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Keywords = protoplast preparation

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14 pages, 3272 KiB  
Article
Optimization of Protoplast Preparation and Establishment of PEG-Mediated Genetic Transformation Method in Cordyceps cicadae
by Haikun Qi, Haihua Ruan, Tao Wu, Hongyang Zhang, Rui Dong and Yanjun Jiang
J. Fungi 2025, 11(3), 219; https://doi.org/10.3390/jof11030219 - 13 Mar 2025
Viewed by 1291
Abstract
Cordyceps cicadae (C. cicadae) is an important edible medicinal fungus; however, owing to its wild growth and lack of genome annotation, construction of a stable genetic transformation system in C. cicadae is greatly limited, impeding the extensive exploitation of C. cicadae [...] Read more.
Cordyceps cicadae (C. cicadae) is an important edible medicinal fungus; however, owing to its wild growth and lack of genome annotation, construction of a stable genetic transformation system in C. cicadae is greatly limited, impeding the extensive exploitation of C. cicadae in industry. Here, we successfully established an efficient plasmid transformation method within protoplasts of C. cicadae by PEG mediation using pCas9-EGFP as a marker plasmid. In order to overcome low transformation efficiency and acquire sufficient protoplasts for transformation, the influence of enzyme species, enzymatic hydrolysis time, enzymatic hydrolysis temperature, and fungal age on protoplast preparation were analyzed sequentially, and the optimal conditions for protoplast preparation were determined as follows: 2-day-old C. cicadae mycelia with 1.5% lywallzyme hydrolysis at 34 °C for 5 h. Our results indicate that no less than 5.1 × 107 CFU/mL protoplasts could be acquired. Additionally, five osmotic pressure stabilizers including potassium chloride (KCl), sodium chloride (NaCl), glucose, mannitol, and sucrose were employed to enhance the regeneration of protoplasts, among which sucrose exhibited the highest regeneration rate of 10.43%. The transformation efficiency of plasmid was 37.3 CFU/µg DNA. On this basis, a genetic transformation method was successfully constructed, laying the foundation for further gene editing and metabolic engineering of C. cicadae. Full article
(This article belongs to the Section Fungal Genomics, Genetics and Molecular Biology)
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19 pages, 4807 KiB  
Article
Optimization of Protoplast Preparation Conditions in Lyophyllum decastes and Transcriptomic Analysis Throughout the Process
by Xiaobin Li, Ying Qin, Yufei Kong, Samantha Chandranath Karunarathna, Yunjiang Liang and Jize Xu
J. Fungi 2024, 10(12), 886; https://doi.org/10.3390/jof10120886 - 21 Dec 2024
Cited by 2 | Viewed by 1357
Abstract
Protoplasts are essential tools for genetic manipulation and functional genomics research in fungi. This study systematically optimized protoplast preparation conditions and examined transcriptional changes throughout the preparation and regeneration processes to elucidate the molecular mechanisms underlying the formation and regeneration of protoplasts in [...] Read more.
