Senior Research Group in Genome Research of Industrial Microorganisms, Center for Biotechnology, Bielefeld University, Universitätsstraße 27, 33615 Bielefeld, Germany.
J Biotechnol. 2013 Aug 20;167(2):178-89. doi: 10.1016/j.jbiotec.2012.08.011. Epub 2012 Aug 31.
The pseudotetrasaccharide acarbose is a medically relevant secondary metabolite produced by strains of the genera Actinoplanes and Streptomyces. In this study gene products involved in acarbose metabolism were identified by analyzing the cytosolic and extracellular proteome of Actinoplanes sp. SE50/110 cultures grown in a high-maltose minimal medium. The analysis by 2D protein gel electrophoresis of cytosolic proteins of Actinoplanes sp. SE50/110 resulted in 318 protein spots and 162 identified proteins. Nine of those were acarbose cluster proteins (Acb-proteins), namely AcbB, AcbD, AcbE, AcbK, AcbL, AcbN, AcbR, AcbV and AcbZ. The analysis of proteins in the extracellular space of Actinoplanes sp. SE50/110 cultures resulted in about 100 protein spots and 22 identified proteins. The identifications included the three acarbose gene cluster proteins AcbD, AcbE and AcbZ. After their identification, proteins were classified into functional groups. The dominant functional groups were the carbohydrate binding, carbohydrate cleavage and carbohydrate transport proteins. The other functional groups included protein cleavage, amino acid degradation, nucleic acid cleavage and a number of functionally uncharacterized proteins. In addition, signal peptide structures of extracellularly found proteins were analyzed. Of the 22 detected proteins 19 contained signal peptides, while 2 had N-terminal transmembrane helices explaining their localization. The only protein having neither of them was enolase. Under the conditions applied, the secretome of Actinoplanes sp. SE50/110 was dominated by seven proteins involved in carbohydrate metabolism (PulA, AcbE, AcbD, MalE, AglE, CbpA and Cgt). Of special interest were the identified extracellular pullulanase PulA and the two solute-binding proteins MalE and AglE. The identifications suggest that Actinoplanes sp. SE50/110 has two maltose/maltodextrin import systems. We postulate the identified MalEFG transport system of Actinoplanes sp. SE50/100 as the missing acarbose-metabolite importer and present a model of acarbose metabolism that is extended by the newly identified gene products.
假四糖阿卡波糖是放线菌属和链霉菌属菌株产生的一种具有医学相关性的次生代谢物。在这项研究中，通过分析在高麦芽糖基础培养基中生长的游动放线菌 SE50/110 培养物的胞质和胞外蛋白质组，鉴定了与阿卡波糖代谢相关的基因产物。通过 2D 蛋白质凝胶电泳分析游动放线菌 SE50/110 的胞质蛋白，得到 318 个蛋白点和 162 个鉴定蛋白。其中 9 个是阿卡波糖簇蛋白（Acb-蛋白），即 AcbB、AcbD、AcbE、AcbK、AcbL、AcbN、AcbR、AcbV 和 AcbZ。分析游动放线菌 SE50/110 培养物胞外空间的蛋白质，得到约 100 个蛋白点和 22 个鉴定蛋白。鉴定结果包括三个阿卡波糖基因簇蛋白 AcbD、AcbE 和 AcbZ。鉴定后，将蛋白质分类为功能组。占主导地位的功能组是碳水化合物结合、碳水化合物裂解和碳水化合物转运蛋白。其他功能组包括蛋白裂解、氨基酸降解、核酸裂解和许多功能尚未确定的蛋白。此外，还分析了胞外发现的蛋白质的信号肽结构。在所检测的 22 种蛋白质中，有 19 种含有信号肽，而 2 种含有 N 端跨膜螺旋，解释了它们的定位。唯一没有这两种结构的蛋白质是烯醇酶。在应用的条件下，游动放线菌 SE50/110 的分泌组主要由参与碳水化合物代谢的七种蛋白组成（PulA、AcbE、AcbD、MalE、AglE、CbpA 和 Cgt）。特别值得注意的是鉴定出的胞外普鲁兰酶 PulA 和两种溶质结合蛋白 MalE 和 AglE。这些鉴定结果表明，游动放线菌 SE50/110 有两种麦芽糖/麦芽糊精进口系统。我们推测，鉴定出的游动放线菌 SE50/100 的 MalEFG 转运系统是缺失的阿卡波糖代谢物进口器，并提出了一个阿卡波糖代谢模型，该模型通过新鉴定的基因产物得到了扩展。
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