Efficient Synthesisof Vitamin B₅ in Escherichiacoli by Engineering Ketopantoate Hydroxymethyltransferaseand Cofactor Supply

d-pantothenic acid (d-PA), also known as vitamin B₅, is an essential precursor of coenzyme A and plays a crucial role in maintaining the physiological functions of organisms. Ketopantoate hydroxymethyltransferase (PanB), encoded by <i>panB</i> gene, serves as a key rate-limiting enzyme in d-PA synthesis. Additionally, the catalytic function of PanB requires the cofactor 5,10-methylenetetrahydrofolate (5,10-CH₂–THF). This study aimed to increase d-PA production by engineering ketopantoate hydroxymethyltransferase and cofactor supply. The key transcription factor <i>bhsA</i> that restricts d-PA production was screened and identified through transcription factor engineering applications. Subsequently, PanB was coexpressed with PanC to regulate expression. Furthermore, the highly catalytic mutant PanBM^V123I/K124W was generated through Km/Kcat algorithm prediction and enzyme engineering, leading to a 2.5-fold increase in d-PA production. The de novo synthesis pathway of 5,10-CH₂–THF was enhanced, whereas its degradation pathway was suppressed to improve cofactor supply. Then, the extracellular transport of d-PA was enhanced by introducing the d-PA transporter PanT from Streptococcus intermedius. The plasmid-free strain DPA23 produced 78.48 g/L of d-PA in a 5-L bioreactor, with a productivity of 2.69 g/L/h after 24 h and a glucose yield of 0.54 g/g. These strategies provided a reference for constructing microbial cell factories for d-PA and its derivatives.