Journal of Microbiology

Review

Progress and challenges in CRISPR/Cas applications in microalgae: Quynh-Giao Tran, Trang Thi Le, Dong-Yun Choi, Dae-Hyun Cho, Jin-Ho Yun, Hong Il Choi, Hee-Sik Kim, Yong Jae Lee; J. Microbiol. 2025;63(3):e2501028. Published online March 28, 2025; DOI: https://doi.org/10.71150/jm.2501028

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Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technologies have emerged as powerful tools for precise genome editing, leading to a revolution in genetic research and biotechnology across diverse organisms including microalgae. Since the 1950s, microalgal production has evolved from initial cultivation under controlled conditions to advanced metabolic engineering to meet industrial demands. However, effective genetic modification in microalgae has faced significant challenges, including issues with transformation efficiency, limited target selection, and genetic differences between species, as interspecies genetic variation limits the use of genetic tools from one species to another. This review summarized recent advancements in CRISPR systems applied to microalgae, with a focus on improving gene editing precision and efficiency, while addressing organism-specific challenges. We also discuss notable successes in utilizing the class 2 CRISPR-associated (Cas) proteins, including Cas9 and Cas12a, as well as emerging CRISPR-based approaches tailored to overcome microalgal cellular barriers. Additionally, we propose future perspectives for utilizing CRISPR/Cas strategies in microalgal biotechnology.

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Advancing microbial engineering through synthetic biology
Ki Jun Jeong
Journal of Microbiology.2025; 63(3): e2503100. CrossRef

Journal Articles

Gentic overexpression increases production of hypocrellin A in Shiraia bambusicola S4201: Dan Li , Ning Zhao , Bing-Jing Guo , Xi Lin , Shuang-Lin Chen , Shu-Zhen Yan; J. Microbiol. 2019;57(2):154-162. Published online January 31, 2019; DOI: https://doi.org/10.1007/s12275-019-8259-8

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Abstract

Hypocrellin A (HA) is a perylenequinone (PQ) isolated from Shiraia bambusicola that shows antiviral and antitumor activities, but its application is limited by the low production from wild fruiting body. A gene overexpressing method was expected to augment the production rate of HA in S. bambusicola. However, the application of this molecular biology technology in S. bambusicola was impeded by a low genetic transformation efficiency and little genomic information. To enhance the plasmid transformant ratio, the Polyethylene Glycol-mediated transformation system was established and optimized. The following green fluorescent protein (GFP) analysis showed that the gene fusion expression system we constructed with a GAPDH promoter Pgpd1 and a rapid 2A peptide was successfully expressed in the S. bambusicola S4201 strain. We successfully obtained the HA high-producing strains by overexpressing O-methyltransferase/FAD-dependent monooxygenase gene (mono) and the hydroxylase gene (hyd), which were the essential genes involved in our putative HA biosynthetic pathway. The overexpression of these two genes increased the production of HA by about 200% and 100%, respectively. In general, this study will provide a basis to identify the genes involved in the hypocrellin A biosynthesis. This improved transformation method can also be used in genetic transformation studies of other fungi.

