Atmospheric Room Temperature Plasma Research Articles

Improving the level of the cytidine biosynthesis in E. coli through atmospheric room temperature plasma mutagenesis and metabolic engineering.

Cytidine, as an important commercial precursor in the chemical synthesis of antiviral and antitumor drugs, is in great demand in the market. Therefore, the purpose of this study is to build a microbial cell factory with high cytidine production. A mutant E. coli NXBG-11-F34 with high tolerance to uridine monophosphate structural analogs and good genetic stability was obtained by atmospheric room temperature plasma (ARTP) mutagenesis combined with high-throughput screening. Then, the udk and rihA genes involved in cytidine catabolism were knocked out by CRISPR/Cas9 gene editing technology, and the recombinant strain E. coli NXBG-13 was constructed. The titer, yield, and productivity of cytidine fermented in a 5l bioreactor were 15.7g l-1, 0.164g g-1, and 0.327g l-1 h-1, respectively. Transcriptome analysis of the original strain and the recombinant strain E. coli NXBG-13 showed that the gene expression profiles of the two strains changed significantly, and the cytidine de novo pathway gene of the recombinant strain was up-regulated significantly. ARTP mutagenesis combined with metabolic engineering is an effective method to construct cytidine-producing strains.

Enhancing high-efficiency breeding and microbial microdroplet cultivation techniques for Ganoderma lucidum.

Ganoderma lucidum is known for its bioactive compounds, such as polysaccharides and triterpenoids, which are crucial in food and medicine. However, liquid fermentation encounters challenges in terms of strain differentiation and stability. In this research, we employed atmospheric room temperature plasma mutation and a microbial microdroplet culture system to identify strains with enhanced biomass and triterpenoid production. The three mutant strains, YB05, YB09, and YB18, exhibited accelerated growth rates and antagonized the initial strain G0023 more effectively than the controls. Notably, YB18 displayed the fastest growth, with a 17.25% increase in colony radius. Shake flask cultivation demonstrated that, compared with the initial strain, YB05 and YB18 had 26.33% and 17.85% greater biomass, respectively. Moreover, the triterpenoid production of YB05 and YB18 surpassed that of the control by 32.10% and 15.72%, respectively, as confirmed by colorimetric detection. Importantly, these mutant strains remained stable for five generations. This study revealed a comprehensive screening system utilizing atmospheric pressure, room temperature plasma mutation technology and microbial droplet cultivation. This innovative approach offers a promising pathway for obtaining advantageous Ganoderma strains for liquid fermentation. The methodology of atmospheric room temperature plasma mutation and microbial microdroplet culture systems is detailed for better comprehension.

Efficient Production of a Thermostable Mutant of Transglutaminase by Streptomyces mobaraensis.

The transglutaminase (TGase) from Streptomyces mobaraensis is widely used to improve the texture of protein-based foods. However, wild-type TGase is not heat-resistant, which is unfavorable for its application. In this study, we successfully constructed a S. mobaraensis strain that can efficiently produce TGm2, a thermostable mutant of S. mobaraensis TGase. First, S. mobaraensis DSM40587 was subjected to atmospheric room temperature plasma mutagenesis, generating mutant smY2022 with a 12.2-fold increase in TGase activity. Then, based on the double-crossover recombination, we replaced the coding sequence of the TGase with that of TGm2 in smY2022, obtaining the strain smY2022-TGm2. The extracellular TGase activity of smY2022-TGm2 reached 61.7 U/mL, 147% higher than that of smY2022. Finally, the catalytic properties of TGm2 were characterized. The half-life time at 60 °C and specific activity of TGm2 reached 64 min and 71.15 U/mg, 35.6- and 2.9-fold higher than those of the wild-type TGase, respectively. As indicated by SDS-PAGE analysis, TGm2 exhibited demonstrably better protein cross-linking ability than the wild-type TGase at 70 °C, although both enzymes shared a similar ability at 40 °C. With improved enzyme production and thermal stability, smY2022-TGm2 could be a competitive strain for the industrial production of transglutaminase.

