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Monascus is a filamentous fungus with a long history of application in China, which can produce a variety of secondary metabolites, including Monascus red pigments, Monascus orange pigments, Monascus yellow pigments, and citrinin. There is widespread attention being paid to natural pigments because of their safety. Among the many natural pigments, orange pigment has a wide range of applications because of its unique color, but current production levels in the orange pigment industry are limited to a certain extent due to the insufficiently wide range of sources and low production. In this study, the ARTP mutation was used to obtain a strain with high-yield orange pigment and low citrinin. The strain RS7 was obtained through two-step mutagenesis, and all three pigments were improved to different degrees. The color value of orange pigment was elevated from the original 108 U/mL to 180 U/mL, an increase of 66.7% compared to the original strain, and the citrinin content was reduced by 69%. The result of microscopic morphology showed that RS7 has more wrinkles and is more convex than the R1 strain, but there was little change between the two strains. Therefore, the ARTP mutation influenced the growth and the biosynthesis of pigments in Monascus. In addition, the conditions of ultrasonic extraction of Monascus pigments were optimized using the response surface, and the separation of pigments was achieved with the method of thin-layer chromatography. Pigment stability results showed that the temperature had no significant effect on orange pigment, while tea polyphenol could improve its stability. This study generated a strain with high-yielding orange pigment and could lay a foundation for the future application of Monascus orange pigment in the food industry. [ABSTRACT FROM AUTHOR]

In order to obtain endophytic selenium-rich yeast from Lycium barbarum with strong ability producing alcohol and aroma, and then promote the selenium-rich fermentation products and improve the flavor, this study took the endophytic yeast of L. barbarum stored in the laboratory as experimental materials, and the yeast with strong selenium-rich ability was obtained through the initial screening of selenium-resistant method and red selenium methods. Atmospheric room temperature plasma (ARTP) technology was used to obtain mutant strains and through TTC medium, Duchenne tubule fermentation, alcohol production, ester and acid production, tolerance and other experiments to screen out the target strains of aroma enhancement and high alcohol production. The content of selenium in yeast cell and fermentation liquid was determined by hydride generation atomic fluorescence spectrometry. The results showed that M1 Saccharomyces cerevisiae and M16 Clavispora lusitaniae were obtained by primary selenium screening, the mutagenic strain M1-5 of S. cerevisiae with higher alcohol production capacity and the mutagenic strain M16-28 of C. lusitaniae with higher aroma production capacity were obtained through ARTP mutagenesis. The alcohol production capacity of M1-5 increased by 38.07% compared with M1, and the ester production capacity of M16-28 increased by 107.62% compared with M16, and the genetic stability was good. The selenium contents in the intracellular and fermentation broth of the M1-5 strains were 204.8 and 1158 μg/L, respectively, and in M16-28 were 666.45μg/L and 1830 μg/L, respectively. Therefore, the selenium enrichment capacity of M16-28 was higher than that of M1-5, and the selenium content of fermentation liquid was extremely significantly higher than that of yeast cells (P

Abstract Background The 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. Results We 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. Conclusions Using 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. Video Abstract

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: The 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. Results: We 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. Conclusions: Using 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. ArN8Xjm3N6Dubzus4DCSQm Video Abstract [ABSTRACT FROM AUTHOR]

Saccharomyces cerevisiae D65 isolated from the Malus asiatica naturally fermented liquid was used as the parental strain for mutation breeding of atmospheric and room temperature plasma (ARTP). The mutant strains with excellent characteristics were obtained through acid resistance test, esterase activity test, and fermentation test multi-step screening. The results showed that the specific activity of total esterase of D65-4D10 was 2428.30 U·g−1, and the ester content in the fermented Malus asiatica wine was 5710.75 ug·L−1, which was 20.69% and 41.64% higher than that of the parental strain, respectively. Moreover, the Malus asiatica wine fermented by D65-4D10 had stronger antioxidant capacity than the control, the highest score in sensory evaluation, and completed fermentation 24 h earlier than commercial yeast. After seven generations of continuous culture, the fermentation performance of D65-4D10 remained stable and had the potential to be applied to Malus asiatica wine fermentation. [ABSTRACT FROM AUTHOR]

Monascus is a filamentous fungus with a long history of application in China, which can produce a variety of secondary metabolites, including Monascus red pigments, Monascus orange pigments, Monascus yellow pigments, and citrinin. There is widespread attention being paid to natural pigments because of their safety. Among the many natural pigments, orange pigment has a wide range of applications because of its unique color, but current production levels in the orange pigment industry are limited to a certain extent due to the insufficiently wide range of sources and low production. In this study, the ARTP mutation was used to obtain a strain with high-yield orange pigment and low citrinin. The strain RS7 was obtained through two-step mutagenesis, and all three pigments were improved to different degrees. The color value of orange pigment was elevated from the original 108 U/mL to 180 U/mL, an increase of 66.7% compared to the original strain, and the citrinin content was reduced by 69%. The result of microscopic morphology showed that RS7 has more wrinkles and is more convex than the R1 strain, but there was little change between the two strains. Therefore, the ARTP mutation influenced the growth and the biosynthesis of pigments in Monascus. In addition, the conditions of ultrasonic extraction of Monascus pigments were optimized using the response surface, and the separation of pigments was achieved with the method of thin-layer chromatography. Pigment stability results showed that the temperature had no significant effect on orange pigment, while tea polyphenol could improve its stability. This study generated a strain with high-yielding orange pigment and could lay a foundation for the future application of Monascus orange pigment in the food industry.

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.

