Biochemical Research of Novel Carbohydrate Metabolic Pathway for Development of Efficient Conversion of Biornass

L-阿拉伯糖是一些植物材料的主要成分，因此L-阿拉伯糖的分解代谢与以植物材料为碳源的微生物有关。L-阿拉伯糖在细菌中的代谢途径已被广泛研究。与已知的细菌代谢途径不同，人们认为细菌L-阿拉伯糖代谢存在两条不涉及磷酸化反应的替代途径。在第一个途径中，L-阿拉伯糖被依赖于NAD(P)的脱氢酶氧化成L-阿拉伯-γ-内酯。内酯被内酯酶裂解成L-阿拉伯酸，然后连续两次脱水反应生成L-2-酮-3-脱氧阿拉伯酸(L-KDA)和α-酮戊二酸半醛。最后一步是依赖于NAD(P)的半醛脱氢生成α-酮戊二酸。第二条途径有三个相同的起始步骤，但L-KDA是通过醛缩酶反应裂解成糖醛和丙酮酸的。到目前为止，还没有鉴定出参与这些L-阿拉伯糖代谢交替途径的基因编码酶。在本研究中，L-阿拉伯糖1-脱氢酶催化L-阿拉伯糖转化为L-阿拉伯-γ-内酯，是L-阿拉伯糖代谢这一交替途径的第一步。用纯化的L-阿拉伯糖1-脱氢酶的部分肽序列克隆了该酶的编码基因，并在大肠杆菌中进行了高效表达。这是首次在细菌中鉴定出参与L-阿拉伯糖代谢的另一条途径的基因。经生化分析，该4种酶及相关基因分别为L-阿拉伯内酯酶、L-阿拉伯酸脱水酶、L-2-酮-3-脱氧阿拉伯酸脱水酶和α-酮基半醛脱氢酶。

L-Arabinose is a major constituent of some plant materials, and L-arabinose catabolism is therefore relevant for microorganisms using plant material as a carbon source. The metabolic pathway from L-arabinose to D-xylulose 5-phosphate in bacterium has been investigated extensively. It is believed that there are two alternative pathways for bacterial L-arabinose metabolism, which do not involve a phosphorylation reaction, in contrast to the known bacterial pathway. In the first pathway, L-arabinose is oxidized to L-arabino-γ-lactone by NAD(P)+-dependent dehydrogenase. The lactone is cleaved by a lactonase to L-arabonate, followed by two successive dehydration reactions forming L-2-keto-3-deoxyarabonate (L-KDA) and α-ketoglutaric semialdehyde. The last step is the NAD(P)+-dependent dehydrogenation of the semialdehyde to α-ketoglutaric acid. The second pathway has the same initial three steps, but L-KDA is cleaved through an aldolase reaction to glycoaldehyde and pyruvate. No gene-encoding enzyme involved in these alternative pathways of L-arabinose metabolism had been identified so far. In this study, L-arabinose 1-dehydrogenase catalyzing the conversion of L-arabinose to L-arabino-γ-lactone was characterized as an enzyme responsible for the first step of this alternative pathway of L-arabinose metabolism. The gene encoding L-arabinose 1-dehydrogenase was cloned using a partial peptide sequence of the purified enzyme and was overexpressed in Escherichia coli as a fully active enzyme. This is the first identification of a gene involved in an alternative pathway of L-arabinose metabolism in bacterium. Furthermore, the four other enzymes and the relevant genes were also characterized as L-arabinonolactonase, L-arabonate dehydratase, L-2-keto-3-deoxyarabonate dehydratase, and α-ketogoutaric semialdehyde dehydrogease by biochemical analysis.