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12 pages, 2276 KiB

Open AccessArticle

Improving and Streamlining Gene Editing in Yarrowia lipolytica via Integration of Engineered Cas9 Protein

by Baixi Zhang and Jiacan Cao

J. Fungi 2024, 10(1), 63; https://doi.org/10.3390/jof10010063 - 12 Jan 2024

Viewed by 1092

The oleaginous yeast Yarrowia lipolytica is a prominent subject of biorefinery research due to its exceptional performance in oil production, exogenous protein secretion, and utilization of various inexpensive carbon sources. Many CRISPR/Cas9 genome-editing systems have been developed for Y. lipolytica to meet the [...] Read more.

The oleaginous yeast Yarrowia lipolytica is a prominent subject of biorefinery research due to its exceptional performance in oil production, exogenous protein secretion, and utilization of various inexpensive carbon sources. Many CRISPR/Cas9 genome-editing systems have been developed for Y. lipolytica to meet the high demand for metabolic engineering studies. However, these systems often necessitate an additional outgrowth step to achieve high gene editing efficiency. In this study, we introduced the eSpCas9 protein, derived from the Streptococcus pyogenes Cas9(SpCas9) protein, into the Y. lipolytica genome to enhance gene editing efficiency and fidelity, and subsequently explored the optimal expression level of eSpCas9 gene by utilizing different promoters and selecting various growth periods for yeast transformation. The results demonstrated that the integrated eSpCas9 gene editing system significantly enhanced gene editing efficiency, increasing from 16.61% to 86.09% on TRP1 and from 33.61% to 95.19% on LIP2, all without the need for a time-consuming outgrowth step. Furthermore, growth curves and dilution assays indicated that the consistent expression of eSpCas9 protein slightly suppressed the growth of Y. lipolytica, revealing that strong inducible promoters may be a potential avenue for future research. This work simplifies the gene editing process in Y. lipolytica, thus advancing its potential as a natural product synthesis chassis and providing valuable insights for other comparable microorganisms. Full article

(This article belongs to the Special Issue Fungal Biotechnology and Application 2.0)

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15 pages, 2481 KiB

Open AccessArticle

Production of Xylanase by Trichoderma Species Growing on Olive Mill Pomace and Barley Bran in a Packed-Bed Bioreactor

by Kholoud M. Alananbeh, Rana Alkfoof, Riyadh Muhaidat and Muhannad Massadeh

J. Fungi 2024, 10(1), 49; https://doi.org/10.3390/jof10010049 - 05 Jan 2024

Viewed by 977

Abstract

Xylanases are hydrolytic enzymes that have tremendous applications in different sectors of life, but the high cost of their production has limited their use. One solution to reduce costs and enhance xylanase production is the use of agro-wastes as a substrate in fungal [...] Read more.

Xylanases are hydrolytic enzymes that have tremendous applications in different sectors of life, but the high cost of their production has limited their use. One solution to reduce costs and enhance xylanase production is the use of agro-wastes as a substrate in fungal cultures. In this study, olive mill pomace (OMP) and barley bran (BB) were used as carbon sources and possible inducers of xylanase production by three species of Trichoderma (atroviride, harzianum, and longibrachiatum), one major xylanase producer. The experiments were conducted under a solid-state fermentation system (SSF) in flask cultures and a packed-bed bioreactor. Cultures of OMP and BB were optimized by examining different ratios of OMP and BB, varied particle sizes, and inoculum size for the three species of Trichoderma. The ratio of 8:2 OMP and BB yielded the highest xylanase activity, with a particle size of 1 mm at 29 °C and an inoculum size of 1 × 10⁷ spores/mL. Studying the time profile of the process revealed that xylanase activity was highest after seven days of incubation in flask SSF cultures (1.779 U/mL) and after three days in a packed-bed bioreactor (1.828 U/mL). The maximum percentage of OMP degradation recorded was about 15% in the cultures of T. harzianum flask SSF cultures, compared to about 11% in T. longibrachiatum bioreactor cultures. Ammonium sulfate precipitation and dialysis experiments showed that Xylane enzyme activity ranged from 0.274 U/mL in T. harzianum to 0.837 U/mL in T. atroviride when crude extract was used, with the highest activity (0.628 U/mL) at 60% saturation. Xylose was the main sugar released in all purified fractions, with the G-50 and G-75 fractions showing the maximum units of xylanase. Full article