Protoplasts are essential tools for genetic manipulation and functional genomics research in fungi. This study systematically optimized protoplast preparation conditions and examined transcriptional changes throughout the preparation and regeneration processes to elucidate the molecular mechanisms underlying the formation and regeneration of protoplasts in Lyophyllum decastes. The results indicated an optimal protoplast yield of 5.475 × 106 cells/mL under conditions of fungal age at 10 days, digestion time of 2.25 h, enzyme concentration of 2%, and digestion temperature of 28 °C. The Z5 medium supplemented with L. decastes mycelial extract achieved a high regeneration rate of 2.86. RNA-seq analysis revealed 2432 differentially expressed genes (DEGs) during protoplast formation and 5825 DEGs during regeneration. Casein kinase I, cytochrome P450 (CYP52), and redox-regulated input receptor (PEX5) were significantly upregulated during the protoplast stage, while β-1,3-glucan synthase (SKN1), chitin synthase (CHS2), hydrophobin-1, and hydrophobin-2 showed significant upregulation during the protoplast regeneration phase. These findings provide a reference for the efficient preparation and regeneration of protoplasts and offer new insights into the molecular mechanisms of protoplast formation and cell wall regeneration in fungi. Full article
(This article belongs to the Special Issue Current Trends in Mycological Research in Southeast Asia)
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28 pages, 4507 KiB  
Article
Development of a Robust Saccharomyces cerevisiae Strain for Efficient Co-Fermentation of Mixed Sugars and Enhanced Inhibitor Tolerance through Protoplast Fusion
by Jianzhi Zhao, Yuping Zhao, Longhao Wu, Ning Yan, Shuo Yang, Lili Xu, Deyun He, Hongxing Li and Xiaoming Bao
Microorganisms 2024, 12(8), 1526; https://doi.org/10.3390/microorganisms12081526 - 25 Jul 2024
Cited by 7 | Viewed by 2191
Abstract
The economical and efficient commercial production of second-generation bioethanol requires fermentation microorganisms capable of entirely and rapidly utilizing all sugars in lignocellulosic hydrolysates. In this study, we developed a recombinant Saccharomyces cerevisiae strain, BLH510, through protoplast fusion and metabolic engineering to enhance its [...] Read more.
The economical and efficient commercial production of second-generation bioethanol requires fermentation microorganisms capable of entirely and rapidly utilizing all sugars in lignocellulosic hydrolysates. In this study, we developed a recombinant Saccharomyces cerevisiae strain, BLH510, through protoplast fusion and metabolic engineering to enhance its ability to co-ferment glucose, xylose, cellobiose, and xylooligosaccharides while tolerating various inhibitors commonly found in lignocellulosic hydrolysates. The parental strains, LF1 and BLN26, were selected for their superior glucose/xylose co-fermentation capabilities and inhibitor tolerance, respectively. The fusion strain BLH510 demonstrated efficient utilization of mixed sugars and high ethanol yield under oxygen-limited conditions. Under low inoculum conditions, strain BLH510 could completely consume all four kinds of sugars in the medium within 84 h. The fermentation produced 33.96 g/L ethanol, achieving 84.3% of the theoretical ethanol yield. Despite the challenging presence of mixed inhibitors, BLH510 successfully metabolized all four sugars above after 120 h of fermentation, producing approximately 30 g/L ethanol and reaching 83% of the theoretical yield. Also, strain BLH510 exhibited increased intracellular trehalose content, particularly under conditions with mixed inhibitors, where the intracellular trehalose reached 239.3 mg/g yeast biomass. This elevated trehalose content contributes to the enhanced stress tolerance of BLH510. The study also optimized conditions for protoplast preparation and fusion, balancing high preparation efficiency and satisfactory regeneration efficiency. The results indicate that BLH510 is a promising candidate for industrial second-generation bioethanol production from lignocellulosic biomass, offering improved performance under challenging fermentation conditions. Our work demonstrates the potential of combining protoplast fusion and metabolic engineering to develop superior S. cerevisiae strains for lignocellulosic bioethanol production. This approach can also be extended to develop robust microbial platforms for producing a wide array of lignocellulosic biomass-based biochemicals. Full article
(This article belongs to the Section Microbial Biotechnology)
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15 pages, 7788 KiB  
Article
Selection and Genetic Analysis of High Polysaccharide-Producing Mutants in Inonotus obliquus
by Lanlan Hua, Hongling Shi, Qing Lin, Haozhong Wang, Yan Gao, Jun Zeng, Kai Lou and Xiangdong Huo
Microorganisms 2024, 12(7), 1335; https://doi.org/10.3390/microorganisms12071335 - 29 Jun 2024
Cited by 5 | Viewed by 1818
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 [...] Read more.
