Cellulose synthase is the enzyme responsible for synthesizing the cellulose microfibril. This enzymatic reaction is twofold of β1→4-glucan synthesis (polymerization) and microfibril formation (crystallization). Mechanism of the polymerization starts to be clarified based on the three dimensional molecular structure of the enzyme, while the crystallization mechanism still remains to be clarified. For investigating the crystallization mechanism as well as polymerization, we conducted the reconstitution of bacterial cellulose synthase to synthesize cellulose in vitro and in vivo, either of which will be a useful platform to analyze the crystallization process of cellulose synthase. Crude enzyme preparation of Acetobacter solubilized by mild detergent like alkylmaltoside successfully produced cellulose from UDP-glucose in vitro. Weight-averaged degree of polymerization (DPw) of the synthesized cellulose was as high as 300, which is higher than the celloligomers synthesized by reversing hydrolase or phosphorylase reaction. This highlights that our crude cellulose synthase preparation has polymerization function. However the synthesized cellulose is globular aggregation and crystallized into cellulose II after washing SDS, meaning that this enzyme preparation is unable to form the microfibril by assembling a bunch of cellulose chains, despite a significant polymerizing activity. We also have conducted in vivo synthesis of cellulose by heterologous expression of Acetobacter cellulose synthase in Escherichia coli. It was successful to produce cellulose polymer as long as 700 of DPw by the recombinant E. coli, which we designated as CESEC (Cellulose synthesis in E. coli). However the product obtained after washing by SDS and dilute NaOH was cellulose II aggregation as well. This indicates that the cellulose synthase expressed in living E. coli cell is not enough for the native activity to produce cellulose I microfibril. We could not, to date, accomplish the reconstitution of the native activity of bacterial cellulose synthase. However these enzyme preparations maintain polymerizing activity, one of the essential functions of cellulose synthase. It then should be possible to recover the native activity of cellulose synthase by supplementing the missing factor(s). We believe that either system, in vitro or in vivo (CESEC), is a useful experimental setup to find out key factors for the microfibril formation of cellulose synthase.

Cellulose II formation by cellulose synthase: Negative data can make themselves positive?

253rd National Meeting (2017)

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