(This article belongs to the Special Issue Fungal Biotechnology and Application 2.0)

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12 pages, 1155 KiB

Open AccessArticle

Ecdysteroid UDP-Glucosyltransferase Expression in Beauveria bassiana Increases Its Pathogenicity against Early Instar Silkworm Larvae

by Xueqin Mao, Dongxu Xing, Die Liu, Haoran Xu, Luyu Hou, Ping Lin, Qingyou Xia, Ying Lin and Guanwang Shen

J. Fungi 2023, 9(10), 987; https://doi.org/10.3390/jof9100987 - 04 Oct 2023

Viewed by 1013

Abstract

Beauveria bassiana (B. bassiana) is a broad-spectrum entomopathogenic fungus that can control pests in agriculture and forestry. In this study, encoding ecdysteroid uridine diphosphate glucosyltransferase gene (egt) was successfully screened in B. bassiana on the medium containing 500μg/mL G418 sulfate [...] Read more.

Beauveria bassiana (B. bassiana) is a broad-spectrum entomopathogenic fungus that can control pests in agriculture and forestry. In this study, encoding ecdysteroid uridine diphosphate glucosyltransferase gene (egt) was successfully screened in B. bassiana on the medium containing 500μg/mL G418 sulfate solution through the protoplast transformation method. This enzyme has the function of 20E (20-hydroxyecdysone) inactivation, thus increasing the mortality of the early instar larvae infected with B. bassiana. In this study, we transformed B. bassiana with the egt gene, which deactivates 20-hydroxyecdysone, a key hormone in insect development. The results showed that transgenic B. bassiana killed more silkworms of the 2nd instar larvae than the wild-type with a shorter LT50 time, which was reduced by approximately 20% (day 1 of the 2nd instar silkworm infection of B. bassiana) and 26.4% (day 2 of the 2nd instar silkworm infection of B. bassiana) compared to the wild-type, and also showed a higher mortality number before molting. The transgenic B. bassiana had a higher coverage of the body surface of silkworms compared to the wild type on the 3rd instar. In summary, improving entomopathogenic fungi using biological methods such as genetic engineering is feasible. Full article

(This article belongs to the Special Issue Fungal Biotechnology and Application 2.0)

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19 pages, 1740 KiB

Open AccessArticle

Antimicrobial and Antioxidant Activities of Endophytic Fungi Associated with Arrabidaea chica (Bignoniaceae)

by Raiana Silveira Gurgel, Dorothy Ívila de Melo Pereira, Ana Vyktória França Garcia, Anne Terezinha Fernandes de Souza, Thaysa Mendes da Silva, Cleudiane Pereira de Andrade, Weison Lima da Silva, Cecilia Veronica Nunez, Cleiton Fantin, Rudi Emerson de Lima Procópio and Patrícia Melchionna Albuquerque

J. Fungi 2023, 9(8), 864; https://doi.org/10.3390/jof9080864 - 21 Aug 2023

Cited by 3 | Viewed by 1710

Abstract

The endophytic fungal community of the Amazonian medicinal plant Arrabidaea chica (Bignoniaceae) was evaluated based on the hypothesis that microbial communities associated with plant species in the Amazon region may produce metabolites with interesting bioactive properties. Therefore, the antimicrobial and antioxidant activities of [...] Read more.

The endophytic fungal community of the Amazonian medicinal plant Arrabidaea chica (Bignoniaceae) was evaluated based on the hypothesis that microbial communities associated with plant species in the Amazon region may produce metabolites with interesting bioactive properties. Therefore, the antimicrobial and antioxidant activities of the fungal extracts were investigated. A total of 107 endophytic fungi were grown in liquid medium and the metabolites were extracted with ethyl acetate. In the screening of fungal extracts for antimicrobial activity, the fungus identified as Botryosphaeria mamane CF2-13 was the most promising, with activity against E. coli, S. epidermidis, P. mirabilis, B. subtilis, S. marcescens, K. pneumoniae, S. enterica, A. brasiliensis, C. albicans, C. tropicalis and, especially, against S. aureus and C. parapsilosis (MIC = 0.312 mg/mL). Screening for antioxidant potential using the DPPH elimination assay showed that the Colletotrichum sp. CG1-7 endophyte extract exhibited potential activity with an EC₅₀ of 11 µg/mL, which is equivalent to quercetin (8 µg/mL). The FRAP method confirmed the antioxidant potential of the fungal extracts. The presence of phenolic compounds and flavonoids in the active extracts was confirmed using TLC. These results indicate that two of the fungi isolated from A. chica exhibit significant antimicrobial and antioxidant potential. Full article