Breeding a novel chlorophyll-deficient mutant of Auxenochlorella pyrenoidosa for high-quality protein production by atmospheric room temperature plasma mutagenesis

In the present work, a novel chlorophyll-deficient mutant of Auxenochlorella pyrenoidosa named A4-1 was generated by atmospheric room temperature plasma (ARTP) mutagenesis. Compared to the green wild type (WT) strain, the A4-1 mutant cultured in the dark displayed yellow colour with a 118-fold decrease of chlorophyll a and no detected chlorophyll b. Higher contents of protein (44.22 % DW), total amino acids (AAs, 34.84 % DW) and essential AAs (17.50 % DW) were also achieved, showing 31 %, 22 % and 30 % increases compared to the WT, respectively (p < 0.05). Metabolite profile analysis revealed that the chlorophyll biosynthesis pathway in the A4-1 mutant was probably inhibited in the dark, while more carbon skeletons might be utilized for de novo AAs synthesis. These results demonstrated that the A4-1 mutant not only has extremely low chlorophyll content, but also has higher protein content, making it a very promising candidate to produce microalgal protein for future foods.

Breeding of a thermostable xylanase-producing strain of Myceliophthora thermophila by atmospheric room temperature plasma (ARTP) mutagenesis.

Introduction: Hemicellulose is an important component in lignocellulose materials, which is second only to cellulose, accounting for 15%-35% of the dry weight of plants. In the current situation of energy shortage, making full use of lignocellulose materials to produce fuel ethanol has become an important way to solve the energy problem. Xylanase plays a crucial role in the utilization of hemicellulose. It is a necessary means to reduce the cost of hemicellulose utilization by improving the activity of xylanase. Moreover, most naturally xylanases are mesophilic enzymes, which limits their industrial application. Methods:In this study, Myceliophthora thermophila was used to produce xylanases and a thermostable mutant M 2103 was obtained by atmospheric room temperature plasma (ARTP) mutagenesis. The research work started with exploring the effects of ARTP mutagenesis on the antioxidase system [superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), polyphenol oxidase (PPO), and antioxidant capacity (AOC)] of M. thermophile, and found that superoxide dismutase activity increased by 221.13%, and polyphenol oxidase activity increased by 486.04% as compared with the original strain when the implantation time was 300s. So as to determine the conditions for subsequent mutagenesis. Results and Discussion:For the mutant M 2103, the reaction temperature for xylanase production remained stable in the range of 70°C-85°C. Its optimum temperature was 75°C, which was 15°C higher than that of the original strain. And its xylanase activity increased by 21.71% as compared with the original strain. M 2103 displayed a significantly higher relative xylanase activity than the original strain in the acidic (pH 4.0-7.0) range, and the xylanase activity was relatively stable in the pH range of 6.0-8.5. These results provide an alternative biocatalyst for the production of xylooligosaccharide, and a potential usage of ARTP in the mutagenesis of thermostable mutant.

ARTP mutagenesis of phospholipase D-producing strain Streptomyces hiroshimensis SK43.001, and its enzymatic properties

Phospholipase D (PLD) is a group of enzymes that act on phospholipid molecules, which is widely used in the fields of food and medicine. PLD is extracted from animals and plants with low transesterification activity and high price. Therefore, it is benefit to screen an efficient PLD producing strain from microorganisms. A highly productive strain of PLD with transphosphatidylation activity, named Streptomyces hiroshimensis SK43.001, was screened from soil in our laboratory and mutated using atmospheric room temperature plasma (ARTP). A mutant strain SK43.001-11 with the highest enzyme activity and superior genetic stability was obtained, and its fermentation enzyme activity was 5.3 U/mL, which was 82% increased comparing to wild strain. The purification of PLD showed that the specific enzyme activity increased to 49.48 U/mg, which was 54.37-fold higher than that of the crude enzyme, with a recovery of 32.31%. In addition, enzymatic properties of PLD have revealed that the optimal pH and temperature were 7.0 and 60 °C, respectively. Metal ion Mg2+ and surfactant Triton X-100 made the enzymatic activity increased by 16% and 100%, respectively. The reaction kinetic parameters showed that the mutant PLD had higher affinity for the substrate of egg PC and better catalytic efficiency with Km, Vmax and Kcat of 30.20 mmol/L, 99.70 μmol/min and 76.33 s−1, respectively. This study may provide important inspiration for obtaining high enzyme activity strains with PLD.

