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Heterologous expression of a three-gene cluster from Streptomyces aurantiacus coding for a cyclodipeptide synthase, a prenyltransferase, and a methyltransferase led to the elucidation of the biosynthetic steps of streptoazine C (2). In vivo biotransformation experiments proved the high flexibility of the prenyltransferase SasB toward tryptophan-containing cyclodipeptides for regular C-3-prenylation. Furthermore, their corresponding dehydrogenated derivatives prepared by using cyclodipeptide oxidases were also used for prenylation. This study provides an enzyme with high substrate promiscuity from a less explored group of prenyltransferases for potential use to generate prenylated derivatives.

Abstract Cytochrome P450 monooxygenases (P450s) are considered nature’s most versatile catalysts and play a crucial role in regio- and stereoselective oxidation reactions on a broad range of organic molecules. The oxyfunctionalisation of unactivated carbon-hydrogen (C-H) bonds, in particular, represents a key step in the biosynthesis of many natural products as it provides substrates with increased reactivity for tailoring reactions. In this study, we investigated the function of the P450 enzyme TraB in the terrestric acid biosynthetic pathway. We firstly deleted the gene coding for the DNA repair subunit protein Ku70 by using split marker-based deletion plasmids for convenient recycling of the selection marker to improve gene targeting in Penicillium crustosum. Hereby, we reduced ectopic DNA integration and facilitated genetic manipulation in P. crustosum. Afterward, gene deletion in the Δku70 mutant of the native producer P. crustosum and heterologous expression in Aspergillus nidulans with precursor feeding proved the involvement of TraB in the formation of crustosic acid by catalysing the essential hydroxylation reaction of viridicatic acid. Key points •Deletion of Ku70 by using split marker approach for selection marker recycling. •Functional identification of the cytochrome P450 enzyme TraB. •Fulfilling the reaction steps in the terrestric acid biosynthesis.

Secondary metabolites derived from microorganism constitute an important part of natural products. Mining of the microbial genomes revealed a large number of uncharacterized biosynthetic gene clusters, indicating their greater potential to synthetize specialized or secondary metabolites (SMs) than identified by classic fermentation and isolation approaches. Various bioinformatics tools have been developed to analyze and identify such gene clusters, thus accelerating significantly the mining process. Heterologous expression of an individual biosynthetic gene cluster has been proven as an efficient way to activate the genes and identify the encoded metabolites that cannot be detected under normal laboratory cultivation conditions. Herein, we describe a concept of genomics-guided approach by performing genome mining and heterologous expression to uncover novel CDPS-derived DKPs and functionally characterize novel tailoring enzymes embedded in the biosynthetic pathways. Recent works focused on the identification of the nucleobase-related and dimeric DKPs are also presented.

Expression of a nonribosomal peptide synthetase-nonreducing polyketide synthase hybrid gene pcr10109 from Penicillium crustosum PRB-2 in Aspergillus nidulans led to the accumulation of 4-hydroxy-6-(4-hydroxyphenyl)-α-pyrone (1). Adding para-hydroxybenzoic acid into the medium in which the overexpressing mutant is growing increased the product yield up to 5-fold. This strategy could be helpful for heterologous gene expression experiments requiring special substrates for product formation.

Accumulation of two benzoyl esters in Aspergillus ustus after feeding with alcohols was reported 30 years ago. To the best of our knowledge, the biosynthesis for these esters has not been elucidated prior to this study. Here, we demonstrate that these compounds are artifical products of the phenethyl polyketide ustethylin A biosynthestic pathway. In addition, four aditional benzoyl esters with different methylation levels were also isolated and identified as shunt products. Feeding experiments provided evidence that the enzyme-bound polyketide acyl intermediates are hijacked by externally fed MeOH or EtOH, leading to the formation of the benzoyl esters. Graphic abstract

2-Alkenyl-tetrahydropyrans belong to a rare class of natural products that exhibit broad antifungal activities. Their structural instability and rareness in nature have restrained their discovery and drug development. In this study, the heterologous expression of a single highly reducing polyketide synthase (HR-PKS, App1) from Trichoderma applanatum in Aspergillus nidulans leads to the formation of seven 2-alkenyl-tetrahydropyran derivatives including one known compound virensol C (1) and six new compounds (2–7). However, introducing App1 into Saccharomyces cerevisiae resulted in the identification of additional two 2-alkenyl-tetrahydropyrans lacking the hydroxyl or methoxyl group at the C-2 position (8 and 9). The structures of the isolated compounds were elucidated by extensive spectroscopic analysis using NMR and HR-ESI-MS.

