Design of inducible expression vectors for improved protein production in Ralstonia eutropha H16 derived host strains.

Ralstonia eutropha H16 (Cupriavidus necator H16) is a Gram-negative, facultative chemolithoautotrophic bacterium which can use H2 and CO2 as sole energy and carbon sources in the absence of organic substrates. The biotechnological use of R. eutropha H16 on an industrial scale has already been established; however, only a small number of tools promoting inducible gene expression is available. Within this study two systems promoting inducible expression were designed on the basis of the strong j5 promoter and the Escherichia coli lacI or the Pseudomonas putida cumate regulatory elements. Both expression vectors display desired regulatory features and further increase the number of suitable inducible expression systems for the production of metabolites and proteins with R. eutropha H16.

[1]  L. Bourget,et al.  Novel, Versatile, and Tightly Regulated Expression System for Escherichia coli Strains , 2010, Applied and Environmental Microbiology.

[2]  B. Witholt,et al.  Production of microbial polyesters: fermentation and downstream processes. , 2001, Advances in biochemical engineering/biotechnology.

[3]  Keiji Matsumoto,et al.  Construction of a stable plasmid vector for industrial production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) by a recombinant Cupriavidus necator H16 strain. , 2013, Journal of bioscience and bioengineering.

[4]  Anne Pohlmann,et al.  Autotrophic Production of Stable-Isotope-Labeled Arginine in Ralstonia eutropha Strain H16 , 2012, Applied and Environmental Microbiology.

[5]  A. Pühler,et al.  A Broad Host Range Mobilization System for In Vivo Genetic Engineering: Transposon Mutagenesis in Gram Negative Bacteria , 1983, Bio/Technology.

[6]  C. Hwang,et al.  High-density Escherichia coli cultivation process for hyperexpression of recombinant porcine growth hormone. , 1992, Enzyme and microbial technology.

[7]  Sriram Srinivasan,et al.  High level recombinant protein expression in Ralstonia eutropha using T7 RNA polymerase based amplification. , 2004, Protein expression and purification.

[8]  Anne Pohlmann,et al.  Genome sequence of the bioplastic-producing “Knallgas” bacterium Ralstonia eutropha H16 , 2006, Nature Biotechnology.

[9]  H. Schwab,et al.  Versatile plasmid-based expression systems for Gram-negative bacteria--General essentials exemplified with the bacterium Ralstonia eutropha H16. , 2015, New biotechnology.

[10]  J. Vorholt,et al.  Cumate-Inducible Gene Expression System for Sphingomonads and Other Alphaproteobacteria , 2013, Applied and Environmental Microbiology.

[11]  E. Lanka,et al.  KorB protein of promiscuous plasmid RP4 recognizes inverted sequence repetitions in regions essential for conjugative plasmid transfer. , 1992, Nucleic acids research.

[12]  R. Cramm Genomic View of Energy Metabolism in Ralstonia eutropha H16 , 2008, Journal of Molecular Microbiology and Biotechnology.

[13]  A. Sinskey,et al.  Kinetic and stoichiometric characterization of organoautotrophic growth of Ralstonia eutropha on formic acid in fed-batch and continuous cultures , 2014, Microbial biotechnology.

[14]  Christopher M Thomas,et al.  Complete Nucleotide Sequence of Birmingham IncPα Plasmids: Compilation and Comparative Analysis , 1994 .

[15]  A. Steinbüchel,et al.  Application of a KDPG-aldolase gene-dependent addiction system for enhanced production of cyanophycin in Ralstonia eutropha strain H16. , 2006, Metabolic engineering.

[16]  D. Roop,et al.  Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. , 1995, Gene.

[17]  B. Bowien,et al.  Genetics and control of CO2 assimilation in the chemoautotroph Ralstoniaeutropha , 2002, Archives of Microbiology.

[18]  S. Enfors,et al.  Modeling of high cell density fed batch cultivation. , 1994, FEMS microbiology reviews.

[19]  Alexander Steinbüchel,et al.  Metabolic engineering of strains of Ralstonia eutropha and Pseudomonas putida for biotechnological production of 2-methylcitric acid. , 2006, Metabolic engineering.

[20]  H. Chang,et al.  Production of poly(3-hydroxybutyrate) by high cell density fed-batch culture of Alcaligenes eutrophus with phospate limitation. , 1997, Biotechnology and bioengineering.

[21]  R. Tait,et al.  Genetic map of the crown gall suppressive IncW plasmid pSa , 2004, Molecular and General Genetics MGG.

[22]  D. Wood,et al.  Integrated Recombinant Protein Expression and Purification Platform Based on Ralstonia eutropha , 2005, Applied and Environmental Microbiology.

[23]  A. Malcolm Campbell,et al.  Improving the Lac System for Synthetic Biology , 2010 .

[24]  R. Lo,et al.  The cumate gene-switch: a system for regulated expression in mammalian cells , 2006 .

[25]  C. Batt,et al.  Comparative study of promoters for the production of polyhydroxyalkanoates in recombinant strains of Wautersia eutropha , 2005, Applied Microbiology and Biotechnology.

[26]  T. Gerngross,et al.  A Novel High-Cell-Density Protein Expression System Based on Ralstonia eutropha , 2002, Applied and Environmental Microbiology.

[27]  S. Srivastava,et al.  Mutagenesis of Alcaligenes eutrophus by insertion of the drug-resistance transposon Tn5 , 1982, Archives of Microbiology.

[28]  G. Ditta,et al.  Plasmids related to the broad host range vector, pRK290, useful for gene cloning and for monitoring gene expression. , 1985, Plasmid.

[29]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[30]  Anthony J. Sinskey,et al.  Production of Poly(3-Hydroxybutyrate-co-3-Hydroxyhexanoate) from Plant Oil by Engineered Ralstonia eutropha Strains , 2011, Applied and Environmental Microbiology.

[31]  J. Liao,et al.  A synthetic anhydrotetracycline-controllable gene expression system in Ralstonia eutropha H16. , 2015, ACS synthetic biology.

[32]  E. Chiellini,et al.  Continuous production of poly([R]-3-hydroxybutyrate) by Cupriavidus necator in a multistage bioreactor cascade , 2011, Applied Microbiology and Biotechnology.

[33]  Nathan J Hillson,et al.  Development of a broad-host synthetic biology toolbox for ralstonia eutropha and its application to engineering hydrocarbon biofuel production , 2013, Microbial Cell Factories.

[34]  T. Fukui,et al.  Evaluation of promoters for gene expression in polyhydroxyalkanoate-producing Cupriavidus necator H16 , 2011, Applied Microbiology and Biotechnology.