Atmospheric And Room Temperature Plasma Research Articles

Enhanced production, artificial intelligence optimized three-phase partitioning extraction, and in silico characterization of extracellular neutral Bacillus cereus proteinase

The present study applied atmospheric and room temperature plasma (ARTP) mutagenesis to improve Bacillus cereus strain for neutral proteinase production. The selected improved stable strain, Phe−Tyr-ARTP-60-E demonstrated a 2.88-fold enhanced proteinase yield and 2.81-fold enhanced proteinase activity with shortened fermentation time. An artificial neural network-embedded genetic algorithm (ANN-GA) was employed to optimize the enzyme's three-phase partitioning (TPP) recovery using Python software. The optimized solution of the hybrid model recommended 38.05% wv−1 (NH4)2SO4, 1.0:2.0 crude extract/tert-butanol ratio, pH 5.84 at 23.4 °C, for the exclusive partitioning of proteinase in the intermediate phase, with 202% recovery, 13-fold purity with specific activity of 922 Umg−1. Sensitivity analysis of the model revealed that enzyme recovery was most sensitive to crude extract/tert-butanol ratio with total sensitivity (ST) of 0.253 while purification factor was most sensitive to pH with an ST of 0.444. In silico analysis using Expasy ProtParam tool revealed that mutant proteinase had 3 amino acid substitutions; Val-to-Cys; Val-to-His and Ile-to-Lys, at positions 152, 288, and 292, respectively, and 39.2 kDa molecular weight, 6.42 isoelectric point, and 33.35 instability index. The 3-Dimensional structure of the enzyme predicted by ModWeb v r273 Server revealed 39% sequence identity with Pseudoalteromonas lipolytica thermolysin. The macromolecule increased transmittance in lysozyme-coated contact lenses by 99.6% in 45 min. We conclude that ARTP mutagenesis and ANN-GA optimized TPP are effective and efficient recalcitrant strain improvement and product recovery methods, respectively. Further genetic tools and analysis of the neutral proteinase-producing gene may be required for increased improvement toward sustainable application.

Mutagenesis selection and large-scale cultivation of non-green Chlamydomonas reinhardtii for food applications.

The green alga Chlamydomonas reinhardtii is an accepted food ingredient in the United States of America (United States), the European Union, Singapore, and China. It can be consumed in unlimited quantities. As this alga is rich in nutrients, proteins, and rough polysaccharides and contains a balanced proportion of various amino acids, it is an excellent raw material for food production. Although various edible brown and green algae are available on the market, their color and strong grassy flavor have constrained their popularity among consumers, thereby limiting their application in food additives and animal feed. Chlorophyll-deficient C. reinhardtii mutants were developed using atmospheric and room temperature plasma (ARTP) technology. A yellow-colored C. reinhardtii variant (A7S80) cultivated in dark conditions was isolated. This light-sensitive variant has a mutation in the chlM gene, and it can grow heterotrophically using acetate as a carbon source. Compared to wild-type C. reinhardtii, A7S80 has significantly lower chlorophyll levels, reduced grassy flavor, and more diverse pigments, with considerable potential for commercial application in human and animal food production, as well as in pharmaceutical and cosmetic industries.

Enhancement on the lipase activity of M. circinelloides by ARTP mutagenesis for biodiesel production from woody plant oil

The objective is to enhance lipase activity of Mucor circinelloides via atmospheric and room temperature plasma (ARTP). ARTP was employed to treat spores of M. circinelloides for the selection of high lipase producers. With an irradiation time of 100 s at a lethal rate of 95 %, 18 mutants with higher lipase activity were initially selected. Measurements of the lipase activity revealed that a mutant strain LA-26 was characterized by a steady hereditary stability with lipase activity increase to 376.7 U/g. Moreover, the mutant LA-26 was immobilized as whole-cell biocatalyst used in solvent-free system to produce biodiesel. Response surface methodology (RSM) was used to optimize the reaction parameters. With the addition of 8 % biocatalyst, 10 % water content (w/w) and the stepwise addition of methanol (methanol to oil ratio of 4:1), the highest biodiesel (FAME) yield up to 98 % was achieved within 48 h. Immobilized LA-26 can be used for repeated batches, maintaining 70 % FAME yield after 6 cycles. The identified mutant strain LA-26 exhibits great potential for biodiesel production.

