Ⅰ. BuPT, A New Aromatic Prenyltransferase From Aspergillus Terreus And Its Amazing Substrate PromiscuityⅡ. Microbial Transformations Of Taxadienes And The Multi-drug Resistant Tumor Reversal Activities Of The Metabolites

Part I. BuPT, a new aromatic prenyltransferase from Aspergillus terreus and its amazing substrate promiscuityAromatic prenyltransferases are the key enzymes involved in the biosynthesis of diverse active prenylated aromatics. A new aromatic prenyltransferase gene BuPT has been cloned from the genome of Aspergillus terreus A8-4.(+)-Butyrolactone II could be prenylated by BuPT to produce (+)-butyrolactone I, which first revealing that BuPT is the prenyltransferase gene involved in the biosynthesis of (+)-butyrolactone I. Further investigation of substrate specificities showed the amazing substrate promiscuity of BuPT. Structurally diverse aromatic acceptors and prenyl donors with different number of C5units can be accepted by BuPT as substrates and converted to their corresponding prenylated derivatives. The prenylation of lignanoids, coumarins, flavonoid glycosides, benzophenones and curcuminoids are all first reported. Enzymatical prenylations of different kinds of aromatic acceptors by BuPT have been performed and diverse prenylated aromatic derivatives have been obtained. The structural basis for the promiscuity of BuPT has been also illustrated through the demonstration of its high-resolution X-ray crystal structures. As a new prenyltransferase, the amazing promiscuity of BuPT endow it not only a versatile tool for chemoenzymatic synthesis of diverse prenylated aromatic derivatives, but also a potentially general functional part for secondary metabolism pathway design and construction in synthetic biology of bioactive natural and unnatural products for lead compounds discovery in drug R&D.1. Gene cloning and functional characterization of a putative prenyltransferase gene BuPT from A. terreus A8-4Eight putative soluble aromatic prenyltransferase genes have been cloned from the genome of A. terreus A8-4under the guidance of bioinformatics analysis and sequence blasting results. One of the putative prenyltransferase genes BuPT was overexpressed in Escherichia coli, and the soluble His6-fusion BuPT was purified to homogeneity and biochemically characterised.(+)-Butyrolactone II and dimethylallyl diphosphate (DMAPP) were identified as the substrates of this enzyme to produce (+)-butyrolactone I. BuPT is the first reported prenyltransferase gene involved in the biosynthesis of (+)-butyrolactone I.2. Substrate promiscuity of prenyltransferase BuPTThe substrate specificities of the new prenyltransferase BuPT were investigated.102compounds belonged to23types of aromatics have been tested as the acceptors, which including (+)/(-)-butyrolactone II, flavonoids (flavones, flavonones, flavonols, flavanonols, isoflavones, isoflavanones, chalcones, dihydrochalcones, flavonoid glycosides), xanthones, benzophenones, lignanoids, coumarins, curcuminoids, phenylethyl chromones, stilbenes, indoles (diketopiperazines, tryptophan derivatives), anthraquinones, hydroxynaphthalenes, naphthoquinones, phenolic acids and aromatic amino acids. Among them,65compounds distributed to21types of aromatics could be accepted by BuPT as substrates and enzymatically converted to their corresponding prenylated derivatives with one or more prenyl groups on different positions in high yields. The prenylations of lignanoids, flavonoid glycosides, benzophenones, coumarins, curcuminoids and phenylethyl chromones are all first reported.Moreover, prenyl donors with different number of C5units such as DMAPP (C5), GPP (C10), FPP (C15), PPP (C20) also could be accepted by BuPT to catalyze the prenylation of diverse aromatics including (+)/(-)-butyrolactone II. Prenyltransferases like BuPT with such amazing substrate promiscuity toward both prenyl donors and prenyl acceptors are very unusual in the reported prenyltransferase so far.3. Enzymatic synthesis of diverse prenylated aromatics by BuPT(+)/(-)-Butyrolactone II (lignanoids), cyclo-(L-Trp-L-Trp)(diketopiperazine), genistin (flavonoid glycoside), maackiain (isoflavanone),7-hydroxycoumarin (coumarin) were selected as the prenyl acceptors, and geranyl diphosphate (GPP) was used as the prenyl donor for the enzymatic prenylations. The corresponding prenylated products were obtained by semi-preparative HPLC and identified on the basis of MS and NMR spectroscopic data analyses. Totally,17enzymatic products were obtained and all of them were new compounds. Based on their structures, it is found that BuPT could introduce one or more prenyl moieties on the different positions of aromatics skeleton yielding both O-and/or C-prenylated products.The structural basis for the promiscuity of BuPT was illustrated through the demonstration of its high-resolution X-ray crystal structures, in which there possesses a large pocket at the site of substrate binding, which leads to its acceptance of various aromatic prenyl acceptors and donors.4. Biochemical properties of BuPTThe enzymatic activity in prenylation of aromatics by BuPT was not dependent on divalent metal ions. Effects of different influent factors on BuPT were tested including pH value, reaction temperature and reaction time. The highest activity of BuPT is under the conditions of pH7.0and25-42℃. When GPP was used as the prenyl donor, the product formation increased linearly in the first60minutes and30minutes when (+)-butyrolactone Ⅱ and (-)-butyrolactone Ⅱ were used as the prenyl acceptor respectively. The Km and Vmax values for (+)/(-)-butyrolactone Ⅱ and DMAPP/GPP were also determined respectively. The Km values of GPP were much