Actinoplanes sp. ATCC 33002 produziert ein fünffach chloriertes Phenylpyrrolderivat, das Pentachlorpseudilin[1], für dessen Biosynthese die Beteiligung FADH2-abhängiger Halogenasen vermutet wurde. Ziel dieser Arbeit war es deshalb, in Actinoplanes sp. nach Genen FADH2-abhängiger Halogenasen zu suchen und diese heterolog zu überexprimieren. Durch Hybridisierungsexperimente mit prnC[2] (Halogenasegen aus Pseudo¬monas fluorescens) als Sonde konnten in Actinoplanes sp. ATCC 33002 zwei potentielle Gene FADH2-abhängiger Halogenasen (halA und halB) detektiert werden. Beide Gene weisen hohe Homologien zu Genen bereits bekannter FADH2-abhängiger Halogenasen auf. Die für HalA und HalB abgeleiteten Aminosäuresequenzen weisen die größten Ähnlichkeiten zur Chlortetra¬cyclin¬halogenase (Cts4)[3] aus Streptomyces aureofaciens und zur Halogenase PrnC[2] aus Pseudomonas fluorescens BL915 auf, welche im Bereich von 55 % bis 61 % liegen. HalA und HalB enthalten die in bekannten FADH2-abhängigen Halogenasen konservierten Sequenzmotive: die FADH2-Bindestelle und das Tryptophanmotiv[4]. Während die Überexpression von halA und halB in Escherichia coli zu Inclusionbodies führte, konnte halB in zwei Pseudomonadenstämmen in löslicher Form überexprimiert werden. In P. aureofaciens pCIBhalB wurde halB und in P. fluorescens pCIBhalBhis halBhis überexprimiert. In Enzymaktivitätstests mit verschiedenen Phenylpyrrolderivaten als potentiellen Substraten wurde im HalB enthaltenden Rohextrakt des Expressionsstammes P. aureofaciens pCIBhalB in vitro chlorierende Aktivität nachgewiesen. HalB gehört somit zur Gruppe der FADH2-abhängigen Halogenasen. [1] Cavalleri, B., Volpe, G., Tuan, G., Berti, M., Parenti, F., Curr. Microbiol. 1 (1978) 319 [2] Hammer, P. E., Hill, D. S., Lam, S. T., van Pée, K.-H., Ligon, J. M., Appl. Environ. Microbiol. 63 (1997) 2147 [3] Dairi, T., Nakano, T., Aisaka, K., Katsumata, R., Hasegawa, M., Biosci. Biotechnol. Biochem. 59 (1995) 1099 [4] van Pée, K.-H., Zehner, S., Enzymology and molecular genetics of biological halogenation. In: G. W. Gribble (Ed.), The Handbook of Environmental Chemistry, Vol. 3, part P. Natural production of organohalogen compounds. Springer-Verlag Berlin, Heidelberg, (2003) 171

Acarbose hat als Inhibitor von Hydrolasen alpha-1,4-glykosidischer Bindungen medizinische Bedeutung. Das Acarbose-Biosynthese-Gencluster (acb) des grampositiven Produzenten Actinoplanes sp. wurde identifiziert und Genprodukte z. T. charakterisiert. Das Modell zum Acarbose-Metabolismus beschreibt einen Acarbosekreislauf, bei dem das Pseudotetrasaccharid als Carbophor fungiert. Das Molekül wird in das umgebende Medium abgegeben und durch das Zusammenwirken zweier extrazellulärer Enzyme nach Stärkehydrolyse mit einer unterschiedlichen Anzahl an Glukosemonomeren beladen. Nach dem vermuteten Re-Import über ein Bindeprotein-abhängiges ABC-Transportsystem AcbHFG stünde dem Organismus dann ein Gewinn an Glukosemolekülen zur Verfügung. Neben diesem Vorteil gegenüber Nahrungskonkurrenten im Habitat fungiert Acarbose ebenso als Hemmer der artfremden extrazellulären a-Amylasen. Die ökologische Funktion des Pseudotetrasaccharids wurde durch Untersuchungen zum Einfluss auf den Maltodextrin-Stoffwechsel von E. coli verifiziert und ausgeweitet. Es lässt sich ein ökonomisch sinnvolleres Konkurrenzverhalten von Actinoplanes sp. ableiten. Von den durch den Acarboseproduzenten selbst bereitgestellten Maltosacchariden aus Stärke profitieren artfremde Mikroorganismen nicht, da neben den Exoenzymen auch die Maltodextrin-Aufnahmesysteme in ihrer Funktion gehemmt sind. Außerdem wurde eine für Actinoplanes sp. geforderte Kapazität zur Aufnahme von Maltose und Maltodextrinen in vivo gefunden und in Transportexperimenten mit radioaktiv markierten Zuckern charakterisiert. Die Transportaktivität wird wahrscheinlich über zwei Bindeprotein-abhängige ABC-Importer mit multiplem Substratspektrum realisiert. Das ABC-Importsystem AcbHFG wurde heterolog in E. coli und S. lividans synthetisiert und z. T. erfolgreich gereinigt. In Substrat-Bindungsstudien konnte für das Bindeprotein AcbH eine Interaktion mit Acarbose und längerkettigen Derivaten, nicht jedoch mit Maltose/Maltodextrinen beobachtet werden. / Acarbose acts as an inhibitor of alpha-glucosidases and is therefore clinically used. The biosynthesis gene cluster (acb) was identified and partly characterized. The proposed model describes a pathway in which acarbose might function as a carbophor. The molecule is secreted into the medium where, after hydrolysis of starch, it is charged with additional glucose moieties. Re-uptake by a binding-protein dependent ABC importer AcbHFG would then result in a net gain of carbon and energy. Besides extracting glucose from the extracellular pool acarbose also acts as an inhibitor of alpha-amylases secreted by competitors in the natural environment. Prompted by the structural similarity between acarbose and maltotetraose, the effects of acarbose on the metabolism of maltose and maltodextrins in whole cells of E. coli and on individual components of the maltose / maltodextrin system were studied. The results demonstrate that acarbose is efficiently transported but not metabolized by E. coli due to its poor performance as a substrate of maltodextrin-degrading enzymes. Thus, besides acting as a carbophor acarbose also severely inhibits growth of competitors on maltodextrins. Actinoplanes using starch as carbon source should be able to import maltose and maltodextrins. Experiments with radioactive sugars indicate the action of two different binding-protein dependent ABC transport systems with a multiple substrate spectrum. Within the acb cluster a putative operon (acbHFG) encoding components of an ABC import system was found. To elucidate gene functions the products were overproduced in E. coli and S. lividans and some of the proteins were purified. Surface plasmon resonance analysis showed that the substrate binding protein AcbH binds acarbose and longer derivatives, but not maltose and maltodextrins.