ARTP mutagenesis of Aureobasidium pullulans RM1603 for high pullulan production and transcriptome analysis of mutants.

Pullulan is a microbial exopolysaccharide produced by Aureobasidium spp. with excellent physical and chemical properties, resulting in great application value. In this study, a novel strain RM1603 of Aureobasidium pullulans with high pullulan production of 51.0 ± 1.0g·L- 1 isolated from rhizosphere soil was subjected to atmospheric and room temperature plasma (ARTP) mutagenesis, followed by selection of mutants to obtain pullulan high-producing strains. Finally, two mutants Mu0816 and Mu1519 were obtained, with polysaccharide productions of 58.7 ± 0.8 and 60.0 ± 0.8g∙L- 1 after 72-h fermentation, representing 15.1 and 17.6% increases compared with the original strain, respectively. Transcriptome analysis of the two mutants and the original strain revealed that the high expression of α/β-hydrolase (ABHD), α-amylase (AMY1), and sugar porter family MFS transporters (SPF-MFS) in the mutants may be related to the synthesis and secretion of pullulan. These results demonstrated the effectiveness of ARTP mutagenesis in A. pullulans, providing a basis for the investigation of genes related to pullulan synthesis and secretion.

Research Progress of ARTP Mutagenesis Technology Based on Citespace Visualization Analysis.

Atmospheric and room temperature plasma (ARTP) mutagenesis technology has been developed rapidly in recent years because of its simple operation, safety, environmental friendliness, high mutation rate, and large mutation library capacity. It has been widely used in traditional fields such as food, agriculture, and medicine, and has been gradually applied in emerging fields such as environmental remediation, bioenergy, and microalgae utilization. In this paper, the Web of Science Core Collection (WOSCC) was used as the data source, and the keywords and core literature of ARTP mutagenesis technology were plotted by citespace software, and the research progress and research hotspots of ARTP mutagenesis technology were analyzed. Through citespace visualization analysis, it is concluded that the country with the largest number of studies is China, the institution with the largest number of studies is Jiangnan University, and the author of the most published papers is Jiangnan University. Through keyword analysis, it is concluded that the most widely used ARTP mutagenesis technology is fermentation-related majors, mainly for biosynthesis and microbial research at the molecular level. Among them, the most widely used microorganisms are Escherichia coli and Saccharomyces cerevisiae.

Improving Agar Degradation Activity of Vibrio natriegens WPAGA4 via Atmospheric and Room Temperature Plasma (ARTP)

Agar oligosaccharides from the degradation of agar harbor great potential in the food and pharmaceutical industries. An agar-degrading bacterium, Vibrio natriegens WPAGA4, was isolated from the deep sea in our previous work. However, the agar-degrading activity of WPAGA4 remains to be improved for more production benefits of this strain. The aim of this study was to enhance the agar-degrading activity of WPAGA4 by using atmospheric and room temperature plasma (ARTP) mutagenesis. Three mutant strains, including T1, T2, and T3, with good genetic stability were obtained, and the agar-degrading activities of these strains increased by 136%, 141%, and 135%, respectively. The optimal temperature and pH for agar degradation were slightly changed in the mutant strains. No sequence mutation was detected in all the agarase genes of WPAGA4, including agaW3418, agaW3419, agaW3420, and agaW3472. However, ARPT mutagenesis increased the relative expression levels of agaW3418, agaW3419, and agaW3420 in the mutant strains, which could be the reason for the improvement of degradation activities in the mutant strains. Furthermore, T3 had the lowest consumption rate of agar oligosaccharide, which was 21% less than the wild-type strain. Therefore, T3 possessed a preferable production value due to its higher degrading activity and lower consumption of agar oligosaccharides. The current work enhanced the agar-degrading activity of WPAGA4 and offered strains with greater potential for agar oligosaccharide production, thereby laying the foundation for industrial applications.

Selection and Genetic Analysis of High Polysaccharide-Producing Mutants in Inonotus obliquus.

