Characterization of 5-aminolevulinate synthase from Agrobacterium radiobacter,screening new inhibitors for 5-aminolevulinate dehydratase from Escherichia coli and their potential use for high 5-aminolevulinate production |
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| Authors: | Jianping Lin Weiqi Fu Peilin Cen |
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| Institution: | 1. Institute of Bioengineering, Zhejiang University of Technology, Hangzhou 310014, People''s Republic of China;2. Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of Technology, Hangzhou 310014, People''s Republic of China;1. College of Veterinary Medicine, Nursing & Allied Health, Department of Pathobiology, Tuskegee University, 1200 Montgomery Road, Tuskegee, AL 36088, USA;2. Kafrelshikh University, College of Veterinary Medicine, Kafrelsheikh, Egypt;3. Middle Technical University, Institute of Medical Technology, Department of Community Health, Baghdad, Iraq;4. Pennsylvania School of Dental Medicine, Department of Microbiology, Philadelphia, PA 19104, USA;5. The Department of Microbiology & Immunology and The Witebsky Center for Microbial Pathogenesis and Immunology, School of Medicine and Biomedical Sciences, 138 Farber Hall, 3435 Main St., University at Buffalo, NY 14214, USA;1. Department of Biotechnology, Korea University, Seoul 136-701, Republic of Korea;2. Department of Chemical and Biological Engineering, Korea University, Seoul 136-701, Republic of Korea;3. Department of Chemical Engineering, Kwangwoon University, Seoul 139-701, Republic of Korea;1. Center for Bioengineering and Biotechnology, China University of Petroleum (East China), Qingdao, 266580, PR China;2. Yantai Institute of Coastal Zone, Chinese Academy of Sciences, Yantai, 264003, PR China |
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| Abstract: | The hemA gene encoding 5-aminolevulinate synthase (ALAS) from Agrobacterium radiobacter zju-0121 showed 92.6% homology with that from A. radiobacter ATCC4718 and contained several rare codons. To enhance the expression of this gene, Escherichia coli Rosetta(DE3), which is a rare codon optimizer strain, was used as the host to construct an efficient recombinant strain. And the encoded protein was over-expressed as fusion protein and was purified by affinity purification on Ni-NTA agarose and by gel filtration chromatography on Sephadex G-25 Medium resin. The recombinant protein was partly characterized, and d-glucose, d-fructose, d-xylose, d-mannose, l-arabinose, d-galactose, lactose, sucrose and maltose were detected to have no distinct inhibition on this recombinant ALAS. Meanwhile, 20 mM d-glucose or d-xylose inhibited about 20% activity of ALA dehydratase (ALAD) from Escherichia coli Rosetta(DE3). Combining d-xylose as a new inhibitor for ALAD with d-glucose in fed-batch culture and based on the optimal culture system using Rosetta(DE3)/pET28a-hemA, the yield of ALA achieved was 7.3 g/l (56 mM) under the appropriate conditions in the fermenter. |
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