Heterologous expression of stearoyl-acyl carrier protein desaturase (S-ACP-DES) from Arabidopsis thaliana in Escherichia coli |
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| Authors: | Yujin Cao Mo Xian Jianming Yang Xin Xu Wei Liu Liangzhi Li |
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| Affiliation: | 1. Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, WI, United States;2. Microbiology Doctoral Training Program, University of Wisconsin-Madison, Madison, WI, United States;3. Department of Biology and Biological Engineering, Chalmers University of Technology, Göteborg, Sweden;1. Department of Agricultural and Life Science, Graduate School of Science and Technology, Shinshu University, 8304 Minamiminowa, Nagano 399-4598, Japan;2. Department of Bioscience and Biotechnology, Faculty of Agriculture, Shinshu University, 8304 Minamiminowa, Nagano 399-4598, Japan;3. Innovative Research Center for Agricultural and Food Industry (CAFI), Shinshu University, 4-17-1 Wakasato, Nagano City, Nagano 380-8553, Japan;4. R&D Center, Kobayashi Pharmaceutical Co., Ltd., 1-30-3, Toyokawa, Ibaraki, Osaka 567-0057, Japan;5. Omics Research Center, National Cerebral and Cardiovascular Center, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan;6. Department of Pharmaceutical Sciences, Health Sciences University of Hokkaido, 1757 Kanazawa, Tobetsu-cho, Ishikari, Hokkaido 061-0293, Japan;1. The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kemitorvet 220, 2800 Kgs, Lyngby, Denmark;2. The Novo Nordisk Foundation Center for Biosustainability, Department of Chemical & Biological Engineering, Chalmers University of Technology, Kemivägen 10, SE412 96 Gothenburg, Sweden |
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| Abstract: | Fatty acid desaturases are enzymes that introduce double bonds into fatty acyl chains, among which stearoyl-acyl carrier protein desaturase (S-ACP-DES) was widely distributed in the plant kingdom. We cloned the cDNA coding for fab2/ssi2, an S-ACP-DES from Arabidopsis thaliana, into the vector pET30a and heterologously expressed this fatty acid desaturase in Escherichia coli BL21 (DE3). After being induced with IPTG, the fusion protein was efficiently expressed in a soluble form. The SSI2 desaturase was purified by nickel ion affinity chromatography and the product obtained showed a single band by SDS–PAGE analysis. The expression of ssi2 modified the fatty acid composition of the recombinant strain. The ratio of palmitic acid (16:0) decreased from 45.2% (the control strain) to 35.2% while palmitoleate (16:1Δ9) and cis-vaccenate (18:1Δ11) levels were enhanced to some extent. The desaturase enzymatic activity was measured in vivo when the enzyme substrate stearic acid was provided in the culture medium. A new fatty acid, oleic acid (18:1Δ9) was found in the recombinant strain which did not exist in wild-type E. coli. These results demonstrated that the cofactors of the host system can complement the requirement of the SSI2 desaturase. |
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