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Optimisation of hypocrellin production in Shiraia -like fungi via genetic modification involving a transcription factor gene and a putative monooxygenase gene
Zi-Min Lu, Run-Tong Zhang, Xiao-Bo Huang, Xue-Ting Cao, Xiao-Ye Shen, Li Fan, Cheng-Lin Hou
Mycology.2024; 15(2): 272. CrossRef
Production of fungal hypocrellin photosensitizers: Exploiting bambusicolous fungi and elicitation strategies in mycelium cultures
Xin Ping Li, Wen Hao Shen, Jian Wen Wang, Li Ping Zheng
Mycology.2024; : 1. CrossRef
Urea-Induced Enhancement of Hypocrellin A Synthesis in Shiraia bambusicola GDMCC 60438: Strategies and Mechanisms
Yanbo Tang, Yongdi Wen, Xiang Zhang, Qian Gao, Fuqiang Yu, Zhenqiang Wu, Xiaofei Tian
Fermentation.2024; 10(8): 381. CrossRef
Advancements and Future Prospects in Hypocrellins Production and Modification for Photodynamic Therapy
Xiang Zhang, Qiulin Wei, Liwen Tian, Zhixian Huang, Yanbo Tang, Yongdi Wen, Fuqiang Yu, Xiaoxiao Yan, Yunchun Zhao, Zhenqiang Wu, Xiaofei Tian
Fermentation.2024; 10(11): 559. CrossRef
Biosynthesis of Natural and Unnatural Perylenequinones for Drug Development
Zengping Su, Yan Zhang, Zhenbo Yuan, Yijian Rao
ChemMedChem.2024;[Epub] CrossRef
Heat stress enhanced perylenequinones biosynthesis of Shiraia sp. Slf14(w) through nitric oxide formation
Chenglong Xu, Wenxi Lin, Yunni Chen, Boliang Gao, Zhibin Zhang, Du Zhu
Applied Microbiology and Biotechnology.2023; 107(11): 3745. CrossRef
Biotechnological production and potential applications of hypocrellins
Zhuanying Bao, Yunchang Xie, Chenglong Xu, Zhibin Zhang, Du Zhu
Applied Microbiology and Biotechnology.2023; 107(21): 6421. CrossRef
L-Arginine enhanced perylenequinone production in the endophytic fungus Shiraia sp. Slf14(w) via NO signaling pathway
Yunni Chen, Chenglong Xu, Huilin Yang, Zhenying Liu, Zhibin Zhang, Riming Yan, Du Zhu
Applied Microbiology and Biotechnology.2022; 106(7): 2619. CrossRef
Advances and perspectives on perylenequinone biosynthesis
Huaxiang Deng, Xinxin Liang, Jinbin Liu, Xiaohui Zheng, Tai-Ping Fan, Yujie Cai
Frontiers in Microbiology.2022;[Epub] CrossRef
Temperature-responsive regulation of the fermentation of hypocrellin A by Shiraia bambusicola (GDMCC 60438)
Yongdi Wen, Baosheng Liao, Xiaoxiao Yan, Zhenqiang Wu, Xiaofei Tian
Microbial Cell Factories.2022;[Epub] CrossRef
Microbial production of nematicidal agents for controlling plant-parasitic nematodes
Jaemin Seong, Jongoh Shin, Kangsan Kim, Byung-Kwan Cho
Process Biochemistry.2021; 108: 69. CrossRef
Nitric oxide donor sodium nitroprusside-induced transcriptional changes and hypocrellin biosynthesis of Shiraia sp. S9
Yan Jun Ma, Xin Ping Li, Yue Wang, Jian Wen Wang
Microbial Cell Factories.2021;[Epub] CrossRef
Nitric oxide regulates perylenequinones biosynthesis in Shiraia bambusicola S4201 induced by hydrogen peroxide
Ning Zhao, Yingying Yu, Yunxia Yue, Mingzhu Dou, Bingjing Guo, Shuzhen Yan, Shuanglin Chen
Scientific Reports.2021;[Epub] CrossRef
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Rosa Sagita, Wim J. Quax, Kristina Haslinger
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Global identification of alternative splicing in Shiraia bambusicola and analysis of its regulation in hypocrellin biosynthesis
Xin-Yao Liu, Li Fan, Jian Gao, Xiao-Ye Shen, Cheng-Lin Hou
Applied Microbiology and Biotechnology.2020; 104(1): 211. CrossRef
Improved A40926 production from Nonomuraea gerenzanensis using the promoter engineering and the co-expression of crucial genes
Huijun Dong, Xue Yue, Bingyu Yan, Wen Gao, Shuai Wang, Yongquan Li
Journal of Biotechnology.2020; 324: 28. CrossRef
Adding bamboo charcoal powder to Shiraia bambusicola preculture improves hypocrellin A production
Xin Ping Li, Yan Jun Ma, Jian Wen Wang
Sustainable Chemistry and Pharmacy.2019; 14: 100191. CrossRef
Efficient agrobacterium-mediated transformation ofShiraia bambusicolaand activation of a specific transcription factor for hypocrellin production
Tong Li, Cheng-Lin Hou, Xiao-Ye Shen
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Response mechanism of hypocrellin colorants biosynthesis by Shiraia bambusicola to elicitor PB90
Wen Du, Chunlong Sun, Baogui Wang, Yanmei Wang, Bin Dong, Junhua Liu, Jiangbao Xia, Wenjun Xie, Jun Wang, Jingkuan Sun, Xuehong Liu, Hongguo Wang
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An efficient Agrobacterium-mediated transformation method for aflatoxin generation fungus Aspergillus flavus: Guomin Han , Qian Shao , Cuiping Li , Kai Zhao , Li Jiang , Jun Fan , Haiyang Jiang , Fang Tao; J. Microbiol. 2018;56(5):356-364. Published online May 2, 2018; DOI: https://doi.org/10.1007/s12275-018-7349-3

