scholarly journals Recombinant Production of Biliverdin IXβ and δ Isomers in the T7 Promoter Compatible Escherichia coli Nissle

2021 ◽  
Vol 12 ◽  
Author(s):  
Elizabeth A. Robinson ◽  
Nicole Frankenberg-Dinkel ◽  
Fengtian Xue ◽  
Angela Wilks
Keyword(s):  
Heme Oxygenase ◽  
Chemical Oxidation ◽  
Heme Biosynthesis ◽  
T7 Promoter ◽  
E Coli ◽  
Recombinant Production ◽  
Alternative Approaches ◽  
Biliverdin Ix ◽  
Scalable Production ◽  
Escherichia Coli Nissle

The ability to obtain purified biliverdin IX (BVIX) isomers other than the commercially available BVIXα is limited due to the low yields obtained by the chemical coupled oxidation of heme. Chemical oxidation requires toxic chemicals, has very poor BVIX yields (<0.05%), and is not conducive to scalable production. Alternative approaches utilizing recombinant E. coli BL21 expressing a cyanobacterial heme oxygenase have been employed for the production BVIXα, but yields are limited by the rate of endogenous heme biosynthesis. Furthermore, the emerging roles of BVIXβ and BVIXδ in biology and their lack of commercial availability has led to a need for an efficient and scalable method with the flexibility to produce all three physiologically relevant BVIX isomers. Herein, we have taken advantage of an optimized non-pathogenic E. coli Nissle (EcN(T7)) strain that encodes an endogenous heme transporter and an integrated T7 polymerase gene. Protein production of the Pseudomonas aeruginosa BVIXβ and BVIXδ selective heme oxygenase (HemO) or its BVIXα producing mutant (HemOα) in the EcN(T7) strain provides a scalable method to obtain all three isomers, that is not limited by the rate of endogenous heme biosynthesis, due to the natural ability of EcN(T7) to transport extracellular heme. Additionally, we have optimized our previous LC-MS/MS protocol for semi-preparative separation and validation of the BVIX isomers. Utilizing this new methodology for scalable production and separation we have increased the yields of the BVIXβ and -δ isomers >300-fold when compared to the chemical oxidation of heme.

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

2020 ◽  
Vol 19 (1) ◽  
Author(s):  
Kerstin Fiege ◽  
Nicole Frankenberg-Dinkel
Keyword(s):  
Rna Polymerase ◽  
Heme Proteins ◽  
T7 Rna Polymerase ◽  
Expression Vectors ◽  
High Yield ◽  
Efficient Production ◽  
T7 Promoter ◽  
E Coli ◽  
Recombinant Production ◽  
Expression Of Genes

Abstract 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.

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

2020 ◽  
Author(s):  
Kerstin Kiege ◽  
Nicole Frankenberg-Dinkel
Keyword(s):  
Rna Polymerase ◽  
Heme Proteins ◽  
Protein Production ◽  
Coli Strain ◽  
T7 Rna Polymerase ◽  
Expression Vectors ◽  
T7 Promoter ◽  
E Coli ◽  
Recombinant Production ◽  
Expression Of Genes

Abstract 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.

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

2020 ◽  
Author(s):  
Kerstin Fiege ◽  
Nicole Frankenberg-Dinkel
Keyword(s):  
Rna Polymerase ◽  
Heme Proteins ◽  
T7 Rna Polymerase ◽  
Expression Vectors ◽  
High Yield ◽  
Efficient Production ◽  
T7 Promoter ◽  
E Coli ◽  
Recombinant Production ◽  
Expression Of Genes

Abstract 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 Duet 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 Duet 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.

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

2020 ◽  
Author(s):  
Kerstin Fiege ◽  
Nicole Frankenberg-Dinkel
Keyword(s):  
Rna Polymerase ◽  
Heme Proteins ◽  
T7 Rna Polymerase ◽  
Expression Vectors ◽  
High Yield ◽  
Efficient Production ◽  
T7 Promoter ◽  
E Coli ◽  
Recombinant Production ◽  
Expression Of Genes

Abstract 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 Duet 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 Duet 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.

