(506c) β-1,3-Glucan Production By Metabolically Engineered Agrobacterium Sp. from Cellulosic Sugar

β-Glucans are polysaccharides of D-glucose monomers linked by β-1,3-glycosidic bonds and have been used for immune and heart health functional ingredients in food industry due to their functions as "biological response modifiers" including activating the immune system. In addition, they can be utilized as growth promoting agent of domestic animals without use of antibiotcs in feed industry and lots of companies currently use β-glucan as a functional ingredient in cosmetics. Production of β-1,3-glucans from cellulosic sugar, such as cellobiose, is important as it establishes a more sustainable technology for the synthesis of this commercial product, and allows a cheaper renewable substrate, cellulose, to be used. While cellobiose could be potentially used through a hydrolysis mechanism, in which the glucose dimer is hydrolyzed into two glucose molecules, we have decided to pursue a more energetically favorable mechanism, which relies on cellobiose phosphorylase. Cellobiose phosphorylase is an enzyme specific to cellobiose and catalyzes the phosphorolytic cleavage of cellobiose, generating glucose and glucose-1-phospahte which can be directly used for biosynthesis of sugar nucleotides and polysaccharides including β-1,3-glucan. In order to construct a strain producing β-1,3-glucan from cellobiose, the gene encocding cellobiose phosphorylase (CBP), , was introduced to Agrobacterium sp. for improved cellobiose utilization in of this bacterium. Five promoters, including three Agrobacterium sp. promoters and two commercial promoters, were tested for expression of CBP. Two engineered strains, with promoters P_HCE and P_ExoC, produced β-glucan 11.6 g/L and 10.3 g/L, respectively, in shake flask cultivation after 5 days. Compared to that of control strain, the two engineered strains produced 2.5~3.0 folds higher β-glucan. The production level in engineered strains are also higher than that from sucrose (7~8 g/L), the best carbon source for β-glucan production. In 5L jar fermenter cultivation, both engineered strains produced 24.5~25.8 g/L of β-glucan from 50 g/L cellobiose, which is as much as twice of the parent strain. Therefore, engineering of Agrobacterium sp. for production of β-glucan via cellobiose phosphorolytic reaction is an effective strategy for high-yield production of β-glucan from cellobiose. The availability of metabolically engineered strain capable of producing β-glucan from cellobiose paves the way for its production from cellulose.