Improved Method for the Incorporation of Heme Cofactors into Recombinant Proteins Using Escherichia coli Nissle 1917

2020

Preprint

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Background: Heme proteins and heme-derived molecules play an important role in several cellular processes. Therefore, their production and functional analysis is of great research interest. For the analysis of these molecules, high production yields are required. We recently reported the use of the probiotic E. coli strain Nissle 1917 (EcN) to sufficiently produce heme proteins. This strain is capable of taking up heme from the growth medium due to the outer membrane heme receptor ChuA which is absent in regular E. coli expression strains. Unfortunately, the strain 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 Duet series. Results: A new T7-promoter compatible EcN strain was constructed. Therefore, the gene for T7-RNA polymerase under the control of a lac UV5 promoter was integrated 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) efficiently resulted in the 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 open-chain tetrapyrroles. We demonstrate successful recombinant production of the phytochromes BphP from Pseudomonas aeruginosa and Cph1 from Synechocystis sp. PCC6803 loaded with their cofactor biliverdin and phycocyanobilin, respectively. Conclusion: We present a new E. coli strain for sufficient 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 Duet expression vectors. Furthermore, the capability of feeding EcN and EcN(T7) with heme overcomes the rate limiting step in the recombinant heme protein production, i.e. heme biosynthesis of E. coli . Therefore, a higher heme saturation of heme proteins and also higher yields of heme-derived molecules is obtained using the constructed strain.

Section: Construction Of the New Ecn(t7) Strainmentioning

confidence: 90%

Section: Introductionmentioning

confidence: 99%

Construction of a new T7 promoter compatible Escherichia coli Nissle 1917 strain for recombinant production of heme-dependent proteins

Kiege

2020

Preprint

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“…While RdmS_sGAF2 displays two distinct peaks in this region, only one peak was detected in the ferrous form of MsmS_sGAF2, suggesting a different heme iron coordination. While the heme in MsmS exists as a mixture of 5c and 6c (Fiege et al, 2018), the heme iron in RdmS_sGAF2 appears to be mainly 6c. Whether this is of functional significance is currently unknown.…”

Section: Rdms Contains a Heme Cofactor In The Second Gaf Domainmentioning

confidence: 99%

“…Expression in EcN was performed as described before (Fiege et al, 2018). For the production of the full-length protein, LB-high salt medium (LB-HS: 10 g/l tryptone, 5 g/l yeast extract, 24.22 g/l NaCl) was used.…”

Section: Recombinant Protein Production In E Coli Nissle 1917 (Ecn)mentioning

confidence: 99%

Thiol‐based redox sensing in the methyltransferase associated sensor kinase RdmS in Methanosarcina acetivorans

Fiege

Environmental Microbiology

2019

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Summary Organisms have evolved signal transduction systems to quickly adapt their lifestyle to internal and environmental changes. While protein kinases and two‐component systems are widely distributed in Bacteria, they are also found in Archaea but are less diversified and abundant. In this work, we analysed the function of the kinase RdmS and its role in a putative two‐component system in the methanogenic archaeon Methanosarcina acetivorans. RdmS is encoded upstream of the regulator MsrF, which activates the expression of the corrinoid/methyltransferase fusion protein MtsD. In contrast to a typical bacterial histidine kinase, RdmS lacks a membrane domain and the conserved histidine residue for phosphorylation, indicating a different mechanism of signal transduction in comparison to bacterial counterparts. RdmS covalently binds a heme cofactor and is thereby able to bind small molecules like CO and dimethyl sulfide. Interestingly, RdmS possesses a redox‐dependent autophosphorylation activity, which, however, is independent of the bound heme cofactor. In fact, our experimental data suggest a thiol‐based redox sensing mechanism by RdmS. Moreover, we were able to show that RdmS interacts with the regulator protein MsrF. From these data, we conclude RdmS to be a thiol‐based kinase sensing redox changes and forming an archaeal multicomponent system with the regulators MsrG/F/C.

“…We have recently shown that E. coli Nissle 1917 (EcN) is a very suitable host for the production of heme proteins [14]. In this study, we went a step further and constructed a T7 promoter compatible strain to overcome the limitation of recombinant protein production in EcN to expression vectors lacking the T7 promoter.…”

Section: Construction Of the New Ecn(t7) Strainmentioning

confidence: 99%

Construction of a new T7 promoter compatible Escherichia coli Nissle 1917 strain for recombinant production of heme-dependent proteins

Fiege

2020

Microb Cell Fact

Self Cite

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 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. Results 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. Conclusion 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.