The echinulin family alkaloids can be grouped into three series depending on the number of the exo double bonds adjacent to the diketopiperazine core structure. Heterologous expression of the putative echinulin biosynthetic gene cluster from Aspergillus ruber in Aspergillus nidulans led to accumulation of echinulin without a double bond and neoechinulin A with one double bond (Δ10) as major products. Their analogues with a different number of prenyl moieties were detected as minor products. Neoechinulin B and analogues with two double bonds (Δ10,14) were not observed. Feeding experiments confirmed that the cytochrome P450 enzyme EchP450 only catalyzes the formation of the double bond between C10 and C11. Coincubation and substrate concentration dependent assays with the prenyltransferase EchPT2 revealed that the reversely C2-prenylated preechinulin without a double bond is a much better substrate than neoechinulin A. These results prove that preechinulin serves as a common substrate for the formation of echinulin by two regiospecific prenylation steps with EchPT2 or for EchP450 to introduce one double bond and subsequent prenylations with low regioselectivity.

Biflavonoids are pharmaceutically important compounds. Prenylation usually improves bioactivity; however, prenylated biflavonoids are rare in nature. Here, we report successful prenylation or geranylation of biflavonoids using fungal prenyltransferase CdpC3PT and its mutants. F253 was identified as a key residue related to donor selectivity, which enables the switching from utilizing DMAPP to GPP precisely at the same C-3''' site of biflavonoids. Furthermore, another residue W181 was discovered to generally increase prenylation activity toward biflavonoids.

A highly oxygenated phenethyl derivative ustethylin A was isolated from Aspergillus ustus. Gene deletion, isotope labeling, and heterologous expression proved that the phenethyl core structure is assembled from malonyl-CoA by a polyketide synthase harboring a methyltransferase domain. Propionate was converted via acetyl-CoA to malonyl-CoA and incorporated into the molecule. Modifications on the core structure by three different oxidoreductases and one O-methyltransferase lead to the final product, ustethylin A.

NotF from Aspergillus sp. MF297-2 and BrePT from Aspergillus versicolor catalyze a reverse C2-prenylation of brevianamide F in the biosynthetic pathway of brevianamides and notoamides. NotF was reported to use only brevianamide F as substrate while BrePT demonstrated broad substrate promiscuity. With high identity at amino acid level, it is interesting to reinvestigate the catalytic activities of these two prenyltransferases in vitro toward 14 cyclodipeptides. Product identification of the in vitro assays by MS proved that NotF and BrePT share similar catalytic ability and substrate promiscuity.

Crustosic acid (1) differs from terrestric acid (2) by a 5β-carboxylmethyl at the tetronate ring instead of a 5α-methyl group in Penicillium crustosum. The formation of 1 via carboxylcrustic and viridicatic acid was confirmed by gene deletion and heterologous expression. The conversion of 1 to 2 requires a decarboxylation-mediated olefination by TraH and subsequent reduction by TraD. The redox-assisted decarboxylation and stereoisomerization proved the biosynthetic relationships of fungal acyltetronates with different stereochemistry.

The cyclo-l-Trp-l-Trp (cWW, 1) tailoring P450 GutD2774 from Streptomyces lavendulae was characterized by expression in Streptomyces coelicolor, precursor feeding and enzyme assays. GutD2774 catalyzes mainly the transfer of hypoxanthine to C2 and C3 of the indole ring of 1. cWW adducts with guanine were detected as minor products. An orthologous cluster was identified in Streptomyces xanthophaeus. These results expand the spectrum of cyclodipeptide derivatives by involvement of an additional nucleobase and identification of new coupling patterns.

Heterologous expression in Streptomyces coelicolor and in vitro enzyme characterization proved that two P450 enzymes, AspB and NasB, from Streptomyces sp. NRRL S-1868 catalyze two new dimerization patterns of tryptophan-containing cyclodipeptides. Structure elucidation of the metabolites revealed an N1-C7' dimer of two cWP molecules as the predominant product of AspB and C3-C7' connected cWP with cWA as that of NasB.