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Abstract

Aspergillus flavus often invade many important corps and produce harmful aflatoxins both in preharvest and during storage stages. The regulation mechanism of aflatoxin biosynthesis in this fungus has not been well explored mainly due to the lack of an efficient transformation method for constructing a genome-wide gene mutant library. This challenge was resolved in this study, where a reliable and efficient Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for A. flavus NRRL 3357 was established. The results showed that removal of multinucleate conidia, to collect a homogenous sample of uninucleate conidia for use as the transformation material, is the key step in this procedure. A. tumefaciens strain AGL-1 harboring the ble gene for zeocin resistance under the control of the gpdA promoter from A. nidulans is suitable for genetic transformation of this fungus. We successfully generated A. flavus transformants with an efficiency of ~ 60 positive transformants per 106 conidia using our protocol. A small-scale insertional mutant library (~ 1,000 mutants) was constructed using this method and the resulting several mutants lacked both production of conidia and aflatoxin biosynthesis capacity. Southern blotting analysis demonstrated that the majority of the transformants contained a single T-DNA insert on the genome. To the best of our knowledge, this is the first report of genetic transformation of A. flavus via ATMT and our protocol provides an effective tool for construction of genome-wide gene mutant libraries for functional analysis of important genes in A. flavus.

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Agrobacterium tumefaciens-mediated transformation for the genetic modification of the biotechnologically relevant fungus Aspergillus vadensis through synthetic biology
Carolina Ropero-Pérez, Paloma Manzanares, Jose F. Marcos, Sandra Garrigues
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Fangyi Ju, Zhongqiang Qi, Jiajin Tan, Tingting Dai
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An efficient targeted gene deletion approach for Cochliobolus heterostrophus using Agrobacterium tumefaciens-mediated transformation
Jiaying Sun, Rui Yang, Yujia Liu, Zengran Zhou, Jiaqi Jia, Hongming Huang, Shuqin Xiao, Chunsheng Xue
Journal of Microbiological Methods.2024; 216: 106863. CrossRef
Establishment of an Agrobacterium tumefaciens-Mediated Transformation System for Hirsutella sinensis
Lijuan Wu, Xinkun Hu, Shen Yan, Zenglin Wu, Xuzhong Tang, Lei Xie, Yujie Qiu, Rui Li, Ji Chen, Mengliang Tian
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Role of Flavohemoglobins in the Development and Aflatoxin Biosynthesis of Aspergillus flavus
Xiaoling Zhou, Dongyue Chen, Min Yu, Yuan Jiao, Fang Tao
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HacA, a key transcription factor for the unfolded protein response, is required for fungal development, aflatoxin biosynthesis and pathogenicity of Aspergillus flavus
Min Yu, Xiaoling Zhou, Dongyue Chen, Yuan Jiao, Guomin Han, Fang Tao
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Hui Yang, Chaonan Song, Chengwei Liu, Pengchao Wang
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Summi Dutta, Gabriella Houdinet, Gitanjali NandaKafle, Arjun Kafle, Christine V. Hawkes, Kevin Garcia
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Systematic Characterization of bZIP Transcription Factors Required for Development and Aflatoxin Generation by High-Throughput Gene Knockout in Aspergillus flavus
Qianqian Zhao, Hao Pei, Xiaoling Zhou, Kai Zhao, Min Yu, Guomin Han, Jun Fan, Fang Tao
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Homologous Expression and Characterization of α-L-rhamnosidase from Aspergillus niger for the Transformation of Flavonoids
Hangyu Ye, Xiaojun Li, Luyuan Li, Yinjun Zhang, Jianyong Zheng
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Ye-Eun Son, Hee-Soo Park
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Fang Tao, Kai Zhao, Qianqian Zhao, Fangzhi Xiang, Guomin Han
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Kristyn A. Robinson, Mallory Dunn, Shane P. Hussey, Lillian K. Fritz-Laylin, Louise A. Rollins-Smith
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Yi Hua, Rui Pan, Xuelian Bai, Bin Wei, Jianwei Chen, Hong Wang, Huawei Zhang
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Xing Xiao, Yu Li, JingLin Qin, Ya He, Wenying Cai, Zhiwen Chen, Liyan Xi, Junmin Zhang
Journal of Microbiological Methods.2020; 170: 105838. CrossRef
Genome-wide association study leads to novel genetic insights into resistance to Aspergillus flavus in maize kernels
Guomin Han, Cuiping Li, Fangzhi Xiang, Qianqian Zhao, Yang Zhao, Ronghao Cai, Beijiu Cheng, Xuewen Wang, Fang Tao
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The Efficacy of Composite Essential Oils against Aflatoxigenic Fungus Aspergillus flavus in Maize
Fangzhi Xiang, Qianqian Zhao, Kai Zhao, Hao Pei, Fang Tao
Toxins.2020; 12(9): 562. CrossRef
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Yongwen Lin, Hongchun Ruan, Komivi Senyo Akutse, Baochun Lai, Yizhang Lin, Youming Hou, Fenglin Zhong
Journal of Agricultural and Food Chemistry.2019; 67(49): 13706. CrossRef