Inonotus obliquus, a medicinal fungus, has garnered significant attention in scientific research and medical applications. In this study, protoplasts of the I. obliquus HS819 strain were prepared using an enzymatic method and achieved a regeneration rate of 5.83%. To enhance polysaccharide production of I. obliquus HS819, atmospheric and room temperature plasma (ARTP) technology was employed for mutagenesis of the protoplasts. Through liquid fermentation, 32 mutant strains exhibiting diverse characteristics in morphology, color of the fermentation broth, mycelial pellet size, and biomass were screened. Secondary screening identified mutant strain A27, which showed a significant increase in polysaccharide production up to 1.67 g/L and a mycelial dry weight of 17.6 g/L, representing 137.67% and 15% increases compared to the HS819 strain, respectively. Furthermore, the fermentation period was reduced by 2 days, and subsequent subculture cultivation demonstrated stable polysaccharide production and mycelial dry weight. The genome resequencing analysis of the HS819 strain and mutant strain A27 revealed 3790 InDel sites and mutations affecting 612 functional genes associated with polysaccharide synthesis. We predict that our findings will be helpful for high polysaccharide production through genetic engineering of I. obliquus.

Whole-genome mutagenesis of whiteleg shrimp (Litopenaeus vannamei) by ARTP and analysis of its mutational characteristics

Artificial induction has been widely employed to generate genetic resources with increased mutation rates for subsequent selection. To assess the potential of physical mutagenesis in shrimp breeding, we utilized Atmospheric and Room Temperature Plasma (ARTP) to induce mutations in fertilized eggs from four different families of Litopenaeus vannamei. Treatment durations ranged from 2 to 12 min. The results revealed a significant decrease in hatching rates among the ARTP-treated groups, ranging from 3.22% to 67.81%, with more substantial reductions observed in longer treatment durations. Following a 90-day mixed rearing test, we found no statistically significant differences (P > 0.05) in the average body weight of 743 shrimps across the 13 mutagenesis groups compared to the control group. However, variations in weight were observed in the 22 largest or smallest sample groups, with 12 groups displaying an average increase of 8.69% and 10 groups showing an average decrease of 9.97% compared to their respective controls. Regarding fitness traits, seven treatment groups from two families exhibited higher median lethal times or mean survival times against WSSV compared to the control group, while two treatment groups demonstrated improved resistance to ammonium nitrogen. Genomic resequencing analysis was conducted on growth sampling groups, identifying a total of 2,718,961 SNPs and 331,172 Indels in the treatment group. The mutation rate in the treatment group was 2.148 × 10−3, significantly higher than the control group's rate of 5.275 × 10−5. Within the treatment group, 17,104 specific non-synonymous SNPs were identified in the exons of 7780 genes, markedly surpassing the control group's count of 121 SNPs and 110 genes. GO annotation analysis revealed an even distribution of mutations induced by ARTP across various biological processes, cellular components, and functions. Furthermore, KEGG enrichment analysis demonstrated a significant enrichment trend in metabolic pathways, both in terms of gene count and FDR value. A total of 42 KEGGs and several potential growth-related genes, including IGF1 and AMY2, previously confirmed to be highly correlated with growth in L. vannamei, were identified. In conclusion, a feasible method for whole-genome mutagenesis in shrimp zygotes was developed in this study. Tests on economic traits revealed that ARTP can generate mutant individuals with either a greater degree of variation or superior traits. Resequencing revealed the presence of numerous mutation SNPs involved in various biological processes and contribute to a higher level of genetic diversity. These results suggest that ARTP can be utilized to enhance the available genetic variation in shrimp breeding, thereby providing valuable materials and methods for genetic breeding in whiteleg shrimp.

Cytokinetic engineering enhances the secretory production of recombinant human lysozyme in Komagataella phaffii

BackgroundHuman lysozyme (hLYZ) is a natural antibacterial protein with broad applications in food and pharmaceutical industries. Recombinant production of hLYZ in Komagataella phaffii (K. phaffii) has attracted considerable attention, but there are very limited strategies for its hyper-production in yeast.ResultsHere through Atmospheric and Room Temperature Plasma (ARTP)-based mutagenesis and transcriptomic analysis, the expression of two genes MYO1 and IQG1 encoding the cytokinesis core proteins was identified downregulated along with higher hLYZ production. Deletion of either gene caused severe cytokinesis defects, but significantly enhanced hLYZ production. The highest hLYZ yield of 1,052,444 ± 23,667 U/mL bioactivity and 4.12 ± 0.11 g/L total protein concentration were obtained after high-density fed-batch fermentation in the Δmyo1 mutant, representing the best production of hLYZ in yeast. Furthermore, O-linked mannose glycans were characterized on this recombinant hLYZ.ConclusionsOur work suggests that cytokinesis-based morphology engineering is an effective way to enhance the production of hLYZ in K. phaffii.

