Atmospheric And Room Temperature Plasma Mutagenesis Research Articles

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

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.

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.

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

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

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

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

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

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

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

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

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

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

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

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

Enhanced Pentostatin Production in Actinomadura sp. by Combining ARTP Mutagenesis, Ribosome Engineering and Subsequent Fermentation Optimization

The special structure of pentostatin causes it to possess a wide spectrum of biological and pharmacological properties, and it has been extensively employed to treat malignant tumors and is the first-line treatment for hairy cell leukemia. Pentostatin is mainly distributed in several actinomycetes and fungi species. However, its low titer in microbes is not able to meet medical needs. Here, we report a strain improvement strategy based on combined atmospheric and room-temperature plasma (ARTP) mutagenesis and ribosome engineering screening, as well as fermentation optimization, for enhanced pentostatin production. The original strain, Actinomadura sp. ATCC 39365, was treated with ARTP and screened by ribosome engineering to obtain one stable pentostatin high-yield mutant Actinomadura sp. S-15, which produced 86.35 mg/L pentostatin, representing a 33.79% increase compared to Actinomadura sp. ATCC 39365. qRT-PCR analysis revealed that pentostatin biosynthesis-related gene expression was significantly upregulated in Actinomadura sp. S-15. Then, to further enhance pentostatin production, the fermentation medium was optimized in flask culture and the pentostatin production of Actinomadura sp. S-15 reached 152.06 mg/L, which is the highest pentostatin production reported so far. These results demonstrate the effectiveness of combined ARTP mutation, ribosome engineering screening, and medium optimization for the enhancement of pentostatin production, and provide a methodology enabling the sustainable production of pentostatin on an industrial scale.

Adaptive evolutionary strategy coupled with an optimized biosynthesis process for the efficient production of pyrroloquinoline quinone from methanol

BackgroundPyrroloquinoline quinone (PQQ), a cofactor for bacterial dehydrogenases, is associated with biological processes such as mitochondriogenesis, reproduction, growth, and aging. Due to the extremely high cost of chemical synthesis and low yield of microbial synthesis, the election of effective strains and the development of dynamic fermentation strategies for enhancing PQQ production are meaningful movements to meet the large-scale industrial requirements.ResultsA high-titer PQQ-producing mutant strain, Hyphomicrobium denitrificans FJNU-A26, was obtained by integrating ARTP (atmospheric and room‑temperature plasma) mutagenesis, adaptive laboratory evolution and high-throughput screening strategies. Afterward, the systematic optimization of the fermentation medium was conducted using a one-factor-at-a-time strategy and response surface methodology to increase the PQQ concentration from 1.02 to 1.37 g/L. The transcriptional analysis using qRT-PCR revealed that the expression of genes involved in PQQ biosynthesis were significantly upregulated when the ARTP-ALE-derived mutant was applied. Furthermore, a novel two-stage pH control strategy was introduced to address the inconsistent effects of the pH value on cell growth and PQQ production. These combined strategies led to a 148% increase in the PQQ concentration compared with that of the initial strain FJNU-6, reaching 1.52 g/L with a yield of 40.3 mg/g DCW after 144 h of fed-batch fermentation in a 5-L fermenter.ConclusionThe characteristics above suggest that FJNU-A26 represents an effective candidate as an industrial PQQ producer, and the integrated strategies can be readily extended to other microorganisms for the large-scale production of PQQ.

Breeding of New Strains of Gracilariopsis lemaneiformis with High Agar Content by ARTP Mutagenesis and High Osmotic Pressure Screening.

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 46s 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.

Construction of automated high-throughput screening method for finding efficient 3-ketosteroid 1,2-dehydrogenating strains.

