Fungi-derived flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenases (FADGDHs) are the most popular and advanced enzymes for SMBG sensors because of their high substrate specificity toward glucose and oxygen insensitivity. However, this type of FADGDH hardly shows direct electron transfer (DET) ability. In this study, we developed a new DET-type FADGDH by harboring Cytochrome b(562) (cyt b(562)) derived from Escherichia coli as the electron transfer domain. The structural genes encoding fusion enzymes composed of cyt b(562) at either the N- or C-terminus of fungal FADGDH, (cyt b(562)-GDH or GDH-cyt b(562)), were constructed, recombinantly expressed, and characteristics of the fusion proteins were investigated. Both constructed fusion enzymes were successfully expressed in E. coli, as the soluble and GDH active proteins, showing cyt b(562) specific redox properties. Thusconstructed fusion proteins showed internal electron transfer between FAD in FADGDH and fused cyt b(562). Consequently, both cyt b(562)-GDH and GDH-cyt b(562) showed DET abilities toward electrode. Interestingly, cyt b(562)-GDH showed much rapid internal electron transfer and higher DET ability than GDH-cyt b(562). Thus, we demonstrated the construction and production of a new DET-type FADGDH using E.coli as the host cells, which is advantageous for future industrial application and further engineering. (C) 2020 Elsevier Inc. All rights reserved.