Mikrobielle Biosynthese
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Date
2015
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Abstract
Diese Arbeit beschäftigt sich anhand ausgewählter Beispiele aus den zwei großen Naturstoffklassen der Terpene und Polyketide mit unterschiedlichen (chemo-)fermentativen und (chemo-)enzymatischen Verfahren, um die Verfügbarkeit von Naturstoffen zu verbessern oder deren Derivatsierbarkeit zu ermöglichen.
Zur Verbesserung der Verfügbarkeit der Naturstofffamilie der Diterpenfusicoccane wurde die präparative, heterologe Fermentation des biosynthetischen Intermediats Fusicocca-2,10(14)-dien in einem durch Metabolic Engineering optimierten Bäckerhefestamm etabliert. Dabei wurde die Ausbeute durch eine systematische Variation der Fermentationsparameter von anfänglich 6 mg/L auf 30 mg/L gesteigert.
Neben der Optimierung äußerer Parameter, wurde sich auch mit der Identifizierung inhärenter, limitierender Schritte beschäftigt. Eine Analyse der unabhängigen Expression der beiden Domänen des bifunktionellen Schlüsselenzyms bei der Biosynthese des Fusicocca-2,10(14)-diens wies darauf hin, dass die lösliche Expression des Gesamtproteins durch die schlechte lösliche Expression der N-terminalen Terpencyclasedomäne limitiert sein könnte. Um die intrinsische Stabilität dieses Enzyms oder der Cyclasedomäne zu erhöhen, wurde ein Verfahren für die gerichtete Proteinevolution diese Enzyms und einzelner Domänen entwickelt. In einer ersten Generation wurden bereits Hot Spots für die Erzeugung löslicherer Enzymvarianten identifiziert.
Zur Erzeugung einfacher Diterpenfusiocaccananaloga, um die phytotoxischen und anticarcinogenen Wirkungen dieser Substanzfamilie nachzuahmen, wurde die enzymatische, oxidative Derivatisierung des Fusicocca-2,10(14)-diens in vitro anvisiert.
Alternativ zu der Ganzzellfermentation wurde auch ein Konzept zur in vitro Totalsynthese von Terpenen mittels thermostabiler Enzyme vorgestellt.
Außerdem wurde sich mit der Substraterkennung in der Polyketidbiosynthese beschäftigt. Durch gezielte Mutagenese und Fütterung unnatürlicher Substratderivate konnten zwei neue Erythromycinderivate erzeugt sowie die Einbaurate der artifiziellen Bausteine verbessert werden. Alternativ konnten durch die Ausnutzung der natürlichen Substrattoleranz der Polyketidsynthase in der Monensinbiosynthese durch die Fütterung artifizieller Bausteine drei neue Monensinderivate massenspektrometrisch nachgewiesen werden.
This work describes different (chemo-)fermentative and (chemo-)enzymatic procedures to enhance the availability or to enable the derivatisation of terpenes or polyketides. To enhance the availability of the natural product family of the diterpene fusicoccanes, the heterologous fermentation of the biosynthetic intermediate fusicocca-2,10(14)-diene in a metabolic engineered baker’s yeast strain was established. Through systematic variation of the fermentation parameters the yield was increased from 6 mg/L to 30 mg/L. Besides the variation of fermentation parameters, it was also intended to enhance the yield by addressing inherent limitations. The individual expression of the two domains of the bifunctional key enzyme responsible for the biosynthesis of fusicocca-2,10(14)-diene, revealed low soluble expression of the N-terminal terpene cyclase domain. To increase folding stability of the whole enzyme or the terpene cyclase domain directed evolution was used. In a first generation already several hot spots for the production of variants with a higher solubility were identified. To mimic the phytotoxic or anticarcinogenic bioactivities of the diterpene fusicoccanes, enzymatic oxidation tools were applied to produce simple fusicoccane analogs starting from fusicocca-2,10(14)-diene. As an alternative to the use of whole cell biocatalysts for the production of complex terpenes, also a concept for the in vitro enzymatic total synthesis with thermostable enzymes was introduced. Furthermore this thesis deals with the substrate recognition in the polyketide biosynthesis. By targeted mutagenesis and feeding of unnatural substrates two new erythromycin derivatives where produced and the incorporation of artificial building blocks was enhanced. Alternatively, by exploiting the natural substrate tolerance of the polyketide synthase of the monensin biosynthesis and feeding of artificial substrates three new monensin derivatives were detected by mass spectrometry.
This work describes different (chemo-)fermentative and (chemo-)enzymatic procedures to enhance the availability or to enable the derivatisation of terpenes or polyketides. To enhance the availability of the natural product family of the diterpene fusicoccanes, the heterologous fermentation of the biosynthetic intermediate fusicocca-2,10(14)-diene in a metabolic engineered baker’s yeast strain was established. Through systematic variation of the fermentation parameters the yield was increased from 6 mg/L to 30 mg/L. Besides the variation of fermentation parameters, it was also intended to enhance the yield by addressing inherent limitations. The individual expression of the two domains of the bifunctional key enzyme responsible for the biosynthesis of fusicocca-2,10(14)-diene, revealed low soluble expression of the N-terminal terpene cyclase domain. To increase folding stability of the whole enzyme or the terpene cyclase domain directed evolution was used. In a first generation already several hot spots for the production of variants with a higher solubility were identified. To mimic the phytotoxic or anticarcinogenic bioactivities of the diterpene fusicoccanes, enzymatic oxidation tools were applied to produce simple fusicoccane analogs starting from fusicocca-2,10(14)-diene. As an alternative to the use of whole cell biocatalysts for the production of complex terpenes, also a concept for the in vitro enzymatic total synthesis with thermostable enzymes was introduced. Furthermore this thesis deals with the substrate recognition in the polyketide biosynthesis. By targeted mutagenesis and feeding of unnatural substrates two new erythromycin derivatives where produced and the incorporation of artificial building blocks was enhanced. Alternatively, by exploiting the natural substrate tolerance of the polyketide synthase of the monensin biosynthesis and feeding of artificial substrates three new monensin derivatives were detected by mass spectrometry.
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Keywords
Terpene, Polyketide, Biosynthese, Biokatalyse, Chemoenzymatische Synthese