Atmospheric And Room Temperature Plasma Research Articles

Key role of K+ and Ca2+ in high-yield ethanol production by S. Cerevisiae from concentrated sugarcane molasses

BackgroundSaccharomyces cerevisiae is an important microorganism in ethanol synthesis, and with sugarcane molasses as the feedstock, ethanol is being synthesized sustainably to meet growing demands. However, high-concentration ethanol fermentation based on high-concentration sugarcane molasses—which is needed for reduced energy consumption of ethanol distillation at industrial scale—is yet to be achieved.ResultsIn the present study, to identify the main limiting factors of this process, adaptive laboratory evolution and high-throughput screening (Py-Fe3+) based on ARTP (atmospheric and room-temperature plasma) mutagenesis were applied. We identified high osmotic pressure, high temperature, high alcohol levels, and high concentrations of K+, Ca2+, K+ and Ca2+ (K+&Ca2+), and sugarcane molasses as the main limiting factors. The robust S. cerevisiae strains of NGT-F1, NGW-F1, NGC-F1, NGK+, NGCa2+ NGK+&Ca2+-F1, and NGTM-F1 exhibited high tolerance to the respective limiting factor and exhibited increased yield. Subsequently, ethanol synthesis, cell morphology, comparative genomics, and gene ontology (GO) enrichment analysis were performed in a molasses broth containing 250 g/L total fermentable sugars (TFS). Additionally, S. cerevisiae NGTM-F1 was used with 250 g/L (TFS) sugarcane molasses to synthesize ethanol in a 5-L fermenter, giving a yield of 111.65 g/L, the conversion of sugar to alcohol reached 95.53%. It is the highest level of physical mutagenesis yield at present.ConclusionOur results showed that K+ and Ca2+ ions primarily limited the efficient production of ethanol. Then, subsequent comparative transcriptomic GO and pathway analyses showed that the co-presence of K+ and Ca2+ exerted the most prominent limitation on efficient ethanol production. The results of this study might prove useful by promoting the development and utilization of green fuel bio-manufactured from molasses.

Unveiling elevated spontaneous mutation rates in Phyllostachys edulis (Moso Bamboo) through whole genome sequencing (WGS) and investigating the impact of atmospheric and room temperature plasma (ARTP) induced mutagenesis

Moso bamboo, recognized for its wide distribution and economic importance, encounters challenges in varietal enhancement owing to its protracted sexual reproduction cycle. This study employed whole-genome resequencing to identify spontaneous mutations in Moso bamboo and investigated mutagenesis using atmospheric and room temperature plasma (ARTP). Through the sequencing results, we identified a population of flowering bamboo as an asexual breeding line. Notably, the flowering Moso bamboo population, exclusively derived from asexual reproduction, exhibited a high spontaneous mutation rate (4.54 × 10−4 to 1.15 × 10−3/bp) during sexual reproduction, considering parental and cross-pollination effects. Genetic disparities between the offspring and parents exhibited a bimodal distribution, indicating a substantial cross-pollination rate. ARTP mutagenesis increased structural variations in offspring, whereas changes in SNPs and INDELs were less pronounced. Sanger sequencing validated the gene subset, providing a foundation for the investigation of spontaneous mutation rates via whole-genome sequencing. These insights, particularly from mutagenized offspring sequencing, can contribute to Moso bamboo breeding strategies.

Mutation breeding of high-stress resistant strains for succinic acid production from corn straw

The production of succinic acid from corn stover is a promising and sustainable route; however, during the pretreatment stage, byproducts such as organic acids, furan-based compounds, and phenolic compounds generated from corn stover inhibit the microbial fermentation process. Selecting strains that are resistant to stress and utilizing nondetoxified corn stover hydrolysate as a feedstock for succinic acid production could be effective. In this study, A. succinogenes CICC11014 was selected as the original strain, and the stress-resistant strain A. succinogenes M4 was obtained by atmospheric and room temperature plasma (ARTP) mutagenesis and further screening. Compared to the original strain, A. succinogenes M4 exhibited a twofold increase in stress resistance and a 113% increase in succinic acid production when hydrolysate was used as the substrate. By conducting whole-genome resequencing of A. succinogenes M4 and comparing it with the original strain, four nonsynonymous gene mutations and two upstream regions with base losses were identified.Key points• A high-stress-resistant strain A. succinogenes M4 was obtained by ARTP mutation• The production of succinic acid increased by 113%• The mutated genes of A. succinogenes M4 were detected and analyzedGraphical

Recent progress of atmospheric and room-temperature plasma as a new and promising mutagenesis technology.