Heterologous expression of secondary metabolite genes and gene clusters has been proven to be a successful strategy for identification of new natural products of cryptic or silent genes hidden in the genome sequences. It is also a useful tool to produce designed compounds by synthetic biology approaches. In this study, we demonstrate the potential usage of the gene locus pcr4401 in the fast-growing filamentous fungus Penicillium crustosum as an integration site for heterologous gene expression. The deduced polyketide synthase (PKS) Pcr4401 is involved in the dihydroxynaphthalene (DHN)-melanin pigment formation, and its deletion in P. crustosum PRB-2 led to an albino phenotype. Heterologous expression of pcr4401 in Aspergillus nidulans proved its function as the melanin precursor YWA1 synthase. To ensure gene expression after genomic integration and to easily identify the potential transformants by visualization, the gene locus of pcr4401 was chosen as an integration site. For heterologous expression in P. crustosum, the expression constructs were created by ligation-independent homologous recombination in Escherichia coli or Saccharomyces cerevisiae. A pyrG deficient strain was also created, so that both the pyrG and hph resistance gene can be used as selection markers. Successful expression in P. crustosum was demonstrated by using one uncharacterized PKS gene from Aspergillus and two from Penicillium strains. All three genes were successfully introduced, heterologously expressed, and their biosynthetic products elucidated. The results presented in this study demonstrated that P. crustosum can be used as a suitable host for heterologous expression of secondary metabolite genes.

Penilactones A and B consist of a γ-butyrolactone and two clavatol moieties. We identified two separate gene clusters for the biosynthesis of these key building blocks in Penicillium crustosum. Gene deletion, feeding experiments, and biochemical investigations proved that a nonreducing PKS ClaF is responsible for the formation of clavatol and the PKS-NRPS hybrid TraA is involved in the formation of crustosic acid, which undergoes decarboxylation and isomerization to the predominant terrestric acid. Both acids are proposed to be converted to γ-butyrolactones with involvement of a cytochrome P450 ClaJ. Oxidation of clavatol to hydroxyclavatol by a nonheme FeII/2-oxoglutarate-dependent oxygenase ClaD and its spontaneous dehydration to an ortho-quinone methide initiate the two nonenzymatic 1,4-Michael addition steps. Spontaneous addition of the methide to the γ-butyrolactones led to peniphenone D and penilactone D, which undergo again stereospecific attacking by methide to give penilactones A/B.

Nonribosomal peptide synthetase (NRPS)-like enzymes comprising A-T-TE architectures catalyze the dimerization of α-keto carboxylic acids leading to the formation of hydroxybenzoquinones or lactones. Domain change experiments with five enzymes revealed that A and A-T domains of phenyl or 4-hydroxyphenyl pyruvate-using enzymes can be effectively used by the TE domains of other enzymes. Even the A and A-T domains of an indolyl hydroxybenzoquinone synthase were successfully recombined with TE domains of a phenyl and a 4-hydroxyphenyl pyruvate-activating enzyme.

Covering: up to July 2020Naturally occurring chalcones carrying up to three modified or unmodified C5-, C10-, and C15-prenyl moieties on both rings A and B as well as at the α- and β-carbons are widely distributed in plants of the families of Fabaceae, Moraceae, Zingiberaceae and Cannabaceae. Xanthohumol and isobavachalcone being the most investigated representatives, exhibit diverse and remarkable biological and pharmacological activities. The present review deals with their structural characters, biological activities and occurrence in the plant kingdom. Biosynthesis of prenylated chalcones and metabolism of xanthohumol are also discussed.

Oxepinamides are fungal oxepine-pyrimidinone-ketopiperazine derivatives. In this study, we elucidated the biosynthetic pathway of oxepinamide D in Aspergillus ustus by gene deletion, heterologous expression, feeding experiments, and enzyme assays. We demonstrated that the cytochrome P450 enzymes catalyzed highly specific and stereoselective oxepin ring formation.

Anthocyanins are a type of well-known natural flavonoids for their various beneficial health effects. However, prenylated anthocyanins are not discovered in nature although prenylation is believed to generally enhance the biological accessibility of flavonoids. In this article, we demonstrate the first example for prenylation of anthocyanins. A chemo-enzymatic approach was achieved for the synthesis of a series of 7-O-prenylated anthocyanins, using the fungal prenyltransferase CdpC3PT from Neosartorya fischeri.