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.

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.

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 TDH4A 1 B 5 was constructed with the chromosomal integration of polyhydroxyalkanoates (PHA) synthesis operon phaCAB and deletion of phaP 1 gene encoding PHA synthesis associated protein phasin, forming H. bluephagenesis TDH4A 1 B 5 P, 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 TDH4A 1 B 5 P 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 TDH4A 1 B 5 P 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 TDH4A 1 B 5 P. This study successfully developed a low-salt tolerant chassis H. bluephagenesis TDH4A 1 B 5 P and revealed salt-stress related genes of halophilic host strains. • H. bluephagenesis was engineered to become a low-salt chassis termed TDH4A 1 B 5 P for convenience of bioproductions. • H. bluephagenesis TDH4A 1 B 5 P was able to produce 75 g/L P34HB grown on 10 g/L NaCl. • 101 genes were identified as salt-stress related gene set, of which 4 were verified. • H. bluephagenesis TDH4A 1 B 5 P exhibited better PHA production using sodium acetate and gluconate. • Economic analysis of P34HB shows 33% cost reduction using H. bluephagenesis TDH4A 1 B 5 P. [ABSTRACT FROM AUTHOR]

Abstract A novel extracellular polymeric substance (EPS) with flocculating activity produced by Pseudomonas fluorescein isolated from soil was studied in this paper. Firstly, atmospheric and room temperature plasma (ARTP) was applied to get a mutant of P. fluorescein with higher EPS production. A mutant T4-2 exhibited a 106.48% increase in flocculating activity compared to the original strain. The maximum EPS yield from T4-2 was enhanced up to 6.42 g/L, nearly 10 times higher than the original strain on a 3.6-L bioreactor with optimized fermentation conditions. Moreover, the flocculating activity of the mutant reached 3023.4 U/mL, 10.96-fold higher than that of T4. Further identification showed that EPS from mutant T4-2 was mainly composed of polysaccharide (76.67%) and protein (15.8%) with a molecular weight of 1.17 × 105 Da. The EPS showed excellent adsorption capacities of 80.13 mg/g for chromium (VI), which was much higher than many reported adsorbents such as chitosan and cellulose. The adsorption results were described by Langmuir isotherm and pseudo-second-order kinetic model. The thermodynamic parameters (ΔG 0, ΔH 0 and ΔS 0) revealed that the adsorption process was spontaneous and exothermic. Adsorption mechanisms were speculated to be electrostatic interaction, reduction, and chelation. Graphical Abstract

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. [ABSTRACT FROM AUTHOR]

Eicosapentaenoic acid (EPA) belonged to the ω-3 series of polyunsaturated fatty acids and had physiological functions lipid as regulating blood lipid and preventing cardiovascular diseases. Schizochytrium sp. was considered to be a potential industrial fermentation strain of EPA because of its fast growth, high oil content, and simple fatty acid composition. However, Schizochytrium sp. produced EPA with low production efficiency and a long synthesis path. This research aims to improve the yield of EPA in Schizochytrium sp. by ARTP mutagenesis and to reveal the mechanism of high-yield EPA through transcriptome analysis. ARTP mutagenesis screening yielded the mutant M12 that whereas the productivity of EPA increased 108% reaching 0.48 g/L, the total fatty acid concentration was 13.82 g/L with an increase of 13.7%. The transcriptomics revealed 2995 differentially expressed genes were identified between M12 and the wild-type strain and transcripts involved in carbohydrate, amino acid, energy, and lipid metabolism were up-regulated. Among them, the hexokinase (HK) and the phosphofructokinase genes (PFK), which can catalyze pyruvate to acetyl-CoA, were increased 2.23-fold and 1.78-fold. Glucose-6-phosphate dehydrogenase (G6PD) and glutamate dehydrogenase (GLDH), which can both generate NADPH, were increased by 1.67-fold and 3.11-fold. Furthermore, in the EPA synthesis module, the expression of 3-oxoacyl-[acyl-carrier protein] reductase(fabG) and carbonyl reductase 4 / 3-oxoacyl-[acyl-carrier protein] reductase beta subunit(CBR4), also up-regulated 1.11-fold and 2.67-fold. These may lead to increases in cell growth. The results provide an important reference for further research on promoting fatty acid and EPA accumulation in Schizochytrium sp. [ABSTRACT FROM AUTHOR]

A novel extracellular polymeric substance (EPS) with flocculating activity produced by Pseudomonas fluorescein isolated from soil was studied in this paper. Firstly, atmospheric and room temperature plasma (ARTP) was applied to get a mutant of P. fluorescein with higher EPS production. A mutant T4-2 exhibited a 106.48% increase in flocculating activity compared to the original strain. The maximum EPS yield from T4-2 was enhanced up to 6.42 g/L, nearly 10 times higher than the original strain on a 3.6-L bioreactor with optimized fermentation conditions. Moreover, the flocculating activity of the mutant reached 3023.4 U/mL, 10.96-fold higher than that of T4. Further identification showed that EPS from mutant T4-2 was mainly composed of polysaccharide (76.67%) and protein (15.8%) with a molecular weight of 1.17 × 105 Da. The EPS showed excellent adsorption capacities of 80.13 mg/g for chromium (VI), which was much higher than many reported adsorbents such as chitosan and cellulose. The adsorption results were described by Langmuir isotherm and pseudo-second-order kinetic model. The thermodynamic parameters (ΔG0, ΔH0 and ΔS0) revealed that the adsorption process was spontaneous and exothermic. Adsorption mechanisms were speculated to be electrostatic interaction, reduction, and chelation. [ABSTRACT FROM AUTHOR]

