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Enhancing recombinant Chaetomium thermophilium Formate Dehydrogenase Expression with CRISPR Technology

Ar, Erhan; Demiroglu, Adem; Yilmaz, Mahmut Selim; Yilmazer, Berin; Aslan, Elif Sibel; Binay, Baris


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        "affiliation": "Gebze Tech Univ, Fac Nat Sci, Dept Mol Biol & Genet, TR-41400 Kocaeli, Turkey", 
        "name": "Ar, Erhan"
      }, 
      {
        "affiliation": "Gebze Tech Univ, Fac Nat Sci, Dept Mol Biol & Genet, TR-41400 Kocaeli, Turkey", 
        "name": "Demiroglu, Adem"
      }, 
      {
        "affiliation": "Gebze Tech Univ, Fac Nat Sci, Dept Mol Biol & Genet, TR-41400 Kocaeli, Turkey", 
        "name": "Yilmaz, Mahmut Selim"
      }, 
      {
        "affiliation": "Gebze Tech Univ, Fac Nat Sci, Dept Mol Biol & Genet, TR-41400 Kocaeli, Turkey", 
        "name": "Yilmazer, Berin"
      }, 
      {
        "affiliation": "Biruni Univ, Dept Mol Biol & Genet, TR-34010 Istanbul, Turkey", 
        "name": "Aslan, Elif Sibel"
      }, 
      {
        "affiliation": "Gebze Tech Univ, Fac Engn, Dept Bioengn, TR-41400 Kocaeli, Turkey", 
        "name": "Binay, Baris"
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    "description": "Genetic manipulation of Escherichia coli influences the regulation of bacterial metabolism, which could be useful for the production of different targeted products. The RpoZ gene encodes for the omega subunit of the RNA polymerase (RNAP) and is involved in the regulation of the relA gene pathway. RelA is responsible for the production of guanosine pentaphosphate (ppGpp), which is a major alarmone in the stringent response. Expression of relA is reduced in the early hours of growth of RpoZ mutant E. coli. In the absence of the omega subunit, ppGpp affinity to RNAP is decreased; thus, rpoZ gene deleted E. coli strains show a modified stringent response. We used the E. coli K-12 MG1655 strain that lacks rpoZ (JEN202) to investigate the effect of the modified stringent response on recombinant protein production. However, the absence of the omega subunit results in diminished stability of the RNA polymerase at the promoter site. To avoid this, we used a deactivated CRISPR system that targets the omega subunit to upstream of the promoter site in the expression plasmid. The expression plasmid encodes for Chaetomium thermophilum formate dehydrogenase (CtFDH), a valuable enzyme for cofactor regeneration and CO2 reduction. A higher amount of CtFDH from the soluble fraction was purified from the JEN202 strain compared to the traditional BL21(DE3) method, thus offering a new strategy for batch-based recombinant enzyme production.", 
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      "issue": "4", 
      "pages": "504-511", 
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    "science_branches": [
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    "title": "Enhancing recombinant Chaetomium thermophilium Formate Dehydrogenase Expression with CRISPR Technology"
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