Strengthening the (R)-pantoate pathway to produce D-pantothenic acid based on systematic metabolic analysis

As the key precursor of coenzyme A synthesis in vivo, D-pantothenic acid plays an important role in maintaining biological function, which is widely applied in the food, pharmaceutical, and cosmetic industries. At present, it was mainly produced by chemical methods, with serious environmental pollution, therefore, it is urgent to develop an economical, practical, and environmentally friendly biological fermentation method based on microbial chassis cells. Herein, based on our previously developed D-pantothenic acid producing strains, we obtained higher yielding D-pantothenic acid producing strains by systematic metabolic engineering. First, according to the results of CRISPRi experiments, lpd were overexpressed, disrupted the non-PTS (non-phosphoenolpyruvate-pyruvate transport system) as well as enhanced the PTS (phosphoenolpyruvate-pyruvate transport system) allowing precursor accumulation. Subsequently, based on the analysis of metabolic intermediates, the pyruvate bypass pathway was blocked as well as the (R)-pantothenic acid pathway was further enhanced, increasing the titer of D-pantothenic acid to 4.60 g/L. Finally, the organic acid and branched-chain amino acid synthesis pathways were blocked, and in order to accelerate the synthesis of acetolactate and the output of D-pantothenic acid, heterologous acetolactate synthase and pantothenate transfer protein were introduced, which significantly increased the yield of D-pantothenic acid, reach to 6.33 g/L. The final strain in a 5 L bioreactor produced 32.32 g/L D-pantothenic acid with a productivity of 2.33 g/L/h and the high-yielding strain provides an effective platform for biological fermentation to produce D-pantothenic acid.

Automated summary

Through systematic metabolic engineering, higher yielding D-pantothenic acid producing strains were obtained based on previously developed strains. Overexpression of lpd gene, disruption of non-PTS pathway, and enhancement of PTS pathway were conducted to increase precursor accumulation. Blocking pyruvate bypass pathway, enhancing (R)-pantothenic acid pathway, introducing heterologous proteins, and blocking other synthesis pathways significantly increased the yield of D-pantothenic acid to 6.33 g/L.