Quantitative evaluation of DNA damage caused by atmospheric and room-temperature plasma (ARTP) and other mutagenesis methods using a rapid umu-microplate test protocol for microbial mutation breeding

Mutagenesis is an important technique for microbial mutation breeding.As the source of mutations,DNA damage extent is a key indicator for the effectiveness of mutagenesis.Therefore,a rapid and easy DNA damage quantification method is required for the comparison of mutagenesis effects and development of mutagenesis tools.Here,we used the umu-microplate test system to quantitatively compare the DNA damage strength caused by atmospheric and room-temperature plasma(ARTP)and other traditional mutagenesis methods including:ultraviolet radiation(UV),diethyl sulfate(DES)and 4-nitroquinoline-1-oxide(4-NQO).The test strain of Salmonella typhimurium TA1535/pSK1002 was used to monitor the time-course profile of b-galactosidase activity induced by DNA damage caused by different mutagenesis methods using a microplate reader.The umu-microplate test results showed that ARTP caused higher extent of DNA damage than UV and chemical mutagens,which agrees well with the result obtained by SOS-FACS-based quantification method as reported previously.This umu-microplate test is accessible for broad researchers who are lack of the expensive FACS instruments and allows the quick quantitative evaluation of DNA damage among living cells for different mutagenesis methods in the study of the microbial mutation breeding.

Combination of ARTP mutagenesis and color-mediated highthroughput screening to enhance 1-naphthol yield from microbial oxidation of naphthalene in aqueous system

Strain QCG of the aerobic bacteria Bacillus cereus is capable of producing l-naphthol from naphtha-lene,this strain was first isolated and characterized in this study.Strain QCG was mutagenized to enhance l-naphthol production,using atmospheric and room temperature plasma(ARTP)technology.Then,a microbial clone screening system was used to accelerate the operation.Meanwhile,a novel color-mediated high-throughput screening using 4-aminoantipyrine was performed to screen mutants.The optimal mutant strain QCG4 produced 19.58土0.34 mg·L-1-naphthol from naphthalene that was 47.32%higher than that of the original strain(13.29+0.28 mg·L-1).In addition,the optimal conditions for l-naphthol production via whole-cell catalysis of strain QCG4 were determined to be an OD600 of40,150 mg.L I naphthalene,and 7.5%dimethyl formamide as a co-solvent at pH 7.5 and 26℃ for 3 h,resulting in 41.18士0.12 mg·L-l-naphthol,i.e.,the mutant strain produces a 2.1-fold higher yield compared to the original strain.

Genomic and metabolomic analysis of Bacillus licheniformis with enhanced poly-γ-glutamic acid production through atmospheric and room temperature plasma mutagenesis

Poly-γ-glutamic acid is an extracellular polymeric substance with various applications owing to its valuable properties of biodegradability,flocculating activity,water solubility,and nontoxicity.However,the ability of natural strains to produce poly-γ-glutamic acid is low.Atmospheric and room temperature plasma was applied in this study to conduct mutation breeding of Bacillus licheniformis CGMCC 2876,and a mutant strain M32 with an 11%increase in poly-γ-glutamic acid was obtained.Genome resequencing analysis identified 7 nonsynonymous mutations of ppsC encoding lipopeptide synthetase associated with poly-γ-glutamic acid metabolic pathways.From molecular docking,more binding sites and higher binding energy were speculated between the mutated plipastatin synthase subunit C and glutamate,which might contribute to the higher poly-γ-glutamic acid production.Moreover,the metabolic mechanism analysis revealed that the upregulated amino acids of M32 provided substrates for glutamate and promoted the conversion between L-and D-glutamate acids.In addition,the glycolytic pathway is enhanced,leading to a better capacity for using glucose.The maximum poly-γ-glutamic acid yield of 14.08 g·L^(–1)was finally reached with 30 g·L^(–1)glutamate.