Flow cytometric sorting of the filamentous fungus Trichoderma reesei for improved strains |
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| Affiliation: | 1. Genencor, a Danisco Division, 925 Page Mill Road, Palo Alto, CA 94304, United States;2. University of Manchester, Faculty of Life Sciences, Manchester, UK;1. Department of Chemistry, Capital Normal University, Beijing 100048, PR China;2. Beijing Key Laboratory of Radioactive Waste Treatment, Institute of Nuclear and New Energy Technology, Tsinghua University, Beijing 100084, PR China;3. Beijing Key Laboratory of Microanalytical Methods and Instrumentation, Department of Chemistry, Tsinghua University, Beijing 100084, PR China;1. Department of Marine Life Sciences, School of Marine Biomedical Sciences, Jeju National University, Jeju Self-Governing Province 63243, Republic of Korea;2. Fish Vaccine Research Center, Jeju National University, Jeju Self-Governing Province 63243, Republic of Korea;3. Department of Veterinary Medicine, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-8570, Japan;4. Informatics Development & Management Group, Korea Institute of Oriental Medicine, 1672 Yuseongdae-ro, Yuseong-gu, Daejeon 34054, Republic of Korea;1. Institute of Aging Research, School of Medicine, Hangzhou Normal University, Hangzhou, Zhejiang Province 311121, China;2. Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Life Sciences College, Nanjing Normal University, Nanjing, Jiangsu Province 210046, China;3. The People''s No 4 Hospital of Xiaoshan, Hangzhou, Zhejiang Province 311225, China;1. Graduate Program in Environmental Health Sciences, Graduate School of Integrated Pharmaceutical and Nutritional Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan;2. Department of Environmental and Life Sciences, School of Food and Nutritional Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan;3. Department of Biological Environment, Akita Prefectural University, Shimoshinjo-Nakano, Akita 010-0195, Japan;4. National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba 305-8506, Japan;1. Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, Ontario M5S 3H6, Canada;2. Department of Chemical and Physical Sciences, University of Toronto Mississauga, 3359 Mississauga Rd., Mississauga, Ontario L5L 1C6, Canada;3. Department of Cell and Systems Biology, University of Toronto, 25 Harbord St., Toronto, Ontario M5S 3G5, Canada;1. Institute for Molecular Bioscience, University of Queensland, Brisbane, 4072, QLD, Australia;2. Australian Institute for Bioengineering and Nanotechnology, University of Queensland, Brisbane, 4072, QLD, Australia;3. Queensland University of Technology (QUT), Brisbane, 4001, QLD, Australia |
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| Abstract: | Metabolic measurements and screening of Trichoderma reesei have conventionally been performed during the hyphal stage of fungal development. To determine if flow cytometric measurements of protein expression could be made on germinating spores we created a gene construct, placing the Renilla reniformis green fluorescent protein gene under control of the cellobiohydrolase I (cbh1) promoter and terminator of T. reesei. This vector was transformed into T. reesei and GFP expression was measured in germlings by flow cytometry. Fluorescence associated with GFP expression was observed in germlings grown under conditions known to induce cellulases in Trichoderma. Spores were mutated using UV light and germinating spores were screened for increased GFP expression using high-speed cell sorting, to select for strains with genetic changes associated with increased protein expression. Secondary screens for cellulase production were conducted in microtitre plates. Flow cytometric screening of germinating spores expressing GFP yielded a mutant with improved ability to hydrolyse biomass. |
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