Biotransformation of (-)-α-pinene and geraniol to α-terpineol and p-menthane-3,8-diol by the white rot fungus, Polyporus brumalis: Su-Yeon Lee , Seon-Hong Kim , Chang-Young Hong , Se-Yeong Park , In-Gyu Choi; J. Microbiol. 2015;53(7):462-467. Published online June 27, 2015; DOI: https://doi.org/10.1007/s12275-015-5081-9

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Abstract

In this study, the monoterpenes, α-pinene and geraniol, were biotransformed to synthesize monoterpene alcohol compounds. Polyporus brumalis which is classified as a white rot fungus was used as a biocatalyst. Consequently α-terpineol was synthesized from α-pinene by P. brumalis mycelium, after three days. Moreover, another substrate, the acyclic monoterpenoids geraniol was transformed into the cyclic compound, p-menthane-3, 8-diol (PMD). The main metabolites, i.e., α-terpineol and PMD, are known to be bioactive monoterpene alcohol compounds. This study highlights the potential of fungal biocatalysts for monoterpene transformation.

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Biotransformation of essential oil composition of Zanthoxylum limonella by the fungus Pleopunctum pseudoellipsoideum provides the products with enhanced antimicrobial activities
Sarunpron Khruengsai, Teerapong Sripahco, Pavaret Sivapornnukul, Patcharee Pripdeevech
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Biotransformation of Geraniol to Geranic Acid Using Fungus Mucor irregularis IIIMF4011
Haseena Shafeeq, Bashir Ahmad Lone, Ananta Ganjoo, Nargis Ayoub, Hema Kumari, Sumeet Gairola, Prasoon Gupta, Vikash Babu, Zabeer Ahmed
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Elison de Souza Sevalho, Bruno Nicolau Paulino, Antonia Queiroz Lima de Souza, Afonso Duarte Leão de Souza
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Rogers J. Nyamwihura, Ifedayo Victor Ogungbe
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Proloy Sankar Dev Roy, Brajeshwar Singh, Vikas Sharma, Chandan Thappa
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An update on the progress of microbial biotransformation of commercial monoterpenes
Ruchika Mittal, Gauri Srivastava, Deepak Ganjewala
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Xin Wang, Jia-wen Hou, Wen-rui Liu, Jia Bao
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Production, Properties, and Applications of α-Terpineol
Adones Sales, Lorena de Oliveira Felipe, Juliano Lemos Bicas
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Biotransformation of terpene and terpenoid derivatives by Aspergillus niger NRRL 326
Cengiz Çorbacı
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Optimization of limonene biotransformation for the production of bulk amounts of α-terpineol
Gustavo Molina, Marina G. Pessôa, Juliano L. Bicas, Pierre Fontanille, Christian Larroche, Gláucia M. Pastore
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Biogeneration of aroma compounds
Adones Sales, Bruno Nicolau Paulino, Glaucia Maria Pastore, Juliano Lemos Bicas
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Transcriptomic analysis of the white rot fungus Polyporus brumalis provides insight into sesquiterpene biosynthesis
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Research Support, Non-U.S. Gov'ts