ARTP (atmospheric and room temperature plasma mutagenesis) mutagenesis was tried on G. lemaneiformis, and mutagenesis conditions were confirmed. An osmotic pressure screening program was established. Mutants were identified and characterized of relevant physiological traits. The aim of the study is to try to use ARTP mutagenesis and osmotic pressure screening for the breeding of high-agar G. lemaneiformis. Treatment time of 46 s was found to be an optimal mutagenesis time. The mutagenized spores were initially screened with 58‰ salinity artificial seawater, and then, the surviving spores were screened twice with 60‰ salinity artificial seawater in their vertical growth phase and branch growth phase, respectively. Four fast-growing and hypertonic resistance gametophytes were selected. The actual photosynthetic efficiency [Y(PSII)], photochemical quenching (qL), and non-photochemical quenching (NPQ) of four mutants were measured. The values of Y(PSII) and qL of HAGL-X3 and HAGL-X5 were higher than those of the control in the early stage of salt stress. NPQs of HAGL-X3 and HAGL-X5 were higher than control in most of the times. The growth rates of the four mutants were higher than that of the control. HAGL-X4 was the highest. The agar content was measured; HAGL-X5 displayed the highest agar content among the tested strains. HAGL-X5 was more in line with expectations, because of its high agar content and good hypertonic resistance. In this study, the mutant of G. lemaneiformis with high agar content was obtained by the procedure, which provided a certain reference for the selection of G. lemaneiformis strains with high agar content. [ABSTRACT FROM AUTHOR]

ABSTRACT Biotransformation of plastics or their depolymerization monomers as raw materials would offer a better end-of-life solutions to the plastic waste dilemma. 1,4-butanediol (BDO) is one of the major depolymerization monomers of many plastics polymers. BDO valorization presents great significance for waste plastic up-recycling and fermenting feedstock exploitation. In the present study, atmospheric pressure room temperature plasma (ARTP)-induced mutation combined with adaptive laboratory evolution (ALE) was used to improve the BDO utilization capability of Pseudomonas putida KT2440. The excellent mutant P. putida NB10 was isolated and stored in the China Typical Culture Preservation Center (CCTCC) with the deposit number M 2021482. Whole-genome resequencing and transcriptome analysis revealed that the BDO degradation process consists of β-oxidation, glyoxylate carboligase (GCL) pathway, glyoxylate cycle and gluconeogenesis pathway. The imbalance between the two key intermediates (acetyl-CoA and glycolyl-CoA) and the accumulation of cytotoxic aldehydes resulted in the weak metabolism performance of KT2440 in the utilization of BDO. The balance of the carbon flux and enhanced tolerance to cytotoxic intermediates endow NB10 with great BDO degradation capability. This study deeply revealed the metabolic mechanism behind BDO degradation and provided an excellent chassis cell for BDO further up-cycling to high-value chemicals. IMPORTANCE Plastic waste represents not only a global pollution problem but also a carbon-rich, low-cost, globally renewable feedstock for industrial biotechnology. BDO is the basic material for polybutylene terephthalate (PBT), poly butylene adipate-co-terephthalate (PBAT), poly (butylene succinate) (PBS), etc. Herein, the construction of BDO valorization cell factory presents great significance for waste plastic up-recycling and novel fermentation feedstock exploitation. However, BDO is hard to be metabolized and its metabolic pathway is unclear. This study presents a P. putida mutant NB10, obtained through the integration of ARTP and ALE, displaying significant growth improvement with BDO as the sole carbon source. Further genome resequencing, transcriptome analysis and genetic engineering deeply revealed the metabolic mechanism behind BDO degradation in P. putida, this study offers an excellent microbial chassis and modification strategy for plastic waste up-cycling.

Abstract Background Mutational technology has been used to achieve genome-wide variations in laboratory and industrial microorganisms. Genetic polymorphisms of natural genome evolution include nucleotide variations and structural variations, which inspired us to suggest that both types of genotypic variations are potentially useful in improving the performance of chassis cells for industrial applications. However, highly efficient approaches that simultaneously generate structural and nucleotide variations are still lacking. Results The aim of this study was to develop a method of increasing biosynthesis of astaxanthin in yeast by Combining Nucleotide variations And Structure variations (CNAS), which were generated by combinations of Atmospheric and room temperature plasma (ARTP) and Synthetic Chromosome Recombination and Modification by LoxP-Mediated Evolution (SCRaMbLE) system. CNAS was applied to increase the biosynthesis of astaxanthin in yeast and resulted in improvements of 2.2- and 7.0-fold in the yield of astaxanthin. Furthermore, this method was shown to be able to generate structures (deletion, duplication, and inversion) as well as nucleotide variations (SNPs and InDels) simultaneously. Additionally, genetic analysis of the genotypic variations of an astaxanthin improved strain revealed that the deletion of YJR116W and the C2481G mutation of YOL084W enhanced yield of astaxanthin, suggesting a genotype-to-phenotype relationship. Conclusions This study demonstrated that the CNAS strategy could generate both structure variations and nucleotide variations, allowing the enhancement of astaxanthin yield by different genotypes in yeast. Overall, this study provided a valuable tool for generating genomic variation diversity that has desirable phenotypes as well as for knowing the relationship between genotypes and phenotypes in evolutionary processes.