(This article belongs to the Special Issue Fungal Biotechnology and Application 2.0)

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11 pages, 1390 KiB

Open AccessArticle

Marine-Derived Fungi as a Valuable Resource for Amylases Activity Screening

by Di Zhang, Lan Liu and Bi-Shuang Chen

J. Fungi 2023, 9(7), 736; https://doi.org/10.3390/jof9070736 - 09 Jul 2023

Cited by 1 | Viewed by 1081

Abstract

Marine microbial enzymes including amylases are important in different industrial production due to their properties and applications. This study was focused on the screening of marine-derived fungi for amylase activities. First, we isolated a number of fungi from the sediments of the South [...] Read more.

Marine microbial enzymes including amylases are important in different industrial production due to their properties and applications. This study was focused on the screening of marine-derived fungi for amylase activities. First, we isolated a number of fungi from the sediments of the South China Sea. By the method of dish screening (in vitro), we subsequently obtained a series of amylase-producing fungal strains. The cell-lysate activities of amylases produced by marine fungi toward starch hydrolysis were achieved with the dinitrosalyicylic acid (DNS) method. In addition, the effect of pH and temperature on amylase activities, including thermal and pH stability were discussed. Results showed that out of the 57 isolates with amylase-producing activities, fungi Aspergillus flavus 9261 was found to produce amylase with the best activity of 10.7482 U/mg (wet mycelia). The amylase of Aspergillus flavus 9261 exhibited remarkable thermostability and pH stability with no activity loss after incubation at 50 °C and pH 5.0 for 1 h, respectively. The results provide advances in discovering enzymes from marine-derived fungi and their biotechnology relevance. Full article

(This article belongs to the Special Issue Fungal Biotechnology and Application 2.0)

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19 pages, 3061 KiB

Open AccessArticle

Investigation on the Influence of Production and Incubation Temperature on the Growth, Virulence, Germination, and Conidial Size of Metarhizium brunneum for Granule Development

by Tanja Seib, Katharina Fischer, Anna Maria Sturm and Dietrich Stephan

J. Fungi 2023, 9(6), 668; https://doi.org/10.3390/jof9060668 - 14 Jun 2023

Cited by 2 | Viewed by 1646

Abstract

Important for the infection of an insect with an entomopathogenic fungus and its use as a plant protection agent are its growth, conidiation, germination, and virulence, which all depend on the environmental temperature. We investigated not only the effect of environmental temperature but [...] Read more.

Important for the infection of an insect with an entomopathogenic fungus and its use as a plant protection agent are its growth, conidiation, germination, and virulence, which all depend on the environmental temperature. We investigated not only the effect of environmental temperature but also that of production temperature of the fungus. For this purpose, Metarhizium brunneum JKI-BI-1450 was produced and incubated at different temperatures, and the factors mentioned as well as conidial size were determined. The temperature at which the fungus was produced affects its subsequent growth and conidiation on granule formulation, the speed of germination, and the conidial width, but not its final germination or virulence. The growth and conidiation was at its highest when the fungus was produced at 25 °C, whereas when the germination was faster, the warmer the fungus was produced. The incubation temperature optimum of JKI-BI-1450 in relation to growth, speed of germination, and survival time was 25–30 °C and for conidiation 20–25 °C. Conidial length decreased with increasing incubation temperature. Although the fungus could not be adapted to unfavorable conditions by the production temperature, it was found that the quality of a biological control agent based on entomopathogenic fungi can be positively influenced by its production temperature. Full article

(This article belongs to the Special Issue Fungal Biotechnology and Application 2.0)

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13 pages, 2804 KiB

Open AccessArticle

Filamentous Fungi Are Potential Bioremediation Agents of Semi-Synthetic Textile Waste

by Rachel Harper and Suzy Clare Moody

J. Fungi 2023, 9(6), 661; https://doi.org/10.3390/jof9060661 - 13 Jun 2023

Viewed by 1697

Abstract

Textile waste contributes to the pollution of both terrestrial and aquatic ecosystems. While natural textile fibres are known to be biodegraded by microbes, the vast majority of textiles now contain a mixture of processed plant-derived polymers and synthetic materials generated from petroleum and [...] Read more.

