CRISPRi enables fast growth followed by stable aerobic pyruvate formation in without auxotrophy.

CRISPR interference (CRISPRi) was applied to enable the aerobic production of pyruvate in MG1655 under glucose excess conditions by targeting the promoter regions of or . Knockdown strains were cultivated in aerobic shaking flasks and the influence of inducer concentration and different sgRNA binding sites on the production of pyruvate was measured. Targeting the promoter regions of or triggered pyruvate production during the exponential phase and reduced expression of . In lab-scale bioreactor fermentations, an silenced strain successfully produced pyruvate under fully aerobic conditions during the exponential phase, but loss of productivity occurred during a subsequent nitrogen-limited phase. Targeting the promoter region of enabled pyruvate production during the growth phase of cultivations, and a continued low-level accumulation during the nitrogen-limited production phase. Combinatorial targeting of the promoter regions of both and in MG1655 pdCas9 psgRNA_aceE_234_pdhR_329 resulted in the stable aerobic production of pyruvate with non-growing cells at Y  =  0.36 ± 0.029 g/g in lab-scale bioreactors throughout an extended nitrogen-limited production phase.