Abstract
Growth decoupling can be used to optimize the 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 to increase the 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 the repression of 11 pathway genes led to the increased green fluorescent protein production and decreased growth. The sgRNA targets pyrF, pyrG, and cmk were selected and further used to improve the 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.
Highlights
T recently published strain design algorithm further highlights the possibilities of using growth decoupling to improve production of a large number of small molecules in Escherichia coli (Venayak, von Kamp, Klamt, & Mahadevan, 2018)
A total of 22 different genes in the nucleotide biosynthesis pathway were selected as targets to investigate the potential of using purine and pyrimidine biosynthesis genes as CRISPR interference (CRISPRi)-based growth switches (Fig. 1a)
4 out of 21 CRISPRi-induced strains had a lower Optical density (OD) compared to the respective uninduced control (Fig S1d); this can to large extent be explained by the decrease in OD that uninduced strains displayed between the 12 and 24 h sample points
Summary
T recently published strain design algorithm further highlights the possibilities of using growth decoupling to improve production of a large number of small molecules in Escherichia coli (Venayak, von Kamp, Klamt, & Mahadevan, 2018). Decoupling is generally achieved by natural or synthetic regulation of growth and/or induction of product expression. By controlling isocitrate lyase expression with a degradable inducer, carbon flux could gradually be routed toward wax ester accumulation, improving wax ester yields almost 4-fold during growth on acetate (Santala, Efimova, & Santala, 2018). It can be induced to target gene(s) or cellular function(s) at a desired time point in order to increase precursor supply (Cress et al, 2017), redirect metabolic flux toward production and away from byproduct formation (Chang, Su, Qi, & Liang, 2016; Tian, Kang, Kang, & Lee, 2019), or to induce growth arrest by inhibition of essential genes The results from this study indicated an enrichment of promising targets among genes involved in biosynthesis of purines and pyrimidines
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