Transformation of Inorganic P Fractions of Soil and Plant Growth Promotion by Phosphate-solubilizing Ability of Penicillium oxalicum I1: Mingbo Gong , Peng Du , Xue Liu , Changxiong Zhu; J. Microbiol. 2014;52(12):1012-1019. Published online November 3, 2014; DOI: https://doi.org/10.1007/s12275-014-4406-4

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Abstract

The solubilization of tricalcium phosphate is often considered as the standard for screening of most phosphate-solubilizing microorganisms (PSMs). However, usually the effect of large-scale application of PSM on the promotion of crop growth varies. This study presents an efficient method for screening and testing phosphate-solubilizing fungus that enhance plant growth. A fungus Penicillium oxalicum I1 (PI1) was isolated and identified that had high ability of phosphate- solubilization and could utilize maize root exudates as sources, and propagate well in vitro and in soil. P-I1 excreted oxalic acid and reached 593.9 μg/ml, and the pH value was decreased from 6.90 to 1.65 in 26 h. The amount of P-I1 increased by 48-fold in 28 d and was maintained for 49 d in soil. PSM showed selectivity on the transformation of the different forms of phosphorus, a wide range of insoluble phosphates, such as Ca8H2(PO4)6·5H2O, AlPO4, FePO4, and Ca10(PO4)6(OH)2, were converted to soluble CaHPO4 in soil, and CaHPO4 was also inhibited from being converted into insoluble phosphate by P-I1. The Ca2-P content reached 27.11 μg/g soil on day 28 at 20°C, which increased by 110.32%, and plant growth promotion was tested and verified, the
results
showed that maize yield increased remarkably than control after inoculated P-I1, maize yield increased maximum by 14.47%. The data presented that P-I1 appear attractive for exploring their plant growth-promoting activity and potential field application.

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Inoculations of phosphate-solubilizing bacteria alter soil microbial community and improve phosphorus bioavailability for moso bamboo (Phyllostachys edulis) growth
Yaohui Liu, Ashrafun Nessa, Qiyuan Zheng, Dongnan Hu, Wenyuan Zhang, Manyun Zhang
Applied Soil Ecology.2023; 189: 104911. CrossRef
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Hafiza Farhat, Faizah Urooj, Muhammed Irfan, Nida Sohail, Saima Majeed, Shahid Ullah, Hafza Asma Shafique
Current Microbiology.2023;[Epub] CrossRef
Effect of Bacillus megaterium var. phosphaticum applied together with rock phosphate on wheat yield and some soil properties in a calcareous soil
Betül BAYRAKLI
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Saheed Adekunle Akinola, Olubukola Oluranti Babalola
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Characterization of Recombinant β-Glucosidase from Arthrobacter chlorophenolicus and Biotransformation of Ginsenosides Rb1, Rb2, Rc, and Rd: Myung Keun Park , Chang-Hao Cui , Sung Chul Park , Seul-Ki Park , Jin-Kwang Kim , Mi-Sun Jung , Suk-Chae Jung , Mi-Sun Jung , Suk-Chae Jung , Sun-Chang Kim , Wan-Taek Im; J. Microbiol. 2014;52(5):399-406. Published online May 9, 2014; DOI: https://doi.org/10.1007/s12275-014-3601-7

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Abstract

The focus of this study was the cloning, expression, and characterization of recombinant ginsenoside hydrolyzing β-glucosidase from Arthrobacter chlorophenolicus with an ultimate objective to more efficiently bio-transform ginse-nosides. The gene bglAch, consisting of 1,260 bp (419 amino acid residues) was cloned and the recombinant enzyme, over-expressed in Escherichia coli BL21 (DE3), was characterized. The GST-fused BglAch was purified using GST·Bind agarose resin and characterized. Under optimal conditions (pH 6.0 and 37°C) BglAch hydrolyzed the outer glucose and arabino-pyranose moieties of ginsenosides Rb1 and Rb2 at the C20 position of the aglycone into ginsenoside Rd. This was fol-lowed by hydrolysis into F2 of the outer glucose moiety of ginsenoside Rd at the C3 position of the aglycone. Additio-nally, BglAch more slowly transformed Rc to F2 via C-Mc1 (compared to hydrolysis of Rb1 or Rb2). These results in-dicate that the recombinant BglAch could be useful for the production of ginsenoside F2 for use in the pharmaceutical and cosmetic industries.