At present, atmospheric and room-temperature plasma (ARTP) is regarded as a new and powerful mutagenesis technology with the advantages of environment-friendliness, operation under mild conditions, and fast mutagenesis speed. Compared with traditional mutagenesis strategies, ARTP is used mainly to change the structure of microbial DNA, enzymes, and proteins through a series of physical, chemical, and electromagnetic effects with the organisms, leading to nucleotide breakage, conversion or inversion, causing various DNA damages, so as to screen out the microbial mutants with better biological characteristics. As a result, in recent years, ARTP mutagenesis and the combination of ARTP with traditional mutagenesis have been widely used in microbiology, showing great potential for application. In this review, the recent progress of ARTP mutagenesis in different application fields and bottlenecks of this technology are systematically summarized, with a view to providing a theoretical basis and technical support for better application. Finally, the outlook of ARTP mutagenesis is presented, and we identify the challenges in the field of microbial mutagenesis by ARTP.

Optimizing mycelial protein yield in Pleurotus djamor via ARTP mutagenesis and hybridization strategies

With the world’s population rapidly increasing, the demand for high-quality protein is on the rise. Edible fungi breeding technology stands as a crucial avenue to obtain strains with high yield, high-quality protein, and robust stress resistance. To address the protein supply gap, Atmospheric and Room Temperature Plasma (ARTP) mutagenesis, and spore hybridization techniques were employed to enhance Pleurotus djamor mycelium protein production. Beginning with the original strain Pleurotus djamor JD-1, ARTP was utilized to mutate spore suspension. The optimal treatment time for Pleurotus djamor spores, determined to achieve optimal mortality, was 240 s. Through primary and secondary screenings, 6 mutant strains out of 39 were selected, exhibiting improved protein yield and growth rates compared to the original strain. Among these mutagenic strains, 240S–4 showcased the highest performance, with a mycelial growth rate of 9.5±0.71 mm/d, a biomass of 21.45±0.54 g/L, a protein content of 28.75±0.92%, and a remarkable protein promotion rate of 128.03±7.29%. Additionally, employing spore hybridization and breeding, 7 single-nuclei strains were selected for pin-two hybridization, resulting in 21 hybrid strains. The biomass and protein content of 9 hybrid strains surpassed those of the original strains. One hybrid strain, H-5, exhibited remarkable mycelial protein production, boasting a mycelial growth rate of 26.5±0.7 mm/d, a biomass of 21.70±0.46 g/L, a protein content of 28.44±0.22%, and a protein promotion rate of 128.02±1.73%. Notably, both strains demonstrated about a 28% higher mycelial protein yield than the original strains, indicating comparable effectiveness between hybrid breeding and mutagenesis breeding. Finally, we analyzed the original and selected strains by molecular biological identification, which further proved the effectiveness of the breeding method. These findings present novel insights and serve as a reference for enhancing edible fungi breeding, offering promising avenues to meet the escalating protein demand.

Multi-mode enhancement and Co-fermentation mechanism of 2-phenylethanol production by Kluyveromyces marxianus

As a valuable aromatic alcohol, 2-phenylethanol has been widely used in the fields of food, medicine and chemistry, but its production has become a key factor limiting microbial industrialisation. Therefore, atmospheric and room temperature plasma (ARTP) mutagenesis was applied to improve host cell tolerance of 2-phenylethanol, and 2-phenylethanol production was increased from 2.23 g/L to 2.51 g/L in Kluyveromyces marxianus. Fermentation conditions of K. marxianus and Pediococcus lactis co-culture were optimised and production of 2-phenylethanol was further increased by 42.63%, reaching 3.58 g/L. Metabonomics analysis revealed that the cell wall of P. lactis could effectively activate Ehrlich pathway-related metabolism and upregulate arginine synthesis pathways to enhance the cellular robustness of K. marxianus, thereby improving the efficiency of 2-phenylethanol synthesis, and providing a novel strategy for cost-effective 2-phenylethanol production.

