High-efficiency enzymatic conversion of cellulose to starch by strengthening glucose-utilization bypass

The in vitro biosynthesis of artificial starch from non-food cellulose biomass represents a sustainable strategy for the valorization of agricultural waste and alleviation of food shortages. However, the previously established in vitro cellulose-to-starch pathway is fundamentally limited, as only half of the glucose units in cellulose can be integrated into amylose, resulting in only 50% of the theoretical amylose yield. Here, we constructed a glucose-utilization bypass consisting of polyphosphate glucokinase and phosphoglucomutase (PGM) to redirect the glucose byproduct toward amylose biosynthesis, thereby increasing the theoretical amylose yield to 100%. To facilitate the thermodynamically unfavorable conversion of glucose-6-phosphate to glucose-1-phosphate catalyzed by PGM in the glucose-utilization bypass, three types of bienzyme complex between PGM and the next-step enzyme potato α-glucan phosphorylase (PGP) were constructed. The results showed that the fusion complex PGP-PGM exhibited the highest promoting effect on strengthening the glucose-utilization bypass with the amylose yield of one-pot cellulose-to-starch biotransformation reaching up to 68.2%, which was 22.4% higher than that achieved with separate PGM and PGP. The newly established cellulose-to-starch pathway 2.0 with a strengthened glucose-utilization bypass significantly enhanced the efficiency of starch production with a 2.3-fold increase in amylose yield compared to the previous pathway, and therefore improved the economic feasibility of valorization of cellulosic biomass into starch.