CRISPR interference of nucleotide biosynthesis improves production of a
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.