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. Full article
(This article belongs to the Section Microbial Biotechnology)
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13 pages, 10711 KiB  
Article
Preparation of Bioaerogel from Iron-Rich Microalgae for the Removal of Water Pollutants
by Xinqi Niu, Junhui Si, Binyi Chen, Qianting Wang, Sen Zeng and Zhixiang Cui
Processes 2024, 12(7), 1313; https://doi.org/10.3390/pr12071313 - 24 Jun 2024
Cited by 5 | Viewed by 1839
Abstract
Microalgae-based materials have gained significant attention considering their rich resources, cost-effectiveness, and environmental friendliness. Herein, iron-rich microalgae (Chlorella pyrenoidosa, CP) were treated by hydrothermal reaction under alkaline conditions to remove the protoplast and obtain a hollow shell with an FexOy [...] Read more.
Microalgae-based materials have gained significant attention considering their rich resources, cost-effectiveness, and environmental friendliness. Herein, iron-rich microalgae (Chlorella pyrenoidosa, CP) were treated by hydrothermal reaction under alkaline conditions to remove the protoplast and obtain a hollow shell with an FexOy core inside. Then, the iron-rich microalgae-based aerogel (Fe-CP aerogel) was fabricated through a freeze-drying process. The as-prepared Fe-CP aerogel exhibited superior adsorption performance, and the maximum adsorption quantity for Cu2+ could reach 208.3 mg/g due to the synergistic adsorption of the hollow shell of CP cells and FexOy core. The Fe-CP aerogel also possessed super-hydrophilicity and displayed high separation efficiency (over 99%) when used for separating different oil/water emulsions. Moreover, the existence of FexOy endowed the Fe-CP aerogel with photo-Fenton activity, thus exhibiting excellent antifouling performance. The prepared Fe-CP aerogel could be considered an ideal adsorbent, with green, efficient, low-cost, and regenerative properties, and has the potential for the practical treatment of wastewater. Full article
(This article belongs to the Special Issue State-of-the-Art Wastewater Treatment Techniques)
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16 pages, 2263 KiB  
Article
Biological Applications of Synthetic Binders Isolated from a Conceptually New Adhiron Library
by Claudia D’Ercole, Matteo De March, Gianluca Veggiani, Sandra Oloketuyi, Rossella Svigelj and Ario de Marco
Biomolecules 2023, 13(10), 1533; https://doi.org/10.3390/biom13101533 - 17 Oct 2023
Cited by 3 | Viewed by 2658
Abstract
Background: Adhirons are small (10 kDa) synthetic ligands that might represent an alternative to antibody fragments and to alternative scaffolds such as DARPins or affibodies. Methods: We prepared a conceptionally new adhiron phage display library that allows the presence of cysteines in the [...] Read more.
Background: Adhirons are small (10 kDa) synthetic ligands that might represent an alternative to antibody fragments and to alternative scaffolds such as DARPins or affibodies. Methods: We prepared a conceptionally new adhiron phage display library that allows the presence of cysteines in the hypervariable loops and successfully panned it against antigens possessing different characteristics. Results: We recovered binders specific for membrane epitopes of plant cells by panning the library directly against pea protoplasts and against soluble C-Reactive Protein and SpyCatcher, a small protein domain for which we failed to isolate binders using pre-immune nanobody libraries. The best binders had a binding constant in the low nM range, were produced easily in bacteria (average yields of 15 mg/L of culture) in combination with different tags, were stable, and had minimal aggregation propensity, independent of the presence or absence of cysteine residues in their loops. Discussion: The isolated adhirons were significantly stronger than those isolated previously from other libraries and as good as nanobodies recovered from a naïve library of comparable theoretical diversity. Moreover, they proved to be suitable reagents for ELISA, flow cytometry, the western blot, and also as capture elements in electrochemical biosensors. Full article
(This article belongs to the Collection Feature Papers in 'Biomacromolecules: Proteins')
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14 pages, 1294 KiB  
Review
Guidelines for Performing CRISPR/Cas9 Genome Editing for Gene Validation and Trait Improvement in Crops
by Nikolaos Tsakirpaloglou, Endang M. Septiningsih and Michael J. Thomson
Plants 2023, 12(20), 3564; https://doi.org/10.3390/plants12203564 - 13 Oct 2023
Cited by 10 | Viewed by 7188
Abstract
With the rapid advances in plant genome editing techniques over the past 10 years, more efficient and powerful crop genome editing applications are now possible. Candidate genes for key traits can be validated using CRISPR/Cas9-based knockouts and through the up- and down-regulation of [...] Read more.
