Construction of a new T7 promoter compatibleEscherichia coliNissle 1917 strain for recombinant production of heme-dependent proteins

Background 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 ofE. colistrains naturally capable of taking up heme from the medium. We recently reported the use of the probioticE. colistrain 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. Results A new T7-promoter compatible EcN strain was constructed by integrating the gene for T7-RNA polymerase under the control of alacUV5 promoter into themalEFGoperon 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, fromPseudomonas aeruginosa,and Cph1, fromSynechocystissp. PCC6803, loaded with their linear tetrapyrrole cofactors, biliverdin and phycocyanobilin, respectively. Conclusion We present a newE. colistrain 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 utilizingE. colistrains EcN and EcN(T7), capable of being fed heme, the rate limiting step of heme biosynthesis inE. coliis eliminated, thereby permitting higher heme saturation of heme proteins and also higher yields of heme-derived molecules.