d-Pantothenic acid (vitamin B-5) has wide applications in the feed, food, chemical, and pharmaceutical industries. Its biological production routes which employ pantothenate synthetase (PS) as the key enzyme are attractive since they avoid the tedious and time-consuming optical resolution process. However, little data is available on the activity and kinetics of this enzyme, hampering the rational selection of an efficient enzyme for the biological production of d-pantothenic acid. In this study, six phylogenetically distant PS-encoding genes, from Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Bacillus thuringiensis, Bacillus cereus, and Enterobacter cloacae, were expressed in E. coli. The PS from C. glutamicum exhibited a specific activity of 205.1 U/mg and a turnover number of 127.6 s(-1), which to our best knowledge are the highest values ever reported. The addition of substrates (d-pantoic acid and beta-alanine) to the E. coli strain harboring this enzyme during the early log phase of fermentation resulted in the production of 97.1 g/L of d-pantothenic acid within 32 h, corresponding to a conversion yield of 99.1% and a productivity of 3.0 g/L/h. To the best of our knowledge, this is the highest productivity reported to date.