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

Abstract

CRISPR interference (CRISPRi) was applied to enable the aerobic production of pyruvate in Escherichia coli MG1655 under glucose excess conditions by targeting the promoter regions of aceE or pdhR. 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 aceE or pdhR triggered pyruvate production during the exponential phase and reduced expression of aceE. In lab‐scale bioreactor fermentations, an aceE 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 pdhR 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 aceE and pdhR in E. coli MG1655 pdCas9 psgRNA_aceE_234_pdhR_329 resulted in the stable aerobic production of pyruvate with non‐growing cells at YP/S = 0.36 ± 0.029 gPyruvate/gGlucose in lab‐scale bioreactors throughout an extended nitrogen‐limited production phase.

Escherichia coli aceE variants coding pyruvate dehydrogenase improve the generation of pyruvate‐derived acetoin

“…Moreover, though the pdhR promoter dominates aceEF expression, promoter sequences upstream of aceEF, independent from PdhR regulation, can also drive aceEF expression [58][59][60]. Thus, expression from PpdhrR and PaceE must both be reduced to modulate PDH activity in E. coli effectively [18]. Complex transcriptional regulation and redundancy of promoters can pose a challenge when engineering weak promoters to decrease carbon flux through a branchpoint.…”

“…For example, the native aceE promoter was replaced with a modified vanABK promoter yielding a strain of Corynebacterium glutamicum in which aceE expression was controlled by the presence of a P vanABK ‐sensitive effector molecule [ 17 ]. Similarly, CRISPR interference decreases the expression of the pyruvate dehydrogenase complex (PDH) by targeting the promoter regions of pdhR and aceE , resulting in pyruvate accumulation by Escherichia coli [ 18 ].…”

Metabolic Engineering of Microorganisms to Produce Pyruvate and Derived Compounds

“…For instance, silencing the aceE gene, an essential gene encoding PDHc in E. coli , and combining it with the deletion of other genes produced 26 g/L pyruvate from glucose in 72 h [ 19 ]. The application of CRISPRi targeting the promoter of aceE or pdhR reduced aceE expression, resulting in a pyruvate yield of 0.36 g/g during glucose fermentation [ 20 ]. Gene silencing technologies are destined to become more widespread with the development of methods for fine-tuning the activity of target enzymes in metabolic engineering.…”

CRISPR Tools in Bacterial Whole-Cell Biocatalysis

“…Additionally, the selection of the target DNA strand must be accounted for. While there seems to be no difference in targeting the template or nontemplate strand when the promoter region is considered, generally, aiming for the nontemplate strand of the coding sequence is the more effective alternative …”

“…Differences in several orders of magnitude can be observed in gene repression level depending on the distance of the binding site to the transcription start, the selection of the target strand, and the occurrence of mismatches between the gRNA and the binding site. 107 In this section of the review, a selection of examples of gene repression tunability tools is presented, highlighting their applicability in biotransformations as well as the advantages and disadvantages that arise from their implementation.…”