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Insight into a novel β-1,4-glucosidase from Streptomyces griseorubens JSD-1
H.-W. Feng, Y.-E. Zhi, Y.-J. Sun, L.-R. Xu, L.-M. Wang, X.-J. Zhan, P. Zhou
Applied Biochemistry and Microbiology.2016; 52(4): 371. CrossRef
Overexpression and characterization of a glycoside hydrolase family 1 enzyme from Cellulosimicrobium cellulans sp. 21 and its application for minor ginsenosides production
Ye Yuan, Yanbo Hu, Chenxing Hu, Jiayi Leng, Honglei Chen, Xuesong Zhao, Juan Gao, Yifa Zhou
Journal of Molecular Catalysis B: Enzymatic.2015; 120: 60. CrossRef

NOTE] Is The Biotransformation of Chlorinated Dibenzo-p-dioxins by Sphingomonas wittichii RW1 Governed by Thermodynamic Factors?: In-Hyun Nam , Hyo-Bong Hong , Stefan Schmidt; J. Microbiol. 2014;52(9):801-804. Published online February 17, 2014; DOI: https://doi.org/10.1007/s12275-014-3424-6

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Abstract

Density functional theory (DFT) calculations were used to explore the relationship between the biotransformation of dibenzo-p-dioxin and selected chlorinated derivatives by resting cells of Sphingomonas wittichii RW1 and measuring the thermodynamic properties of the biotransformation substrates. Sphingomonas wittichii RW1 can aerobically catabolize dibenzo-p-dioxin as well as 2,7-dichloro-, 1,2,3-trichloro-, 1,2,3,4-tetrachloro-, and 1,2,3,4,7,8-hexachlorodibenzo-pdioxin; however, neither the 2,3,7-trichloro- nor the 1,2,3,7,8-pentachlorodibenzo-p-dioxin was transformed to its corresponding metabolic intermediate. The experimental biotransformation rates established were apparently governed by the selected thermodynamic properties of the substrates tested.

Citations

Citations to this article as recorded by

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Qihong Lu, Qi Liang, Shanquan Wang
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Development of a two-stage washing and biodegradation system to remediate octachlorinated dibenzo-p-dioxin-contaminated soils
J. L. Lin, C. D. Dong, C. W. Chen, S. H. Chen, T. E. Hsieh, C. M. Kao
International Journal of Environmental Science and Technology.2017; 14(9): 1919. CrossRef
Aerobic bacterial catabolism of persistent organic pollutants — potential impact of biotic and abiotic interaction
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Current Opinion in Biotechnology.2016; 38: 71. CrossRef

Optimized Transformation of Streptomyces sp. ATCC 39366 Producing Leptomycin by Electroporation: Yong-Qiang Fan , Hong-Jian Liu , Li Yan , Yu-Shi Luan , Hai-Meng Zhou , Jun-Mo Yang , Shang-Jun Yin , Yu-Long Wang; J. Microbiol. 2013;51(3):318-322. Published online April 26, 2013; DOI: https://doi.org/10.1007/s12275-013-2428-y

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Abstract: Streptomyces sp. ATCC 39366 produces leptomycin derivatives. Leptomycin B, a potent and specific inhibitor against the export of nuclear proteins, is the main product; however, the introduction of DNA into this strain is almost impossible, which has impeded its further use. We developed a Streptomyces sp. ATCC 39366 transformation protocol to introduce foreign DNA via electroporation. Various conditions were examined, including treatments of the cell wall with weakening agents, electroporation parameters, and DNA content. We found that only plasmid DNA isolated from a dam- ET12567 strain resulted in successful transformation. The mycelium growing in a yeast-peptone-dextrose medium supplemented with 1% glycine at 28°C on a rotary shaker (220 rpm) was more dispersed than those without supplementation and prone to electroporation. The maximum transformation efficiency of 8×102 CFU/μg plasmid DNA was obtained at a field strength of 13 kV/cm with a time constant of 13 ms (25-μF capacitor; parallel resistance, 600 Ω) using 1-mm electrocuvettes. The results of the transformations of two other Streptomyces species indicated that the optimized conditions established in this study might only be applicable to Streptomyces sp. ATCC 39366. However, this is the first report of successful transformation of Streptomyces sp. ATCC 39366, and will facilitate the construction of a gene knockout mutant in Streptomyces sp. ATCC 39366 to produce series of new leptomycin derivatives.