Prenylation is a process widely prevalent in primary and secondary metabolism, contributing to functionality and chemical diversity in natural systems. Due to their high regio- and chemoselectivities, prenyltransferases are also valuable tools for creation of new compounds by chemoenzymatic synthesis and synthetic biology. Over the last ten years, biochemical and structural investigations shed light on the mechanism and key residues that control the catalytic process, but to date crucial information on how certain prenyltransferases control regioselectivity and chemoselectivity is still lacking. Here, we advance a general understanding of the enzyme family by contributing the first structure of a tryptophan C5-prenyltransferase 5-DMATS. Additinally, the structure of a bacterial tryptophan C6-prenyltransferase 6-DMATS was solved. Analysis and comparison of both substrate-bound complexes led to the identification of key residues for catalysis. Next, site-directed mutagenesis was successfully implemented to not only modify the prenyl donor specificity but also to redirect the prenylation, thereby switching the regioselectivity of 6-DMATS to that of 5-DMATS. The general strategy of structure-guided protein engineering should be applicable to other related prenyltransferases, thus enabling the production of novel prenylated compounds.

Heterologous expression has been proven to be a successful strategy for the identification of metabolites encoded by cryptic/silent genes. Expression of a nonreducing polyketide synthase (NR-PKS) gene from Penicillium crustosum in Aspergillus nidulans led to the accumulation of three isocoumarins 1-3. Feeding experiments revealed that the PKS product 1 can be converted by the host enzymes to its hydroxylated (2) and methylated (3) derivatives. These results provided one additional example that unexpected further modifications of an enzyme product can take place in a heterologous host.

The attachment of a dimethylallyl moiety to C4 of 1,3-dihydroxynaphthalene led to spontaneous oxidative cyclisations, resulting in the formation of two tetrahydrobenzofuran and one bicyclo[3.3.1]nonane derivatives. Incubation under an 18O-rich atmosphere proved that both the incorporated oxygen atoms originated from O2. A radical-involved mechanism is proposed for these cyclisations.

The biosynthetic pathway of the prenylated salicylaldehyde flavoglaucin and congeners in Aspergillus ruber was elucidated by genome mining, heterologous expression, precursor feeding, and biochemical characterization. The polyketide skeleton was released as alkylated salicyl alcohols, which is a prerequisite for consecutive hydroxylation and prenylation, before reoxidation to the final aldehyde products. Our results provide an excellent example for a highly programmed machinery in natural product biosynthesis.

Prenylation of cyclodipeptides contributes largely to the structure diversification and biological activity. The prenylated products can be further metabolized by modifications like hydroxylation with cytochrome P450 enzymes or nonheme FeII/2-oxoglutarate-dependent oxygenases. Herein, we cloned and overexpressed NFIA_045530 from Neosartorya fischeri, which shares high sequence similarity with the nonheme FeII/2-oxoglutarate-dependent oxygenase FtmOx1Af from Aspergillus fumigatus on the amino acid level. FtmOx1Af is a member of the biosynthetic enzymes for fumitremorgin-type mycotoxins and catalyzes the conversion of fumitremorgin B to verruculogen by insertion of an oxygen molecule into the two prenyl moieties. The recombinant protein EAW25734 encoded by NFIA_045530 was purified to apparent homogeneity and then was used for incubation with intermediates of the fumitremorgin biosynthetic pathway. LC-MS analysis revealed no consumption of fumitremorgin B but good conversion with its biosynthetic precursor t...

In this study, fibrous core–shell silica particles were successfully synthesized via a one-step oil–water biphase stratification coating strategy. The core–shell silica particles were composed of 3-µm non-pore silica cores and thin shells (50–100 nm), which have radial-like direct channels and a large pore size (19.89 nm). The fibrous core–shell silica particles were further modified by n-octadecyltrichlorosilane and used as stationary-phase media in high-performance liquid chromatography (HPLC). The chromatographic properties of the particles were systematically studied in small-molecule and protein separation processes. The results showed that the back pressure was as low as 8.5 MPa under the 1.0-mL min−1 flow velocity. Furthermore, fibrous core–shell silica particles with an 80-nm shell were used for separating seven small molecules within 10 min and six proteins within 6 min. This work demonstrates that the fibrous core–shell silica particles could be used as an HPLC stationary phase with good performance and low back pressure, and that they have great potential for application to HPLC separation in the future.

Expression of a cyclodipeptide synthase gene from Nocardiopsis prasina (CDPS-Np) in Escherichia coli resulted in the formation of cyclo-(l-Tyr-l-Tyr) (1) as the minor and cyclo-(l-Tyr-l-Phe) (2) as the major products. Site-directed mutagenesis revealed a strong influence on product accumulation of the amino acid residues in pocket P1. An 8-fold increase in product formation for 1 and 10-fold for 2 were detected in the double mutant T82V_Y196F compared with the wild type.

