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利用植物材料作为碳源的微生物有关。细菌从l -阿拉伯糖到d - 5-磷酸木糖的代谢途径已被广泛研究。人们认为细菌l -阿拉伯糖代谢有两种替代途径，与已知的细菌途径相比，它们不涉及磷酸化反应。在第一个途径中，l -阿拉伯糖被NAD(P)+依赖的脱氢酶氧化为l -阿拉伯糖-γ-内酯。内酯被内酯酶裂解为l -阿拉伯酸酯，然后连续两个脱水反应形成l -2-酮-3-脱氧阿拉伯酸酯（L-KDA）和α-酮戊二酸半醛。最后一步是NAD(P)+依赖的半醛脱氢成α-酮戊二酸。第二种途径具有相同的初始三个步骤，但L-KDA通过醛缩酶反应裂解为糖醛酸和丙酮酸。到目前为止，还没有基因编码酶参与l -阿拉伯糖代谢的这些替代途径。在本研究中，催化l -阿拉伯糖转化为l -阿拉伯糖-γ-内酯的l -阿拉伯糖1-脱氢酶被认为是l -阿拉伯糖代谢这一替代途径的第一步酶。利用l -阿拉伯糖1-脱氢酶的部分肽序列克隆了l -阿拉伯糖1-脱氢酶编码基因，并在大肠杆菌中过表达为全活性酶。这是第一次鉴定一个基因参与l -阿拉伯糖在细菌代谢的替代途径。另外4种酶及其相关基因也通过生化分析鉴定为l -阿拉伯糖内酯酶、l -阿拉伯酸脱水酶、l -2-酮-3-脱氧阿拉伯酸脱水酶和α-酮苦荞半醛脱氢酶。

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.