Construction and co-expression of polycistronic plasmids encoding thermophilic l-arabinose isomerase and hyperthermophilic β-galactosidase for single-step

d-Tagatose is a naturally occurred hexoketose. It is rarely found in nature and shows advantages as a low calorie sweetener and potentials as the substitute of sucrose. The biological production of d-tagatose involves hydrolysis of lactose by β-galactosidase and isomerization of released d-galactose into d-tagatose by l-arabinose isomerase (AI). In this study, two thermostable enzymes including an AI from Lactobacillus fermentum CGMCC2921 and a β-galactosidase from Thermus thermophilus HB27 were co-expressed in Escherichia coli. The order of the two genes, the downstream Shine–Dalgarno (SD) sequence and the aligned spacing (AS) length were optimized to balance their expression levels. One recombinant E. coli strain showed balanced activities for the two enzymes using GAAGGAGA as the downstream SD sequence and 10 nucleotides (nt) for AS. During whole cell bio-catalysis, the recombinant E. coli cells showed optimal catalytic temperature and pH at 70 °C and 7.0, respectively. With the addition of borate, d-tagatose was produced directly from cheap raw material lactose in 16 h in a concentration of 101 g/L, a yield of 20.2%, and a productivity of 6.3 g/L h. This study shed light on the efficient and economical single-step production of d-tagatose in a thermophilic bio-conversion system.