游动放线菌Actinoplanes sp.SE50/110中阿卡波糖脱氧氨基糖单元的生物合成

Biosynthetic pathway of deoxyaminosugar moiety in acarbose from Actinoplanes sp. SE50/110

摘要:

[目的] 解析Actinoplanes sp. SE50/110（简称SE50/110）中阿卡波糖脱氧氨基糖单元的生物合成机制。[方法] 经过BLASTp分析，推测了AcbA、AcbB和AcbV负责阿卡波糖脱氧氨基糖单元的生物合成。首先，本研究在SE50/110中分别构建了acbA、acbB和acbV的同框缺失和回补突变株。然后，利用大肠杆菌BL21（DE3）/pGro7分别对AcbA、AcbB和AcbV成功实现了可溶性表达。最后，以D-葡萄糖-1-磷酸为起始底物，通过体外催化反应，研究脱氧氨基糖单元的生物合成过程和相关蛋白的酶学性质。[结果] 在SE50/110中分别缺失acbA、acbB和acbV基因后，相应突变株均丧失了阿卡波糖的合成能力，将acbA、acbB和acbV基因分别回补后，各菌株又恢复了阿卡波糖的合成能力，证明了它们均为阿卡波糖生物合成的必需基因。在体外酶促反应中，D-葡萄糖-1-磷酸-胸腺嘧啶转移酶AcbA催化D-葡萄糖-1-磷酸和dTTP合成dTDP-D-葡萄糖，对D-葡萄糖-1-磷酸的K_m值为（0.185±0.053）mmol/L，V_max为（2.366±0.217）μmol/（min·mg）；对dTTP的K_m值为（4.964±1.089）mmol/L，V_max为（60.310±5.419）μmol/（min·mg）。dTDP-D-葡萄糖-4，6-脱水酶AcbB催化dTDP-D-葡萄糖转化为dTDP-4-酮基-6-脱氧-D-葡萄糖，K_m值和V_max分别为（0.353±0.089）mmol/L和（306.401±28.740）μmol/（min·mg）。氨基转移酶AcbV催化dTDP-4-酮基-6-脱氧-D-葡萄糖生成dTDP-4-氨基-4，6-双脱氧-D-葡萄糖，K_m值和V_max分别为（1.411±0.293）mmol/L和（3.447±0.279）μmol/（min·mg）。[结论] 本研究阐明了阿卡波糖脱氧氨基糖单元的生物合成过程，为全面解析阿卡波糖生物合成途径奠定了基础。同时，测定了相关酶的动力学参数，为代谢工程改造SE50/110，提高阿卡波糖产量提供了重要的理论依据。

Abstract:

[Objective] Elucidation of the biosynthetic mechanism of the deoxyaminosugar moiety in acarbose from Actinoplanes sp. SE50/110. [Methods] On the basis of BlastP analysis, AcbA, AcbB and AcbV were proposed to be associated with the biosynthesis of deoxyaminosugar moiety. Firstly, the in-frame deletion and trans-complementation of acbA, acbB and acbV were performed in SE50/110 to investigate their involvement in acarbose biosynthesis. Then, AcbA, AcbB and AcbV were heterologously expressed in E. coli BL21(DE3)/pGro7 and purified by Ni affinity chromatography. Finally, using D-glucose-1-phosphate as the starting substrate, the biosynthetic process of deoxyaminosugar moiety was elucidated by enzymatic assay. In addition, the properties of these involved enzymes were characterized. [Results] Deletion mutants of acbA, acbB and acbV in SE50/110 were named as ZD03, ZD04 and ZD05, respectively, all of which lost the productivity of acarbose. And then, the production of acarbose was recovered through trans-complementation of acbA, acbB and acbV in ZD03, ZD04 and ZD05, respectively. In vitro enzymatic analysis suggested that AcbA, a D-glucose-1-phosphate thymidylyltransferase, is responsible for the biosynthesis of dTDP-D-glucose from D-glucose-1-phosphate and dTTP. It showed a K_m of (0.185±0.053) mmol/L and a V_max of (2.366±0.217) μmol/(min·mg) with D-glucose-1-phosphate, as well as a K_m of (4.964±1.089) mmol/L and a V_max of (60.310±5.419) μmol/(min·mg) with dTTP. AcbB, a TDP-D-glucose-4,6-dehydratase, catalyzed the dehydration of dTDP-D-glucose to dTDP-4-keto-6-deoxy-D-glucose. The K_m and V_max of AcbB are (0.353±0.089) mmol/L and (306.401±28.740) μmol/(min·mg), respectively. AcbV is a dTDP-4-keto-6-deoxy-D-glucose-aminotransferase and catalyzes the transamination of dTDP-4-keto-6-deoxy-D-glucose to dTDP-4-amino-4,6-dideoxy-D-glucose using glutamic acid as amino donor. The K_m and V_max of AcbV are (1.411±0.293) mmol/L and (3.447±0.279) μmol/(min·mg), respectively. [Conclusion] This study elucidated the biosynthetic pathway of deoxyaminosugar moiety in acarbose, which paved a solide way for a full elucidation of acarbose biosynthesis. Meanwhile, the characterization of the involved enzymes provided important information for the metabolic engineering of SE50/110 to improve acarbose production.