Mining key genes for biosynthesis of D-pantothenic acid in Escherichia coli by CRISPRi

Abstract:

[Objective] D-pantothenic acid (DPA) is a functional compound widely used in medical care, cosmetics, animal food and feed and other fields, with good market prospects. This study aims to use CRISPRi to mine the endogenous gene targets affecting the biosynthesis of DPA by the lab-stored Escherichia coli strain DPAP10. [Methods] We constructed a dual-plasmid CRISPRi system with pTarget and pdCas9 to achieve the inhibition of gene expression individually or in combination. Shaking flask fermentation was carried out to validate the effect of gene blocking on DPA production. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) was employed to determine the transcription level after gene blocking. The changes of metabolic pathways were analyzed by high performance liquid chromatography (HPLC). [Results] We screened out 5 key genes (pgk, gltA, ptsH, ptsI, and crp) and 7 gene combinations (pgk-gltA, pgk-ptsH, gltA-ptsH, pgk-ptsI, gltA-ptsI, pgk-crp, and gltA-crp) from 126 target genes. The engineered strain DPAP10/pdCas9+pT-gltA-ptsH showed the DPA yield of 5.3 g/L in a shaking flask, which increased by 49.5% compared with that by the start strain DPAP10. Furthermore, we down-regulated the expression of gltA and ptsH to construct an engineered strain DPAP10-gltA^TTG-ptsH^TTG. This strain showed the DPA yield of 75.4g/L in 5 L fermentor which compared with the control strain DPAP10 under the same culture conditions increased by 19.5%. [Conclusion] We confirmed that CRISPRi could mine the genes involved in DPA synthesis. Changing the pyruvate flux and reducing the tricarboxylic acid (TCA) cycling rate would promote the carbon flow into the DPA synthesis, thus increasing DPA production. This finding provides a new idea for constructing higher yielding strains.