Degradation of Endocrine Disrupting Chemicals by Genetic Transformants with Two Lignin Degrading Enzymes in Phlebia tremellosa: Hyunwoo Kum , Sungsuk Lee , Sunhwa Ryu , Hyoung T. Choi; J. Microbiol. 2011;49(5):824-827. Published online November 9, 2011; DOI: https://doi.org/10.1007/s12275-011-1230-y

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Abstract: A white rot fungus Phlebia tremellosa produced lignin degrading enzymes, which showed degrading activity against various recalcitrant compounds. However, manganese peroxidase (MnP) activity, one of lignin degrading enzymes, was very low in this fungus under various culture conditions. An expression vector that carried both the laccase and MnP genes was constructed using laccase genomic DNA of P. tremellosa and MnP cDNA from Polyporus brumalis. P. tremellosa was genetically transformed using the expression vector to obtain fungal transformants showing increased laccase and MnP activity. Many transformants showed highly increased laccase and MnP activity at the same time in liquid medium, and three of them were used to degrade endocrine disrupting chemicals. The transformant not only degraded bisphenol A and nonylphenol more rapidly but also removed the estrogenic activities of the chemicals faster than the wild type strain.

Journal Article

High Efficiency Transformation by Electroporation of Yarrowia lipolytica: Jia-Hung Wang , Wenpin Hung , Shu-Hsien Tsai; J. Microbiol. 2011;49(3):469-472. Published online June 30, 2011; DOI: https://doi.org/10.1007/s12275-011-0433-6

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Abstract: Yarrowia lipolytica was usually transformed by heat shock, but linearized integrative vectors always resulted in a low transformation efficiency when electroporation was used. To develop a high efficiency integrative transformation method by electroporation of Y. lipolytica, we report here that pretreatment of Y. lipolytica with 150 mM LiAc for 1 h before electroporation will approximately 30-fold of increase transformation efficiency. A cell concentration of 1010/ml and instrument settings of 1.5 kV will generate the highest transformation efficiencies. We have developed a procedure to transform Y. lipolytica that will be able to yield an efficiency of 2.1×104 transformants/μg for integrative linear DNA. With our modifications, the electroporation procedures became a very efficient and reliable tool for Y. lipolytica transformation.

Research Support, Non-U.S. Gov'ts

Genetic Introduction of Foreign Genes to Pleurotus eryngii by Restriction Enzyme-Mediated Integration: Won Noh , Sang-Woo Kim , Dong-Won Bae , Jae-Yean Kim , Hyeon-Su Ro; J. Microbiol. 2010;48(2):253-256. Published online May 1, 2010; DOI: https://doi.org/10.1007/s12275-010-9278-7

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Abstract: Pleurotus eryngii was transformed via restriction enzyme-mediated integration. In order to construct the transformation plasmid, the enhanced cyan fluorescent protein (ECFP) gene was ligated next to the gpd promoter of the plasmid pAN7-1. Transformation was facilitated via the heat treatment of a transformation mixture containing 1 μg of the HindIII-digested plasmid DNA and 106 mushroom protoplasts in 40% polyethyleneglycol solution, resulting in 10-40 hygromycin-resistant transformants. Successful transformation was evidenced by PCR, Southern blot, and confocal fluorescence microscopic analyses on the selected transformants. To date, this is the first report on the transformation of P. eryngii by REMI technique.

Cloning and Sequence Analysis of a Glyceraldehyde-3-phosphate Dehydrogenase Gene from Ganoderma lucidum: Xu Fei , Ming Wen Zhao , Yu Xiang Li; J. Microbiol. 2006;44(5):515-522.; DOI: https://doi.org/2446 [pii]

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Abstract: A cDNA library of Ganoderma lucidum has been constructed using a Zap Express cloning vector. A glyceraldehyde-3-phosphate dehydrogenase gene (gpd) was isolated from this library by hybridization of the recombinant phage clones with a gpd-specific gene probe generated by PCR. By comparison of the cDNA and the genomic DNA sequences, it was found that the complete nucleotide sequence encodes a putative polypeptide chain of 338 amino acids interrupted by 6 introns. The predicted amino acid sequence of this gene shows a high degree of sequence similarity to the GPD proteins from yeast and filamentous fungi. The promoter region contains a CT-rich stretch, two CAAT boxes, and a consensus TATA box. The possibility of using the gpd promoter in the construction of new transformation vectors is discussed.

