Review
Biochemical Research Methods
Jia-Yu Shen, Qunfei Zhao, Qing-Li He
Summary: Fungi, especially filamentous fungi and macrofungi, have a strong ability to produce secondary metabolites and can be used as excellent chassis cells for producing valuable enzymes or natural products in synthetic biology. Establishing simple, reliable, and efficient techniques for genetic modification of fungi is crucial. The CRISPR/Cas9 system has been widely used for gene editing in life science research and has shown great potential in the genetic modification of filamentous and macrofungi. This paper discusses the various components and development of the CRISPR/Cas9 system, as well as the challenges and potential in using this system in filamentous fungi and macrofungi.
ACS SYNTHETIC BIOLOGY
(2023)
Article
Biochemical Research Methods
Zofia Dorota Jarczynska, Katherina Garcia Vanegas, Marcus Deichmann, Christina Norskov Jensen, Marouschka Jasmijn Scheeper, Malgorzata Ewa Futyma, Tomas Strucko, Fabiano Jares Contesini, Tue Sparholt Jorgensen, Jakob Blaesbjerg Hoof, Uffe Hasbro Mortensen
Summary: This study developed a novel fungal in vivo DNA assembly toolbox for genetic engineering of filamentous fungi, based on research on fusion reactions of DNA fragments in fungi.
ACS SYNTHETIC BIOLOGY
(2022)
Review
Microbiology
Chunmiao Jiang, Gongbo Lv, Yayi Tu, Xiaojie Cheng, Yitian Duan, Bin Zeng, Bin He
Summary: Filamentous fungi have the ability to produce a wide variety of secondary metabolites, but research on industrially important secondary metabolites has been hindered by the lack of efficient genetic manipulation techniques. The emergence of CRISPR/Cas9 technology offers potential solutions to this issue, revolutionizing genetic research and enabling the discovery of new bioactive compounds in filamentous fungi.
FRONTIERS IN MICROBIOLOGY
(2021)
Article
Microbiology
Andrew S. Urquhart, Donald M. Gardiner
Summary: Fungal spore killers are genetic elements that promote their own inheritance by killing non-inheriting gametes. However, little is known about how they kill other spores within the fungal ascus. In this study, we investigate the spore killer protein SPOK1 in Podospora anserina and demonstrate that it kills by targeting DNA. This finding provides insights into the mechanisms of spore killers and their potential applications.
Article
Biochemistry & Molecular Biology
Vendula Horackova, Lubos Voleman, Kari D. Hagen, Marketa Petru, Martina Vinopalova, Filip Weisz, Natalia Janowicz, Lenka Markova, Alzbeta Motyckova, Vladimira Najdrova, Pavla Tumova, Scott C. Dawson, Pavel Dolezal
Summary: This study demonstrated successful genome editing of G. intestinalis using CRISPR/Cas9 technology, achieving effective gene knockout and providing important tools for further research on parasitic protists.
Article
Biology
Zhiquan Liu, Siyu Chen, Wanhua Xie, Hao Yu, Liangxue Lai, Zhanjun Li
Summary: Researchers have revisited and engineered a compact Cas9 orthologue derived from Neisseria cinerea (NcCas9) for efficient genome editing in mammal cells. NcCas9 can recognize a PAM sequence (N4GYAT) that existing Cas9s cannot, and by optimizing its architecture and spacer length, editing efficacy is improved. NcCas9-derived Base editors can efficiently generate base conversions, and six anti-CRISPR proteins were identified as off-switches for NcCas9. NcCas9 successfully generated efficient editing of mouse embryos by microinjection of NcCas9 mRNA and the corresponding sgRNA.
COMMUNICATIONS BIOLOGY
(2022)
Article
Biotechnology & Applied Microbiology
Dede Abdulrachman, Lily Eurwilaichitr, Verawat Champreda, Duriya Chantasingh, Kusol Pootanakit
Summary: A new class 2 CRISPR-Cas system, Cpf1, optimized from Francisella tularensis subsp. novicida U112, was developed as a versatile platform for precise genome modification in filamentous fungi like Aspergillus aculeatus. The study demonstrated that FnCpf1 efficiently induced site-specific double-strand breaks and insertions or deletions through the endogenous NHEJ DNA repair pathway, highlighting the potential of CRISPR/Cpf1 technology for strain improvements and functional genomics in fungi.