Prenyltransferases of the dimethylallyltryptophan synthase (DMATS) superfamily catalyze the attachment of prenyl or prenyl-like moieties to diverse acceptor compounds. These acceptor molecules are generally aromatic in nature and mostly indole or indole-like. Their catalytic transformation represents a major skeletal diversification step in the biosynthesis of secondary metabolites, including the indole alkaloids. DMATS enzymes thus contribute significantly to the biological and pharmacological diversity of small molecule metabolites. Understanding the substrate specificity of these enzymes could create opportunities for their biocatalytic use in preparing complex synthetic scaffolds. However, there has been no framework to achieve this in a rational way. Here, we report a chemoinformatic pipeline to enable prenyltransferase substrate prediction. We systematically catalogued 32 unique prenyltransferases and 167 unique substrates to create possible reaction matrices and compiled these data into a browsable database named PrenDB. We then used a newly developed algorithm based on molecular fragmentation to automatically extract reactive chemical epitopes. The analysis of the collected data sheds light on the thus far explored substrate space of DMATS enzymes. To assess the predictive performance of our virtual reaction extraction tool, 38 potential substrates were tested as prenyl acceptors in assays with three prenyltransferases, and we were able to detect turnover in >55% of the cases. The database, PrenDB (www.kolblab.org/prendb.php), enables the prediction of potential substrates for chemoenzymatic synthesis through substructure similarity and virtual chemical transformation techniques. It aims at making prenyltransferases and their highly regio- and stereoselective reactions accessible to the research community for integration in synthetic work flows.

Aspergillus terreus aromatic prenyltransferase (AtaPT) catalyzes predominantly C-monoprenylation of acylphloroglucinols in the presence of different prenyl diphosphates. With dimethylallyl diphosphate (DMAPP) as prenyl donor, gem-diprenylated products 1D3, 2D3, and 3D3 were also detected. High conversion of 1D1 to 1D3, 2D1 to 2D3, and 3D1 to 3D3 was demonstrated by incubation with AtaPT and DMAPP. The first example of gem-diprenylation by a member of the dimethylallyltryptophan synthase superfamily is provided.

Prenylation increases the bioactivity of flavonoids. Herein, we report the first examples of regioselective enzymatic geranylation of biflavonoids using Aspergillus terreus aromatic prenyltransferase (AtaPT). For biflavonoids 1-3 dimerized through a diphenyl linkage, geranylation occurs at the hydrogen bond involving C5''-OH group, which is less chemically accessible than other OH groups in the molecule. This study would be referential for developing green synthetic solutions for prenylated biflavonoids.

Mutation of the gatekeeping residues for prenyl donor selectivity in six dimethylallyl transferases significantly increased their activities toward geranyl diphosphate. Forty-two geranylated derivatives were obtained from 15 cyclic dipeptides by using the engineered enzymes. Taking cyclo-l-Trp-l-Trp as an example, the geranyl moiety can be attached to all seven possible positions of the indole nucleus. This study demonstrates a convenient way to increase the structural diversity of geranylated products by structure-based engineering of the available dimethylallyl transferases.

Microorganisms provide valuable enzyme machinery to assemble complex molecules. Fungal prenyltransferases (PTs) typically catalyse highly regiospecific prenylation reactions that are of significant pharmaceutical interest. While the majority of PTs accepts dimethylallyl diphosphate (DMAPP), very few such enzymes can use geranyl diphosphate (GPP) or farnesyl diphosphate (FPP) as donors. This catalytic gap prohibits the wide application of PTs for structural diversification. Structure-guided molecular modelling and site-directed mutagenesis of FgaPT2 from Aspergillus fumigatus led to the identification of the gatekeeping residue Met328 responsible for the prenyl selectivity and sets the basis for creation of GPP- and FPP-accepting enzymes. Site-saturation mutagenesis of the gatekeeping residue at position 328 in FgaPT2 revealed that the size of this side chain is the determining factor for prenyl selectivity, while its hydrophobicity is crucial for allowing DMAPP and GPP to bind.

