Metabolic engineering of Pseudomonas putida for production of the natural sweetener 5‐ketofructose from fructose or sucrose by periplasmic oxidation with a heterologous fructose dehydrogenase

5‐Ketofructose (5‐KF) is a promising low‐calorie natural sweetener with the potential to reduce health problems caused by excessive sugar consumption. It is formed by periplasmic oxidation of fructose by fructose dehydrogenase (Fdh) of Gluconobacter japonicus, a membrane‐bound three‐subunit enzyme containing FAD and three haemes c as prosthetic groups. This study aimed at establishing Pseudomonas putida KT2440 as a new cell factory for 5‐KF production, as this host offers a number of advantages compared with the established host Gluconobacter oxydans. Genomic expression of the fdhSCL genes from G. japonicus enabled synthesis of functional Fdh in P. putida and successful oxidation of fructose to 5‐KF. In a batch fermentation, 129 g l(−1) 5‐KF were formed from 150 g l(−1) fructose within 23 h, corresponding to a space‐time yield of 5.6 g l(−1) h(−1). Besides fructose, also sucrose could be used as substrate for 5‐KF production by plasmid‐based expression of the invertase gene inv1417 from G. japonicus. In a bioreactor cultivation with pulsed sucrose feeding, 144 g 5‐KF were produced from 358 g sucrose within 48 h. These results demonstrate that P. putida is an attractive host for 5‐KF production.