Abstract
In the present study, a novel breeding strategy of atmospheric and room temperature plasma (ARTP) mutagenesis was used to improve the uridine production of engineered Bacillus subtilis TD12np. A high-throughput screening method was established using both resistant plates and 96-well microplates to select the ideal mutants with diverse phenotypes. Mutant F126 accumulated 5.7 and 30.3 g/L uridine after 30 h in shake-flask and 48 h in fed-batch fermentation, respectively, which represented a 4.4- and 8.7-fold increase over the parent strain. Sequence analysis of the pyrimidine nucleotide biosynthetic operon in the representative mutants showed that proline 1016 and glutamate 949 in the large subunit of B. subtilis carbamoyl phosphate synthetase were of importance for the allosteric regulation caused by uridine 5′-monophosphate. The proposed mutation method with efficient high-throughput screening assay was proved to be an appropriate strategy to obtain uridine-overproducing strain.
Highlights
Uridine and its nucleotide derivatives play an important role in the biochemical and physiological processes including the synthesis of DNA, RNA, membrane constituents and glycosylation [1]
The parent strain B. subtilis TD 12np was previously constructed in Tianjin university [12]. This strain was derived from B. subtilis 168 by modification of a series of genes involved in pyrimidine nucleoside biosynthesis
When the cells suspension was treated with ARTP for 10, 30, and 45 s, the lethality rates increased to 60.7%, 92.7% and 99.5% respectively (S2 Fig)
Summary
Uridine and its nucleotide derivatives play an important role in the biochemical and physiological processes including the synthesis of DNA, RNA, membrane constituents and glycosylation [1]. Uridine has been increasingly used as precursors for antivirus and antitumor drugs in pharmaceutical industry [2, 3]. Chemical synthesis is the commercial method for uridine production by the condensation reaction of uracil and D-ribose, the expensive raw materials increase the production cost. Other methods such as enzymatic decomposition of ribonucleic acid and salvage synthesis from uracil or orotic acid are used for uridine production [4].
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