With the rapid advances in plant genome editing techniques over the past 10 years, more efficient and powerful crop genome editing applications are now possible. Candidate genes for key traits can be validated using CRISPR/Cas9-based knockouts and through the up- and down-regulation of gene expression. Likewise, new trait improvement approaches can take advantage of targeted editing to improve stress tolerance, disease resistance, and nutritional traits. However, several key steps in the process can prove tricky for researchers who might be new to plant genome editing. Here, we present step-by-step guidelines and best practices for a crop genome editing pipeline that should help to improve the rate of success. Important factors in the process include proper target sequence analysis and single guide RNA (sgRNA) design, sequencing of the target site in the genotypes of interest, performing an in vitro CRISPR/Cas9 ribonucleoprotein (RNP) assay to validate the designed sgRNAs, preparing the transformation constructs, considering a protoplast editing step as further validation, and, finally, stable plant transformation and mutation detection by Sanger and/or next-generation sequencing. With these detailed guidelines, a new user should be able to quickly set up a genome editing pipeline in their crop of interest and start making progress with the different CRISPR/Cas-based editing variants for gene validation and trait improvement purposes. Full article
(This article belongs to the Special Issue Plant Genetic Engineering and Biotechnology)
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13 pages, 1388 KiB  
Article
Enhancement of Triterpenoid Synthesis in Antrodia cinnamomea through Homologous Expression of the Key Synthetic Pathway Genes AcLSS and AcERG4
by Siqi Zheng, Mingyue Fang, Jiaxin Huang, Yanbin Li and Yuxia Mei
Fermentation 2023, 9(10), 880; https://doi.org/10.3390/fermentation9100880 - 29 Sep 2023
Cited by 2 | Viewed by 1795
Abstract
Antrodia cinnamomea (AC), a rare fungus endemic to Taiwan, contains high levels of various secondary metabolites, notably triterpenoids, having useful medicinal and pharmacological properties. Techniques for increasing the production of AC triterpenoids (ACT) for medicinal purposes are a high research priority. We measured [...] Read more.
Antrodia cinnamomea (AC), a rare fungus endemic to Taiwan, contains high levels of various secondary metabolites, notably triterpenoids, having useful medicinal and pharmacological properties. Techniques for increasing the production of AC triterpenoids (ACT) for medicinal purposes are a high research priority. We measured and compared the biomass and ACT content of AC mycelia under various liquid fermentation culture conditions. Relative gene expression levels of ten enzymes involved in the mevalonate (MVA) pathway and “subsequent group modification pathway” were determined, and correlation analysis was performed to evaluate the roles of these enzyme genes in ACT synthesis. Two representative genes encoding the enzymes lanosterol synthase (AcLSS) and sterol C-24 reductase (AcERG4), whose activity is closely associated with ACT content, were selected for homologous expression. AcLSS and AcERG4 were separately linked to plasmid pCT74, and transformed into prepared AC protoplasts to obtain two recombinant strains, termed RpLSS and RpERG4, by polyethylene glycol (PEG)-CaCl2-mediated protoplast transformation. Upregulated expression levels of AcLSS and AcERG4 (1.78- and 1.41-fold, respectively) were associated with significantly higher (1.82- and 1.37-fold, respectively) ACT content in the recombinant strains in comparison with the wild-type. Our findings provide a theoretical and practical basis for the enhancement of ACT production using homologous expression techniques. Full article
(This article belongs to the Section Microbial Metabolism, Physiology & Genetics)
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14 pages, 2198 KiB  
Article
Optimization of Protoplast Preparation System from Leaves and Establishment of a Transient Transformation System in Apium graveolens
by Jiageng Du, Huitao Zhang, Weilong Li, Xiaoyan Li, Zhuo Wang, Ying Zhang, Aisheng Xiong and Mengyao Li
Agronomy 2023, 13(8), 2154; https://doi.org/10.3390/agronomy13082154 - 17 Aug 2023
Cited by 9 | Viewed by 3698
Abstract
Protoplast culture and transformation technology offer a novel method for developing new plant varieties. Nonetheless, the effective preparation of protoplasts and transformation technology specific to celery has yet to be achieved. This study utilized celery seedling leaves as the primary materials to examine [...] Read more.
