Room Temperature Plasma Research Articles

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.

Enhanced oxygen reduction activity of α-MnO2 by NH3 plasma treatment

Oxygen vacancies and heteroatom doping play important role in oxygen reduction activity of metal oxides. Developing efficient modification method is one of the key issues in catalysts research. Room temperature plasma treatment, with the advantages of mild working conditions, no emissions and high efficiency, is a new catalyst modification method developed in recent years. In this work, hydrothermal synthesized α-MnO2 nanorods are treated in NH3 plasma at room temperature. In the reducing atmosphere, oxygen vacancies and N doping are achieved simultaneously on the surface. The NH3 plasma etched MnO2 demonstrate a significant enhanced oxygen reduction activity with half-wave potential of 0.84 V, limiting current density of 6.32 mA cm−2 and transferred electrons number of 3.9. The Mg–air battery with N-MnO2 display a maximum power density of 76.3 mW cm−2 as well as stable discharge performance. This work provides new ideas for preparing efficient and cost-effective method to boost the catalysts activity.

One-step constructing advanced N-doped carbon@metal nitride as ultra-stable electrocatalysts via urea plasma under room temperature

Highly active transition metal nitrides are desirable for electrocatalytic reactions, but their long-term stability is still unsatisfactory and thus limiting commercial applications. Herein, for the first time, we report a unique and universal room-temperature urea plasma method for controllable synthesis of N-doped carbon coated metal (Fe, Co, Ni, etc.) nitrides arrays electrocatalysts. The preformed metal oxides arrays can be successfully converted into metal nitrides arrays with preserved nanostructures and a thin layer of N-doped carbon (NC) via one-step urea plasma. Typically, as a representative case, NC@CoN nanowire arrays are illustrated and corresponding formation mechanism by plasma is proposed. Notably, the designed NC@CoN catalysts deliver excellent electrocatalytic activity and long-term stability both in oxygen evolution reaction (OER) and urea oxidation reaction (UOR). For OER, a low overpotential (264 mV at 10 mA/cm2) and high stability (>50 h at 20 mA/cm2) are acquired. For UOR, a current density of 100 mA/cm2 is achieved at only 1.39 V and maintain over 100 h. Theoretical calculations reveal that the synergetic coupling effect of CoN and NC can significantly facilitate the charge-transfer process, optimize adsorbed intermediates binding strength and further greatly decrease the energy barrier. This strategy provides a novel method for fabrication of NC@ metal nitrides as highly active and stable catalysts.

Comparative transcriptome analysis reveals the redirection of metabolic flux from cell growth to astaxanthin biosynthesis in Yarrowia lipolytica.

Engineering Yarrowia lipolytica to produce astaxanthin provides a promising route. Here, Y. lipolytica M2 producing a titer of 181 mg/L astaxanthin was isolated by iterative atmospheric and room-temperature plasma mutagenesis and diphenylamine-mediated screening. Interestingly, a negative correlation was observed between cell biomass and astaxanthin production. To reveal the underlying mechanism, RNA-seq analysis of transcriptional changes was performed in high producer M2 and reference strain M1, and a total of 1379 differentially expressed genes were obtained. Data analysis revealed that carbon flux was elevated through lipid metabolism, acetyl-CoA and mevalonate supply, but restrained through central carbon metabolism in strain M2. Moreover, upregulation of other pathways such as ATP-binding cassette transporter and thiamine pyrophosphate possibly provided more cofactors for carotenoid hydroxylase and relieved cell membrane stress caused by astaxanthin insertion. These results suggest that balancing cell growth and astaxanthin production may be important to promote efficient biosynthesis of astaxanthin in Y. lipolytica.

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.

Improvement of ribonucleic acid production in Cyberlindnera jadinii and optimization of fermentation medium

To enhance the ribonucleic acid (RNA) productivity for industrial applications, this study employed strain screening and medium optimization to improve the content of RNA in Cyberlindnera jadinii. A rapid screening method, combining atmospheric and room temperature plasma mutagenesis, 48-deep-well plates fermentation, and microplate reader detection, was developed. A mutant strain named WB15 with high RNA content was successfully obtained, exhibiting the RNA content of 156 ± 4.5 mg/g DCW, 1.4 times of the starting strain CCTCC AY 92020. Furthermore, Plackett–Burman design and response surface methodology were employed to identify three significant factors (yeast extract, soybean peptone, and KH2PO4) affecting the RNA content. By utilizing the optimal medium composed of 13.43 g/L yeast extract, 12.12 g/L soybean peptone and 2.78 g/L KH2PO4, the RNA content of WB15 further increased to 184 ± 4.9 mg/g DCW. Additionally, the mutant strain WB15 exhibited a greater cellular width compared to AY 92020, along with increased growth rate and single-cell RNA content by 22% and 48.9%, respectively. Perturbations in ribosome assembly, specifically a reduction in the ratio of ribosomal proteins to ribosomal RNA of the large subunit, might indirectly contribute to the higher RNA content in the WB15 strain. Overall, the combination of rapid screening with fermentation medium optimization proved to be an effective approach for improving the RNA content of C. jadinii, thus facilitating the industrial production of RNA.

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

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

Synergistic application of atmospheric and room temperature plasma mutagenesis and adaptive laboratory evolution improves the tolerance of Escherichia coli to L-cysteine.

L-Cysteine production through fermentation stands as a promising technology. However, excessive accumulation of L-cysteine poses a challenge due to the potential to inflict damage on cellular DNA. In this study, we employed a synergistic approach encompassing atmospheric and room temperature plasma mutagenesis (ARTP) and adaptive laboratory evolution (ALE) to improve L-cysteine tolerance in Escherichia coli. ARTP-treated populations obtained substantial enhancement in L-cysteine tolerance by ALE. Whole-genome sequencing, transcription analysis, and reverse engineering, revealed the pivotal role of an effective export mechanism mediated by gene eamB in augmenting L-cysteine resistance. The isolated tolerant strain, 60AP03/pTrc-cysEf , achieved a 2.2-fold increase in L-cysteine titer by overexpressing the critical gene cysEf during batch fermentation, underscoring its enormous potential for L-cysteine production. The production evaluations, supplemented with L-serine, further demonstrated the stability and superiority of tolerant strains in L-cysteine production. Overall, our work highlighted the substantial impact of the combined ARTP and ALE strategy in increasing the tolerance of E. coli to L-cysteine, providing valuable insights into improving L-cysteine overproduction, and further emphasized the potential of biotechnology in industrial production.

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

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

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

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

Investigations in mutation breeding and culturing media by Xanthophyllomyces dendrorhous

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