Heme proteins and heme-derived molecules are essential in numerous cellular processes. Research into their in vitro functionality requires the production of large amounts of protein. Unfortunately, high yield expression is hampered by the lack of E. coli strains naturally capable of taking up heme from the medium. We recently reported the use of the probiotic E. coli strain Nissle 1917 (EcN) to sufficiently produce heme containing proteins, as it encodes the outer membrane heme receptor, ChuA, which allows for natural uptake of heme. The EcN strain however lacks the gene for T7 RNA polymerase, which is necessary for the expression of genes under the control of the T7-promotor, widely used in expression vectors like the pET or pDuet series. A new T7-promoter compatible EcN strain was constructed by integrating the gene for T7-RNA polymerase under the control of a lacUV5 promoter into the malEFG operon of EcN. Test expressions of genes via T7 promoter-based vectors in the new EcN(T7) strain were successful. Expression in EcN(T7) resulted in the efficient production of recombinant heme proteins in which the heme cofactor was incorporated during protein production. In addition, the new EcN(T7) strain can be used to co-express genes for the production of heme-derived molecules like biliverdin or other linear tetrapyrroles. We demonstrate the successful recombinant production of the phytochromes BphP, from Pseudomonas aeruginosa, and Cph1, from Synechocystis sp. PCC6803, loaded with their linear tetrapyrrole cofactors, biliverdin and phycocyanobilin, respectively. We present a new E. coli strain for efficient production of heme proteins and heme-derived molecules using T7-promoter based expression vectors. The new EcN(T7) strain enables the use of a broader spectrum of expression vectors, as well as the co-expression of genes using the pDuet expression vectors, for expressing heme containing proteins. By utilizing E. coli strains EcN and EcN(T7), capable of being fed heme, the rate limiting step of heme biosynthesis in E. coli is eliminated, thereby permitting higher heme saturation of heme proteins and also higher yields of heme-derived molecules.

中文翻译：

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新型T7启动子兼容的大肠杆菌Nissle 1917菌株的构建，用于重组生产血红素依赖性蛋白

血红素蛋白和血红素衍生的分子在许多细胞过程中至关重要。研究它们的体外功能需要大量蛋白质的产生。不幸的是，由于缺乏天然能够从培养基中吸收血红素的大肠杆菌菌株，阻碍了高产量表达。我们最近报道了益生菌大肠杆菌Nissle 1917（EcN）菌株的使用，可充分产生含血红素的蛋白质，因为它编码外膜血红素受体ChuA，可以自然吸收血红素。然而，EcN菌株缺少T7 RNA聚合酶的基因，这是在T7启动子控制下表达基因所必需的基因，该基因广泛用于pET或pDuet系列等表达载体。通过将lacUV5启动子控制下的T7-RNA聚合酶基因整合到EcN的malEFG操纵子中，构建了一个新的T7启动子兼容EcN菌株。通过基于T7启动子的载体在新的EcN（T7）菌株中测试基因表达成功。在EcN（T7）中的表达导致重组血红素蛋白质的高效生产，其中血红素辅因子在蛋白质生产过程中被掺入。此外，新的EcN（T7）菌株可用于共表达基因，以产生血红素衍生的分子，如biliverdin或其他线性四吡咯。我们证明了从铜绿假单胞菌，和Cph1，从集胞藻sp的植物色素BphP的成功重组生产。PCC6803分别装有线性四吡咯辅助因子biliverdin和phycocyanobilin。我们提出一个新的E。使用基于T7启动子的表达载体高效生产血红素蛋白和血红素衍生分子的大肠杆菌菌株。新的EcN（T7）菌株能够使用更广泛的表达载体，以及使用pDuet表达载体共表达基因，以表达含血红素的蛋白质。通过利用能够喂入血红素的大肠杆菌EcN和EcN（T7）菌株，消除了大肠杆菌中血红素生物合成的限速步骤，从而允许血红素蛋白具有更高的血红素饱和度，并且血红素衍生分子的产量更高。 。表达含血红素的蛋白质。通过利用能够喂入血红素的大肠杆菌EcN和EcN（T7）菌株，消除了大肠杆菌中血红素生物合成的限速步骤，从而允许血红素蛋白具有更高的血红素饱和度，并且血红素衍生分子的产量更高。 。表达含血红素的蛋白质。通过利用能够喂入血红素的大肠杆菌EcN和EcN（T7）菌株，消除了大肠杆菌中血红素生物合成的限速步骤，从而允许血红素蛋白具有更高的血红素饱和度，并且血红素衍生分子的产量更高。 。