Improvement of uridine production of Bacillus subtilis by atmospheric and room temperature plasma mutagenesis and high-throughput screening

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.

Insights into the molecular-level effects of atmospheric and room-temperature plasma on mononucleotides and single-stranded homo- and hetero-oligonucleotides

“…More and more experimental results have also showed that the ARTP as a new kind of physical mutation tool has a bright prospect for the genome mutation of the organisms with rapid mutation, high diversity of mutants and high safety 1,4 . Up to now, ARTP as a novel powerful mutagenesis method has been successfully used to mutation breeding of more than 110 kinds of microorganisms and plants as well animals [19][20][21][22]25 . Generally, mutagenesis of organisms is a complex process, dependent on the cellular DNA damage strength and the subsequent repair system of damaged DNA, which allows mutation to occur.…”

“…1). We have employed this system to mutate the genomes of various microbes and plants as well as animals, including bacteria, microalgae, fungi, and yeast 1,4,[19][20][21][22][23][24] . The results of those studies indicated that ARTP mutation is a rapid, effective, convenient, and multifaceted means of generating mutant libraries with sufficient diversity for the improvement of phenotypes.…”

Improved Neomycin Sulfate Potency in Streptomyces fradiae Using Atmospheric and Room Temperature Plasma (ARTP) Mutagenesis and Fermentation Medium Optimization

“…This incurs DNA damage which is subsequently repaired by the SOS ("Save Our Soul") system leading to DNA alterations [8,9]. ARTP technology generates high mutation frequencies, is simple and safe, and is widely used for bacteria [10][11][12][13], fungi [14,15], and microalgae [16][17][18]. On the other hand, mutations in strains due to streptomycin resistance are closely related to their ability to produce antibiotics [19].…”

Improving the Level of the Tyrosine Biosynthesis Pathway in Saccharomyces cerevisiae through HTZ1 Knockout and Atmospheric and Room Temperature Plasma (ARTP) Mutagenesis

“…Atmospheric and room temperature plasma (ARTP) mutagenesis can induce breakage in plasmid DNA and oligonucleotides through treatment with a diverse range of plasma doses . Compared to conventional mutation methods, ARTP mutagenesis provides highly homogeneous plasma jets under mild conditions, induces mutations at a higher rate, and is environmentally friendly, efficient, and easy to operate. − Recently, this method has been applied on multiple microorganisms to obtain targeted phenotypes. , Fan et al treated Bacillus subtilis with ARTP and obtained a high uridine-producing strain, in which production was 8.7-fold higher than the parent strain . This technology has also been applied on yeast.…”

“…18,19 Fan et al treated Bacillus subtilis with ARTP and obtained a high uridine-producing strain, in which production was 8.7-fold higher than the parent strain. 20 This technology has also been applied on yeast. Luo et al used this mutagenesis method along with a screening strategy to screen pyruvate-producing Candida glabrata mutant strains and obtained 35.4% higher titer than that obtained from the wild-type strain.…”