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. • This is the first report in which laccase yield was enhanced in A. niger through this strategy. • The findings of this study delineate an efficient method for the heterologous expression of laccase in A. niger. • The findings of this study furnish insights for amplifying protein production within heterologous platforms. • Our optimized strain is exceptionally suited for applications in the industrial and food sectors. [ABSTRACT FROM AUTHOR]

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. • Identified flowering Moso bamboo as a clonal lineage. • Revealed high spontaneous mutation rate in sexual reproduction of Moso bamboo. • Predicted high cross-pollination rates in Moso bamboo. • ARTP mutagenesis enhances structural variations in Moso bamboo offspring. [ABSTRACT FROM AUTHOR]

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. • ARTP mutagenesis was optimal at 85 s and yielded stable S. cerevisiae mutant strains, SCZ40. • The mutant strain SCZ40 enhanced GSH synthesis with a 71.1% increase in rice wine. • Up-regulation of key genes for GSH synthesis and reduction in GSH consumption pathways. [ABSTRACT FROM AUTHOR]

The original strain ASAGF 2-G4 was applied to undergo Atmospheric Room Temperature Plasma (ARTP) mutagenesis of various doses and 2 mg/mL NTG compound mutagenesis of different action durations. Five high-yield mutant strains were obtained after preliminary screening through 96-orifice-plate fermentation and re-screening in shake flasks. At last, a high-yielding mutant strain 2-A9 was selected, which increased the yield by 30.92% compared with the original strain after genetic stability verification. A high-yield mutant ASAGF 2-A9 was applied as the starting strain, the experiment design Plackett-Burman was used to screen the factors affecting the fermentation media, with a conclusion that factor glucose, dextrin, cottonseed protein and corn starch is a significant factor affecting butenyl-spinosyns. The experimental design Box-Behnken was applied to optimize the factors significantly affecting the experiment Plackett-Burman. The optimal growing media components were deduced as follows (g/L): glucose 60.0, dextrin 25.0, cottonseed protein 25.0, coen steep dry powder 12.5, peptone milk 10.0, MgSO4 1.0, NaCl 2.0, CaCO3 5.0. It’s verified that the average output of butenyl- spinosyns is close to the predicted value, indicating that the model is feasible and effective.

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 300 s. 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.

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.

(1) Background: The degradation products of ribonucleic acid (RNA)are widely used in the food and pharmaceutical industry for their flavoring and nutritional enhancement functions. Yeast is the main source for commercial RNA production, and an efficient strain is the key to reducing production costs; (2) Methods: A mutant Saccharomyces pastorianus G03H8 with a high RNA yield was developed via ARTP mutagenesis and fed-batch fermentation was applied to optimize production capacity. Genome sequencing analysis was used to reveal the underlying mechanism of higher RNA production genetic differences in the preferred mutant; (3) Results: Compared with the highest RNA content of the mutant strain, G03H8 increased by 40% compared with the parental strain G03 after response surface model optimization. Meanwhile, in fed-batch fermentation, G03H8′s dry cell weight (DCW) reached 60.58 g/L in 5 L fermenter by molasses flowing and RNA production reached up to 3.58 g/L. Genome sequencing showed that the ribosome biogenesis, yeast meiosis, RNA transport, and longevity regulating pathway were closely related to the metabolism of high RNA production; (4) Conclusion: S. pastorianus G03H8 was developed for RNA production and had the potential to greatly reduce the cost of RNA production and shorten the fermentation cycle. This work lays the foundation for efficient RNA content using S. pastorianus. [ABSTRACT FROM AUTHOR]

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.

Copyright of Journal of East China University of Science & Technology is the property of Journal of East China University of Science & Technology Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

A high production mutated strain Bacillus thuringiensis X023PN (BtX023PN) was screened from the wild strain Bacillus thuringiensis X023 (BtX023) after atmospheric and room temperature plasma (ARTP) and nitrosoguanidine (NTG) mutation. BtX023PN grows faster than the wild strain, and its lysis of mother cell was 6 h ahead BtX023, but the ability of sporulation was significantly reduced. Bioassay indicated that compared with the wild type strain, the virulence of BtX023PN against Plutella xylostella (P. xylostella) and Mythimna seperata (M. seperata) increased to 2.33-fold and 2.13-fold respectively. qRT-PCR and SDS-PAGE demonstrated that the production of Cry1Ac increased by 61%. Resequence indicated that the mutated sites enriched on the key carbohydrate metabolism and amino acid metabolism. This study provides a new strain resource for the development of Bt insecticides and a feasible technical strategy for the breeding of Bt. Key points: • Atmospheric and room temperature plasma used in breeding of Bacillus thuringiensis. • Less stationary phase time with more ICP production. • Semi-lethal concentration against Plutella xylostella reduced by about 57% [ABSTRACT FROM AUTHOR]

The threonine gene-engineered strains with the resistance to pha-ges were screened by atmospheric room temperature plasma (ARTP). The mixed phage (P-1) was isolated from the broth of threonine fermentation. P-1 was used as screening factor to mutate threonine producing strain YPT by atmospheric plasma at room temperature, preliminary screening of resistance, lysogenicity verification, genetic stability verification of phage resistance and acid production verification in fermentation tank. The results showed that a threonine producing strain with stable resistance to P-1 was obtained, which named YPT-1. The mechanism of the strain YPT-1 against phage was investigated based on molecular level. The knockout of fhuA gene was carried out by CRISPR, which indicated that the deletion of fhuA could lead to the achievement of phages resistance. The study indicates that compared with the control strain (YPT), the acid production index of the strain doesn't decrease. and the acid production is stable, which has good industrial application value. [ABSTRACT FROM AUTHOR]