Protoplast culture and transformation technology offer a novel method for developing new plant varieties. Nonetheless, the effective preparation of protoplasts and transformation technology specific to celery has yet to be achieved. This study utilized celery seedling leaves as the primary materials to examine the key factors influencing protoplast isolation. The aim was to prepare leaf protoplasts with a high yield and of high quality and subsequently conduct transient gene transformation and expression. The findings indicated that the most effective procedure for isolating and purifying protoplasts was enzymatic digestion using an enzyme solution consisting of 2.0% cellulase, 0.1% pectolase, and 0.6 M mannitol for a duration of 8 h. Subsequently, the protoplasts were filtered through a 400-mesh sieve and purified through centrifugation at 200× g. Within this system, the overall protoplast yield was exceptionally high, reaching a viability rate of up to 95%. The transient transformation system yielded a maximum transformation efficiency of approximately 53%, as evaluated using the green fluorescent protein (GFP) as a reporter gene. The parameters of the transient transformation system were as follows: a protoplast concentration of 5 × 105 cells·mL−1, exogenous DNA concentration of 500 μg·mL−1, final concentration of PEG4000 at 40%, and transformation duration of 15 min. The transient transformation system was also utilized to further analyze the protein localization characteristics of the celery transcription factor AgMYB80. The findings indicated that AgMYB80 predominantly localizes in the nucleus, thereby confirming the reliability and effectiveness of the transient transformation system. This study successfully established an efficient system for isolating, purifying, and transforming celery protoplasts, and will serve as a basis for future studies on molecular biology and gene function. Full article
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15 pages, 43230 KiB  
Article
Establishment of PEG-Mediated Transient Gene Expression in Protoplasts Isolated from the Callus of Cunninghamia lanceolata
by Kaiyue Hong, Zhenyu Chen, Yasmina Radani, Renhua Zheng, Xueyan Zheng, Yong Li, Jinhui Chen and Liming Yang
Forests 2023, 14(6), 1168; https://doi.org/10.3390/f14061168 - 6 Jun 2023
Cited by 5 | Viewed by 2627
Abstract
Cunninghamia lanceolata (C. lanceolata) is an important timber tree species in southern China that requires gene function studies to understand its traits. In this study, we investigated the callus induction rates of immature zygotic embryos from reciprocal hybrids between genotypes B46 [...] Read more.
