Akhtar M Kalim, Jones Patrik R
Research and Development Division, Fujirebio Incorporated, 51 Komiya-cho, Hachioji-shi, Tokyo 192-0031, Japan.
Appl Microbiol Biotechnol. 2008 Apr;78(5):853-62. doi: 10.1007/s00253-008-1377-6. Epub 2008 Mar 5.
Proteins that catalyze H2-pathways often contain iron-sulfur (Fe-S) clusters and are sensitive to O2. 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 H2-pathway components and stimulate ferredoxin-dependent H2-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) O2 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 DeltaiscR strain (700-fold lower than anoxic conditions, 180-fold greater than the limit of detection). Under closed batch conditions starting with 5% (v/v) O2, DeltaiscR strains displayed fivefold greater levels of total hydrogenase activity and recombinant ferredoxin-dependent H2-accumulation relative to the control strain. In cultures starting with 10% (v/v) O2, H2-accumulation was stimulated 35-fold relative to the control. DeltaiscR strains displayed enhanced synthesis and activity of integral H2-pathway components under all tested conditions and enhanced H2-accumulation under partially oxic conditions. Deletion of iscR is, therefore, a useful strategy to stimulate H2-production, particularly if the hydrogenase catalyzes the rate-limiting reaction.
催化H2途径的蛋白质通常含有铁硫（Fe-S）簇，并且对O2敏感。我们测试了编码转录负调节因子IscR的基因缺失是否能增强大肠杆菌BL21在有氧或无氧条件下合成活性重组H2途径成分以及刺激铁氧化还原蛋白依赖性H2积累的能力。在缺氧条件下，iscR基因的缺失使重组Fe-Fe氢化酶活性提高了三倍，而基于质粒的isc操纵子过表达对氢化酶活性没有影响。在顶空含有21%（v/v）O2的条件下培养后，野生型BL21的可溶性提取物中未观察到重组氢化酶活性，尽管在DeltaiscR菌株中可观察到低水平的活性（比缺氧条件下低700倍，比检测限高180倍）。在以5%（v/v）O2开始的密闭分批条件下，DeltaiscR菌株的总氢化酶活性和重组铁氧化还原蛋白依赖性H2积累水平相对于对照菌株提高了五倍。在以10%（v/v）O2开始的培养物中，H2积累相对于对照被刺激了35倍。DeltaiscR菌株在所有测试条件下均表现出整体H2途径成分的合成和活性增强，以及在部分有氧条件下H2积累增强。因此，删除iscR是刺激H2产生的一种有用策略，特别是当氢化酶催化限速反应时。