In order to improve the production of acarbose, the fermentation medium of acarbose-producing strain Actinoplanes sp. KCTC 9161 – L14 mutant was optimized in this internship. Fractional factorial design was employ to investigate the influences of glucose, maltose and corn power on acarbose production (by a-glucosidase inhibitory ability). Two significant factors: glucose and maltose have significant and positive effects on acarbose amount. In addition, a model was obtained from the regression results of fractional factorial experiment. Other success, we demonstrated that chromatography by active charcoal column can used to purify acarbose from fermentation broth. Acarbose amount in purification solution was 191.5 g/L and an acarbose - purification process was inducted. / Nhằm mục đích nâng cao khả năng sinh tổng hợp hoạt chất acarbose từ chủng đột biến Actinoplanes sp. KCTC 9161-L14, môi trường lên men của chủng dùng để sản xuất acarbose đã được tối ưu hóa. Một phần mềm thiết kế đã được thiết lập để khảo sát ảnh hưởng của glucose, maltose và bột ngô đến khả năng sản xuất acarbose (thông qua hoạt tính ức chế a-glucosidase). Kết quả đã cho thấy, hai yếu tố glucose và maltose có ý nghĩa quan trọng và ảnh hưởng trực tiếp đến khả năng sinh tổng hợp acarbose. Một phương trình đã được hình thành từ kết quả tối ưu. Bên cạnh đó, chúng tôi đã chứng minh được cột sắc ký sử dụng than hoạt tính có thể tinh sạch acarbose từ dịch lên men. Hàm lượng acarbose trong dung dịch tinh sạch đạt 191,5 g/l và một quy trình tinh sạch acarbose được đề xuất.

In order to improve the production of acarbose, the fermentation medium of acarbose-producing strain Actinoplanes sp. KCTC 9161 – L14 mutant was optimized in this internship. Fractional factorial design was employ to investigate the influences of glucose, maltose and corn power on acarbose production (by a-glucosidase inhibitory ability). Two significant factors: glucose and maltose have significant and positive effects on acarbose amount. In addition, a model was obtained from the regression results of fractional factorial experiment. Other success, we demonstrated that chromatography by active charcoal column can used to purify acarbose from fermentation broth. Acarbose amount in purification solution was 191.5 g/L and an acarbose - purification process was inducted. / Nhằm mục đích nâng cao khả năng sinh tổng hợp hoạt chất acarbose từ chủng đột biến Actinoplanes sp. KCTC 9161-L14, môi trường lên men của chủng dùng để sản xuất acarbose đã được tối ưu hóa. Một phần mềm thiết kế đã được thiết lập để khảo sát ảnh hưởng của glucose, maltose và bột ngô đến khả năng sản xuất acarbose (thông qua hoạt tính ức chế a-glucosidase). Kết quả đã cho thấy, hai yếu tố glucose và maltose có ý nghĩa quan trọng và ảnh hưởng trực tiếp đến khả năng sinh tổng hợp acarbose. Một phương trình đã được hình thành từ kết quả tối ưu. Bên cạnh đó, chúng tôi đã chứng minh được cột sắc ký sử dụng than hoạt tính có thể tinh sạch acarbose từ dịch lên men. Hàm lượng acarbose trong dung dịch tinh sạch đạt 191,5 g/l và một quy trình tinh sạch acarbose được đề xuất.