Cunninghamia lanceolata (C. lanceolata) is an important timber tree species in southern China that requires gene function studies to understand its traits. In this study, we investigated the callus induction rates of immature zygotic embryos from reciprocal hybrids between genotypes B46 and B49. With zygotic embryo development, the callus induction rates showed an increasing trend, followed by a decreasing trend. Moreover, the rate of callus induction in genotype B46 × B49 immature zygotic embryos was greater than in genotype B49 × B46. Callus from C. lanceolata with genotype B46 × B49 was selected as the donor material for protoplast isolation. By using an enzymatic digestion solution containing cellulase, macerozyme, and pectinase, combined with an osmotic stabilizer, we obtained 9.76 × 106 protoplasts/mL with 92.7% viability. We subsequently transformed plasmids into C. lanceolata callus protoplasts and observed the location of the H2B-eYFP fusion protein in the nucleus. To achieve transient transfection of C. lanceolata callus protoplasts, we compared transfection efficiencies at different concentrations of PEG4000, PEG6000, or PEG8000 in a modified MMg solution. We found that 20% (w/v) PEG6000 mediated the transient transfection of C. lanceolata callus protoplasts with a 6.70% efficiency. This study provides a technical foundation for future research on transient transfection and functional analysis of C. lanceolata genes. Full article
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17 pages, 1632 KiB  
Review
Protoplast Preparation for Algal Single-Cell Omics Sequencing
by Junran Ye, Cuiqiyun Yang, Luojia Xia, Yinjie Zhu, Li Liu, Huansheng Cao and Yi Tao
Microorganisms 2023, 11(2), 538; https://doi.org/10.3390/microorganisms11020538 - 20 Feb 2023
Cited by 9 | Viewed by 4482
Abstract
Single-cell sequencing (SCS) is an evolutionary technique for conducting life science research, providing the highest genome-sale throughput and single-cell resolution and unprecedented capabilities in addressing mechanistic and operational questions. Unfortunately, the current SCS pipeline cannot be directly applied to algal research as algal [...] Read more.
Single-cell sequencing (SCS) is an evolutionary technique for conducting life science research, providing the highest genome-sale throughput and single-cell resolution and unprecedented capabilities in addressing mechanistic and operational questions. Unfortunately, the current SCS pipeline cannot be directly applied to algal research as algal cells have cell walls, which makes RNA extraction hard for the current SCS platforms. Fortunately, effective methods are available for producing algal protoplasts (cells without cell walls), which can be directly fed into current SCS pipelines. In this review, we first summarize the cell wall structure and chemical composition of algal cell walls, particularly in Chlorophyta, then summarize the advances made in preparing algal protoplasts using physical, chemical, and biological methods, followed by specific cases of algal protoplast production in some commonly used eukaryotic algae. This review provides a timely primer to those interested in applying SCS in eukaryotic algal research. Full article
(This article belongs to the Section Microbial Biotechnology)
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12 pages, 2094 KiB  
Article
CRISPR/Cas9-Mediated Multiplexed Genome Editing in Aspergillus oryzae
by Qinghua Li, Jinchang Lu, Guoqiang Zhang, Jingwen Zhou, Jianghua Li, Guocheng Du and Jian Chen
J. Fungi 2023, 9(1), 109; https://doi.org/10.3390/jof9010109 - 13 Jan 2023
Cited by 22 | Viewed by 5762
Abstract
Aspergillus oryzae has great potential and competitive advantages to be developed as an excellent expression system, owing to its powerful protein secretion ability, complex post-translational modification, and safety characteristics. However, the low efficiency of genetic modification and gene function analysis is an urgent [...] Read more.