Article
Biotechnology & Applied Microbiology
Xinran Xu, Jin Feng, Peng Zhang, Jie Fan, Wen-Bing Yin
Summary: This study developed a fungal BGC-capturing system based on CRISPR/Cas9 cleavage, allowing for efficient and precise manipulation of fungal genomic DNA, providing a simple and effective approach to accelerate mining of fungal SMs.
JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY
(2021)
Article
Chemistry, Physical
Helen B. Belato, Carmelissa Norbrun, Jinping Luo, Chinmai Pindi, Souvik Sinha, Alexandra M. D'Ordine, Gerwald Jogl, Giulia Palermo, George P. Lisi
Summary: Allosteric signaling within multidomain proteins is a driving force for communication between functional sites. This study used nuclear magnetic resonance (NMR) and computational approaches to identify an allosteric hotspot in the HNH domain of GeoCas9. Mutation of a specific amino acid disrupted a salt-bridge network and altered the structure, timescale of allosteric motions, and thermostability of the domain. This finding may have implications for the specificity enhancement of Cas9.
JOURNAL OF CHEMICAL PHYSICS
(2022)
Review
Biochemistry & Molecular Biology
Amrita C. Bhagwat, Amrita M. Patil, Sunil D. Saroj
Summary: Plants, fungi, and bacteria produce secondary metabolites with diverse biological activities, but there is a need for more efficient synthesis methods. The lack of reliable gene manipulation techniques has hindered the broader study of industrially important secondary metabolites. The CRISPR/Cas system has revolutionized genetic engineering, offering simplicity and ease of use in applications such as plant breeding and metabolic pathway engineering for the production of valuable bioactives.
MOLECULAR BIOTECHNOLOGY
(2022)
Article
Plant Sciences
Yang Liu, Chunling Zhang, Xiaofei Wang, Xiuming Li, Chunxiang You
Summary: The CRISPR/Cas9 system is an efficient, precise, and easy-to-use genome editing technology that has been widely adopted in horticultural breeding for increasing yield and improving quality. This article provides a comprehensive understanding of the construction and optimization of the CRISPR/Cas9 system, as well as its application in enhancing stress responses, improving fruit quality, and modifying cultivation traits in horticultural plants.
HORTICULTURAL PLANT JOURNAL
(2022)
Review
Microbiology
Sanaz Nargesi, Saeed Kaboli, Jose Thekkiniath, Somayeh Heidari, Fatemeh Keramati, Seyedmojtaba Seyedmousavi, Mohammad Taghi Hedayati
Summary: Manipulating fungal genomes through genetic manipulation techniques helps to understand gene function, pathobiology, virulence, pathogenicity, and develop diagnostics and treatments. Various strategies are used by fungi to adapt and survive, including mutations in key genes. In recent years, genome editing techniques such as CRISPR/Cas9 have become important tools in this field.
Article
Biochemistry & Molecular Biology
Yuya Liang, Sudip Biswas, Backki Kim, Julia Bailey-Serres, Endang M. Septiningsih
Summary: This study optimized Agrobacterium-mediated and biolistic transformation methods for efficient CRISPR-Cas9 gene editing of the submergence tolerance regulator SUBMERGENCE 1A-1 gene in the indica rice cultivar Ciherang-Sub1. High regeneration and editing efficiencies were achieved from immature embryo-derived calli, indicating potential for rapid gene editing in recalcitrant indica rice cultivars. Phenotypic validation of disrupted SUB1A-1 function in T-1 seeds under submergence stress further demonstrated the success of these optimized methods.
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
(2021)
Review
Environmental Sciences
Misbah Naz, Adalberto Benavides-Mendoza, Muhammad Tariq, Jianyu Zhou, Jiahao Wang, Shanshan Qi, Zhicong Dai, Daolin Du
Summary: Phytoremediation is an active research field that focuses on identifying and characterizing novel super-accumulators with high chelation action. Molecular tools and CRISPR-Cas9 technology are widely employed to enhance metal absorption, translocation, and tolerance mechanisms in plants for detoxification. Future research in adaptive phytoremediation ability and sustainable ecosystem will be triggered by advancements in molecular science.
