loading page

CRISPR interference of nucleotide biosynthesis improves production of a single-domain antibody
  • +2
  • Jenny Landberg,
  • Naia Wright,
  • Tune Wulff,
  • Markus Herrgard,
  • Alex Nielsen
Jenny Landberg
Technical University of Denmark

Corresponding Author:[email protected]

Author Profile
Naia Wright
Technical University of Denmark
Author Profile
Tune Wulff
Technical University of Denmark
Author Profile
Markus Herrgard
Technical University of Denmark, Technical University of Denmark
Author Profile
Alex Nielsen
Technical University of Denmark
Author Profile


Growth decoupling can be used to optimize production of biochemicals and proteins in cell factories. Inhibition of excess biomass formation allows for carbon to be utilized efficiently for product formation instead of growth, resulting in increased product yields and titers. Here, we used CRISPR interference (CRISPRi) to increase production of a single domain antibody (sdAb) by inhibiting growth during production. First, we screened 21 sgRNA targets in the purine and pyrimidine biosynthesis pathways, and found that repression of 11 pathway genes led to increased GFP production and decreased growth. The sgRNA targets pyrF, pyrG, and cmk were selected and further used to improve production of two versions of an expression-optimized sdAb. Proteomics analysis of the sdAb-producing pyrF, pyrG, and cmk growth decoupling strains showed significantly decreased RpoS levels and an increase of ribosome-associated proteins, indicating that the growth decoupling strains do not enter stationary phase and maintain their capacity for protein synthesis upon growth inhibition. Finally, sdAb production was scaled up to shake-flask fermentation where the product yield was improved 2.6-fold compared to the control strain with no sgRNA target sequence. An sdAb content of 14.6% was reached in the best-performing pyrG growth decoupling strain.
26 May 2020Submitted to Biotechnology and Bioengineering
27 May 2020Submission Checks Completed
27 May 2020Assigned to Editor
01 Jun 2020Reviewer(s) Assigned
22 Jun 2020Review(s) Completed, Editorial Evaluation Pending
22 Jun 2020Editorial Decision: Revise Major
27 Jul 20201st Revision Received
27 Jul 2020Assigned to Editor
27 Jul 2020Submission Checks Completed
03 Aug 2020Reviewer(s) Assigned
12 Aug 2020Review(s) Completed, Editorial Evaluation Pending
12 Aug 2020Editorial Decision: Revise Minor
13 Aug 20202nd Revision Received
13 Aug 2020Assigned to Editor
13 Aug 2020Submission Checks Completed
14 Aug 2020Review(s) Completed, Editorial Evaluation Pending
14 Aug 2020Editorial Decision: Accept