Textile waste contributes to the pollution of both terrestrial and aquatic ecosystems. While natural textile fibres are known to be biodegraded by microbes, the vast majority of textiles now contain a mixture of processed plant-derived polymers and synthetic materials generated from petroleum and are commonly dyed with azo dyes. This presents a complex recycling problem as the separation of threads and removal of dye are challenging and costly. As a result, the majority of textile waste is sent to landfill or incinerated. This project sought to assess the potential of fungal bioremediation of textile-based dye as a step towards sustainable and environmentally-friendly means of disposal of textile waste. Successful development of an agar-independent microcosm enabled the assessment of the ability of two fungal species to grow on a range of textiles containing an increasing percentage of elastane. The white rot fungus Hypholoma fasciculare was shown to grow well on semi-synthetic textiles, and for the first time, bioremediation of dye from textiles was demonstrated. Volatile analysis enabled preliminary assessment of the safety profile of this process and showed that industrial scale-up may require consideration of volatile capture in the design process. This study is the first to address the potential of fungi as bioremediation agents for solid textile waste, and the results suggest this is an avenue worthy of further exploration. Full article

(This article belongs to the Special Issue Fungal Biotechnology and Application 2.0)

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26 pages, 4681 KiB

Open AccessArticle

Assessment of Tannin Tolerant Non-Saccharomyces Yeasts Isolated from Miang for Production of Health-Targeted Beverage Using Miang Processing Byproducts

by Pratthana Kodchasee, Nattanicha Pharin, Nakarin Suwannarach, Kridsada Unban, Chalermpong Saenjum, Apinun Kanpiengjai, Dipayan Sakar, Kalidas Shetty, Martin Zarnkow and Chartchai Khanongnuch

J. Fungi 2023, 9(2), 165; https://doi.org/10.3390/jof9020165 - 27 Jan 2023

Cited by 1 | Viewed by 1566

Abstract

This research demonstrated an excellent potential approach for utilizing Miang fermentation broth (MF-broth), a liquid residual byproduct from the Miang fermentation process as a health-targeted beverage. One hundred and twenty yeast strains isolated from Miang samples were screened for their potential to ferment [...] Read more.

This research demonstrated an excellent potential approach for utilizing Miang fermentation broth (MF-broth), a liquid residual byproduct from the Miang fermentation process as a health-targeted beverage. One hundred and twenty yeast strains isolated from Miang samples were screened for their potential to ferment MF-broth and four isolates, P2, P3, P7 and P9 were selected, based on the characteristics of low alcoholic production, probiotic properties, and tannin tolerance. Based on a D1/D2 rDNA sequence analysis, P2 and P7 were identified to be Wikerhamomyces anomalus, while P3 and P9 were Cyberlindnera rhodanensis. Based on the production of unique volatile organic compounds (VOCs), W. anomalus P2 and C. rhodanensis P3 were selected for evaluation of MF-broth fermentation via the single culture fermentation (SF) and co-fermentation (CF) in combination with Saccharomyces cerevisiae TISTR 5088. All selected yeasts showed a capability for growth with 6 to 7 log CFU/mL and the average pH value range of 3.91–4.09. The ethanol content of the fermented MF-broth ranged between 11.56 ± 0.00 and 24.91 ± 0.01 g/L after 120 h fermentation, which is categorized as a low alcoholic beverage. Acetic, citric, glucuronic, lactic, succinic, oxalic and gallic acids slightly increased from initial levels in MF-broth, whereas the bioactive compounds and antioxidant activity were retained. The fermented MF-broth showed distinct VOCs profiles between the yeast groups. High titer of isoamyl alcohol was found in all treatments fermented with S. cerevisiae TISTR 5088 and W. anomalus P2. Meanwhile, C. rhodanensis P3 fermented products showed a higher quantity of ester groups, ethyl acetate and isoamyl acetate in both SF and CF. The results of this study confirmed the high possibilities of utilizing MF-broth residual byproduct in for development of health-targeted beverages using the selected non-Saccharomyces yeast. Full article

