Metabolic engineering of Escherichia coli Nissle 1917 for efficient de novo biosynthesis of carnosine.

Carnosine is increasingly needed in pharmaceutical, nutraceutical, and cosmetic industries, but its production in bacteria is severely hampered. In this study, Escherichia coli Nissle 1917 has been engineered for efficient carnosine production. Firstly, the peptidase PepD that degrades carnosine was identified, and its mutant WYT03 was constructed. Secondly, an aspartate α-decarboxylase PanD from Bacillus subtilis and ATP phosphoribosyltransferase HisG from Bacteroides thetaiotaomicron were identified for optimum synthesis of β-alanine and l-histidine in E. coli, respectively; plasmid pWT-Y2DG which overexpress BsPanD, BtHisG, and l-amino acid ligase BsBacDN108E H378K was constructed and transformed in WYT03, resulting in carnosine production. Thirdly, a point mutation W332F in BsBacDN108E H378K that can increase the carnosine yield by 51% was identified through saturation mutagenesis. Fourthly, an optimum combination of the promoter and RBS, Ptac-R2, was screened out and used to replace the leader peptide hisL of his operon and to activate prs expression; the genes edd, amn, and purF were deleted, resulting in WYT10 producing more l-histidine; WYT09 was further modified by replacing purF with Cgppk2 with Ptac-R2, and replacing ldha with BsrocG with Ptac-R2, inserting Ptac-R2 upstream of yfcc, deleting pitA resulting WYT17. Finally, plasmid pWT-Y3DG which overexpress BsPanD, BtHisG, and BsBacDN108E H378K W332F was transformed in WYT17, and the final strain WYT17/pWT-Y3DG could produce 14.39 g/L carnosine in a 5-L fed-batch fermentation with a yield of 0.145 g/g glucose and a productivity of 0.133 g/(L·h). To the best of our knowledge, this represents the highest carnosine titer from microbial fermentation reported to date.