COMPOSITIONS AND METHODS OF PRODUCING (R)-RETICULIN AND ITS PRECURSORS

FIELD: pharmaceutical industry. SUBSTANCE: invention relates to a recombinant expression vector and methods of producing (R)-reticulin or its precursor of formula (II). Disclosed is a method of producing a compound of formula (II) from a corresponding derivative of formula (I): ， ， where R1 is a methoxy group， R2 is hydroxyl， R3 is hydroxyl， R4 is a methoxy group， R5 is methyl， or R1 is a methoxy group， R2 is a hydroxyl， R3 is a hydrogen atom， R4 is a hydroxyl， R5 is methyl， or R1 is a methoxy group， R2 is hydroxyl， R3 is hydroxyl， R4 is hydroxyl， R5 is methyl， where the method involves contacting said derivative with a microorganism expressing a CYP450 and AKR fusion protein， which is SEQ ID NO: 323， growing said microorganism with producing (R)-reticulin or precursor thereof of formula II and recovering (R)-reticulin or precursor thereof of formula II; or the method comprises (a) providing a chimeric nucleic acid sequence capable of producing said CYP450-AKR fusion protein containing， as functionally related components， a first nucleic acid sequence encoding a CYP450 polypeptide， a second nucleic acid sequence encoding an AKR polypeptide， and one or more nucleic acid sequences capable of controlling expression in a host cell; (b) introducing said chimeric nucleic acid sequence into a host cell and growing said host cell to produce said fusion protein CYP450 and AKR to produce (R)-reticulin or a precursor thereof of formula II， where the host cell under natural conditions is able to produce said derivative or derivative is provided to cells as part of cell growth medium， (c) extraction of (R)-reticulin or precursor (R)-reticulin of formula II. Disclosed is a recombinant expression vector suitable for expression in a host cell， containing as functionally related components a nucleic acid sequence， capable of controlling expression in said host cell and a nucleic acid sequence encoding described in 1 CYP450 and AKR fusion proteins. EFFECT: novel efficient methods of producing (R)-reticulin or its precursor of formula II， as well as an effective recombinant expression vector for their preparation. 10 cl， 1 tbl， 13 dwg， 8 ex

技术领域：制药工业。
发明内容：本发明涉及重组表达载体及(R)-网状菌素((R)-reticulin)或其式(II)前体的制备方法。本发明公开了一种由式(I)对应衍生物制备式(II)化合物的方法：[此处省略化学式]，其中：当R1为甲氧基、R2为羟基、R3为羟基、R4为甲氧基、R5为甲基；或R1为甲氧基、R2为羟基、R3为氢原子、R4为羟基、R5为甲基；或R1为甲氧基、R2为羟基、R3为羟基、R4为羟基、R5为甲基时，所述方法包括使所述衍生物与表达细胞色素P450(CYP450)与醛酮还原酶(AKR)融合蛋白（SEQ ID NO:323）的微生物接触，培养所述微生物以产生(R)-网状菌素或其式(II)前体，并回收该产物；或所述方法包括如下步骤：(a)提供可编码所述CYP450-AKR融合蛋白的嵌合核酸序列，其作为功能相关组分包含：编码CYP450多肽的第一核酸序列、编码AKR多肽的第二核酸序列，以及一种或多种可在宿主细胞中调控基因表达的核酸序列；(b)将所述嵌合核酸序列导入宿主细胞并培养该宿主细胞，以产生所述CYP450与AKR融合蛋白，进而生成(R)-网状菌素或其式(II)前体，其中所述宿主细胞在自然状态下可合成所述衍生物，或所述衍生物以细胞生长培养基组分的形式添加至培养体系中；(c)提取(R)-网状菌素或其式(II)前体。本发明还公开了一种可在宿主细胞中表达的重组表达载体，其作为功能相关组分包含：可在所述宿主细胞中调控基因表达的核酸序列，以及编码前述CYP450与AKR融合蛋白的核酸序列。
有益效果：本发明提供了制备(R)-网状菌素或其式(II)前体的新型高效方法，以及用于其制备的有效重组表达载体。本申请包含10项权利要求、1个表格、13幅附图、8个实施例。