Improving tolerance and 1,3-propanediol production of Clostridium butyricum using physical mutagenesis, adaptive evolution and genome shuffling

Bioconversion efficiency of glycerol to 1,3-propanediol (1,3-PD) by Clostridium butyricum is bottlenecked by its low tolerance to various stressors, especially glycerol as the substrate, 1,3-PD as the end product, and butyric acid as a by-product, which eventually decreases 1,3-PD yield. This study aimed at improving the tolerance and 1,3-PD production capability of C. butyricum using random mutagenesis and evolutionary techniques. Mutagenesis of wild strain by atmospheric room temperature plasma (ARTP) provided the first population with maximum tolerance to 160 g/L glycerol, while microbial microdroplet culture system (MMC)-mediated adaptive laboratory evolution (ALE) generated the second population with tolerance to 100 g/L 1,3-PD. Subsequently, genome shuffling of both populations yielded a final strain, GJH-418, which generated 60.12 g/L1,3-PD with a productivity of 1.72 g/L/h. The transcript analysis of the mutant and wild strains revealed the possible involvement of 8 genes in high tolerance and high 1,3-PD production through either up- or down-regulation.

Combinatorial mutagenesis of Bacillus amyloliquefaciens for efficient production of protease

As an important industrial enzyme, protease is widely used in feed, food and other fields. At present, the insufficient protease activity obtained from microorganisms cannot meet the purpose of industrial production. In this study, Bacillus amyloliquefaciens with high protease production was screened from animal feces by plate transparent circle method. To improve the production of protease, atmospheric room temperature plasma (ARTP) mutagenesis was used in the first round, protease activity reached 315.0 U/mL. Then, to enhance production of protease, 60Co-γ irradiation was used for combined mutagenesis, leading to protease activity of B. amyloliquefaciens FMME ZK003 up to 355.0 U/mL. Furthermore, to realize the efficient production of protease, after optimization of fermentation conditions, protease activity was increased to 456.9 U/mL. Finally, protease activity of B. amyloliquefaciens FMME ZK003 reached 823.0 U/mL in a 5 L fermenter. These results indicate that B. amyloliquefaciens can efficiently produce protease, which provides a good foundation for the industrial production of protease.

Analysis of secondary metabolite gene clusters and chitin biosynthesis pathways of Monascus purpureus with high production of pigment and citrinin based on whole-genome sequencing.

Monascus is a filamentous fungus that is widely used for producing Monascus pigments in the food industry in Southeast Asia. While the development of bioinformatics has helped elucidate the molecular mechanism underlying metabolic engineering of secondary metabolite biosynthesis, the biological information on the metabolic engineering of the morphology of Monascus remains unclear. In this study, the whole genome of M. purpureus CSU-M183 strain was sequenced using combined single-molecule real-time DNA sequencing and next-generation sequencing platforms. The length of the genome assembly was 23.75 Mb in size with a GC content of 49.13%, 69 genomic contigs and encoded 7305 putative predicted genes. In addition, we identified the secondary metabolite biosynthetic gene clusters and the chitin synthesis pathway in the genome of the high pigment-producing M. purpureus CSU-M183 strain. Furthermore, it is shown that the expression levels of most Monascus pigment and citrinin clusters located genes were significantly enhanced via atmospheric room temperature plasma mutagenesis. The results provide a basis for understanding the secondary metabolite biosynthesis, and constructing the metabolic engineering of the morphology of Monascus.

Mutation breeding of Aspergillus niger by atmospheric room temperature plasma to enhance phosphorus solubilization ability.

