Mutation Breeding Of Uridine-producing Strains By Method Combining Atmospheric And Room Temperature Plasma And High-throughput Screen

Uridine is widely used in food,health care products.cosmetics and pharmaceutical industry.Particularly,it has important applications as raw material for the synthesis of some antivirus and antitumor drugs in the pharmaceutical industry.Producing of uridine by microbial fenrmentation has many advantages,but there are no high yield strains applied to this technology for industrial production.Undoubtedly,a release from regulation of carbamoyl phosphate synthetase(CPS)is the most critical factor to the accumulation of a large amount of uridinc in culture.To obtain mutant strains with higher uridine production in an efficient and rapid manner.the protocol for rapid mutation by a novel mutagenesis technique with atmospheric and room temperature plasma(ARTP)and high-throughput screening(HTS)method of the mutants using the 96-well microplatc and microplate reader was established in this study.Acquiring mutants subjected to high concentration of pyrimidine analogs was used to be a pre-selection step in the process of the protocol.As a ressult,four mutant strains,B.subtilis A219,B.subtilis A260,B.subtilis A566 and B.subtil is F126,were isolated.Compared with the original strain,B.subtilis TD131,which accumulated 3.5 g/L of uridine in a 5L fermentor under unoptimized conditions,the four mutants had significant improvement in uridine productivity,which amounted to 12 g/L,14.5 g/L,16 g/L and 18 g/L respectively at the same conditions.With good ability of uridine-producing and genetic stability,they own great potential for industrial application.Sequence analysis of the pyrimidine nucleotide biosynthetic operon from the original strain and the four mutants indicated that two vital mutations on the large subunit of CPS,P1016L and E949*,might be the key elements that contribute to increase in uridine production by relieving UMP's feedback inhibition of CPS.The approaches and strategies involved in this paper as well as the discovery of two key control sites of CPS open up new perspectives for combinatorial strategies to derive B.subtilis strains with obviously improved uridine production or other nucleotides-producing microbes.