FgaPT2 from Aspergillus fumigatus catalyzes a regular C4- and its mutant K174A a reverse C3-prenylation of l-tryptophan in the presence of dimethylallyl diphosphate. FgaPT2 also uses tryptophan-containing cyclic dipeptides for C4-prenylation, while FgaPT2_K174A showed almost no activity toward these substrates. In contrast, Arg244 mutants of FgaPT2 accept very well cyclic dipeptides for regular C4-prenylation. In this study, we demonstrate that FgaPT2_K174F, which catalyzes a regular C3-prenylation on tyrosine, can also use cyclo-l-Trp-l-Ala, cyclo-l-Trp-l-Trp, cyclo-l-Trp-Gly, cyclo-l-Trp-l-Phe, cyclo-l-Trp-l-Pro, and cyclo-l-Trp-l-Tyr as substrates, but only with low activity. Combinational mutation on Lys174 and Arg244 increases significantly the acceptance of these cyclic dipeptides. With the exception of cyclo-l-Trp-l-Trp, the tested dipeptides were much better accepted by FgaPT2_K174F_R244X (X = L, N, Q, Y) than FgaPT2, with an increase of two- to six-fold activity. In comparison to FgaPT2_K174F, even two- to ten-fold conversion yields were calculated for the double mutants. Isolation and structural elucidation of the enzyme products revealed stereospecific reverse C3-prenylation on the indole ring, resulting in the formation of syn-cis configured hexahydropyrroloindole derivatives. The results presented in this study highlight the convenience of site-directed mutagenesis for creating new biocatalysts.

Prenylated aromatic compounds are important intermediates in the biosynthesis of bioactive molecules such as 3-chromanols from plants, ubiquinones from prokaryotes and meroterpenoids from sponges. Biosynthesis of prenylated aromatic compounds using prokaryotic microorganisms has attracted increasing attention in the field of synthetic biology. In this study, we demonstrated that the production of 3-geranyl-4-hydroxybenzoic acid (GBA) and a variety of GBA analogues was feasible in a metabolically engineered E. coli by using XimB, a special prenyltransferase involved in the biosynthesis of xiamenmycin A in Streptomyces xiamenensis 318. XimB exhibits broad substrate specificity and can catalyze the transfer reaction of prenyl moieties with different carbon chain lengths to both the natural substrate 4-hydroxybenzoate (4-HBA) and to different substituted 4-HBA derivatives at C-2 and C-3. Feeding 4-HBA to an engineered E. coli equipped with a hybrid mevalonate pathway increased the production of GBA up to 94.30 mg/L. Considerable amounts of other GBA derivatives, compounds 4, 5, 6, 7, and 9, can be achieved by feeding precursors. The plug-and-play design for inserting C5, C15, and C20 prenyl diphosphate synthetases under the control of the T7 promoter resulted in targeted production of 3-dimethylallyl, 3-farnesyl-, and 3-geranylgeranyl-4-hydroxybenzoic acid, respectively. Furthermore, the valuable benzopyran xiamenmycin B was successfully produced in E. coli R7-MVA by coexpression of a complete biosynthetic gene cluster, which contains ximBDE.

A cyclo-l-Trp-l-Trp tailoring P450 with novel function from Streptomyces purpureus was identified by heterologous expression in S. coelicolor and in vitro assays the recombinant protein. Structural elucidation revealed that this enzyme catalyzes the transfer of a guanine moiety to the indole ring of the cyclodipeptide via a C–N bond. Adduct products of CDP and guanine are unprecedented in nature, and CDP modification by coupling with guanine has not been reported prior to this study.

The original version of this article contained mistake. After careful re-examination of the LC-MS data, the products of CDPSs “WP_031028810” and “BAU83478” should be cFL instead of cPY. We apologize for any inconvenience that this may have caused.

Using various chromatographic methods, a new hexacyclic triterpenoid, 2β, 3β, 24β-trihydroxy-12, 13-cyclo-taraxer-l4-en-28oic acid (1), together with ten known compounds, 2α, 3α, 23-trihydroxyurs-12, 20(30)-dien-28oic acid (2), 6, 7-dehydroroyleanone (3), horminone (4), 7-O-methylhorminone (5), sugiol (6), demethylcryptojaponol (7), 14-deoxycoleon U (8), 5, 6-didehydro-7-hydroxy-taxodone (9), ferruginol (10) and dichroanone (11), were isolated from the roots of Salvia deserta. Their structures were identified on the basis of spectroscopic analysis and comparison with the reported data. The individual compounds (1, 3–8) were screened for cytotoxic activity, using the sulforhodamine B bioassay (SRB) method. As the results, Compounds 3, 5 and 8 showed cytotoxic potency against A549, MDA-MB-231, KB, KB-VIN, and MCF7 cell lines with IC(50) values ranging from 6.5 to 10.2 μM.

