Applications of Fungal Prenyltransferases in the Chemoenzymatic Synthesis

Plants, bacteria, and fungi provide diverse structures derived from their primary and the secondary metabolism. Representative substances from secondary metabolite pathways are flavonoids, coumarins, xanthones, and indole alkaloids. The attachment of isoprene units (n × C5) such as dimethylallyl (DM...

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Main Author: Zhou, Kang
Contributors: Li, Shu-Ming (Prof. Dr.) (Thesis advisor)
Format: Dissertation
Language:English
Published: Philipps-Universität Marburg 2017
Pharmazeutische Technologie und Biopharmazie
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Summary:Plants, bacteria, and fungi provide diverse structures derived from their primary and the secondary metabolism. Representative substances from secondary metabolite pathways are flavonoids, coumarins, xanthones, and indole alkaloids. The attachment of isoprene units (n × C5) such as dimethylallyl (DMAPP), geranyl (GPP), or farnesyl (FPP) moieties to the backbones of aromatic secondary metabolites is a further step for the broad diversification. Prenylated natural products often exhibit stronger pharmacological activities than their non-prenylated precursors. The prenyltransferases (PTs) accomplish these prenyl transfer reactions in nature. Therefore, the investigation on the applications of prenyltransferases could be used for structural modification of aromatic substances to produce biologically active compounds. Prenylated acylphloroglucinols (APs), which have remarkable chemical structures and intriguing biological and pharmacological activities, are characteristic constituents of several plant families. Main structural features of prenylated APs are highly oxygenated and densely decorated with prenyl moieties, such as dimethylallyl and geranyl moieties. Phlorisobutyrophenone (PIBP), phlorisovalerophenone (PIVP), and phlorbenzophenone (PBZP) serve as precursors of most prenylated APs. In the first part of this thesis, the acceptance of APs catalyzed by thirteen fungal prenyltransferases in the presence of DMAPP was elucidated. Nine regular prenylated products were obtained from the reactions with AnaPT. The results indicated that AnaPT catalyzes the same prenylation of PIBP and PIVP as the membrane-bound prenyltransferases like HIPT-1 involved in the biosynthesis of the prenylated APs in plants, but with much higher conversion yields than HIPT-1. However, only monoprenylated derivatives were obtained in the presence of DMAPP and the conversion yields of PIBP, PIVP, and PBZP with GPP as prenyl donor were very low in AnaPT reactions. Recently, a fungal prenyltransferase AtaPT was demonstrated to carry an unprecedented promiscuity toward diverse drug-like aromatic acceptors and prenyl donors including DMAPP, GPP, and FPP. On the availability of AtaPT, we investigated the behavior of AtaPT toward PIBP, PIVP, and PBZP. Twenty-one prenylated APs were isolated and their structures were elucidated by NMR and MS analyses. Total conversion yields were calculated for the three APs with AtaPT and DMAPP, which are significantly higher than those of AnaPT. C-prenylated products are in consistent with the AnaPT products. O-prenylated products were also obtained from the reaction mixtures of PIBP and PIVP. Also Gem-diprenylated derivatives were identified in the reaction mixtures of these substrates respectively. C-monoprenylated products were converted into gem-diprenylated derivatives as predominant products in the presence of DMAPP. GPP and FPP also served as good prenyl donors for the reaction of AtaPT. Only one C-prenylated derivative and one Oprenylated derivative each were identified from these incubation mixtures. Subsequently, prenylation of different flavonoids including flavanones and isoflavones by AnaPT at C-6 of the A ring or C-3′ of the B ring was demonstrated. Twelve prenylated flavonoids from incubation mixtures of flavonoids with AnaPT in the presence of DMAPP were produced. Previous studies found that 7-DMATS accepted chalcones, isoflavonoids, and flavanones much better than flavones and flavonols and mainly catalyzed prenylation at C-6. AnaPT and 7-DMATS show different substrate preferences and prenylation positions. In the third part of this thesis, we identified a key amino acid residue Tyr205 in FtmPT1 for the interaction with its aromatic substrate brevianamide F. Saturation mutagenesis on this position resulted in all nineteen possible mutants. FtmPT1_Y205N and FtmPT1_Y205L differ from FtmPT1 in behaviors toward four cyclo-Trp-Pro isomers. Regularly C2- prenylated derivatives were detected as main products of FtmPT1 reactions with all these isomers. In contrast, the reversely C3-prenylated products were found to be the main products in Y205N and Y205L reactions with cyclo-D-Trp-D-Pro, cyclo-D-Trp-L-Pro, and cyclo-L-Trp- D-Pro, while regularly C2- and C3-prenylated derivatives were identified in their reaction mixtures with cyclo-L-Trp-L-Pro. These results indicated that the isomers are in different positions and orientations in the reaction chamber and Tyr205 is important for the prenyl transfer reaction, but can be replaced by other amino acids. The results obtained during this thesis demonstrate that AtaPT and AnaPT could be promising candidates for production of prenylated APs like -bitter acids by synthetic biology. AnaPT and 7-DMATS could be used complementarily for prenylation of flavonoids. The mutants of FtmPT1 can be used for production of regularly C3-prenylated brevianamide F in synthetic biology.
DOI:https://doi.org/10.17192/z2017.0238