Enhancing D-pantothenate production in Escherichia coli through multiplex combinatorial strategies.

D-pantothenate, universally acknowledged as vitamin B5, has garnered considerable interest owing to its crucial functionality in the feed, pharmaceutical, and cosmeceutical sectors. Development of microbial strains for D-pantothenate hyperproducer has emerged as a prominent research direction in recent years. Herein, we converted an engineered Escherichia coli with low yield to a plasmid-free hyperproducer of D-pantothenate using multiplex combinatorial strategies. First, an initial strain was obtained through prolonging the cell lifespan. To promote the accumulation of D-pantothenic acid, the supply of cofactors was adaptively enhanced. Additionally, the heterologous gene panE from Pseudomonas aeruginosa, which encodes ketopantoate reductase (EC 1.1.1.169) catalyzing the synthesis of d-pantoate from α-ketopantoate, was screened and integrated into the chromosome. Subsequently, a strategy of acetate recycling and NOG pathway reconstruction were introduced and successfully to improve the D-pantothenate titer to 5.48 g/L. Additionally, we screened the regulatory factors and optimized its second codon to further increase the DPA yield of the engineered strains to 6.02 g/L in shake flask. The final engineered strain DS6 could efficiently produce 72.40 g/L D-pantothenate, which is 3.18-fold higher than the original strain. This study proposed a novel multiplex combination strategy for developing microbial cell factory of D-pantothenate, which was beneficial for the advancement of efficient D-pantothenate production.