A Comparison of the Phenotypic and Genetic Stability of Recombinant Trichoderma spp. Generated by Protoplast- and Agrobacterium-Mediated Transformation: Rosa Elena Cardoza , Juan Antonio Vizcaino , Maria Rosa Hermosa , Enrique Monte , Santiago Gutierrez; J. Microbiol. 2006;44(4):383-395.; DOI: https://doi.org/2416 [pii]

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Abstract: Four different Trichoderma strains, T. harzianum CECT 2413, T. asperellum T53, T. atroviride T11 and T. longibrachiatum T52, which represent three of the four sections contained in this genus, were transformed by two different techniques: a protocol based on the isolation of protoplasts and a protocol based on Agrobacterium-mediated transformation. Both methods were set up using hygromycin B or phleomycin resistance as the selection markers. Using these techniques, we obtained phenotypically stable transformants of these four different strains. The highest transformation efficiencies were obtained with the T. longibrachiatum T52 strain: 65-70 transformants/μg DNA when transformed with the plasmid pAN7-1 (hygromycin B resistance) and 280 transformants/107 spores when the Agrobacterium-mediated transformation was performed with the plasmid pUR5750 (hygromycin B resistance). Overall, the genetic analysis of the transformants showed that some of the strains integrated and maintained the transforming DNA in their genome throughout the entire transformation and selection process. In other cases, the integrated DNA was lost.

The Effect of Transformation on the Virulence of Streptococcus pneumoniae: Xue-Mei Zhang , Yi-Bing Yin , Dan Zhu , Bao-De Chen , Jin-Yong Luo , Yi-Ping Deng , Ming-Fang Liu , Shu-Hui Chen , Jiang-Ping Meng , Kai Lan , Yuan-Shuai Huang , Ge-Fei Kang; J. Microbiol. 2005;43(4):337-344.; DOI: https://doi.org/2256 [pii]

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Abstract: Although pneumococcus is one of the most frequently encountered opportunistic pathogen in the world, the mechanisms responsible for its infectiveness have not yet been fully understood. In this paper, we have attempted to characterize the effects of pneumococcal transformation on the pathogenesis of the organism. We constructed three transformation-deficient pneumococcal strains, which were designated as Nos. 1d, 2d, and 22d. The construction of these altered strains was achieved via the insertion of the inactivated gene, comE, to strains 1, 2 and 22. We then conducted a comparison between the virulence of the transformation-deficient strains and that of the wild-type strains, via an evaluation of the ability of each strain to adhere to endothelial cells, and also assessed psaA mRNA expression, and the survival of hosts after bacterial challenge. Compared to what was observed with the wild-type strains, our results indicated that the ability of all of the transformation-deficient strains to adhere to the ECV304 cells had been significantly reduced (p < 0.05), the expression of psaA mRNA was reduced significantly (p < 0.05) in strains 2d and 22d, and the median survival time of mice infected with strains 1d and 2d was increased significantly after intraperitoneal bacterial challenge (p < 0.05). The results of our study also clearly indicated that transformation exerts significant effects on the virulence characteristics of S. pneumoniae, although the degree to which this effect is noted appears to depend primarily on the genetic background of the bacteria.

Chlorothalonil-Biotransformation by Glutathione S-Transferase of Escherichia coli: Young-Mog Kim , Kunbawui Park , Soon-Hyun Jung , Jun-Ho Choi , Won-Chan Kim , Gil-Jae Joo , In-Koo Rhee; J. Microbiol. 2004;42(1):42-46.; DOI: https://doi.org/2002 [pii]

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Abstract: It has recently been reported that one of the most important factors of yeast resistance to the fungicide chlorothalonil is the glutathione contents and the catalytic efficiency of glutathione S-transferase (GST) (Shin et al., 2003). GST is known to catalyze the conjugation of glutathione to a wide variety of xenobiotics, resulting in detoxification. In an attempt to elucidate the relation between chlorothalonil detoxification and GST, the GST of Escherichia coli was expressed and purified. The drug hypersensitive E. coli KAM3 cells harboring a plasmid for the overexpression of the GST gene can grow in the presence of chlorothalonil. The purified GST showed chlorothalonil-biotransformation activity in the presence of glutathione. Thus, chlorothalonil is detoxified by the mechanism of glutathione conjugation catalyzed by GST.

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