Substrate Promiscuity Engineering of ScALDO Enables a Versatile Minimized Enzyme Cascade for Efficient Utilization of Various Sugars

The depletion of fossil fuels has turned researchers’ attention toward utilizing waste biomass resources and their monomeric sugars to produce chemicals. In this study, we reshaped the active site pocket of alditol oxidase from Streptomyces coelicolor (ScALDO) through substrate promiscuity engineering, and a mutant ScALDO/Q288G was identified with high promiscuity toward C3 to C6 aldoses and alditols. Active site pocket volume analysis, spatial steric hindrance analysis, molecular dynamics simulations, and kinetic parameter studies revealed that ScALDO/Q288G achieves oxidation of cyclic d -glucose through an expanded active site pocket, reduces spatial hindrance for substrates, and facilitates hydride transfer. To further clarify the catalytic mechanism of ScALDO/Q288G in oxidizing cyclic aldoses, we proposed a possible mechanism that involves initial hydride transfer followed by spontaneous hydration and ring opening. Further combinatorial mutations produced an optimal variant, ScALDO/Q288G/E53D/V256E/E348P (M4), showing over a 68.8-fold increase in activity for D-xylose and 268-fold for D-ribose, while also demonstrating significant d -glucose oxidation activity. M4 also maintains significant activity toward smaller substrates, such as d -glyceraldehyde, and exhibits superior thermal stability ( T m = 68.7 °C). Using M4, we established a minimized glycolytic cascade (MGC). MGC efficiently utilizes diverse substrates, including d -glucose, D-xylose, l -arabinose, D-galactose, and xylitol, to produce pyruvate, with a conversion of 5 mM d -glucose to 9.08 mM pyruvate within 24 h, achieving a yield of 90.8%. In conclusion, we created M4, which exhibits high catalytic promiscuity toward C3 to C6 aldoses and alditols, thus enabling the establishment of an MGC for the utilization of various sugars. Beyond its application in MGC, M4 holds the potential to be used in all cascades involving aldoses and alditols of various sizes to reduce the number of enzymes required.