Enhanced Biocatalytic Activity of Recombinant Lipase Immobilized on Gold Nanoparticles

2019 ◽  
Vol 20 (6) ◽  
pp. 497-505 ◽  
Author(s):  
Abeer M. Abd El-Aziz ◽  
Mohamed A. Shaker ◽  
Mona I. Shaaban
Keyword(s):  
Gold Nanoparticles ◽  
Lipolytic Activity ◽  
Market Demand ◽  
Biotechnological Applications ◽  
Lipase Gene ◽  
Expression Plasmid ◽  
E Coli ◽  
Nickel Affinity Chromatography ◽  
Recombinant Production ◽  
Sds Page

Background: Bacterial lipases especially Pseudomonas lipases are extensively used for different biotechnological applications. Objectives: With the better understanding and progressive needs for improving its activity in accordance with the growing market demand, we aimed in this study to improve the recombinant production and biocatalytic activity of lipases via surface conjugation on gold nanoparticles. Methods: The full length coding sequences of lipase gene (lipA), lipase specific foldase gene (lipf) and dual cassette (lipAf) gene were amplified from the genomic DNA of Pseudomonas aeruginosa PA14 and cloned into the bacterial expression vector pRSET-B. Recombinant lipases were expressed in E. coli BL-21 (DE3) pLysS then purified using nickel affinity chromatography and the protein identity was confirmed using SDS-PAGE and Western blot analysis. The purified recombinant lipases were immobilized through surface conjugation with gold nanoparticles and enzymatic activity was colorimetrically quantified. Results: Here, two single expression plasmid systems pRSET-B-lipA and pRSET-B-lipf and one dual cassette expression plasmid system pRSET-B-lipAf were successfully constructed. The lipolytic activities of recombinant lipases LipA, Lipf and LipAf were 4870, 426 and 6740 IUmg-1, respectively. However, upon immobilization of these recombinant lipases on prepared gold nanoparticles (GNPs), the activities were 7417, 822 and 13035 IUmg-1, for LipA-GNPs, Lipf-GNPs and LipAf-GNPs, respectively. The activities after immobilization have been increased 1.52 and 1.93 -fold for LipA and LipAf, respectively. Conclusion: The lipolytic activity of recombinant lipases in the bioconjugate was significantly increased relative to the free recombinant enzyme where immobilization had made the enzyme attain its optimum performance.

scholarly journals Isolation of a Gene Responsible for the Oxidation oftrans-Anethole topara-Anisaldehyde by Pseudomonas putida JYR-1 and Its Expression in Escherichia coli

2012 ◽  
Vol 78 (15) ◽  
pp. 5238-5246 ◽  
Author(s):  
Dongfei Han ◽  
Ji-Young Ryu ◽  
Robert A. Kanaly ◽  
Hor-Gil Hur
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Putida ◽  
Hypothetical Protein ◽  
Aldehyde Group ◽  
Agrobacterium Vitis ◽  
T7 Promoter ◽  
Content Type ◽  
E Coli ◽  
Cell Extracts ◽  
Isoeugenol Monooxygenase

ABSTRACTA plasmid, pTA163, inEscherichia colicontained an approximately 34-kb gene fragment fromPseudomonas putidaJYR-1 that included the genes responsible for the metabolism oftrans-anethole to protocatechuic acid. Three Tn5-disrupted open reading frame 10 (ORF 10) mutants of plasmid pTA163 lost their abilities to catalyzetrans-anethole. Heterologously expressed ORF 10 (1,047 nucleotides [nt]) under a T7 promoter inE. colicatalyzed oxidative cleavage of a propenyl group oftrans-anethole to an aldehyde group, resulting in the production ofpara-anisaldehyde, and this gene was designatedtao(trans-anetholeoxygenase). The deduced amino acid sequence of TAO had the highest identity (34%) to a hypothetical protein ofAgrobacterium vitisS4 and likely contained a flavin-binding site. Preferred incorporation of an oxygen molecule from water intop-anisaldehyde using18O-labeling experiments indicated stereo preference of TAO for hydrolysis of the epoxide group. Interestingly, unlike the narrow substrate range of isoeugenol monooxygenase fromPseudomonas putidaIE27 andPseudomonas nitroreducensJin1, TAO fromP. putidaJYR-1 catalyzed isoeugenol,O-methyl isoeugenol, and isosafrole, all of which contain the 2-propenyl functional group on the aromatic ring structure. Addition of NAD(P)H to the ultrafiltered cell extracts ofE. coli(pTA163) increased the activity of TAO. Due to the relaxed substrate range of TAO, it may be utilized for the production of various fragrance compounds from plant phenylpropanoids in the future.