Dehydrogenation reaction at C1(2) positions is typical and representative of industrial production of steroid drugs. Anti-inflammatory activity can be doubled when the nucleus of the anti-inflammatory steroid hormone drug introduces double bonds at the C1(2) positions. Arthrobacter simplex is currently the most widely studied and used strain for C1(2) dehydrogenation. Therefore, breeding Arthrobacter simplex with high-efficiency dehydrogenation ability is of great significance. In order to obtain high-efficiency strains, the research proposed a new screening strategy based on image process technique: firstly, a color reaction between 2,4-dinitrophenylhydrazine (DNPH) and 9α-hydroxyandrost-4-ene-3,17-dione (9α-OH-AD) was established to characterize the dehydrogenation ability of the strain; secondly, the color data of strains mutated by atmospheric and room temperature plasma (ARTP) in the "color reaction" were automated and analyzed for dehydrogenation ability prediction using optimized support vector machine model. Result showed that the prediction accuracy reached as high as 96% in verification experiments. After a series of mutagenesis, including breaking the bottleneck of a single mutation in ARTP, the dominant strain ARLU-146 was finally obtained from 5168 strains. Its initial conversion rate was 0.8059g/L/h, with a conversion of 94.41% at 24h, compared to the original strain ASP which increased the transformation rate by more than 10%. By further process optimization, a high conversion (94.34% within 20h) with high substrate (85g/L cortisone acetate) was achieved. According to literature research, it is the highest conversion at this substrate concentration. KEY POINTS: • A high-throughput screening method was developed by using image processing and machine learning technique. • "Mutation bottleneck" of single ARTP mutagenesis was surpassed by complex mutagenesis. • A high substrate (85g/L CA) and high transformation rate craft (94.34% within 20h) were built.

ARTP mutagenesis promotes selenium accumulation in Saccharomyces boulardii

Atmospheric and room temperature plasma (ARTP) is a rapid method for strain to obtain desirable mutants. In this study, Saccharomyces boulardii was used for mutagenesis through ARTP mutagenesis and the medium for cultivation was further optimized by Box–Behnken design experiments. The biomass of the optimal mutant MSBV reached 42.8 g/L wet cell weight (WCW), and the total selenium (Se) was 13.96 mg/g dry cell weight with the addition of 300 μg/mL of sodium selenite at 0 h medium for 48 h cultivation. In the same conditions, the biomass of wild type (WT) only reached 29.68 g/L WCW, and the Se content was 8.79 mg/g. In addition, MSBV bound to selenium compounds accounted for 76.84% of the total protein, while WT accounted for 79.24%, which was consistent with the energy dispersive spectroscopic analysis. Transmission electron microscopy images of Se-enriched yeast showed that elemental Se0 had a desirable size of approximately 50–200 nm and Se nanoparticles were possibly coated by protein. Therefore, it is worth characterizing the Se nanoparticles and studying the mechanism in the future work. In this study, due to its high Se accumulation and high biomass content, the mutant yeast shows great potential applications in food, medical and pharmaceutic industries.

Microfluidic screening and genomic mutation identification for enhancing cellulase production in Pichia pastoris

BackgroundPichia pastoris is a widely used host organism for heterologous production of industrial proteins, such as cellulases. Although great progress has been achieved in improving protein expression in P. pastoris, the potential of the P. pastoris expression system has not been fully explored due to unknown genomic impact factors. Recently, whole-cell directed evolution, employing iterative rounds of genome-wide diversity generation and high-throughput screening (HTS), has been considered to be a promising strategy in strain improvement at the genome level.ResultsIn this study, whole-cell directed evolution of P. pastoris, employing atmospheric and room temperature plasma (ARTP) mutagenesis and droplet-based microfluidic HTS, was developed to improve heterogenous cellulase production. The droplet-based microfluidic platform based on a cellulase-catalyzed reaction of releasing fluorescence was established to be suitable for methanol-grown P. pastoris. The validation experiment showed a positive sorting efficiency of 94.4% at a sorting rate of 300 droplets per second. After five rounds of iterative ARTP mutagenesis and microfluidic screening, the best mutant strain was obtained and exhibited the cellulase activity of 11,110 ± 523 U/mL, an approximately twofold increase compared to the starting strain. Whole-genome resequencing analysis further uncovered three accumulated genomic alterations in coding region. The effects of point mutations and mutant genes on cellulase production were verified using reconstruction of point mutations and gene deletions. Intriguingly, the point mutation Rsc1G22V was observed in all the top-performing producers selected from each round, and gene deletion analysis confirmed that Rsc1, a component of the RSC chromatin remodeling complex, might play an important role in cellulase production.ConclusionsWe established a droplet-based microfluidic HTS system, thereby facilitating whole-cell directed evolution of P. pastoris for enhancing cellulase production, and meanwhile identified genomic alterations by whole-genome resequencing and genetic validation. Our approaches and findings would provide guides to accelerate whole-cell directed evolution of host strains and enzymes of high industrial interest.