BackgroundPhosphorus (P) is abundant in soils, including organic and inorganic forms. Nevertheless, most of P compounds cannot be absorbed and used by plants. Aspergillus niger v. Tiegh is a strain that can efficiently degrade P compounds in soils.MethodsIn this study, A. niger xj strain was mutated using Atmospheric Room Temperature Plasma (ARTP) technology and the strains were screened by Mo-Sb Colorimetry with strong P-solubilizing abilities.ResultsCompared with the A. niger xj strain, setting the treatment time of mutagenesis to 120 s, four positive mutant strains marked as xj 90–32, xj120–12, xj120–31, and xj180–22 had higher P-solubilizing rates by 50.3%, 57.5%, 55.9%, and 61.4%, respectively. Among them, the xj120–12 is a highly efficient P solubilizing and growth-promoting strain with good application prospects. The growth characteristics such as plant height, root length, and dry and fresh biomass of peanut (Arachis hypogaea L.) increased by 33.5%, 43.8%, 43.4%, and 33.6%, respectively. Besides available P, the chlorophyll and soluble protein contents also vary degrees of increase in the P-solubilizing mutant strains.ConclusionsThe results showed that the ARTP mutagenesis technology can improve the P solubilization abilities of the A. niger mutant strains and make the biomass of peanut plants was enhanced of mutant strains.

Multi-omics Comparative Analysis of Streptomyces Mutants Obtained by Iterative Atmosphere and Room-Temperature Plasma Mutagenesis.

Sponges, the most primitive multicellular animals, contain a large number of unique microbial communities. Sponge-associated microorganisms, particularly actinomyces, have the potential to produce diverse active natural products. However, a large number of silent secondary metabolic gene clusters have failed to be revived under laboratory culture conditions. In this study, iterative atmospheric room-temperature plasma. (ARTP) mutagenesis coupled with multi-omics conjoint analysis was adopted to activate the inactive wild Streptomyces strain. The desirable exposure time employed in this study was 75 s to obtain the appropriate lethality rate (94%) and mutation positive rate (40.94%). After three iterations of ARTP mutagenesis, the proportion of mutants exhibiting antibacterial activities significantly increased by 75%. Transcriptome analysis further demonstrated that the differential gene expression levels of encoding type I lasso peptide aborycin had a significant upward trend in active mutants compared with wild-type strains, which was confirmed by LC-MS results with a relative molecular mass of 1082.43 ([M + 2H]2+ at m/z = 2164.86). Moreover, metabolome comparative analysis of the mutant and wild-type strains showed that four spectra or mass peaks presented obvious differences in terms of the total ion count or extracting ion current profiles with each peak corresponding to a specific compound exhibiting moderate antibacterial activity against Gram-positive indicators. Taken together, our data suggest that the ARTP treatment method coupled with multi-omics profiling analysis could be used to estimate the valid active molecules of metabolites from microbial crudes without requiring a time-consuming isolation process.

Rapid improvement in the macrolactins production of Bacillus sp. combining atmospheric room temperature plasma with the specific growth rate index

Macrolactins (MLNs) have attracted considerable attention due to their antibacterial and antiviral properties. Here, the MLN production of Bacillus sp. strain IMDGX0108 was improved using a breeding strategy of atmospheric room temperature plasma (ARTP) technique. Combining with a selection procedure based on the colony morphology and specific growth rate index (SGRI), two genetically stable mutants A29 and A72 were identified. The MLN production of A29 and A72 was 35.2% and 52.8% greater than that of the parent strain, respectively. The best-performing mutant A72 was subjected to RNA-sequence analysis. Five pathways were significantly enriched, and fatty acid bioprocesses might play an important role in improving the production of MLNs. The combined strategy developed herein (i.e., ARTP mutation plus an efficient screening procedure) might be an appropriate method by which to obtain strains overproducing MLNs.

Development and application of a droplet-based microfluidic high-throughput screening of Pichia pastoris