• New perspectives for actinomycetes breeding. • Atmospheric and room temperature plasma used in breeding of actinomycetes. • Promoting source innovation and sustainable industrial development of actinomycetes. • Comparisons between ARTP mutation and traditional breeding methods in actinomycetes. Atmospheric and room temperature plasma (ARTP) is an emerging artificial mutagenesis breeding technology. In comparison to traditional physical and chemical methods, ARTP technology can induce DNA damage more effectively and obtain mutation strains with stable heredity more easily after screening. It possesses advantages such as simplicity, safety, non-toxicity, and cost-effectiveness, showing high application value in microbial breeding. This article focuses on ARTP mutagenesis breeding of actinomycetes, specifically highlighting the application of ARTP mutagenesis technology in improving the performance of strains and enhancing the biosynthetic capabilities of actinomycetes. We analyzed the advantages and challenges of ARTP technology in actinomycetes breeding and summarized the common features, specific mutation sites and metabolic pathways of ARTP mutagenic strains, which could give guidance for genetic modification. It suggested that the future research work should focus on the establishment of high throughput rapid screening methods and integrate transcriptomics, proteomics, metabonomics and other omics to delve into the genetic regulations and synthetic mechanisms of the bioactive substances in ARTP mutated actinomycetes. This article aims to provide new perspectives for actinomycetes breeding through the establishment and application of ARTP mutagenesis technology, thereby promoting source innovation and the sustainable industrial development of actinomycetes. [ABSTRACT FROM AUTHOR]

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. • High lipase activity of Mucor circinelloides LA-26 obtained by ARTP mutagenesis. • LA-26 showed genetic stability, and lipase activity increased to 376.7 U/g. • Biodiesel yield reached 98 % after 48 h using immobilized LA-26 as biocatalyst. • Whole-cell catalyst LA-26 retained 70 % FAME yield after 6th reuse. [ABSTRACT FROM AUTHOR]

BACKGROUND: Monascus pigments (MPs) produced by the genus Monascus, have been utilized for more than 2000 years in the food industry. In the present study, by submerged batch‐fermentation (SBF), we were able to obtain a mutant strain with a high tolerance of inhibitory compounds generated from rice husk hydrolysate, allowing the production of MPs. RESULTS: The mutant strain, M. Purpureus M523 with high rice husk hydrolysate tolerance was obtained using the atmospheric and room temperature plasma (ARTP) screening system, producing 39.48 U mL−1 extracellular total MPs (yellow and orange MPs), using non‐detoxified rice husk diluted sulfuric acid hydrolysate (RHSAH) as the carbon source in SBF. Extracellular MPs (exMPs) production was enhanced to 72.1 and 80.7 U mL−1 in supplemented SBF (SSBF) and immobilized fermentation (IF) using non‐detoxified RHSAH, with productivities of 0.16 and 0.37 U mL−1 h−1, respectively. In addition, our findings revealed that despite having a high RHSAH tolerance, the mutant strain was unable to degrade phenolic compounds. Furthermore, we discovered that inhibitory compounds, including furfural (Fur) and 5′‐hydroxymethyl furfural (5′‐HMF), not only inhibit MP biosynthesis, but also regulate the conversion of pigment components. CONCLUSION: The low‐cost agricultural by‐product, rice husk, can serve as an efficient substitute for MP production with high productivity via IF by Monascus spp. © 2021 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

4-Hydroxyphenylacetic acid (4HPAA) is an important building block for synthesizing drugs, agrochemicals, and biochemicals, and requires sustainable production to meet increasing demand. Here, we use a 4HPAA biosensor to overcome the difficulty of conventional library screening in identification of preferred mutants. Strains with higher 4HPAA production and tolerance are successfully obtained by atmospheric and room temperature plasma (ARTP) mutagenesis coupled with adaptive laboratory evolution using this biosensor. Genome shuffling integrates preferred properties in the strain GS-2-4, which produces 25.42 g/L 4HPAA. Chromosomal mutations of the strain GS-2-4 are identified by whole genome sequencing. Through comprehensive analysis and experimental validation, important genes, pathways and regulations are revealed. The best gene combination in inverse engineering, acrD - aroG , increases 4HPAA production of strain GS-2-4 by 37% further. These results emphasize precursor supply and stress resistance are keys to efficient 4HPAA biosynthesis. Our work shows the power of biosensor-assisted screening of mutants from libraries. The methods developed here can be easily adapted to construct cell factories for the production of other aromatic chemicals. Our work also provides many valuable target genes to build cell factories for efficient 4HPAA production in the future. • ARTP mutagenesis, adaptive evolution and genome shuffling were used for strain improvement. • Biosensor-assisted screening identified E. coli mutants with enhanced 4HPAA production. • Mutant genes linking to 4HPAA biosynthesis were revealed and verified by experiments. • Precursor supply and stress resistance are keys to efficient 4HPAA biosynthesis. [ABSTRACT FROM AUTHOR]

Copyright of Science & Technology of Cereals, Oils & Foods is the property of Science & Technology of Cereals, Oils & Foods Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

