Deletion of iscR stimulates recombinant clostridial Fe-Fe hydrogenase activity and H2-accumulation in Escherichia coli BL21(DE3)
Deletion of iscR stimulates recombinant clostridial Fe-Fe hydrogenase activity and H2-accumulation in Escherichia coli BL21(DE3)
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DOI：
10.1007/s00253-008-1377-6
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发表时间：
2008-04-01
影响因子：
5
通讯作者：
Jones, Patrik R.
中科院分区：
文献类型：
--
作者：
Akhtar, M. Kalim;Jones, Patrik R.
催化H-2-途径的蛋白质通常含有铁-硫（Fe-S）簇，对O-2敏感。我们测试了是否删除编码转录负调控因子IscR的基因可以增强大肠杆菌BL 21合成活性重组H-2-途径组分的能力，并在存在或不存在氧气的情况下刺激铁氧还蛋白依赖的H-2-积累。在缺氧条件下，删除iscR刺激重组铁-铁氢化酶活性的三倍，而质粒为基础的过度表达的isc操纵子氢化酶活性没有影响。在顶空中用21%（v/v）O-2培养后，在野生型BL 21的可溶性提取物中未观察到重组氢化酶活性，尽管在Delta iscR菌株中可观察到低水平的活性（比缺氧条件低700倍，比检测限高180倍）。在封闭的批处理条件下，5%（v/v）的O-2开始，德尔塔iscR菌株显示五倍以上的总氢化酶活性和重组铁氧还蛋白依赖性H-2积累的水平相对于对照菌株。在10%（v/v）的O-2，H-2-积累的文化开始刺激35倍，相对于控制。Delta iscR菌株显示增强的合成和活性的完整的H-2-途径组件在所有测试条件下，并增强H-2-积累在部分好氧条件下。因此，缺失iscR是刺激H-2-产生的有用策略，特别是如果氢化酶催化限速反应。
Proteins that catalyze H-2-pathways often contain iron-sulfur (Fe-S) clusters and are sensitive to O-2. We tested whether deletion of the gene encoding the transcriptional negative regulator, IscR, could enhance the ability of Escherichia coli BL21 to synthesize active recombinant H-2-pathway components and stimulate ferredoxin-dependent H-2-accumulation in the presence or absence of oxygen. Under anoxic conditions, deletion of iscR stimulated recombinant Fe-Fe hydrogenase activity threefold, whilst plasmid-based overexpression of the isc operon had no effect on hydrogenase activity. After cultivation with 21% (v/v) O-2 in the headspace, no recombinant hydrogenase activity was observed in soluble extracts of wild-type BL21, although low levels of activity could be observed in the Delta iscR strain (700-fold lower than anoxic conditions, 180-fold greater than the limit of detection). Under closed batch conditions starting with 5% (v/v) O-2, Delta iscR strains displayed fivefold greater levels of total hydrogenase activity and recombinant ferredoxin-dependent H-2-accumulation relative to the control strain. In cultures starting with 10% (v/v) O-2, H-2-accumulation was stimulated 35-fold relative to the control. Delta iscR strains displayed enhanced synthesis and activity of integral H-2-pathway components under all tested conditions and enhanced H-2-accumulation under partially oxic conditions. Deletion of iscR is, therefore, a useful strategy to stimulate H-2-production, particularly if the hydrogenase catalyzes the rate-limiting reaction.