共表达极端菌伴侣蛋白prefoldin提高P450 BM3突变体催化效率

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共表达极端菌伴侣蛋白prefoldin提高P450 BM3突变体催化效率
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作者单位:华东理工大学生物反应器工程国家重点实验室,中国科学院上海生命科学研究院;中国科学院合成生物学重点实验室,中国科学院上海生命科学研究院;中国科学院合成生物学重点实验室,中国科学院上海生命科学研究院;中国科学院合成生物学重点实验室,华东理工大学生物反应器工程国家重点实验室,中国科学院上海生命科学研究院;中国科学院合成生物学重点实验室,中国科学院上海生命科学研究院;中国科学院合成生物学重点实验室
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基金项目:国家重点基础研究发展计划(973)资助项目(2012CB721103)

Co-expression of Prefoldin From Hyperthermophilic Archaea Pyrococcus furiosus in Escherichia coli Enhances The Catalytic Efficiency of Modified Cytochrome P450 BM3

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State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology,Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences; Key Laboratory of Synthetic Biology, Chinese Academy of Sciences,Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences; Key Laboratory of Synthetic Biology, Chinese Academy of Sciences,Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences; Key Laboratory of Synthetic Biology, Chinese Academy of Sciences,State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology,Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences; Key Laboratory of Synthetic Biology, Chinese Academy of Sciences,Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences; Key Laboratory of Synthetic Biology, Chinese Academy of Sciences

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This work was supported by a grant from National Basic Research Program of China (2012CB721103)

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P450 BM3来源于巨大芽孢杆菌，其突变体(A74G、F87V、L188Q、D168H)能够在大肠杆菌细胞内催化吲哚合成靛蓝．然而在常规的培养条件下(37℃，250 r/min)，大肠杆菌细胞内靛蓝的生物转化量极低．本文将极端嗜热古菌Pyrococcus furiosus的分子伴侣蛋白与P450 BM3突变体在大肠杆菌进行共表达，以研究分子伴侣蛋白是否能够提高靛蓝的生物转化量．实验结果表明，极端嗜热古菌的分子伴侣蛋白prefoldin能够显著提高靛蓝的产量．同时，实验结果发现prefoldin能够明显提高大肠杆菌细胞内的NADPH/NADP+比率．鉴于NADPH是参与靛蓝生物转化过程的重要因素，靛蓝生物转化量的显著增加可能与该比率的提高有关．

Abstract:

P450 BM3 is derived from Bacillus megaterium, and its mutant (A74G, F87V, L188Q, D168H) could hydroxylate indole to indigo in Escherichia coli whereas the bioconversion yield is low under normal culture conditions (37℃, 250 r/min). Chaperones from hyperthermophilic archaea Pyrococcus furiosus were co-expressed with P450 BM3 mutant in E. coli to investigate whether they could improve the production of indigo, and results indicated that prefoldin could significantly increase the indigo production. Our research also revealed that enzyme activity was not the restrictive factor in the indigo bioconversion process. We also demonstrated that prefoldin could increase the intracellular nicotinamide adenine dinucleotide phosphate (NADPH)/NADP⁺ ratio, and this ratio was probably related to the increase of the indigo bioconversion yield.

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彭帅英,褚仲梅,陆坚峰,李东晓,王永红,杨胜利,张毅.共表达极端菌伴侣蛋白prefoldin提高P450 BM3突变体催化效率[J].生物化学与生物物理进展,2017,44(12):1125-1131

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收稿日期:2017-04-10
最后修改日期:2017-08-23
接受日期:2017-09-04
在线发布日期: 2017-12-19
出版日期: 2017-12-20

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