Pichia pastoris is one of the most convenient and widely used heterologous protein expression systems. To further improve its ability to express heterologous proteins, we developed a high-throughput P. pastoris screening method based on droplet microfluidic and demonstrated the method by screening and obtaining mutants with enhanced xylanase expression and secretion abilities. We used PCR (Polymerase Chain Reaction) amplification to obtain a fusion fragment of xylanase xyn5 gene and green fluorescent protein gfp gene, and cloned this fragment into pPIC9K, the expression vector of Pichia pastoris, to construct the plasmid pPIC9K-xyn5-gfp that recombined the DNA fragments of xylanase and green fluorescent protein. After this plasmid entered P. pastoris GS115 by electroporation, the P. pastoris SG strain that could express xylanase and green fluorescent protein was obtained. The above-said strains were then mutagenized by atmospheric room temperature plasma and subsequently encapsulated to form single-cell droplets. After 24-hour cultivation of the droplets, microfluidic screening was carried out to obtain the mutant strain with high xylanase expression for further construction and screening of the next mutagenesis library. After five rounds of droplet microfluidic screening, a highly productive strain P. pastoris SG-m5 was obtained. The activity of the expressed xylanase was 149.17 U/mg, 300% higher than that of those expressed by the original strain SG. This strain's ability to secrete heterologous protein was 160% higher than that of the original strain. With a screening throughput of 100 000 strains per hour, the high-throughput P. pastoris screening system based on single-cell droplet microfluidic developed by the present study screens a library with million strains in only 10 hours and consumes only 100 μL of fluorescent reagent, thus reducing the reagent cost by millions of times compared with the traditional microplate screening and more importantly, providing a novel method to obtain P. pastoris with high abilities to express and secret heterologous proteins by efficient and low-cost screening.

Isolation of oleaginous yeast (Rhodosporidium toruloides) mutants tolerant of sugarcane bagasse hydrolysate

Rhodosporidium toruloides is a lipid-producing yeast, the growth of which is severely suppressed when hydrolysates of lignocellulosic biomass are used as carbon source. This is probably due to the toxic substances, such as organic acids, furans, and phenolic compounds produced during the preparation of the hydrolysates. In order to solve this problem, R. toruloides cultures were subjected to atmospheric room-temperature plasma mutagenesis, resulting in the isolation of mutants showing tolerance to sugarcane bagasse hydrolysate (SBH). Three mutant strains, M11, M13, and M18, were found to grow with producing lipids with SBH as carbon source. M11 in particular appeared to accumulate higher levels (up to 60% of dry cell weight) of intracellular lipids. Further, all three mutant strains showed tolerance of vanillin, furfural, and acetic acid, with different spectra, suggesting that different genetic determinants are involved in SBH tolerance.

Novel mutant strains of Rhodosporidium toruloides by plasma mutagenesis approach and their tolerance for inhibitors in lignocellulosic hydrolyzate

AbstractBACKGROUNDRhodosporidium toruloides can transform carbohydrates from lignocellulosic hydrolyzate into long‐chain fatty acids that contribute to biodiesel production. However R. toruloides cannot survive in lignocellulosic hydrolyzate due to the inhibitory effects of the byproducts co‐produced by hydrolysis.RESULTSTo circumvent the limitation, atmospheric room temperature plasma (ARTP) mutagenesis was utilized to obtain R. toruloides mutant strains M11, M14, and M18 that had strong tolerance for the inhibitory compounds and could grow in lignocellulosic hydrolyzate without detoxification. It was demonstrated that acetic acid and vanillin (phenolic compounds) were major inhibitors that decreased lipid productivity by 30%. Furthermore, acetic acid and vanillin changed the fatty acid composition of the lipids. Among the mutants, M18 exhibited the highest tolerance for all the inhibitory compounds and had near 50% lipid content.CONCLUSIONSThis work provides novel potential strains for biodiesel production using lignocellulosic biomass and a useful foundation for optimization of the pretreatment of lignocellulose. © 2013 Society of Chemical Industry

Surface modification of the nanoparticles by an atmospheric room-temperature plasma fluidized bed

Using hexamethyldisiloxane (HMDSO) monomer, the magnetic nanoparticles (NPs) of nickel oxide (NiO) were modified by using an atmospheric room-temperature plasma fluidized bed (ARPFB). The plasma gas temperature of the ARPFB was not higher than 325 K, which was favorable for organic polymerization. The plasma optical emission spectrum (OES) of the gas mixture consisting of argon (Ar) and HMDSO was recorded by a UV–visible monochromator. The as-treated NPs were characterized by means of scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS). The results show that the assembling NPs were isolated greatly after modified by the organosilicon polymer. Moreover, this treatment process changed the wettability of the NPs from super-hydrophilicity to super-hydrophobicity, and the contact angle (CA) of water on the modified NPs surface exceeded 150°. Therefore, the ARPFB is a prospective technology for the NPs surface modification according to the different requirements.