JOURNAL OF ENVIRONMENTAL MANAGEMENT
(2022)
Article
Microbiology
Binyou Liao, Xi Chen, Xuedong Zhou, Yujie Zhou, Yangyang Shi, Xingchen Ye, Min Liao, Ziyi Zhou, Lei Cheng, Biao Ren
Summary: Genome editing technology has made rapid progress in recent years. The limitations of traditional gene-editing methods have led to the development of the CRISPR/Cas system, which has become the dominant genomic editing technology in academia and the pharmaceutical industry due to its high efficiency, simple design, and wide applications.
ARCHIVES OF MICROBIOLOGY
(2022)
Article
Biotechnology & Applied Microbiology
Gen Zou, Meili Xiao, Shunxing Chai, Zhihua Zhu, Ying Wang, Zhihua Zhou
Summary: This study presents an optimized genome editing approach for filamentous fungi based on RNPs, which significantly increased the transformation efficiency by adding chemical reagents. The optimized RNP-based method achieved efficient homologous recombination integration and gene disruption in Trichoderma reesei, with potential applications in the food and medical industries.
MICROBIAL BIOTECHNOLOGY
(2021)
Article
Agricultural Engineering
Gen Zou, Dapeng Bao, Ying Wang, Sichi Zhou, Meili Xiao, Zhanshan Yang, Yinmei Wang, Zhihua Zhou
Summary: Introducing product-activated cellulase genes significantly increases enzyme activity and saccharification efficiency. Improving thermostability of cellulases was achieved through mutants and enhanced enzyme activity. Introducing product-activated cellulase genes is an effective approach to alleviate product inhibition of cellulases.
BIORESOURCE TECHNOLOGY
(2021)
Article
Biotechnology & Applied Microbiology
Hongguan Xing, Gen Zou, Chunyan Liu, Shunxing Chai, Xing Yan, Xinliang Li, Rui Liu, Yi Yang, Zhihua Zhou
Summary: Through error-prone PCR and B-factor analysis, the thermostability of a xylanase from GH family 11 was enhanced in this study. Mutants with longer half-lives were screened, and substitutions in the palm region and newly formed hydrogen bonds likely contributed to the improved thermostability. The significantly improved mutants may find applications in various industrial sectors.
ENZYME AND MICROBIAL TECHNOLOGY
(2021)
Article
Microbiology
Ying Wang, Zhanshan Yang, Dapeng Bao, Bo Li, Xin Yin, Yingying Wu, Hongyu Chen, Guirong Tang, Nanyi Li, Gen Zou
Summary: Cordyceps militaris, a valuable bioresource, shows significant differences in growth and bioactive compounds synthesis under different cultivation methods. The adaptation to hypoxic conditions plays a crucial role in the growth and synthesis of bioactive compounds in C. militaris. Strategies to improve hypoxia adaption can have distinct effects on the production of bioactive compounds in C. militaris, offering insights for rational strain engineering and optimization strategies for cultivation and fermentation.
FRONTIERS IN MICROBIOLOGY
(2021)
Article
Biochemical Research Methods
Shunxing Chai, Zhihua Zhu, Ernuo Tian, Meili Xiao, Yan Wang, Gen Zou, Zhihua Zhou
Summary: By employing a genome editing system and iterative gene deletion, marker-free T. reesei chassis were constructed, resulting in reduced secretion of native proteins and extracellular protease activity, leading to increased production levels of heterologous proteins.
ACS SYNTHETIC BIOLOGY
(2022)
Article
Agronomy
Ming Gong, Ying Wang, Erzheng Su, Jianguo Zhang, Lihua Tang, Zhengpeng Li, Lanlan Zhang, Gen Zou, Jianing Wan, Dapeng Bao
Summary: The study found that using DGlucono-1,5-lactone as a food additive can effectively prevent cryogenic autolysis of Volvariella volvacea at low temperatures, showing promising application in postharvest management.
POSTHARVEST BIOLOGY AND TECHNOLOGY
(2022)
Article
Biotechnology & Applied Microbiology
Lu Yu, Meili Xiao, Zhihua Zhu, Yinmei Wang, Zhihua Zhou, Pingping Wang, Gen Zou
Summary: An efficient genome-editing system based on in vitro assembled CRISPR/Cas9 gRNA ribonucleoprotein complexes was developed in C. purpurea. Three target genes were successfully knocked out using this system, resulting in mutants with different phenotypes. This study is of great significance for elucidating the EAS biosynthetic pathway and future basic and applied research on Claviceps purpurea.
