Metabolic engineering of Escherichia coli for the production of <sc>d</sc>-panthenol from 3-aminopropanol and glucose

d-Panthenol is an important chemical widely utilized in the feed, medicine and cosmetic industries. For industrial production, d-panthenol is converted by chemical condensation with 3-aminopropanol and d-pantolactone, in which d-pantolactone is produced by enzymatic racemization from chemically synthesized dl-pantolactone. In this study, the de novo biosynthesis of d-panthenol in engineered Escherichia coli from glucose with the supplementation of 3-aminopropanol was established for the first time. Upon confirmation that the d-pantothenate biosynthetic pathway could be used for d-panthenol biosynthesis, a fermentation medium and pyruvate pool of the strain were first optimized, enhancing the d-pantothenol production from 31.6 mg L-1 to 184.2 mg L-1. Moreover, structure-guided rational protein engineering of pantothenate synthase (PS) was also applied for improving the catalytic activities towards 3-aminopropanol, variant M3 (F62L/R123Q/R189I), which destroyed the hydrogen network between the residues R123/R189 and the carboxyl group of beta-alanine and showed a 3.77-fold increase in d-panthenol production compared to that of the wild-type PS. In order to enhance the supply of NADPH, genes for cofactor regeneration and the pentose phosphate pathway (PPP) were enhanced, along with the knockdown of the NADPH depletion pathway. It was found that the reduction of the pgi gene resulted in the enhancement of the transcription level in the PPP pathway, which led to a relatively balanced ratio of NADPH/NADP+. The obtained strain DPN13 reached a peak of 1469.3 mg L-1d-pantothenol in the shake flask. Fed-batch fermentation was then carried out, and the titers of d-pantoate and d-pantothenol of the final strain in a 5 L bioreactor reached 24.1 g L-1 and 13.2 g L-1, respectively, which were the highest of biosynthesized d-pantoate and d-pantothenol reported to date. Overall, with the systematic metabolic engineering of the strain for the de novo biosynthesis of d-panthenol, the green production of d-panthenol with industrial sustainability would be easily achieved.