Metabolome and ROAV analysis reveal the effective flavor improvement in the golden biomass of chlorophyll-deficient mutant of Auxenochlorella pyrenoidosa

The green microalga Auxenochlorella pyrenoidosa is realized a promising source of alternative protein due to its high protein content and fermentable property, but the deep green color and off-odor are the bottlenecks for its application in foods. Using atmospheric and room temperature plasma (ARTP) mutagenesis, a novel chlorophyll-deficient mutant, A4-1 strain with golden color and better flavor, was successfully obtained in our previous work. In this study, to reveal the formation of the improved flavor in A4-1 strain compared to the wild type (WT), the volatiles basis was analyzed by HS-SPME Arrow-GC-MS, and the potential precursors causing flavor improvement were explored by UPLC-ESI-MS/MS. Results showed the volatiles were mainly composed of hydrocarbons, terpenoids, esters, ketones, alcohols, and aldehydes, and 29 of which were significantly different between the two biomass, largely attributed to variations of primary metabolite content, particularly those enriched in the fatty acids and amino acids. The analysis of relative odor activity value (ROAV) verified that β-ionone, dimethyl trisulfide and (Z)-6-nonenal were the main odorants, in which the lower contributions of dimethyl trisulfide (off-flavor, rancid or cooked oniony) and (Z)-6-nonenal (off-flavor, stink or fishy) were found in A4-1, which made A4-1 biomass become more neutral and superior to WT biomass in terms of odor perception. It is the first report of flavor improvement for green microalgae through breeding technology, and the novel biomass of A4-1 strain will broaden A. pyrenoidosa biomass in food application.

Enhanced Heat Resistance in Morchella eximia by Atmospheric and Room Temperature Plasma.

This study focuses on optimizing the mutagenesis process for Morchella eximia (Mel-7) mycelia through atmospheric and room temperature plasma (ARTP) mutation and explores the resultant thermal adaptability and physiological responses of mutant strains. This research demonstrated a clear relationship between ARTP mutagenesis exposure duration and lethality rate, indicating that an exposure time of 40 s resulted in the optimal balance of inducing mutations without causing excessive mortality. Additionally, this study established 43 °C as the ideal screening temperature for identifying mutant strains with enhanced heat resistance, as this temperature significantly challenges the mycelia while allowing thermotolerant strains to be distinguishable. Among the screened mutants, strains L21, L23, L44, and L47 exhibited superior growth and high-temperature tolerance, with notable resilience at 30 °C, highlighting their enhanced adaptability to above-optimal temperatures. Furthermore, this research delved into biochemical responses, including lipid peroxidation and non-enzymatic antioxidant content, highlighting the diverse mechanisms, such as enhanced lipid peroxidation resistance and increased antioxidant content, employed by mutant strains to adapt to temperature fluctuations. The activities of antioxidant enzymes, including peroxidase (POD) and superoxide dismutase (SOD), were shown to be significantly influenced by temperature elevations, illustrating their critical roles in the thermal adaptation of mutant strains. These findings shed light on the importance of considering mutation duration and temperature screening in the development of thermotolerant fungal strains with potential applications in various industries. This study's breakthrough lies in its comprehensive understanding of the thermal adaptability of Mel-7 mycelia and the identification of promising mutant strains, offering valuable insights for both academic and industrial purposes.

Screening of astaxanthin-overproducing Xanthophyllomyces dendrorhous strains via iterative ARTP mutagenesis and cell sorting by flow cytometry.