In order to select a high cellulase producing strain, Trichoderma pseudokoningii was used as the starting strain, the compound physical and chemical mutagenesis of normal temperature and pressure plasma (ARTP) and diethyl sulfate (DES) was used. The compound mutagenesis conditions were determined as ARTP irradiation time of 110 s and DES concentration of 0.8%. Congo red staining was used to screen the mutant strains, and the ratio of transparent circle to colony diameter of 19 mutant strains was greater than that of the original strain. The cellulase activity of 19 mutant strains was determined, and a strain AD-55 with higher cellulase activity was obtained. The filter paper enzyme activity, exonuclease activity and endonuclease activity were 2.16, 7.04 and 3.98 IU/mL, respectively. Through single factor and orthogonal experiments, the optimum enzyme production medium conditions were microcrystalline cellulose 1.25%, corn pulp powder 1.20% and (NH4)2SO4 0.20%. Under the condition, the filter paper enzyme activity, exonuclease activity and endonuclease activity of strain AD-55 were 2.89, 8.82 and 5.10 IU/mL, respectively. The strain AD-55 was genetically stable. [ABSTRACT FROM AUTHOR]

: Flavor stability is important for beer quality and extensive efforts have been undertaken to improve this. In our previous work, we proved a concept whereby metabolic engineering lager yeast with increased cellular nicotinamide adenine dinucleotide hydride (NADH) availability could enhance the flavor stability of beer. However, the method for breeding non-genetically modified strains with higher NADH levels remains unsolved. In the current study, we reported a novel approach to develop such strains based on atmospheric and room temperature plasma (ARTP) mutagenesis coupled with 2,4-dinitrophenol (DNP) selection. As a result, we obtained a serial of strains with higher NADH levels as well as improved flavor stability. For screening an optimal strain with industrial application potential, we examined the other fermentation characteristics of the mutants and ultimately obtained the optimal strain, YDR-63. The overall fermentation performance of the strain YDR-63 in pilot-scale fermentation was similar to that of the parental strain YJ-002, but the acetaldehyde production was decreased by 53.7% and the resistance staling value of beer was improved by 99.8%. The forced beer aging assay further demonstrated that the favor stability was indeed improved as the contents of 5-hydroxymethylfurfural in YDR-63 was less than that in YJ-002 and the sensory notes of staling was weaker in YDR-63. We also employed this novel approach to another industrial strain, M14, and succeeded in improving its flavor stability. All the findings demonstrated the efficiency and versatility of this new approach in developing strains with improved flavor stability for the beer industry [ABSTRACT FROM AUTHOR]

[Display omitted] • Development of protocols for ARTP mutagenesis and high-throughput screening strategies. • Three strains of Corynebacterium glutamicum with high yield of heterologous protein were obtained. • Compared with the original strain, the yield of MA6 was increased by nearly 91 % to approximately 862 mg/L. • Genome analysis was performed to locate the target gene that caused the increase in protein production. Atmospheric and room temperature plasma (ARTP) is a new and efficient mutation breeding technique. In this study, we discuss a strategy combining ARTP mutagenesis and high-throughput screening to engineer Corynebacterium glutamicum towards high yield production of heterologous proteins. First, three target strains, MC2, MA8, and MA6, were screened from the mutant library with enhanced green fluorescent protein (EGFP) as the reporter protein, and their growth stability and the influence of heterologous protein production were verified. Second, genes encoding three high-value medicinal proteins (glycoprotein D, gD; endoxylanase, XynA; and variable domain of heavy chain of heavy-chain antibody, VHH) were expressed in the mutagenized strain, which confirmed its applicability for an increased biosynthesis of other heterologous proteins. During the large-scale fermentation of C. glutamicum for VHH production, the fermentation characteristics of the best mutant MA6 were verified. Compared to the original strain, the yield of VHH obtained with strain MA6 was increased by nearly 91 % to approximately 862 mg/L. Finally, through systematic genome analysis mutations in five genes were obtained. These genes code for putative proteases or are potentially related to the bacterial restriction repair systems. These findings will help to obtain optimized chassis cells and provide a direction for in-depth research on genetic targets that can increase protein production. [ABSTRACT FROM AUTHOR]

BACKGROUND: The non-grain crop cassava has attracted intense attention in the biorefinery process. However, efficient biorefinery of whole cassava is faced with some challenges due to the existence of strain inhibition and refractory cellulose during the citrate production process. RESULTS: Here, a novel breeding method -- atmospheric and room temperature plasma (ARTP) -- was applied for strain improvement of citrate-producing strain Aspergillus niger from whole cassava. The citrate yield of the mutant obtained using ARTP mutagenesis increased by 36.5% in comparison with the original strain. Moreover, citric acid fermentation was further improved on the basis of an enhanced co-saccharification strategy by supplementing glucoamylase and cellulase. The fermentation efficiency increased by 35.8% with a 17.0 g L-1 reduction in residual sugar on a pilot scale. CONCLUSIONS: All these results confirmed that a combination of the novel breeding method and enhanced co-saccharification strategy could be used to efficiently refine whole cassava. The results also provide inspiration for the production of value-added products and waste disposal in agro-based industries. [ABSTRACT FROM AUTHOR]

(1) Background: The degradation products of ribonucleic acid (RNA)are widely used in the food and pharmaceutical industry for their flavoring and nutritional enhancement functions. Yeast is the main source for commercial RNA production, and an efficient strain is the key to reducing production costs; (2) Methods: A mutant Saccharomyces pastorianus G03H8 with a high RNA yield was developed via ARTP mutagenesis and fed-batch fermentation was applied to optimize production capacity. Genome sequencing analysis was used to reveal the underlying mechanism of higher RNA production genetic differences in the preferred mutant; (3) Results: Compared with the highest RNA content of the mutant strain, G03H8 increased by 40% compared with the parental strain G03 after response surface model optimization. Meanwhile, in fed-batch fermentation, G03H8′s dry cell weight (DCW) reached 60.58 g/L in 5 L fermenter by molasses flowing and RNA production reached up to 3.58 g/L. Genome sequencing showed that the ribosome biogenesis, yeast meiosis, RNA transport, and longevity regulating pathway were closely related to the metabolism of high RNA production; (4) Conclusion: S. pastorianus G03H8 was developed for RNA production and had the potential to greatly reduce the cost of RNA production and shorten the fermentation cycle. This work lays the foundation for efficient RNA content using S. pastorianus.

