lac.O_lac-ideal-P_trc/O_lac-ideal and O_lac-ideal-P_trc/O_lac-ideal/O_lac were the most tightly repressed, whereas their transcription efficiencies were no higher than 88% and 80% of the maximum values, resp., upon the partial induction by IPTG.The possible perspectives of the practical applications of the new regulatory elements in biotechnol. are discussed.

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