SYNTHETIC AND SYSTEMS BIOTECHNOLOGY
(2022)
Review
Biotechnology & Applied Microbiology
Ying Wang, Hongyu Chen, Liang Ma, Ming Gong, Yingying Wu, Dapeng Bao, Gen Zou
Summary: This article provides a brief overview of traditional approaches to genetic manipulation of Trichoderma fungi, different strategies employed in establishing CRISPR-Cas systems, utilization of these systems to develop engineered strains of Trichoderma for desired applications, and future trends in biotechnology.
MICROBIAL BIOTECHNOLOGY
(2022)
Article
Microbiology
Ming Gong, Tianyu Huang, Yan Li, Jinxin Li, Lihua Tang, Erzheng Su, Gen Zou, Dapeng Bao
Summary: This study conducted a comprehensive multi-omics analysis to elucidate the molecular mechanism of low-temperature fruiting after a long postripening (LFLP) in Hypsizygus marmoreus. The results revealed that the changes in pH were closely correlated with variations in citrate and arginine. By enhancing the enzyme activity of the arginine synthesis branch pathway in the citrate cycle, the levels of citric acid and arginine were synergistically increased, providing enriched materials for amino acid biosynthesis. Furthermore, GCN2 mediated metabolic adaptation through enhanced protein translation. Exogenously added citric acid and arginine shortened the postripening period and increased the yield.
Editorial Material
Biotechnology & Applied Microbiology
Gen Zou, Jens B. Nielsen, Yongjun Wei
Summary: Recent advances in synthetic biology have revolutionized mushroom farming by shifting the focus from traditional cultivation to comprehensive applications using cutting-edge biotechnologies. Promising applications in this field include precision breeding, exploration of biosynthetic gene clusters, development of mushroom chassis cells, and construction of cell factories for high-value products.
TRENDS IN BIOTECHNOLOGY
(2023)
Article
Microbiology
Jianyu Liu, Haiyang Cui, Ruijuan Wang, Zhen Xu, Hailong Yu, Chunyan Song, Huan Lu, Qiaozhen Li, Danrun Xing, Qi Tan, Weiming Sun, Gen Zou, Xiaodong Shang
Summary: This study developed an optimized CRISPR/Cas9 genome-editing method using in vitro assembled ribonucleoprotein complexes (RNPs) in Flammulina filiformis. The method achieved a 100% targeting efficiency and avoided the use of foreign DNA. This research has significant theoretical and practical implications.
Article
Agricultural Engineering
Ming Gong, Ying Wang, Dapeng Bao, Shan Jiang, Hongyu Chen, Junjun Shang, Xiaojun Wang, Hnin Hnin Yu, Gen Zou
Summary: A new cold-active cellobiohydrolase (VvCBHI-I) derived from Volvariella volvacea showed excellent activity at 10-30 degrees C, which enables efficient cellulose degradation and saccharification at low temperatures. The expression of VvCBHI-I in a transformant significantly improved the activity of cellulase at 10 degrees C, leading to stable silage fermentation with rice straw and reduction of cellulose and hemicellulose contents. These findings highlight the potential of mushroom-derived cold-active cellobiohydrolases in enhancing ensiling efficiency in cold climate regions.
BIORESOURCE TECHNOLOGY
(2023)
Editorial Material
Microbiology
Gen Zou, Jing Zhu, Mingwen Zhao
Article
Chemistry, Multidisciplinary
Meili Xiao, Yinmei Wang, Yan Wang, Xing Yan, Zhihua Zhu, Ernuo Tian, Chengshuai Yang, Erdong Ma, Gen Zou, Zhihua Zhou, Pingping Wang
Summary: In this study, we successfully synthesized different types of terpenes efficiently using Trichoderma reesei. By optimizing the genetic architecture and substrate selection, we were able to significantly increase the production of the target compounds. This has important implications for terpene biosynthesis and sustainable biofabrication.
Article
Chemistry, Multidisciplinary
Gen Zou, Bo Li, Ying Wang, Xin Yin, Ming Gong, Junjun Shang, Yongjun Wei, Xiaoling Li, Dapeng Bao
Summary: Engineered Cordyceps militaris can convert spent mushroom substrate (SMS) into the anticancer drug pentostatin, utilizing lignocellulosic waste for high-value product biosynthesis. Featuring strong hydrolytic activity towards cellulose and increased pentostatin production, this biological strategy offers a sustainable solution for SMS pollution removal while producing valuable bioactive compounds.