The astaxanthin-producing yeast Xanthophyllomyces dendrorhous is widely used in aquaculture. Due to the production of carotenoid, this yeast shows visible color; however, high-throughput approaches for identification of astaxanthin-overproducing strains remain rare. This study verified an effective approach to identify astaxanthin-overproducing mutants of X. dendrorhous by flow cytometry (FCM) and cell sorting. First, the mutant libraries were generated by atmospheric and room-temperature plasma (ARTP) mutagenesis. Second, a highly direct correlation between the concentrations of intracellular astaxanthin and the levels of emitting fluorescence was constructed by testing a variety of astaxanthin-contained populations via FCM and cell sorting. Third, iterative cell sorting efficiently improves the identification of astaxanthin-overproducing strains. Finally, two mutants producing 4.96mg astaxanthin g-1 DCW (dry cell weight) and 5.30mg astaxanthin g-1 DCW were obtained, which were 25.3% and 33.8% higher than that of the original strain, respectively. This study demonstrated that iterative ARTP mutagenesis along with cell sorting by FCM is effective for identifying astaxanthin-overproduction strains.

Synchronous enhancement of astaxanthin and lipid accumulation in Haematococcus lacustris through co-mutation of ethanol and atmospheric and room temperature plasma: Exploration of characteristics and underlying mechanisms

Haematococcus lacustris is a precious algal species renowned for its ability to simultaneous production of astaxanthin and lipid. However, its slow growth rate necessitates the development of appropriate mutagenesis methodologies to effectively enhance its synchronous production of both astaxanthin and lipid. This study introduced the co-mutation of Atmospheric and Room Temperature Plasma (ARTP) and ethanol. The performance and preliminary mechanisms underlying the combined accumulation of astaxanthin and lipid in H. lacustris under both mutations by ARTP and ethanol were comparatively analyzed. Combined astaxanthin and lipid contents relative to total cell mass in the 110–2 strain reached 54.4%, surpassing that of strain 0–3 and the control by 17.0% and 47.6% respectively. Transcriptome level analysis revealed how both ethanol and ARTP induction promote the expressions of carotenoid and lipid synthesis genes and related enzymatic activities. Upregulation of genes associated with cell activity contributed to lipid and astaxanthin metabolism in multi pathways.

Investigations in mutation breeding and culturing media by Xanthophyllomyces dendrorhous

Astaxanthin is a red-orange carotenoid compound that has been used as antioxidant, pigment, and immunopotentiator. Xanthophyllomyces dendrorhous is a promising source for natural astaxanthin. In order to improve the astaxanthin production capacity of X. dendrorhous AS2.1557 (designated WT), nitrosoguanidine (NTG), ultraviolet (UV), and atmospheric and room temperature plasma (ARTP) mutagenesis techniques were applied for mutation breeding. The astaxanthin yield was elevated stepwisely after three-staged mutagenesis: Firstly, WT strain was treated with NTG and thus resulted in a positive mutagenic strain As1-3 with higher astaxanthin production; subsequent, strain As1-3 was exposed to UV irradiation, thereby generating a positive mutagenic strain As2-3; finally, a positive mutant As3-8 was developed by ARTP-induced mutagenesis of strain As2-3. After 72 h of cultivation in yeast extract-peptone-dextrose (YPD) medium, the astaxanthin titer and content produced by strain As3-8 were determined to be 4.45 mg·L−1 and 0.96 mg·g−11, which were 8.24 and 11.95 times that of WT strain, respectively. Afterward, the culture medium composition was optimized using the Plackett-Burman design (PBD), Box-Behnken design (BBD) and response surface methodology (RSM), the astaxanthin titer and content of As3-8 were determined to be 13.05 mg·L−1 and 2.04 mg·g−1 dry biomass after 96 h of cultivation in the optimized medium TM2, 24.17-/25.36-fold of the amounts of WT strain. This study provides a good reference for the enhancement of microbial metabolite production by using mutation breeding and medium optimization, the obtained mutant As3-8 would be an interesting candidate for comparative genomics and transcriptomics analysis-guided metabolic engineering in the future.