Improved laccase production in Pleurotus djamor RP by atmospheric and room temperature plasma (ARTP) mutagenesis

BackgroundPleurotus djamor is an edible mushroom with medicinal properties, and degrades lignocellulose by producing enzymes that have a variety of industrial applications. The aim of this study was to improve laccase producing potential of P. djamor that may easily convert lignocellulose waste into high-value products. ResultsIn the present study, protoplast of the newly isolated fungus P. djamor RP was mutated by atmospheric pressure and room temperature plasma (ARTP) method. Guaiacol was used as an indicator for the rapid visual appearance of laccase positives in the screening process. From the results of preliminary screening, 10 promising positive mutants with a maximum laccase production were selected for further experimentation. Lac activity by the ABTS method showed that the mutant P. djamor strain 51-4 exhibited the maximum laccase production of 494.44 U/L in shake flask cultivation, which was 86.36% higher than wild strain. Notably, the other mutant P. djamor strain 62-27 exhibits a higher laccase production of 26.06 U/g which was 30.28% higher than wild strain when grown on solid-state fermentation. In addition to a higher yield of laccase, all mutants showed better growth and genetic stability than the wild strain. ConclusionsP. djamor RP mutants with high laccase-producing potential were developed by ARTP Mutagenesis technique, and these isolates could be used as a possible candidate for lignocellulose conversion into high-value products.How to cite: Zhang Z, Shah AM, Mohamed H, et al. Improved laccase production in Pleurotus djamor RP by atmospheric and room temperature plasma (ARTP) mutagenesis. Electron J Biotechnol 2022;58. https://doi.org/10.1016/j.ejbt.2022.03.005

ARTP Mutagenesis of Schizochytrium sp. PKU#Mn4 and Clethodim-Based Mutant Screening for Enhanced Docosahexaenoic Acid Accumulation.

Schizochytrium species are one of the best oleaginous thraustochytrids for high-yield production of docosahexaenoic acid (DHA, 22:6). However, the DHA yields from most wild-type (WT) strains of Schizochytrium are unsatisfactory for large-scale production. In this study, we applied the atmospheric and room-temperature plasma (ARTP) tool to obtain the mutant library of a previously isolated strain of Schizochytrium (i.e., PKU#Mn4). Two rounds of ARTP mutagenesis coupled with the acetyl-CoA carboxylase (ACCase) inhibitor (clethodim)-based screening yielded the mutant A78 that not only displayed better growth, glucose uptake and ACCase activity, but also increased (54.1%) DHA content than that of the WT strain. Subsequent optimization of medium components and supplementation improved the DHA content by 75.5 and 37.2%, respectively, compared with that of mutant A78 cultivated in the unoptimized medium. Interestingly, the ACCase activity of mutant A78 in a medium supplemented with biotin, citric acid or sodium citrate was significantly greater than that in a medium without supplementation. This study provides an effective bioengineering approach for improving the DHA accumulation in oleaginous microbes.

Ameliorating end-product inhibition to improve cadaverine production in engineered Escherichia coli and its application in the synthesis of bio-based diisocyanates

Cadaverine is an important C5 platform chemical with a wide range of industrial applications. However, the cadaverine inhibition on the fermenting strain limited its industrial efficiency of the strain. In this study, we report an engineered Escherichia coli strain with high cadaverine productivity that was generated by developing a robust host coupled with metabolic engineering to mitigate cadaverine inhibition. First, a lysine producing E. coli was treated with a combination of radiation (ultraviolet and visible spectrum) and ARTP (atmospheric and room temperature plasma) mutagenesis to obtain a robust host with high cadaverine tolerance. Three mutant targets including HokD, PhnI and PuuR are identified for improved cadaverine tolerance. Further transcriptome analysis suggested that cadaverine suppressed the synthesis of ATP and lysine precursor. Accordingly, the related genes involved in glycolysis and lysine precursor, as well as cadaverine exporter was engineered to release the cadaverine inhibition. The final engineered strain was fed-batch cultured and a titer of 58.7 g/L cadaverine was achieved with a yield of 0.396 g/g, both of which were the highest level reported to date in E. coli. The bio-based cadaverine was purified to >99.6% purity, and successfully used for the synthesis of polyurethane precursor 1,5-pentamethylene diisocyanate (PDI) through the approach of carbamate decomposition.