scholarly journals Streptococcus pneumoniae DNA Gyrase and Topoisomerase IV: Overexpression, Purification, and Differential Inhibition by Fluoroquinolones

1999 ◽  
Vol 43 (5) ◽  
pp. 1129-1136 ◽  
Author(s):  
Xiao-Su Pan ◽  
L. Mark Fisher
Keyword(s):  
Streptococcus Pneumoniae ◽  
Dna Gyrase ◽  
Dna Supercoiling ◽  
Topoisomerase Iv ◽  
T7 Promoter ◽  
Bacterial Killing ◽  
E Coli ◽  
Apparent Homogeneity ◽  
Comparable Activity

ABSTRACT Streptococcus pneumoniae gyrA and gyrBgenes specifying the DNA gyrase subunits have been cloned into pET plasmid vectors under the control of an inducible T7 promoter and have been separately expressed in Escherichia coli. Soluble 97-kDa GyrA and 72-kDa GyrB proteins bearing polyhistidine tags at their respective C-terminal and N-terminal ends were purified to apparent homogeneity by one-step nickel chelate column chromatography and were free of host E. coli topoisomerase activity. Equimolar amounts of the gyrase subunits reconstituted ATP-dependent DNA supercoiling with comparable activity to gyrase of E. coli and Staphylococcus aureus. In parallel, S. pneumoniae topoisomerase IV ParC and ParE subunits were similarly expressed in E. coli, purified to near homogeneity as 93- and 73-kDa proteins, and shown to generate efficient ATP-dependent DNA relaxation and DNA decatenation activities. Using the purified enzymes, we examined the inhibitory effects of three paradigm fluoroquinolones—ciprofloxacin, sparfloxacin, and clinafloxacin—which previous genetic studies with S. pneumoniae suggested act preferentially through topoisomerase IV, through gyrase, and through both enzymes, respectively. Surprisingly, all three quinolones were more active in inhibiting purified topoisomerase IV than gyrase, with clinafloxacin showing the greatest inhibitory potency. Moreover, the tested agents were at least 25-fold more effective in stabilizing a cleavable complex (the relevant cytotoxic lesion) with topoisomerase IV than with gyrase, with clinafloxacin some 10- to 32-fold more potent against either enzyme, in line with its superior activity againstS. pneumoniae. The uniform target preference of the three fluoroquinolones for topoisomerase IV in vitro is in apparent contrast to the genetic data. We interpret these results in terms of a model for bacterial killing by quinolones in which cellular factors can modulate the effects of target affinity to determine the cytotoxic pathway.

Recombinant production of anti-HIV protein, Griffithsin, in E. coli

2011 ◽  
Vol 44 (13) ◽  
pp. S349
Author(s):  
Afshar Shandiz Mohammad ◽  
Vafaee Yavar ◽  
Babalar Mesbah ◽  
Alizadeh Houshang
Keyword(s):  
E Coli ◽  
Recombinant Production ◽  
Anti Hiv

scholarly journals Strain improvement of Escherichia coli K-12 for recombinant production of deuterated proteins

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Vinardas Kelpšas ◽  
Claes von Wachenfeldt
Keyword(s):  
Escherichia Coli ◽  
Structural Biology ◽  
Strain Improvement ◽  
Growth Media ◽  
Genomic Changes ◽  
E Coli ◽  
Recombinant Production ◽  
High Concentrations ◽  
Neutron Protein Crystallography ◽  
K 12

AbstractDeuterium isotope labelling is important for structural biology methods such as neutron protein crystallography, nuclear magnetic resonance and small angle neutron scattering studies of proteins. Deuterium is a natural low abundance stable hydrogen isotope that in high concentrations negatively affect growth of cells. The generation time for Escherichia coli K-12 in deuterated medium is substantially increased compared to cells grown in hydrogenated (protiated) medium. By using a mutagenesis plasmid based approach we have isolated an E. coli strain derived from E. coli K-12 substrain MG1655 that show increased fitness in deuterium based growth media, without general adaptation to media components. By whole-genome sequencing we identified the genomic changes in the obtained strain and show that it can be used for recombinant production of perdeuterated proteins in amounts typically needed for structural biology studies.

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