Molekularbiologische und biochemische Untersuchungen zu Enzymen in der Biosynthese von prenylierten Indolalkaloiden

Fungi, bacteria and plants produce a large number of secondary metabolites. Their advantage in the organism itself is often not yet clear, nevertheless numerous secondary metabolites have an extraordinary significance for humanity. Penicillin has shaped medicine to this day, but lovastatin is also a good example of the potential of secondary metabolites in fungi. Among others the prenylated indole alkaloids are also an interesting group of secondary metabolites. Compared to the respective unprenylated substance, the introduction of a prenyl moiety and the associated increase in lipophilicity can improve biological activity. Indole alkaloids can be composed of tryptophan-containing cyclic dipeptides. Due to the heteroatoms and the versatility in the side chains of the amino acids, the diketopiperazine scaffold of the cyclic dipeptides provides an ideal prerequisite for further modifications such as acetylations, methylations, cyclizations or oxidations. The resulting modifications and functional groups lead to a variety of simple and complex natural products with interesting biological activities. The natural product group of ardeemines from Aspergillus fischeri is a fumiquinazoline (FQ) tripeptide framework of anthranilic acid, D-Ala and L-Trp. A very efficient biosynthetic pathway consisting of the NRPS ArdA and the prenyltransferase ArdB leads to a hexacyclic system (ardeemin) and the acetyltransferase ArdC subsequently forms 5-N-acetylardeemine. Both structures, ardeemin and 5-N-acetylardeemin, exhibit interesting biological activity as a multi-drug resistance export pump inhibitor. Due to the pharmaceutically relevant and promising activities as chemotherapy drugs, fumiquinazolins and related structures have been part of research for years. Through the use of the cyclic dipeptide prenyltransferases FtmPT1, BrePT, CdpNPT, CdpC3PT and AnaPT in this work, 14 different ardeemin FQ and ent Ardeemin FQ derivatives were identified and characterized by LC-MS and NMR analyses. These enzymes catalyzed the prenyl transfer to all the six different positions N1, C2, C3, C5, C6 and C7 of the indole ring. Ardeemin FQ was much better accepted with conversions of up to 77% than its enantiomer ent Ardeemin FQ with total conversions of maximum 22%, which were usually spread over several products. In a second project, 5-DMATS and FgaPT2 mutants were generated by site-directed mutagenesis to enable the production of C5-prenylated cyclo-L-Trp-L-Pro. Despite the successfully produced mutants 5-DMATS_R243L, 5-DMATS_R243L_L327G and FgaPT2_R244L_Y398F, they hardly showed any activity or only little of the desired product was detected. Parallel to these mutants, the multiple prenylating enzyme EchPT2 was mutated with the aim of controlling the multiprenylations and expanding the acceptance of the donor GPP. The successfully obtained mutants EchPT2_A404W, EchPT2_A404F, EchPT2_A404Y and EchPT2_A404L showed no activity towards C2-reverse prenylated cyclo-L-Trp-L-Pro, C2-reverse prenylated cyclo-L-Trp-L-Ala or Tryprostatin B. The mutant EchPT2_A404Y, however, accepted the unprenylated substrate cyclo L-Trp-L-Pro leading to two product peaks. The mutant EchPT2_L334G catalyzed the transfer of GPP to C2-revers prenylated cyclo-L-Trp-L-Pro substrate with a moderate conversion of 14% spread over at least three products. In the third project the substrate specificity of cytochrome P450 (CYP) enzymes were investigated by heterologous expression of the CYP genes ftmP450-1 and ftmP450-2 from Neosartorya fischeri in Saccharomyces cerevisiae (S. cerevisiae) and subsequent feeding of prenylated substrates such as mainly cyclo L-Trp-L-Pro derivatives. The prenylated substrates were either produced by co-expression of a prenyltransferase together with the respective CYP genes in S. cerevisiae or chemo-enzymatically with recombined proteins and then fed. The following heterologous expression systems were successfully established in S. cerevisiae KO3: for monoexpression of ftmP450-1, ftmP450-2, ftmPT1 and brePT as well as for co-expression of ftmPT1 with ftmP450-1, brePT with ftmP450-1 and ftmPT1 with ftmP450-2. For the heterologous expression systems in S. cerevisiae containing a prenyltransferase, unprenylated cyclic dipeptides were fed. Overall these cyclic dipeptides were well accepted and converted to the prenylated products. For the heterologous expression systems with ftmP450-1 or ftmP450-2, but without a prenyltransferase, the regular C2-prenylated cyclic dipeptides cyclo-L-Trp-L-Pro (tryprostatin B), cyclo-D-Trp-D-Pro, cyclo-L-Trp-D-Pro, cyclo-D-Trp-L-Pro, cyclo-L-Trp-L-Leu and cyclo-L-Trp-L-Ala were tested. In comparison ftmP450-2 showed to be more tolerant towards these substrates than ftmP450-1, which mostly showed no conversion. Nevertheless, ftmP450-1 could also be used to test substrates that contained modifications in the prenyl moiety, as it is not directly involved in the oxidation reaction. For this reason, it was possible to feed cyclo-L-Trp-L-Pro derivatives with reverse prenylation at C2, regular prenylation at C4 – C7 or regular geranylation at C2. The shift of the prenyl group to the positions C4 - C7 did not lead to any conversion with ftmP450-1, however, the substrates that carried a modified prenyl unit at C2 were also well accepted, showing the necessity of the prenylation at C2. The products of ftmP450-1 were characterized by LC-MS and NMR analysis.