3.1 Designing the oxidoreductive pathways forD-tagatose production
We constructed the oxidoreductive pathways for producing tagatose from lactose. The synthesis pathway was composed of three modules, as shown in Figure 1: (1) lactose hydrolysis module (withβ -galactosidase); (2) galactose reduction module (with aldose reductase, EC: 1.1.1.307); and (3) galactitol oxidation module (with galactitol dehydrogenase, EC: 1.1.1.14). A three-enzyme cascade converting lactose into tagatose (Pathway I) was designed usingβ -galactosidase, NADPH-preferring Ps XR from P. stipites (GenBank ID: XP_001385181), NAD+-dependentPd PDH from P. denitrificans (GenBank ID: WP_011751060), or the NAD+-dependent Rl GDH from R. leguminosarum (GenBank ID: WP_011650422.1) (Figure 1A). The coenzyme dependence of Ps XR and Rl GDH or Pd PDH is different. Furthermore, we designed a novel Pathway II for constructing a cofactor cycle between the reduction and oxidation modules (Figure 1B).