Pyrimidine nucleoside high-yielding strain and carbamyl phosphate synthetase adjusting site thereof

Abstract

The invention belongs to the technical field of enzyme engineering, and concretely relates to a pyrimidine nucleoside high-yielding strain and a carbamyl phosphate synthetase adjusting site thereof. The invention provides a pyrimidine nucleoside production Bacillus subtilis mutant strain and a carbamyl phosphate synthetase encoding gene. A key regulation site related to carbamyl phosphate synthetase end product feedback inhibition is defined, and provides reference for breeding of later pyrimidine nucleoside high-yielding strains. The Bacillus subtilis mutant strain allows the output of fermentation process produced nucleoside pyrimidine uridine to reach 14-16g / L, and has substantially improved tolerance to different pyrimidine nucleoside structure analogs.

Description

Technical field: [0001] The invention belongs to the technical field of enzyme engineering, and specifically relates to a high-yield pyrimidine nucleoside strain and its carbamoyl phosphate synthetase regulatory site. Background technique: [0002] Pyrimidine nucleosides include uridine and cytidine, which are important components of all organisms. They are widely used, especially as intermediates of antiviral and antitumor drugs, and occupy an important position in the field of medicine. The production methods of pyrimidine nucleosides include chemical synthesis, RNA hydrolysis and microbial fermentation. Based on the advantages and disadvantages of several methods, microbial fermentation has the advantages of short cycle, easy control, high yield and low pollution. The potential of industrialization is therefore increasingly valued by people. [0003] The breeding of pyrimidine nucleoside-producing strains began in the 1960s. The breeding of pyrimidine nucleoside high-yie...

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Problems solved by technology

[0009] The difficulty in the breeding of pyrimidine nucleoside high-yielding strains is to release the feedback repression and feedback inhibition of the final product, especially the release of feedback inhibition. Although researchers have revealed the specific regulation mechanism of pyrimidine nucleoside synthesis and metabolism, and from the molecular level The mechanism by which the pyrimidine operon is repressed by the end product is elucidated, but the specific regulatory site of the carbamoyl phosphate synthase that is repressed by the end product feedback is not clear

[0010] With the application of molecular biology in microbial breeding, relevant researchers have also tried to use PCR to introduce point mutations to release the feedback inhibition of carbamoyl phosphate synthase, but due to the structure and function of carbamoyl phosphate synthase The research is not too in-depth. The method of introducing point mutations by PCR is limited in the application of releasing the feedback inhibition of carbamoyl phosphate synthase. The effect of the reported site on the release of feedback inhibition is not too obvious.

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