Development of cyclic AMP receptor protein-based artificial transcription factor for intensifying gene expression.

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  • Author(s): Zhao P;Zhao P; Wang W; Wang W; Tian P; Tian P
  • Source:
    Applied microbiology and biotechnology [Appl Microbiol Biotechnol] 2018 Feb; Vol. 102 (4), pp. 1673-1685. Date of Electronic Publication: 2018 Jan 16.
  • Publication Type:
    Journal Article
  • Language:
    English
  • Additional Information
    • Source:
      Publisher: Springer International Country of Publication: Germany NLM ID: 8406612 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1432-0614 (Electronic) Linking ISSN: 01757598 NLM ISO Abbreviation: Appl Microbiol Biotechnol Subsets: MEDLINE
    • Publication Information:
      Original Publication: Berlin ; New York : Springer International, c1984-
    • Subject Terms:
      Gene Expression*; Cyclic AMP Receptor Protein/*metabolism ; Lac Repressors/*metabolism ; Recombinant Fusion Proteins/*metabolism ; Transcription Factors/*metabolism; Aldehyde Dehydrogenase/biosynthesis ; Chromatography, High Pressure Liquid ; Cyclic AMP Receptor Protein/genetics ; DNA-Directed RNA Polymerases/metabolism ; Electrophoresis, Polyacrylamide Gel ; Glyceraldehyde/analogs & derivatives ; Glyceraldehyde/metabolism ; Klebsiella pneumoniae/genetics ; Klebsiella pneumoniae/metabolism ; Lac Repressors/genetics ; Lactic Acid/analogs & derivatives ; Lactic Acid/metabolism ; Operator Regions, Genetic ; Propane/metabolism ; Protein Binding ; Recombinant Fusion Proteins/genetics ; Transcription Factors/genetics
    • Abstract:
      Vector-dependent gene overexpression typically relies on an efficient operon and sufficient RNA polymerases (RNAPs). The lac (lactose) operon is a paradigm of transcription control, and cyclic AMP receptor protein (CRP) is a global regulator capable of recruiting RNAPs. However, the gap between lac operon and CRP has not been well bridged. In this work, CRP was fused to lac repressor protein (lacI) to form an artificial transcription factor (ATF) with the expectation that when LacI acted on the lacO-positioned upstream of gene of interest, the LacI-tethered CRP would trap RNAPs and thus improve the expression of PuuC, an aldehyde dehydrogenase catalyzing 3-hydroxypropionaldehyde (3-HPA) to 3-hydroxypropionic acid (3-HP) in Klebsiella pneumoniae. As expected, SDS-PAGE and HPLC showed enhanced PuuC expression and 3-HP production, respectively, compared to the control strain without expressing chimeric protein LacI-CRP. Moreover, quantitative real-time PCR demonstrated increased transcription levels of both PuuC and RNAP coding genes. In shake-flask cultivation, the recombinant K. pneumoniae strain coexpressing LacI-CRP and PuuC produced 1.67-fold of 3-HP relative to the stain only overexpressing PuuC. In bioreactor cultivation, the strain coexpressing LacI-CRP and PuuC produced 35.1 g/L 3-HP, whereas the strain without expressing LacI-CRP generated only 9.8 g/L 3-HP. Overall, these results indicated that as an ATF, LacI-CRP significantly boosted PuuC expression and 3-HP production. We envision that LacI-CRP as a plug-and-play part can be used for regulating gene expression.
    • Grant Information:
      21276014, 21476011 National Natural Science Foundation of China; 2015AA021003 National High Technology Research and Development Program
    • Contributed Indexing:
      Keywords: 3-Hydroxypropionic acid; Cyclic AMP receptor protein; Klebsiella pneumoniae; RNA polymerase; lac operon
    • Accession Number:
      0 (Cyclic AMP Receptor Protein)
      0 (Lac Repressors)
      0 (Recombinant Fusion Proteins)
      0 (Transcription Factors)
      2134-29-4 (3-hydroxypropionaldehyde)
      33X04XA5AT (Lactic Acid)
      367-47-5 (Glyceraldehyde)
      C4ZF6XLD2X (hydracrylic acid)
      EC 1.2.1.3 (Aldehyde Dehydrogenase)
      EC 2.7.7.6 (DNA-Directed RNA Polymerases)
      T75W9911L6 (Propane)
    • Publication Date:
      Date Created: 20180117 Date Completed: 20180806 Latest Revision: 20180806
    • Publication Date:
      20221213
    • Accession Number:
      10.1007/s00253-018-8750-x
    • Accession Number:
      29335877