Aspergillus oryzae has great potential and competitive advantages to be developed as an excellent expression system, owing to its powerful protein secretion ability, complex post-translational modification, and safety characteristics. However, the low efficiency of genetic modification and gene function analysis is an urgent problem to be solved in A. oryzae and other filamentous fungal systems. Therefore, establishing efficient genetic transformation and multiplexed genome editing tools is significant for developing A. oryzae expression systems, and revealing its intrinsic mechanisms. In this study, the high-efficiency transformation of A. oryzae was achieved by optimizing the preparation conditions of protoplasts, and the random editing efficiency of the CRISPR/Cas9 system in A. oryzae for single and double genes reached 37.6% and 19.8%, respectively. With the aid of the selection marker, such as color or resistance, the editing efficiency of single and double genes can reach 100%. Based on the developed CRISPR/Cas9 genome editing method, the heterologous lipase gene (TLL) achieves precise integration at different genetic loci in one step. The efficient and accurate acquisition of positive transformants indicated that the morphological gene yA could be used as a helpful selection marker for genome editing in A. oryzae. In conclusion, the developed system improves the efficiency of transformation and multiplexed genome editing for A. oryzae. It provides a practical method for developing the A. oryzae high-efficiency expression system for heterologous proteins. Full article
(This article belongs to the Special Issue Metabolic Engineering of Aspergillus via CRISPR-Based Systems)
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25 pages, 3124 KiB  
Article
Media Optimization by Response Surface Methodology for the Enhanced Production of Acidic Extracellular Pectinase by the Indigenously Isolated Novel Strain Aspergillus cervinus ARS2 Using Solid-State Fermentation
by Anil R. Shet, Abdullatif Bin Muhsinah, Abdulrahman Alsayari, Sharanappa Achappa, Shivalingsarj V. Desai, Mater H. Mahnashi, Uday M. Muddapur, Ibrahim Ahmed Shaikh, Basheerahmed Abdulaziz Mannasaheb and Aejaz Abdullatif Khan
Fermentation 2022, 8(10), 485; https://doi.org/10.3390/fermentation8100485 - 26 Sep 2022
Cited by 15 | Viewed by 4365
Abstract
Pectinolytic enzymes are related enzymes that hydrolyze pectic substances. Pectinolytic enzymes are of great interest in industrial applications for softening fruits, extracting and clarifying juices, extracting olive oil, retting textile fibers, preparing gel, and isolating protoplasts. The current work presents acidic extracellular pectinase [...] Read more.
Pectinolytic enzymes are related enzymes that hydrolyze pectic substances. Pectinolytic enzymes are of great interest in industrial applications for softening fruits, extracting and clarifying juices, extracting olive oil, retting textile fibers, preparing gel, and isolating protoplasts. The current work presents acidic extracellular pectinase production using low-cost agro-industrial waste with the indigenously isolated novel strain Aspergillus cervinus. Two fungal isolates, ARS2 and ARS8, with maximum pectinase activity, 41.88 ± 1.57 IU/mL and 39.27 ± 1.14 IU/mL, respectively, were screened out of 27 isolates from decayed fruit peels (orange, banana, and lemon) and soil containing decomposed vegetables. The isolate ARS2, identified as Aspergillus cervinus by molecular characterization, showed the highest pectinase activity of 43.05 ± 1.38IU/mL during screening and was further used for media component screening and optimization studies. To understand their effect on pectinase activity, one-factor-at-a-time (OFAT) studies were conducted on carbon sources, nitrogen sources, and mineral salts. The OFAT results showed the highest pectinase activity for orange peel (carbon source) at 44.51 ± 1.33 IU/mL, peptone (nitrogen source) at 45.05 ± 1.04 IU/mL, and NaH2PO4 (mineral salts) at 43.21 ± 1.12 IU/mL. The most significant media components screened by the Plackett–Burman (PB) design based on the p-value, Pareto chart, and main effect plot, were orange peel (p < 0.001), peptone (p < 0.001), NaH2PO4 (p < 0.001), and KH2PO4 (p < 0.001), which were further optimized using Response Surface Methodology (RSM) and Central Composite Design (CCD). The optimization results for the media components showed a maximum pectinase activity of 105.65 ± 0.31 IU/mL for 10.63 g orange peel, 3.96 g/L peptone, 2.07 g/L KH2PO4, and 2.10 g/L NaH2PO4. Thus, it was discovered that the indigenously isolated novel strain Aspergillus cervinus ARS2 was able to successfully produce a significant amount of pectinase using agro-industrial waste. Therefore, it can be considered for the large-scale optimized production of pectinase to meet industrial demands. Full article
(This article belongs to the Section Microbial Metabolism, Physiology & Genetics)
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9 pages, 1285 KiB  
Communication
Optimization of an Efficient Protoplast Transformation System for Transient Expression Analysis Using Leaves of Torenia fournieri
by Ling Zhang, Wai-Shing Yung, Zhili Wang, Man-Wah Li and Mingkun Huang
Plants 2022, 11(16), 2106; https://doi.org/10.3390/plants11162106 - 12 Aug 2022
Cited by 7 | Viewed by 3059
Abstract
Torenia fournieri (T. fournieri) is one of the most widely used horticultural flowers and is considered a potential model plant for the genetic investigation of ornamental traits. In this study, we optimized an efficient protocol for high efficiency preparation and transformation [...] Read more.