(This article belongs to the Special Issue Fungal Biotechnology and Application 2.0)

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13 pages, 2065 KiB

Open AccessArticle

Isolation and Characterization of Ruminal Yeast Strain with Probiotic Potential and Its Effects on Growth Performance, Nutrients Digestibility, Rumen Fermentation and Microbiota of Hu Sheep

by Yao Wang, Zihao Li, Wei Jin and Shengyong Mao

J. Fungi 2022, 8(12), 1260; https://doi.org/10.3390/jof8121260 - 29 Nov 2022

Cited by 2 | Viewed by 1724

Abstract

Yeast strains are widely used in ruminant production. However, knowledge about the effects of rumen native yeasts on ruminants is limited. Therefore, this study aimed to obtain a rumen native yeast isolate and investigate its effects on growth performance, nutrient digestibility, rumen fermentation [...] Read more.

Yeast strains are widely used in ruminant production. However, knowledge about the effects of rumen native yeasts on ruminants is limited. Therefore, this study aimed to obtain a rumen native yeast isolate and investigate its effects on growth performance, nutrient digestibility, rumen fermentation and microbiota in Hu sheep. Yeasts were isolated by picking up colonies from agar plates, and identified by sequencing the ITS sequences. One isolate belonging to Pichia kudriavzevii had the highest optical density among these isolates obtained. This isolate was prepared to perform an animal feeding trial. A randomized block design was used for the animal trial. Sixteen Hu sheep were randomly assigned to the control (CON, fed basal diet, n = 8) and treatment group (LPK, fed basal diet plus P. kudriavzevii, CFU = 8 × 10⁹ head/d, n = 8). Sheep were housed individually and treated for 4 weeks. Compared to CON, LPK increased final body weight, nutrient digestibility and rumen acetate concentration and acetate-to-propionate ratio in sheep. The results of Illumina MiSeq PE 300 sequencing showed that LPK increased the relative abundance of lipolytic bacteria (Anaerovibrio spp. and Pseudomonas spp.) and probiotic bacteria (Faecalibacterium spp. and Bifidobacterium spp.). For rumen eukaryotes, LPK increased the genera associated with fiber degradation, including protozoan Polyplastron and fungus Pichia. Our results discovered that rumen native yeast isolate P. kudriavzevii might promote the digestion of fibers and lipids by modulating specific microbial populations with enhancing acetate-type fermentation. Full article

(This article belongs to the Special Issue Fungal Biotechnology and Application 2.0)

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Review

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34 pages, 4859 KiB

Open AccessReview

Unlocking the Power: New Insights into the Anti-Aging Properties of Mushrooms

by Jing Luo, Kumar Ganesan and Baojun Xu

J. Fungi 2024, 10(3), 215; https://doi.org/10.3390/jof10030215 - 14 Mar 2024

Viewed by 2062

Abstract

Aging is a complex biological process that is influenced by both intrinsic and extrinsic factors. Recently, it has been discovered that reactive oxygen species can accelerate the aging process, leading to an increased incidence of age-related diseases that are characteristic of aging. This [...] Read more.

Aging is a complex biological process that is influenced by both intrinsic and extrinsic factors. Recently, it has been discovered that reactive oxygen species can accelerate the aging process, leading to an increased incidence of age-related diseases that are characteristic of aging. This review aims to discuss the potential of mushrooms as a dietary intervention for anti-aging, focusing on their nutritional perspective. Mushrooms contain various bioactive compounds, including carbohydrates, bioactive proteins, fungal lipids, and phenolic compounds. These compounds have shown promising effectiveness in combating skin aging and age-related diseases. In vitro and in vivo studies have demonstrated that treatments with mushrooms or their extracts can significantly extend lifespan and improve health span. Furthermore, studies have aimed to elucidate the precise cellular and molecular mechanisms of action and the structure–activity relationship of mushroom bioactive compounds. These findings provide a strong basis for further research, including human clinical trials and nutritional investigations, to explore the potential benefits of mushrooms in real-life anti-aging practices. By exploring the anti-aging effects of mushrooms, this review aims to provide valuable insights that can contribute to the development of broader strategies for healthy aging. Full article

(This article belongs to the Special Issue Fungal Biotechnology and Application 2.0)

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