Copyright of Acta Edulis Fungi is the property of Acta Edulis Fungi and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Journal of East China University of Science & Technology is the property of Journal of East China University of Science & Technology Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The mantle of pearl oyster is one of the indispensable tissues for pearl cultivation. In order to obtain highactivity mantle cells, the effects of different gas volume groups [10, 12 and 15 SLM (standard liter per minute; SLM)] and treatment time on cell activity and biomineralizated functions of mantle cells of Hyriopsis cumingii were analyzed using ARTP mutagenesis and flow cytometry. The results showed that the activity of mantle cells of Hyriopsis cumingii increased significantly in 360--900s by ARTP, and reached the maximum value at 900s (P<0.05), which was consistent with the cell activity results of osmotic stabilizer groups. Mantle cells activity in 12 and 15 SLM groups increased significantly at 360--900s (P<0.05), and the proliferation index (PI) of mantle cells reached the maximum value in 12 SLM group (P<0.05); in addition, cells were cultured in vitro for 24h after mutagenesis, the viability of mantle cells in 12 SLM group at 720s was the highest (P<0.05), and the SOD activity (mutagenesis time was 720s, the same as below) showed a significant decreasing trend with the increase of mutagenesis gas volume, and it decreased significantly to the lowest level in the 15 SLM group (P<0.05). On the contrary, the micronucleus rate reached the maximum value; biomineralization analysis showed that the concentration of Ca2+, the key enzymes related to biomineralization (carbonic anhydrase, alkaline phosphatase) and calmodulin (CAM) genes of mantle cells reached the maximum in the 12 SLM group (P<0.05), but expression levels of EFCB1 (EF-hand calcium-binding domain-containing protein 1) gene reached the maximum in the 10 SLM group (P<0.05), followed by 12 SLM group. The above findings indicated that ARTP mutation had the most significant effect on the cell activity and other biological activity of mantle cells when the gas volume is 12 SLM for 720s and the osmotic pressure stabilizer is used together with ARTP, indicating that ARTP mutagenesis can be effective in vitro culture of mantle cells to provide a biological basis and a new idea for the establishment of cell lines. [ABSTRACT FROM AUTHOR]

Naringinase was mainly obtained by microbial fermentation, and mutagenesis was a major way for obtaining excellent mutants. The aim of this study was to screen out a high naringinase yielding mutant to enhance the potential application value of its industrialization and compare the effects of different mutagenic methods on the enzyme activity of the strain. A novel producing naringinase strain, Aspergillus tubingensis MN589840, was isolated from mildewed pomelo peel, later subjected to mutagenesis including UV, ARTP and UV‐ARTP. After five rounds iterative mutagenesis, the mutants U1, A6 and UA13 were screened out with 1448·49, 1848·71, 2475·16 U mg−1 enzyme activity, the naringinase productivity raised by 79·08, 123·56 and 206%, respectively. In addition, the naringinase activity of three mutants rose after each round of iterative mutagenesis. These results indicated that the mutagenesis efficiency of UV‐ARTP was higher than that of single ARTP, and both are better than UV. In summary, the iterative UV‐ARTP mutagenesis is an effective strategy for screening high naringinase‐producing strains. [ABSTRACT FROM AUTHOR]

During second‐generation bioethanol production from lignocellulosic biomass, the desired traits for fermenting microorganisms, such as Saccharomyces cerevisiae, are high xylose utilization and high robustness to inhibitors in lignocellulosic hydrolysates. However, as observed previously, these two traits easily showed the antagonism, one rising and the other falling, in the C6/C5 co‐fermenting S. cerevisiae strain. In this study, LF1 obtained in our previous study is an engineered budding yeast strain with a superior co‐fermentation capacity of glucose and xylose, and was then mutated by atmospheric and room temperature plasma (ARTP) mutagenesis to improve its robustness. The ARTP‐treated cells were grown in 50% (v/v) leachate from lignocellulose pretreatment with high inhibitors content for adaptive evolution. After 30 days, the generated mutant LF1‐6 showed significantly enhanced tolerance, with a six‐fold increase in cell density in the above leachate. Unfortunately, its xylose utilization dropped markedly, indicating the recurrence of the negative correlation between xylose utilization and robustness. To alleviate this antagonism, LF1‐6 cells were iteratively mutated with ARTP mutagenesis and then anaerobically grown using xylose as the sole carbon source, and xylose utilization was restored in the resulting strain 6M‐15. 6M‐15 also exhibited increased co‐fermentation performance of xylose and glucose with the highest ethanol productivity reported to date (0.525 g g−1 h−1) in high‐level mixed sugars (80 g L−1 glucose and 40 g L−1 xylose) with no inhibitors. Meanwhile, its fermentation time was shortened by 8 h compared to that of LF1. During the fermentation of non‐detoxified lignocellulosic hydrolysate with high inhibitor concentrations at pH ~3.5, 6M‐15 can efficiently convert glucose and xylose with an ethanol yield of 0.43 g g−1. 6M‐15 is also regarded as a potential chassis cell for further design of a customized strain suitable for production of second‐generation bioethanol or other high value‐added products from lignocellulosic biomass. [ABSTRACT FROM AUTHOR]