lower than that of DMAPP, which indicated that BuPT had stronger affinity to GPP as prenyl donor than to. DMAPP. Part Ⅱ. Mierobial transformations of taxadienes and the multi-drug resistant tumor reversal activities of the metabolitesThe clinical treatment of cancer with chemotherapeutic drugs is frequently hindered by either intrinsic or acquired resistance of the tumor cells. When tumor cells acquire resistance against a single chemotherapeutic drug, they often show cross resistance to a variety of antitumor drugs with different structures and/or action mechanisms, a state termed multi-drug resistance (MDR). Discovering novel MDR reversal agents is an effective strategy to overcome this clinical problem. In our previous investigations, structural modifications were performed on sinenxan A and its anologues, a type of4(20),11(12)-taxadiene produced from cell cultures of Taxus chinensis with high yield. Several derivatives with potent reversal activities against MDR tumor cells were obtained. On the basis of these results, in an effort to find more potent derivatives, further structural modification of two derivatives of sinenxan A by enzymatic approach was performed in this dissertation. Sixteen new derivatives were obtained including one with an unusual ring system. Several derivatives showed significant reversal activity toward MDR tumor cell line A549/taxol. The occurred reactions exhibited diversity and many of them were difficult to access by chemical means. These results suggest that biotransformation is a powerful approach to the structural diversification of bioactive natural products with complex structures.1. The biotransformation screening experiment of A1and A2Fourteen strains of filamentous fungi and four strains of actinomycetes were employed as biocatalysts for the screening biotransformation of A1and A2. After analysis by TLC and HPLC-UV, four microbes were selected as follows:the biotransformation of A1by Cunninghamella echinulata CGMCC3.3400, Streptomyces griseus CACC200300, Nocardia purpurea CGMCC4.1182, and the biotransformation of A2by C. echinulata CGMCC3.3400, S. griseus CACC200300, N. purpurea CGMCC4.1182and Aspergillus niger Van Tieghem CGMCC3.1858. 2. Preparative biotransformation of AlThe transformations of A1by the above three microbial strains were scaled-up and ten new products (A3-A12) were obtained by a combination of open silica gel column chromatography and semi-preparative HPLC. On the basis of IR, HR-MS,1D-NMR,2D-NMR and/or single crystal X-ray diffraction analysis, their structures were established as5a-hydroxy-10-oxo-2a,14β-diacetoxytaxa-4(20),11(12)-diene (A3),5a,6a-dihydroxy-10-oxo-2α,14β-diacetoxytaxa-4(20),11(12)-diene (A4),5α-hydroxy-10-oxo-2α,6a,14β-triacetoxytaxa-4(20),11(12)-diene (A5),7β-hydroxy-10-oxo-2a,5α,14β-triacetoxytaxa-4(20),11(12)-diene (A6),5α,7p-dihydroxy-10-oxo-2a,14β-diacetoxytaxa-4(20),11(12)-diene (A7),9a-hydroxy-10-oxo-2a,5α,14β-triacetoxytaxa-4(20),11(12)-diene (A8),9β-hydroxy-10-oxo-2a,5α,14β-triacetoxytaxa-4(20),11(12)-diene (A9),9β,18-dihydroxy-10-oxo-2a,5α,14β-triacetoxytaxa-4(20),11(12)-diene (A10),10α,18-epoxy-10β-hydroxy-9-oxo-2α,5α,14β-triacetoxytaxa-4(20),11(12)-diene (All) and5a,14β-dihydroxy-10-oxo-2a-acetoxytaxa-4(20),11(12)-diene (A12); The reactions exhibited diversity, including selective hydroxylation, acetylation, deacetylation, oxidation and acetalization.It is worth to mention that a Furantaxane (All) with an unusual6/8/6/5ring system was obtained from the biotransformation of Al by S. griseus. Single-crystal X-ray diffraction revealed that an additional furan ring comprising C12-C18-O-C10-C11was formed which was first reported in either natural obtained or chemical synthesized taxanes. The plausible bioconversion routes of A1were proposed.3. Preparative biotransformation of A2The transformations of A2by the above four microbial strains were scaled-up and fourteen products including six new compounds (A13-A14, A19-A20, A22, A24) were obtained by a combination of open silica gel column chromatography and semi-preparative HPLC. On the basis of IR, HR-MS,1D-NMR,2D-NMR analysis, their structures were established as5α,9a-dihydroxy-10β-methoxy-2α,14β-diacetoxytaxa-4(20),11(12)-diene (A13),5a,9a,10β-trihydroxy-2a,14p-diacetoxytaxa-4(20),11(12)- diene (A14),5α,18-dihydroxy-10β-methoxy-2a,14β-diacetoxytaxa-4(20),11(12)diene (A19),5α,10β,18-trihydroxy-2α,14β-diacetoxytaxa-4(20),11(12)-diene(A20),5α-hydroxy-2α,14β-diacetoxytaxa-4(20),11(12)-diene-10β-O-(butan-2-ol)-ether (A22),10p-methoxy-2α,5α,14β-trihydroxytaxa-4(20),11(12)-diene (24). The reactions exhibited diversity, including selective hydroxylation, acetylation, deacetylation, methylation, demethylation, epoxidation, oxidation, and O-alkylation. The plausible bioconversion routes for metabolites were hypothesized.4. MDR reversal activities for metabolites in vitroTwenty-four metabolites along with substrates Al, A2were subjected to the evaluation of MDR reversal activity toward MDR tumor cell line A549/taxol when co-administered with paclitaxel at10μM. Several of the metabolites exhibited higher reversal activities than A1and A2. Compound A18had approximately two-fold higher activity than verapamil and five times higher activity than A2. Compound A26had comparable activity to verapamil and two times higher activity than A2. Furantaxane (All) exhibited significant reversal activity which was comparable to verapamil and two times higher than Al. All of the metabolites exhibited weak cytotoxicity, which is a good property for a reversing agent. Thus, the results showed that these compounds might be promising lead compounds for reversal agents against A549/taxol tumor MDR cells.