Enhancement of cellulose production by expression of sucrose synthase in Acetobacter xylinum

Abstract

Higher plants efficiently conserve energy ATP in cellulose biosynthesis by expression of sucrose synthase, in which the high free energy between glucose and fructose in sucrose can be conserved and used for the synthesis of UDP-glucose. A mixture of sucrose synthase and bacterial cellulose synthase proceeded to form UDP-glucose from sucrose plus UDP and to synthesize 1,4-beta-glucan from the sugar nucleotide. The mutant sucrose synthase, which mimics phosphorylated sucrose synthase, enhanced the reaction efficiency (Vmax/Km) on 1,4-beta-glucan synthesis, in which the incorporation of glucose from sucrose was increased at low concentrations of UDP. Because UDP formed after glucosyl transfer can be directly recycled with sucrose synthase, UDP-glucose formed appears to show high turnover with cellulose synthase in the coupled reaction. The expression of sucrose synthase in Acetobacter xylinum not only changed sucrose metabolism but also enhanced cellulose production, in which UDP-glucose was efficiently formed from sucrose. Although the level of UDP-glucose in the transformant with mutant sucrose synthase cDNA was only 1.6-fold higher than that in plasmid-free cells, the level of UDP was markedly decreased in the transformant. The results show that sucrose synthase serves to channel carbon directly from sucrose to cellulose and recycles UDP, which prevents UDP build-up in cellulose biosynthesis.

Figure 1

Effect of time and recombinant sucrose synthase on cellulose formation from sucrose by A. xylinum membrane preparations. •, Mutant sucrose synthase; ○, wild-type sucrose synthase; ▵, cellulose formation from UDP-glucose without sucrose synthase.

Figure 2

Production of cellulose in transformants of A. xylinum. •, Transformant with mutant sucrose synthase cDNA; ○, transformant with wild-type sucrose synthase cDNA; ▵, plasmid-free cells. Because the solid and open symbols overlapped for 2 days, only the open symbols were shown.

Figure 3

Formation of radioactive UDP-glucose and cellulose from [glucose-¹⁴C]sucrose in transformants of A. xylinum. (A) UDP-[¹⁴C]glucose formed from [glucose-¹⁴C]sucrose. •, Transformant with mutant sucrose synthase cDNA; ○, the transformant with wild-type sucrose synthase cDNA; ▵, plasmid-free cells. (B) [¹⁴C]Cellulose formed from [glucose-¹⁴C]sucrose, in which the amount of cellulose is shown as the height of bar due to total radioactive plus nonradioactive cellulose in each fraction, the solid part of which is due to the radioactive cellulose equivalent to specific radioactivity of [¹⁴C]glucose moiety: ■, mutant sucrose synthase cDNA; , wild-type sucrose synthase cDNA; ▨, plasmid-free cells.