Phage infection is common during the production of L‐threonine by E. coli, and low L‐threonine production and glucose conversion percentage are bottlenecks for the efficient commercial production of L‐threonine. In this study, 20 antiphage mutants producing high concentration of L‐threonine were obtained by atmospheric and room temperature plasma (ARTP) mutagenesis, and an antiphage E. coli variant was characterized that exhibited the highest production of L‐threonine Escherichia coli ([E. coli] TRFC‐AP). The elimination of fhuA expression in E. coli TRFC‐AP was responsible for phage resistance. The biomass and cell growth of E. coli TRFC‐AP showed no significant differences from those of the parent strain (E. coli TRFC), and the production of L‐threonine (159.3 g L−1) and glucose conversion percentage (51.4%) were increased by 10.9% and 9.1%, respectively, compared with those of E. coli TRFC. During threonine production (culture time of 20 h), E. coli TRFC‐AP exhibited higher activities of key enzymes for glucose utilization (hexokinase, glucose phosphate dehydrogenase, phosphofructokinase, phosphoenolpyruvate carboxylase, and PYK) and threonine synthesis (glutamate synthase, aspartokinase, homoserine dehydrogenase, homoserine kinase and threonine synthase) compared to those of E. coli TRFC. The analysis of metabolic flux distribution indicated that the flux of threonine with E. coli TRFC‐AP reached 69.8%, an increase of 16.0% compared with that of E. coli TRFC. Overall, higher L‐threonine production and glucose conversion percentage were obtained with E. coli TRFC‐AP due to increased activities of key enzymes and improved carbon flux for threonine synthesis. [ABSTRACT FROM AUTHOR]

Natural red pigments have been widely used as food and cosmetics additives. However, due to toxic byproducts or allergen issues, it is still necessary to look for some other red pigment products. This study proposed combinatorial strategies to improve production of a new kind of red pigments from the fungus Geomyces WNF‐15A, isolated from Antarctica. A high‐production medium was developed by statistical experimental design, which was further simplified for industrial use by single‐factor experiments. Strain breeding by atmospheric room temperature plasma mutagenesis generated a mutant, Geomyces sp. WNF‐15A‐M210, which increased production of red pigments by 24.4% and shortened culture phase by 33.3% comparing with the wild‐type. The production of red pigments by this mutant favored a weak alkaline condition but required only mild dissolved oxygen tension. Control of initial pH 8.5 (process pH around 7.5) increased red pigments production by 19% comparing with natural condition. Precursor and inhibitor addition experiments indicated that the red pigments were synthesized by polyketide pathway, and feeding 6 mmol/L precursor of sodium acetate by three aliquots at days 3 to 5 improved biosynthesis of red pigments by 27%. Finally, the developed culture process was verified in a 5‐L stirred tank bioreactor. The red pigments production of the pH regulation group reached 1.11‐fold of the control and 1.95‐fold of the precursor regulation group, respectively. This study provides high‐production strain, optimized medium, and bioprocess for the possible industrial production of Antarctic Geomyces red pigments in future. Practical Application: Antarctic Geomyces red pigments showed high color value, nontoxic characteristic, and good water solubility. It holds potential for industrial use and is under development for food additive in China currently. This study provides an optional manufacturing process for this new kind of red pigments. [ABSTRACT FROM AUTHOR]

d-Arabitol is an important pentitol that is widely used in the food, pharmaceutical and chemical industries. It is mainly produced by yeasts during the biotransformation of glucose. To obtain strains with high d-arabitol production, Candida parapsilosis was mutated using atmospheric and room temperature plasma (ARTP). Among the screened mutants, mutant A6 had the highest yield at 32.92 g/L, a 53.98% increase compared with the original strain (21.38 g/L). Furthermore, metabolic regulators were added to the medium to improve d-arabitol production. Pyrithioxin dihydrochloride increased d-arabitol production by 34.4% by regulating glucose-6-phosphate dehydrogenase, and 4-methylpyrazole increased d-arabitol production by 77.4% compared with the control group by inhibiting alcohol dehydrogenase activity. Amphotericin B and Triton X-100 increased d-arabitol production by 23.8% and 42.2% by improving the membrane permeability and dissolved oxygen content, respectively. This study may provide important implications for obtaining high-yield d-arabitol strains. [ABSTRACT FROM AUTHOR]

Acid tolerance and aroma profile are crucial factors for wine production in Saccharomyces cerevisiae. However, most wine yeasts to date fail to endure low-pH environments, therefore resulting in problems such as lengthened fermentation and poor flavor during acidic fruit wine production. In the present study, we established a multiple-step screening strategy, which was composed of atmospheric and room temperature plasma (ARTP), high-throughput screening (HTS), and laboratory adaptive evolution (ALE), to screen yeast strains for potential wine-producing with enhanced performances during low pH conditions. Importantly, we obtained the S. cerevisiae strain from the mutant library, ET008-c54, which displayed exhibited excellent performances in survival rate, fermentation time, aroma profile, and genetic stability. More specifically, the survival rate of ET008-c54 at low pH was increased by 10-fold, the fermentation time of greengage plum wine was shortened by about 70%, and the content of main aroma compounds was significantly increased by 52%. Collectively, we demonstrate the practical application of the screening platform designed for discovering mutant strains in winemaking technology. Key Points • A novel multi-step method was developed for screening wine-producing yeast. • Acid tolerance and aroma profiles of yeast were improved by the proposed method. • ET008-c54 showed excellent fermenting performance and stability at low pH. [ABSTRACT FROM AUTHOR]