Biosensor-assisted titratable CRISPRi high-throughput (BATCH) screening for over-production phenotypes.

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  • Author(s): Wang J;Wang J; Li C; Li C; Jiang T; Jiang T; Yan Y; Yan Y
  • Source:
    Metabolic engineering [Metab Eng] 2023 Jan; Vol. 75, pp. 58-67. Date of Electronic Publication: 2022 Nov 12.
  • Publication Type:
    Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't
  • Language:
    English
  • Additional Information
    • Source:
      Publisher: Academic Press Country of Publication: Belgium NLM ID: 9815657 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1096-7184 (Electronic) Linking ISSN: 10967176 NLM ISO Abbreviation: Metab Eng Subsets: MEDLINE
    • Publication Information:
      Original Publication: Brugge, Belgium ; Orlando, FL : Academic Press, c1999-
    • Subject Terms:
      Escherichia coli*/genetics ; Escherichia coli*/metabolism ; High-Throughput Screening Assays*; Coumaric Acids ; Phenotype ; CRISPR-Cas Systems/genetics ; Metabolic Engineering
    • Abstract:
      With rapid advances in the development of metabolic pathways and synthetic biology toolkits, a persisting challenge in microbial bioproduction is how to optimally rewire metabolic fluxes and accelerate the concomitant high-throughput phenotype screening. Here we developed a biosensor-assisted titratable CRISPRi high-throughput (BATCH) screening approach that combines a titratable mismatch CRISPR interference and a biosensor mediated screening for high-production phenotypes in Escherichia coli. We first developed a programmable mismatch CRISPRi that could afford multiple levels of interference efficacy with a one-pot sgRNA pool (a total of 16 variants for each target gene) harboring two consecutive random mismatches in the seed region of sgRNA spacers. The mismatch CRISPRi was demonstrated to enable almost a full range of gene knockdown when targeting different positions on genes. As a proof-of-principle demonstration of the BATCH screening system, we designed doubly mismatched sgRNA pools targeting 20 relevant genes in E. coli and optimized a PadR-based p-coumaric acid biosensor with broad dynamic range for the eGFP fluorescence guided high-production screening. Using sgRNA variants for the combinatorial knockdown of pfkA and ptsI, the p-coumaric acid titer was increased by 40.6% to o 1308.6 mg/l from glycerol in shake flasks. To further demonstrate the general applicability of the BATCH screening system, we recruited a HpdR-based butyrate biosensor that facilitated the screening of E. coli strains achieving 19.0% and 25.2% increase of butyrate titer in shake flasks with sgRNA variants targeting sucA and ldhA, respectively. This work reported the establishment of a plug-and-play approach that enables multilevel modulation of metabolic fluxes and high-throughput screening of high-production phenotypes.
      Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest.
      (Copyright © 2022 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)
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    • Grant Information:
      R35 GM128620 United States GM NIGMS NIH HHS
    • Contributed Indexing:
      Keywords: Biosensor; Butyrate; Mismatch CRISPRi; Titratable repression; p-Coumaric acid
    • Accession Number:
      IBS9D1EU3J (p-coumaric acid)
      0 (Coumaric Acids)
    • Publication Date:
      Date Created: 20221114 Date Completed: 20230117 Latest Revision: 20240102
    • Publication Date:
      20240102
    • Accession Number:
      PMC9845192
    • Accession Number:
      10.1016/j.ymben.2022.11.004
    • Accession Number:
      36375746