Comprehensive Analysis of Catalytic Characteristics and Molecular Mechanisms in Mutant Trametes versicolor Strains with Enhanced Laccase Activities

The commercial potential of Trametes versicolor laccases in the degradation of various persistent contaminants is significant. Despite numerous attempts through rational metabolic engineering to enhance the properties of laccases, the outcomes have proven unsatisfactory for practical implementation. The present study successfully generated two novel mutants, namely, TA-04 and TA-15, derived from Trametes versicolor ATCC20869, utilizing atmospheric and room temperature plasma (ARTP). The laccase activities of TA-04 and TA-15 showed a significant increase to 136.507 ± 4.827 U/mg DCW and 153.804 ± 6.884 U/mg DCW, respectively, which were 1.201 and 1.354 times than that of the original strain. The laccase activities of the mutant strains TA-04 and TA-15 surpassed that of the original strain by 10.372% and 18.914%, respectively, at a higher pH level of five. Sequencing analysis of TA-04 and TA-15 revealed that several alternative amino acids within their active regions may enhance their catalytic characteristics under a higher temperature and pH condition. This study employed ARTP mutagenesis to propose two highly efficient microbial mutants derived from Trametes versicolor ATCC20869, exhibiting enhanced laccase activities. These mutants hold promising potential for the degradation of diverse environmental pollutants.

Engineered salt-tolerant yeast to improve the physicochemical properties and volatiles of sweet flour paste

Sweet flour paste is a traditional condiment and fermented in high brine solution (14∼18%). Aroma-producing yeast plays a crucial role in the flavor and taste of sweet flour paste, but high salt concentrations often inhibit the action of the yeast. In this study, yeast Zygosaccharomyces rouxii AS2.18 was used as the parent strain to breed a salt-tolerant aroma-producing yeast strain by a combination of atmospheric and room temperature plasma (ARTP) mutagenesis and adaptive laboratory evolution (ALE). The engineered strain AMS79 showed the good growth and fermentation capacity at 18% NaCl concentration. The salt tolerance was closely related to intracellular glycerol production, Na+K+-ATPase activity enhancement and cell wall integrity improvement of the strain. During sweet flour paste fermentation, the maximum amino nitrogen content of the AMS79 group was 1.07- and 1.11-fold higher than that of the AS2.18 group and blank group, respectively. Compared to Blank group, the inoculation of AS2.18 enhanced the relative contents of alcohols, acids, ketones and furan(one)s, and the content of all 7 volatile compound types increased when AMS79 was inoculated for fermentation. In particular, the contents of 2-methyl-butanoic acid, benzeneacetaldehyde, 5-methyl-2-phenyl-2-hexenal and furfural were significantly enhanced by the salt-tolerant strain AMS79 compared to the parent strain AS2.18.

Selection of Salt-Tolerance and Ester-Producing Mutant Saccharomyces cerevisiae to Improve Flavour Formation of Soy Sauce during Co-Fermentation with Torulopsis globosa.

Soy sauce, as a traditional seasoning, is widely favoured by Chinese and other Asian people for its unique colour, smell, and taste. In this study, a salt-tolerance Saccharomyces cerevisiae strain HF-130 was obtained via three rounds of ARTP (Atmospheric and Room Temperature Plasma) mutagenesis and high-salt based screening. The ethanol production of mutant HF-130 was increased by 98.8% in very high gravity fermentation. Furthermore, ATF1 gene was overexpressed in strain HF-130, generating ester-producing strain HF-130-ATF1. The ethyl acetate concentration of strain HF-130-ATF1 was increased by 130% compared to the strain HF-130. Finally, the soy sauce fermentation performance of Torulopsis globosa and HF-130-ATF1 was compared with T. globosa, HF-130, HF-130-ATF1, and Torulopsis and HF-130. Results showed ethyl acetate and isoamyl acetate concentrations in co-fermentation of T. globosa and HF-130-ATF1 were increased by 2.8-fold and 3.3-fold, respectively. In addition, the concentrations of ethyl propionate, ethyl caprylate, phenylethyl acetate, ethyl caprate, isobutyl acetate, isoamyl alcohol, phenylethyl alcohol, and phenylacetaldehyde were also improved. Notably, other three important flavour components, trimethylsilyl decyl ester, 2-methylbutanol, and octanoic acid were also detected in the co-fermentation of T. globosa and HF-130-ATF1, but not detected in the control strain T. globosa. This work is of great significance for improving the traditional soy sauce fermentation mode, and thus improving the flavour formation of soy sauce.