Flavonoids are found mainly in plants and exhibit diverse biological and pharmacological activities, which can often be enhanced by prenylations. In plants, such reactions are catalyzed by membrane-bound prenyltransferases. In this study, the prenylation of nine flavonoids from different classes by a soluble fungal prenyltransferase (AnaPT) involved in the biosynthesis of the prenylated indole alkaloid acetylaszonalenin is demonstrated. The behavior of AnaPT toward flavonoids regarding substrate acceptance and prenylation positions clearly differs from that of the indole prenyltransferase 7-DMATS. The two enzymes are therefore complementary in flavonoid prenylations.

Ergot alkaloids are important as mycotoxins or as drugs. Naturally occurring ergot alkaloids as well as their semisynthetic derivatives have been used as pharmaceuticals in modern medicine for decades. We identified 196 putative ergot alkaloid biosynthetic genes belonging to at least 31 putative gene clusters in 31 fungal species by genome mining of the 360 available genome sequences of ascomycetous fungi with known proteins. Detailed analysis showed that these fungi belong to the families Aspergillaceae, Clavicipitaceae, Arthrodermataceae, Helotiaceae and Thermoascaceae. Within the identified families, only a small number of taxa are represented. Literature search revealed a large diversity of ergot alkaloid structures in different fungi of the phylum Ascomycota. However, ergot alkaloid accumulation was only observed in 15 of the sequenced species. Therefore, this study provides genetic basis for further study on ergot alkaloid production in the sequenced strains.

Naturally occurring prenylated acylphloroglucinol derivatives are plant metabolites with diverse biological and pharmacological activities. Prenylation of acylphloroglucinols plays an important role in the formation of these intriguing natural products and is catalyzed in plants by membrane-bound enzymes. In this study, we demonstrate the prenylation of such compounds by a soluble fungal prenyltransferase AnaPT involved in the biosynthesis of prenylated indole alkaloids. The observed activities of AnaPT toward these substrates are much higher than that of a microsomal fraction containing an overproduced prenyltransferase from the plant hop.

Tryptophan prenyltransferases FgaPT2, 5-DMATS, 6-DMATSSv and 7-DMATS catalyse regiospecific C-prenylations on the indole ring, while tyrosine prenyltransferases SirD and TyrPT catalyse the O-prenylation of the phenolic hydroxyl group. In this study, we report the Friedel-Crafts alkylation of L-o-tyrosine by these enzymes. Surprisingly, no conversion was detected with SirD and three tryptophan prenyltransferases showed significantly higher activity than another tyrosine prenyltransferase TyrPT. C5-prenylated L-o-tyrosine was identified as a unique product of these enzymes. Using L-m-tyrosine as the prenylation substrate, product formation was only observed with the tryptophan prenyltransferases FgaPT2 and 7-DMATS. C4- and C6-prenylated derivatives were identified in the reaction mixture of FgaPT2. These results provided additional evidence for the similarities and differences between these two subgroups within the DMATS superfamily in their catalytic behaviours.

Indolosesquiterpene xiamycin A features a pentacyclic core structure. The chemical synthesis of two key precursors, 3-farnesylindole and 3-(epoxyfarnesyl)-indole, allowed elucidation of the enzymatic cascades forming the pentacyclic ring system of xiamycin A by XiaO-catalyzed epoxidation and the membrane protein XiaH-catalyzed terpene cyclization. The substrate flexibility of XiaI, an indole oxygenase for assembly of the central ring, was also demonstrated.

Non-ribosomal peptide synthetases (NRPSs) are key enzymes in microorganisms for the assembly of peptide backbones of biologically and pharmacologically active natural products. The monomodular NRPS-like enzymes comprise often an adenylation (A), a thiolation (T), and a thioesterase (TE) domain. In contrast to the NRPSs, they do not contain any condensation domain and usually catalyze a dimerization of α-keto carboxylic acids and thereby provide diverse scaffolds for further modifications. In this study, we established an expression system for NRPS-like genes in Saccharomyces cerevisiae. By expression of four known genes from Aspergillus terreus, their predicted function was confirmed and product yields of up to 35 mg per liter culture were achieved. Furthermore, expression of ATEG_03090 from the same fungus, encoding for the last uncharacterized NRPS-like enzyme with an A-T-TE domain structure, led to the formation of the benzoquinone derivative atromentin. All the accumulated products were isolated and their structures were elucidated by NMR and MS analyses. This study provides a convenient system for proof of gene function as well as a basis for synthetic biology, since additional genes encoding modification enzymes can be introduced.