Torenia fournieri (T. fournieri) is one of the most widely used horticultural flowers and is considered a potential model plant for the genetic investigation of ornamental traits. In this study, we optimized an efficient protocol for high efficiency preparation and transformation of T. fournieri protoplast. The transformation rate reached ~75% when a 35S:GFP construct was used for the transformation. Using this system, we characterized the subcellular localization of several TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) transcription factors (TFs), and found a distinct localization pattern between the CIN and CYC classes of TCP TFs. Furthermore, we also demonstrated the feasibility of the expression of dual luciferase assay system in T. fournieri protoplasts for the measurement of the activity of cis-regulatory elements. Taken together, a well-optimized transient expression system in T. fournieri protoplasts would be crucial for rapid exploration of the gene function or cis-regulatory elements. Full article
(This article belongs to the Special Issue Advancement of Ornamental Flowering Plants)
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19 pages, 5916 KiB  
Article
Transcriptome Analysis of Sugarcane Young Leaves and Protoplasts after Enzymatic Digestion
by Demei Zhang, Rui Wang, Shijian Han, Zhigang Li, Jiming Xiao, Yangrui Li, Lingqiang Wang and Suli Li
Life 2022, 12(8), 1210; https://doi.org/10.3390/life12081210 - 9 Aug 2022
Cited by 6 | Viewed by 2644
Abstract
Sugarcane somatic cell hybridization can break through the barrier of genetic incompatibility between distantly related species in traditional breeding. However, the molecular mechanisms of sugarcane protoplast regeneration and the conditions for protoplast preparation remain largely unknown. In this study, young sugarcane (ROC22) leaves [...] Read more.
Sugarcane somatic cell hybridization can break through the barrier of genetic incompatibility between distantly related species in traditional breeding. However, the molecular mechanisms of sugarcane protoplast regeneration and the conditions for protoplast preparation remain largely unknown. In this study, young sugarcane (ROC22) leaves were enzymatically digested, and the viability of protoplasts reached more than 90% after enzymatic digestion (Enzymatic combination: 2% cellulase + 0.5% pectinase + 0.1% dissociative enzyme + 0.3% hemicellulase, pH = 5.8). Transcriptome sequencing was performed on young sugarcane leaves and protoplasts after enzymatic digestion to analyze the differences in gene expression in somatic cells before and after enzymatic digestion. A total of 117,411 unigenes and 43,460 differentially expressed genes were obtained, of which 21,123 were up-regulated and 22,337 down-regulated. The GO terms for the 43,460 differentially expressed genes (DEGs) were classified into three main categories: biological process, cellular component and molecular function, which related to developmental process, growth, cell proliferation, transcription regulator activity, signal transducer activity, antioxidant activity, oxidative stress, kinase activity, cell cycle, cell differentiation, plant hormone signal transduction, and so on. After enzymatic digestion of young sugarcane leaves, the expressions of GAUT, CESA, PSK, CyclinB, CyclinA, CyclinD3 and cdc2 genes associated with plant regeneration were significantly down-regulated to 65%, 47%, 2%, 18.60%, 21.32%, 52% and 45% of young leaves, respectively. After enzymatic digestion, Aux/IAA expression was up-regulated compared with young leaves, and Aux/IAA expression was 3.53 times higher than that of young leaves. Compared with young leaves, these key genes were significantly changed after enzymatic digestion. These results indicate that the process of somatic enzymatic digestion process may affect the regeneration of heterozygous cells to a certain extent. Full article
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