Bioremediation of domestic wastewaters integrated with enhanced biodiesel production using Desmodesmus sp. mutated by atmospheric and room temperature plasma

In this work, we analyzed the biochemical changes and cultivation feasibility of microalgae Desmodesmus sp. mutated by atmospheric and room temperature plasma (ARTP) technology in mixed wastewater. The mutant strain AT151 was cultivated in different types of pretreated wastewaters for suitable pretreatment, resulting in the highest biomass content of > 354 mg/L, with a > 52% increase in lipid production in the filtered wastewater, and the difference between 0.22-μm and 0.45-um filtration was not significant (P > 0.05). The fatty acids of AT151 indicated a majority of C16 and C18 at > 86%, and the monounsaturated fatty acid content exceeded 18%, which was beneficial for high-quality biodiesel. Nutrient removal was found to be visible in wastewater by cultivating mutant microalgae AT151. The removal rates of total nitrogen (TN), total phosphorus (TP), and total organic carbon (TOC) for AT151 cultivated in wastewater were more than 87%, 94% and 47%, respectively. Furthermore, the relationship between the microalgae and bacteria was studied, showing significant differences in bacterial community (P < 0.05). The obvious phenomenon consisted of dominant bacteria changes, where Brevundimonas balnearis and Synechocystis sp. served as stable strains during cultivation, forming a symbiotic relationship with mutant microalgae AT151. The dominant bacteria showed obvious metabolism according to functional KEGG prediction, mainly affecting the factors of amino acids, carbohydrates, and growth factor metabolism. Therefore, enhancing microalgae biomass and lipid production through wastewater cultivation holds both scientific significance and practical feasibility.

Engineering low-salt growth Halomonas Bluephagenesis for cost-effective bioproduction combined with adaptive evolution

Halophilic Halomonas bluephagenesis has been engineered to produce various added-value bio-compounds with reduced costs. However, the salt-stress regulatory mechanism remained unclear. H. bluephagenesis was randomly mutated to obtain low-salt growing mutants via atmospheric and room temperature plasma (ARTP). The resulted H. bluephagenesis TDH4A1B5 was constructed with the chromosomal integration of polyhydroxyalkanoates (PHA) synthesis operon phaCAB and deletion of phaP1 gene encoding PHA synthesis associated protein phasin, forming H. bluephagenesis TDH4A1B5P, which led to increased production of poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-4-hydrobutyrate) (P34HB) by over 1.4-fold. H. bluephagenesis TDH4A1B5P also enhanced production of ectoine and threonine by 50% and 77%, respectively. A total 101 genes related to salinity tolerance was identified and verified via comparative genomic analysis among four ARTP mutated H. bluephagenesis strains. Recombinant H. bluephagenesis TDH4A1B5P was further engineered for PHA production utilizing sodium acetate or gluconate as sole carbon source. Over 33% cost reduction of PHA production could be achieved using recombinant H. bluephagenesis TDH4A1B5P. This study successfully developed a low-salt tolerant chassis H. bluephagenesis TDH4A1B5P and revealed salt-stress related genes of halophilic host strains.

Application of Atmospheric and Room-Temperature Plasma (ARTP) to Microbial Breeding.

Atmospheric and room-temperature plasma (ARTP) is an efficient microbial mutagenesis method with broad application prospects. Compared to traditional methods, ARTP technology can more effectively induce DNA damage and generate stable mutant strains. It is characterized by its simplicity, cost-effectiveness, and avoidance of hazardous chemicals, presenting a vast potential for application. The ARTP technology is widely used in bacterial, fungal, and microalgal mutagenesis for increasing productivity and improving characteristics. In conclusion, ARTP technology holds significant promise in the field of microbial breeding. Through ARTP technology, we can create mutant strains with specific genetic traits and improved performance, thereby increasing yield, improving quality, and meeting market demands. The field of microbial breeding will witness further innovation and progress with continuous refinement and optimization of ARTP technology.

Atmospheric and Room Temperature Plasma (ARTP) Mutagenesis Improved the Anti-MRSA Activity of Brevibacillus sp. SPR20.

