游动放线菌Actinoplanes sp. SE50/110阿卡波糖胞外代谢相关基因功能的初步研究

Functions of Genes Involved in Extracellular Acarbose Metabolic Pathway From Actinoplanes sp. SE50/110

游动放线菌Actinoplanes sp. SE50/110(以下简称SE50/110)是重要的抗2型糖尿病药物阿卡波糖的生产菌株。在阿卡波糖的生物合成途径中,其胞内合成代谢途径的部分关键步骤已被详细解析,而与类似物形成相关的胞外代谢过程却少有研究。本研究敲除了阿卡波糖胞外代谢相关基因,利用高效液相色谱检测阿卡波糖产量变化,通过淀粉酶-淀粉-碘(α-amylase-starch-iodine, ASI)显色法检测α-淀粉酶的受抑制程度,间接反映阿卡波糖类似物含量变化。结果显示,与出发菌株SE50/110相比,阿卡维基转移酶基因acbD的缺失导致阿卡波糖产量下降42%,淀粉酶基因acbE和acbZ缺失不影响阿卡波糖生产。在类似物积累方面,敲除acbE使细胞中阿卡波糖类似物的相对含量降低至野生型的14%,敲除acbZ使其积累量显著提升至野生型的3.75倍;在acbE缺失突变株的基础上继续敲除acbD后,类似物相对含量进一步下降至野生型的9%,为利用ASI显色法筛选高产突变株提供了背景清晰的衍生菌株。本研究通过体内基因敲除实验,初步说明了游动放线菌阿卡波糖胞外代谢相关基因与阿卡波糖合成、类似物积累之间的关系,为解析阿卡波糖的胞外代谢途径奠定了基础。

Acarbose, one of the main drugs for type 2 diabetes, currently is produced by Actinoplanes sp. SE50/110 in the industry. Whereas some important intracellular steps of acarbose biosynthesis have been elucidated, less attention has been paid to the extracellular pathways, which would lead to the accumulation of acarbose analogs. In this work, in-frame deletions of acbD, acbE and acbZ were performed in Actinoplanes sp. SE50/110, and their involvements in the biosynthesis of acarbose and its analogs were analyzed by HPLC and α-amylase-starch-iodine(ASI) assay. As detected by HPLC, the titer of acarbose decreased by 42% in the acbD deleted mutant and not significantly changed in the acbE or acbZ deleted mutants. However, as analyzed by the ASI assay, the accumulation of acarbose analogs decreased to 14% with acbE deletion and increased to 375% with acbZ deletion. Furthermore, the combinatory deletions of acbE and acbD resulted in a decrease of acarbose analogs accumulation to 9% compared with the wild-type strain. This might provide us a derivative strain with a clean background for high throughput screening using ASI assay. As a summary, three genes belonging to the extracellular metabolism of acarbose that can cause the accumulation of acarbose and its analogs were preliminarily investigated in this study, which would pave a way for the full elucidation of the extracellular acarbose metabolic pathway.