Improved expression of Cerrena unicolor Laccase in Aspergillus niger via combined strategies and its applications

The application of laccases in biotechnology and industry is an expanding research field. Laccases derived from Cerrena unicolor (C. unicolor) exhibit notable potential in the food and industrial sectors. However, generating sufficient yields of this laccase through heterologous expression to meet commercial demands remains a challenge. Therefore, we heterologously expressed LACCASE2 (Lac2), a laccase gene from C. unicolor, in Aspergillus niger. By incorporating strategies such as codon optimization, molecular chaperone-assisted expression, and atmospheric and room-temperature plasma (ARTP) mutagenesis, we successfully screened the strain OPT-HacA-M10 with the highest laccase yield, which exhibited a remarkable enzymatic activity of 416.8 U/mL in a 50-L fermenter. We further assessed the enzymatic properties of the recombinant laccase rLac2, focusing on its capabilities in dye decolorization and mycotoxin degradation. In the absence of a mediator, rLac2 exhibited extremely high degradation efficiency for Remazol Brilliant Blue R and indigo. Impressively, outstanding results were achieved with this laccase in denim bleaching. In mycotoxin degradation tests, with methyl syringate as a mediator, rLac2 achieved degradation rates of over 94% for aflatoxin B1 and zearalenone. In summary, due to its heightened laccase activity and versatile applications, our enhanced strain is highly suitable for industrial and food sectors.

Identification of the mutual gliding locus as a factor for gut colonization in non-native bee hosts using the ARTP mutagenesis

BackgroundThe gut microbiota and their hosts profoundly affect each other’s physiology and evolution. Identifying host-selected traits is crucial to understanding the processes that govern the evolving interactions between animals and symbiotic microbes. Current experimental approaches mainly focus on the model bacteria, like hypermutating Escherichia coli or the evolutionary changes of wild stains by host transmissions. A method called atmospheric and room temperature plasma (ARTP) may overcome the bottleneck of low spontaneous mutation rates while maintaining mild conditions for the gut bacteria.ResultsWe established an experimental symbiotic system with gnotobiotic bee models to unravel the molecular mechanisms promoting host colonization. By in vivo serial passage, we tracked the genetic changes of ARTP-treated Snodgrassella strains from Bombus terrestris in the non-native honeybee host. We observed that passaged isolates showing genetic changes in the mutual gliding locus have a competitive advantage in the non-native host. Specifically, alleles in the orphan mglB, the GTPase activating protein, promoted colonization potentially by altering the type IV pili-dependent motility of the cells. Finally, competition assays confirmed that the mutations out-competed the ancestral strain in the non-native honeybee gut but not in the native host.ConclusionsUsing the ARTP mutagenesis to generate a mutation library of gut symbionts, we explored the potential genetic mechanisms for improved gut colonization in non-native hosts. Our findings demonstrate the implication of the cell mutual-gliding motility in host association and provide an experimental system for future study on host-microbe interactions.ArN8Xjm3N6Dubzus4DCSQmVideo

Genome-wide transcriptome analyses reveal changes in glutathione-overproducing yeast obtained by ARTP mutagenesis for rice wine brewing

The atmospheric and room temperature plasma (ARTP) technique efficiently enhances yeast glutathione (GSH) yield. This study focused on obtaining a GSH over-producing strain, Saccharomyces cerevisiae SCZ40, through mutagenesis and stability testing. SCZ40 exhibited a remarkable 71.1% increase in GSH content during rice wine fermentation. Scanning electron microscopy (SEM) and whole-genome sequencing identified ARTP-induced changes in cell morphology and alterations in the protein-serine/threonine kinase and isocitrate dehydrogenase pathways linked to GSH synthesis. Transcriptomic analysis revealed 507 differentially expressed genes, highlighting the impact of ARTP on S. cerevisiae. Up/down-regulated genes associated with glycolysis/gluconeogenesis suggested a shift in glucose distribution favoring the pentose phosphate pathway and positively influencing GSH production. Pathway analysis further elucidated changes in glycolysis/gluconeogenesis, tricarboxylic acid cycle (TCA), and GSH biosynthesis pathways. Validation through quantitative real-time polymerase chain reaction (qRT-PCR) confirmed RNA-seq data reliability.

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.