Identification of a lactose-oxidizing enzyme in Escherichia coli and improvement of lactobionic acid production by recombinant expression of a quinoprotein glucose dehydrogenase from Pseudomonas taetrolens.

Lactobionic acid (LBA), an aldonic acid prepared by oxidation of the free aldehyde group of lactose, has been broadly used in cosmetic, food, and pharmaceutical industries. Although Escherichia coli is unable to produce LBA naturally, a wild-type E. coli strain successfully produced LBA from lactose upon pyrroloquinoline quinone (PQQ) supplementation, indicating that E. coli contains at least one lactose-oxidizing enzyme as an apo-form. By inactivating the candidate genes in the E. coli chromosome, we found that the lactose-oxidizing enzyme of E. coli was the quinoprotein glucose dehydrogenase (GCD). To improve the LBA production ability of the E. coli strain, quinoprotein glucose dehydrogenase (GDH) from Pseudomonas taetrolens was recombinantly expressed and culture conditions such as growth temperature, initial lactose concentration, PQQ concentration, and isopropyl-β-D-1-thiogalactopyranoside induction concentration were optimized. We performed batch fermentation using a 5-L bioreactor under the optimized culture conditions determined in flask culture experiments. After batch fermentation, the LBA production titer, yield, and productivity of the recombinant E. coli strain were 200 g/L, 100 %, and 1.28 g/L/h, respectively. To the best our knowledge, this is the first report to identify the lactose-oxidizing enzyme of E. coli and to produce LBA using a recombinant E. coli strain as the production host. Because E. coli is one of the most easily genetically manipulated bacteria, our result provides the groundwork to further enhance LBA production by metabolic engineering of LBA-producing E. coli.