Investigation on Cyclodipeptide Formation and Metabolism in Actinomycetes and Application of Fungal Prenyltransferases in the Chemoenzymatic Synthesis

Secondary metabolites originated from plants, bacteria and fungi constitute a large group of compounds, which are not directly related to the growth, development and reproduction of the organism. However, the producer can benefit from these metabolites to defend itself against natural enemies, compe...

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1. Verfasser: Yu, Huili
Beteiligte: Li, Shu-Ming (Prof. Dr.) (BetreuerIn (Doktorarbeit))
Format: Dissertation
Sprache:Englisch
Veröffentlicht: Philipps-Universität Marburg 2018
Pharmazeutische Biologie
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Zusammenfassung:Secondary metabolites originated from plants, bacteria and fungi constitute a large group of compounds, which are not directly related to the growth, development and reproduction of the organism. However, the producer can benefit from these metabolites to defend itself against natural enemies, competitors or environmental pressure. Furthermore, they often exhibit useful and interesting biological and pharmaceutical activities, which provide benefits to human health and life. For example, cyclodipeptides (CDPs) and their derivatives have gained increasing attention for their interesting and diverse pharmaceutical activities. Natural CDPs are mainly formed by nonribosomal peptide synthetases (NRPSs) or cyclodipeptide synthases (CDPSs). The CDP cores resulted from the NRPS or CDPS pathways are often further modified by varieties of tailoring enzymes leading to the formation of sophisticated natural products. The small size of CDPSs (200−300 residues) makes them genetically easily manipulated in synthetic biology. In the first project of this thesis, a cooperation project with Jing Liu, nine new CDPS genes from eight Streptomyces strains were functionally characterized after cloning into pET28a vector and expressing in E. coli. The products were analyzed on LC-MS and then isolated for NMR analysis. The products were identified as tryptophan-containing cyclodipeptides (cWXs) that are the most common precursors of diketopiperazines (DKPs) with pharmaceutical interest, due to the various modification possibilities on the indole ring. The nine identified CDPSs include six specific CDP synthases consisting of one cWL synthase, two cWP synthases and three new cWW synthases as well as three unspecific CDPSs producing several products with cWA or cWY as the major cyclodipeptide. Total product yields of CDPs were calculated based on HPLC analysis to be 46 to 211 mg/L E. coli culture. Our findings represent rare examples of cWXs derived from CDPS pathways in actinobacteria. This study also provides a valid experimental basis for further combination of CDPSs with other tailoring enzymes in biosynthesis of interesting compounds. The three identified cWW synthases from Streptomyces purpureus NRRL B-5737, Streptomyces lavendulae NRRL B-2774, and Streptomyces xanthophaeus NRRL B-5414 are genetically associated with a putative cytochrome P450. The three P450 homologues share sequence identities of more than 75% with each other on the amino acid level, indicating same or very similar function. As a representative, the function of P450NB5737 from S. purpureus was identified by heterologous expression in Streptomyces coelicolor and in vitro assays with the recombinant protein produced in E. coli. Isolation and structure elucidation confirmed that P450NB5737 catalyzes the transfer of a guanine moiety onto the indole ring of cWW via a C–N bond, leading to unprecedented adducts of cyclodipeptide with a nucleobase. This represents one additional example of rare P450 enzymes catalyzing the coupling of two different substrates. Besides CDPs, prenylated aromatic products often possess various biological and pharmacological activities, in comparison to their non-prenylated precursors. The prenyltransferases of the DMATS superfamily show broad flexibility toward aromatic substrates, but relatively strict prenyl donor tolerance. They usually accept only DMAPP, but not GPP or other prenyl donors with a longer chain length. To broaden the prenyl donor spectra, the acceptance of tyrosine O-prenyltransferases TyrPT and SirD towards unnatural DMAPP analogs such as MAPP, 2-pen-PP, and benzyl-PP was tested in the presence of tyrosine and its derivatives. The results revealed that TyrPT and SirD also use the unnatural donors and exhibit similar behavior as their natural prenyl donor DMAPP. Alkylated or benzylated products at the same positions of that with DMAPP have therefore been identified. This study increased significantly the diversity of prenylated products by chemoenzymatic synthesis. The results presented in this thesis contributed significantly to the formation and metabolism of tryptophan-containing CDPs in nature and expanded the potential application of prenyltransferases. They provide a solid basis for applications in the synthetic biology in the future. 
Beschreibung:147 pages.
DOI:https://doi.org/10.17192/z2018.0497