海洋多糖解重合モニタリングのための遺伝子コード化L-ラムノースバイオセンサーの開発

Marine macroalgae, particularly their complex polysaccharides, are an untapped renewable source of high-quality monosaccharides and related building blocks. To utilize this feedstock for industrial applications, the enzymatic depolymerization by marine microorganisms has been shown to be effective. A prime example is the common green alga Ulva, with its storage polysaccharide ulvan, which contains high quantities of L-rhamnose and D-glucuronic acid. As suitable high-throughput methods for analyzing the enzymatic degradation of complex polysaccharides are still lacking, a transcription factor-based biosensor is described here that utilizes the P_rhaBAD promoter native to E. coli, which is specific for L-rhamnose. This biosensor exhibited a linear response, enabling the quantification of L-rhamnose within a concentration range of 10-1000 µM. The introduction of a T7 stem-loop improved the performance, and various fluorescent reporter genes were studied. The optimized system was then used to evaluate various stages of the ulvan degradation cascade in terms of L-rhamnose release, confirming its applicability to complex sugar mixtures. A detectable fluorescence signal was only generated when all the necessary enzymes for breaking down the polymer into undecorated monosaccharides were present, highlighting the biosensor's specificity. The application of this method to the degradation of Ulva sp. biomass samples of various origins was also successfully demonstrated. This establishes the biosensor as a promising method for further high-throughput investigations. KEY POINTS: • Development of an improved transcription factor-based biosensor for L-rhamnose. • Biosensor application for the analysis of enzymatic polysaccharide degradation. • Reliable quantification of L-rhamnose in complex carbohydrate mixtures.