The invention relates to genetic engineering bacteria capable of producing pantothenic acid at high yield without addition of beta-alanine, a construction method of the genetic engineering bacteria, and application of the genetic engineering bacteria in preparation of D-pantothenic acid by microbial fermentation. According to the invention, (1), the final step of an escherichia coli D-pantothenicacid synthesis pathway is enhanced, and the utilizing ability of escherichia coli to extracellular beta-alanine, (2), a pantoic acid synthesis pathway is enhanced, (3), ilvG gene is repaired, and thefeedback inhibition effect of by-products on a pantoic acid synthesis pathway is weakened, (4), according to the change in cell phenotype, flux of a valine synthesis pathway is weakened, (5), a CRISPRi technique is used to screen metabolic modification sites of TCA cycle, a PPP pathway and a by-product metabolic pathway, according to the result, an isoleucine synthesis pathway is blocked, and thecompetition of 2-butanoic acid for reaction of acetolactate synthesized from pyruvic acid under catalysis of acetolactate synthase is relieved, and (6), aspartate decarboxylase from other strains is subjected to heterologous expression to obtain a genetical engineering strain capable of producing the pantothenic acid at high yield without addition of the beta-alanine. By combined expression of panB and panC which are derived from corynebacterium glutamicum and panD derived from bacillus subtilis together on pTrc99A plasmids, 1.2g / L of D- pantothenic acid is obtained without adding the beta-alanine.