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Recombinant Klebsiella oxytoca Strains with Improved Efficiency in Removal of High Nitrate Loads

机译:重组产酸克雷伯菌具有提高去除高硝酸盐负荷的效率

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摘要

Klebsiella oxytoca CECT 4460 removes high nitrate loads from industrial wastewaters without accumulation of nitrite under optimal culture conditions; however, under nonoptimal conditions nitrite accumulates. This situation reflects an in vivo-limited functioning of nitrite reductase in this strain. As a way to overcome this limitation, an increase in the nitrite reductase gene dose in K. oxytoca CECT 4460 was considered. To achieve this, we cloned and transferred into this strain the Klebsiella pneumoniae nasB gene, which encodes assimilatory nitrite reductase (Lin et al., J. Bacteriol. 176:2551–2559, 1994). The delivery vector was either the wide-host-range plasmid pUPE2, in which the nasB gene is expressed from the Escherichia coli Plac promoter, or a mini-Tn5-Km vector, which upon random insertion in the host chromosome allowed expression of the nasB gene from an unidentified chromosomal host promoter. The effect of the increase in the dose of the nasB gene in K. oxytoca CECT 4460 on the accumulation of nitrite in the culture medium was tested in two recombinant strains. The results obtained showed that K. oxytoca CECT 4460 bearing pUPE2 accumulated 88% less nitrite than the wild-type strain, while the recombinant strain bearing the K. pneumoniae nasB gene in the host chromosome showed a 25% lower level of nitrite accumulation in the culture medium than that of the wild type.
机译:产酸克雷伯菌CECT 4460可在最佳培养条件下从工业废水中去除高硝酸盐量,而不会产生亚硝酸盐积累;但是,在非最佳条件下,亚硝酸盐会累积。这种情况反映了该菌株中亚硝酸还原酶的体内有限功能。作为克服此限制的一种方法,考虑增加产氧假单胞菌CECT 4460中亚硝酸还原酶基因的剂量。为此,我们克隆了肺炎克雷伯氏菌nasB基因并将其转移到该菌株中,该基因编码同化亚硝酸盐还原酶(Lin等人,细菌学杂志176:2551-2559,1994)。递送载体是宽宿主范围的质粒pUPE2(其中从大肠杆菌Plac启动子表达nasB基因)或mini-Tn5-Km载体(在宿主染色体中随机插入后允许nasB表达)来自未知的染色体宿主启动子的基因。在两个重组菌株中测试了产氧假单胞菌CECT 4460中nasB基因剂量的增加对培养基中亚硝酸盐积累的影响。获得的结果表明,带有pUPE2的产氧假单胞菌CECT 4460比野生型菌株积累的亚硝酸盐少88%,而在宿主染色体上带有肺炎克雷伯菌nasB基因的重组菌株在其染色体中的亚硝酸盐积累水平降低了25%。培养基要比野生型培养基大。

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