Two prenyltransferases from Aspergillus ruber control the echinulin biosynthesis via exceptional sequential prenylations. EchPT1 catalyzes the first prenylation step, leading to preechinulin. The unique EchPT2 attaches, in a consecutive prenylation cascade, up to three dimethylallyl moieties to preechinulin and its dehydro forms neoechinulins A and B, resulting in the formation of at least 23 2- to 4-fold prenylated derivatives. Confirming these products in fungal extracts unravels the unprecedented catalytic relevance of EchPT2 for structural diversity.

The blue cheese-making fungus Penicillium roqueforti produces isofumigaclavine A as the main ergot alkaloid. Recently, genome mining revealed the presence of two DNA loci bearing the genetic potential for its biosynthesis. In this study, a short-chain dehydrogenase/reductase (SDR) from one of the loci was proved to be responsible for the conversion of chanoclavine-I to its aldehyde. Furthermore, a putative gene coding for an enzyme with high homology to Old Yellow Enzymes (OYEs) involved in the ergot alkaloid biosynthesis was found outside the two clusters. Biochemical characterisation of this enzyme, named FgaOx3Pr3, showed that it can indeed catalyse the formation of festuclavine in the presence of a festuclavine synthase FgaFS, as had been observed for other OYEs in ergot alkaloid biosynthesis. Differing from other homologues, FgaOx3Pr3 does not convert chanoclavine-I aldehyde to its shunt products in the absence of FgaFS. Instead, it increases significantly the product yields of several SDRs for the conversion of chanoclavine-I to its aldehyde. Kinetic studies proved that overcoming the product inhibition is responsible for the observed enhancement. To the best of our knowledge, this is the first report on the bifunctionality of an OYE and its synergistic effect with SDRs.

The tryptophan derivative 1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (MTCA) is present in many plants and foods including fermentation products of the baker's yeast Saccharomyces cerevisiae. MTCA is formed from tryptophan and acetaldehyde via a Pictet-Spengler reaction. In this study, up to 9 mg/L of MTCA were detected as a mixture of (1S,3S) and (1R,3S) isomers in a ratio of 2.2:1 in Saccharomyces cerevisiae cultures. To the best of our knowledge, this is the first report on the presence of MTCA in laboratory baker's yeast cultures. Expression of three fungal tryptophan prenyltransferase genes, fgaPT2, 5-dmats, and 7-dmats in S. cerevisiae resulted in the formation of MTCA derivatives with prenyl moieties at different positions of the indole ring. Expression of these genes in dimethylallyl diphosphate and tryptophan overproducing strains led to generation of up to 400 mg/L of prenylated MTCAs as mixtures of (1S,3S) and (1R,3S) diastereomers in ratios similar to that of unprenylated MTCA. The structures of the described substances including their stereochemistry were unequivocally elucidated by mass spectrometry as well as one- and two-dimensional NMR spectroscopy. The results of this study provide a convenient system for the production of high amounts of designed prenylated MTCAs in S. cerevisiae. Furthermore, our work can be considered as an excellent example for the construction of more complex molecules by introducing just one key gene.

Two new phenolic acids, ethyl pro-lithospermate (1), n-butyl pro-lithospermate (2) were isolated from Salvia yunnanensis C.H.Wright, along with nineteen known compounds (3–21). The structures of the isolated compounds were elucidated on the basis of extensive spectrometry and by comparing their physical and spectroscopic data to the literature. Among them, compounds 11, 12 and 14–16 were firstly isolated from S. yunnanensis C.H.Wright. Some of the isolated compounds were evaluated for their neuroprotection. Compounds 10–12 showed significant neuroprotective effects in PC12 cells and compounds 1, 4–7 displayed moderate neuroprotective effects.

7-DMATS from Aspergillus fumigatus and SirD from Leptosphaeria maculans catalyse a C7-prenylation of l-tryptophan and an O-prenylation of l-tyrosine in nature, respectively. SirD was reported to catalyse the C7-prenylation of l-tryptophan and some derivatives thereof in vitro. We report here the O-prenylation of tyrosine and O- or N-prenylation of its derivatives by 7-DMATS. These results provide experimental evidence for the close relationship of tyrosine O- and tryptophan C7-prenyltransferases regarding their substrate and catalytic promiscuity.