Brevibacillus sp. SPR20 produced potentially antibacterial substances against methicillin-resistant Staphylococcus aureus (MRSA). The synthesis of these substances is controlled by their biosynthetic gene clusters. Several mutagenesis methods are used to overcome the restriction of gene regulations when genetic information is absent. Atmospheric and room temperature plasma (ARTP) is a powerful technique to initiate random mutagenesis for microbial strain improvement. This study utilized an argon-based ARTP to conduct the mutations on SPR20. The positive mutants of 40% occurred. The M27 mutant exhibited an increase in anti-MRSA activity when compared to the wild-type strain, with the MIC values of 250-500 and 500 μg/mL, respectively. M27 had genetic stability because it exhibited constant activity throughout fifteen generations. This mutant had similar morphology and antibiotic susceptibility to the wild type. Comparative proteomic analysis identified some specific proteins that were upregulated in M27. These proteins were involved in the metabolism of amino acids, cell structure and movement, and catalytic enzymes. These might result in the enhancement of the anti-MRSA activity of the ARTP-treated SPR20 mutant. This study supports the ARTP technology designed to increase the production of valuable antibacterial agents.

Improved Glutamic Acid Production Capacity of Corynebacterium glutamicum by the ARTP Mutagenesis Method

Glutamic acid is an important amino acid that is used widely in the fields of food, medicine, and agriculture. One of the methods of glutamic acid production is direct microbial fermentation, so the genetic stability and glutamic-acid-producing capacity of the producing strain are the keys to improving glutamic acid concentration. Experiments were carried out using Corynebacterium glutamicum GL−6 as the parental strain, with two iterations of mutagenesis by atmospheric and room temperature plasma (ARTP) and screening with agar plates tolerant to high sugar and malonic acid, and the best strains with stable phenotypes were verified by fermentation in 20 L tanks. The results show that the optimal mutagenesis time of ARTP was 140 s, with lethality and positive mutation rates of 93.0% and 15.6%, respectively. The concentrations of the high-sugar and malonic acid agar plates were 240 g/L and 35 g/L, respectively. A mutant strain, P−45, with improved glutamic acid production capacity and genetic stability, was obtained through two rounds of iterative mutagenesis screening. The concentration of this strain in the Erlenmeyer flasks was 17.7 g/L, which was 18.8% higher than that of the parental strain, GL−6, and could be inherited stably for 10 generations. In the glutamic acid synthesis pathway, the upregulation of the gene encoding citrate synthase (cs), gene encoding isocitrate dehydrogenase (icdh), and gene encoding glutamate dehydrogenase (gdh), and the downregulation of the gene encoding oxoglutarate dehydrogenase complex (odhc) increased the carbon flows of the TCA cycle and its branch metabolic flow to glutamic acid synthesis. P−45 showed a glutamic acid concentration of 147.0 g/L under fed-batch fermentation conditions in 20 L tanks, which was 81.5% higher than the starting strain, GL−6. This study provides a new technical solution for improving microbial metabolites and genetic stability.

Enhancing Protease and Amylase Activities in Bacillus licheniformis XS-4 for Traditional Soy Sauce Fermentation Using ARTP Mutagenesis.

This study was conducted to increase the enzymatic activity of Bacillus licheniformis XS-4, which was isolated from the traditional fermented mash of Xianshi soy sauce. The mutation was induced by atmospheric and room-temperature plasma (ARTP), and a mutant strain, mut80, was obtained. mut80 exhibited significant increases in protease and amylase activity by 90.54% and 143.10%, respectively, and the enhanced enzymatic activities were stably maintained after 20 consecutive incubations. Re-sequencing analysis of mut80 revealed that the mutation sites were located in 1518447(AT-T) and 4253106(G-A) in its genome, which was involved in the metabolic pathways of amino acids. The expression of the protease synthetic gene (aprX) increased 1.54 times, while that of the amylase gene (amyA) increased 11.26 times, as confirmed via RT-qPCR. Using ARTP mutagenesis, the present study proposes a highly efficient microbial resource with enhanced protease and amylase activity provided by B. licheniformis, which can potentially be used to improve the efficiency of traditional soy sauce fermentation.