Strategies for the Discovery of New Natural Products by Genome Mining

New drugs from silent gene clusters : Analysis of genome sequence data has identified numerous “cryptic” gene clusters encoding novel natural product biosynthetic assembly lines; this suggests that many new bioactive metabolites remain to be discovered, even in extensively investigated organisms. Several related and complementary strategies for identifying the products of these clusters have emerged recently and revitalized the search for novel bioactive natural products.

     

The Role of a Nonribosomal Peptide Synthetase in l‐Lysine Lactamization During Capuramycin Biosynthesis

Capuramycins are one of several known classes of natural products that contain an l ‐Lys‐derived l ‐α‐amino‐?‐caprolactam (l ‐ACL) unit. The α‐amino group of l ‐ACL in a capuramycin is linked to an unsaturated hexuronic acid component through an amide bond that was previously shown to originate by an ATP‐independent enzymatic route. With the aid of a combined in vivo and in vitro approach, a predicted tridomain nonribosomal peptide synthetase CapU is functionally characterized here as the ATP‐dependent amide‐bond‐forming catalyst responsible for the biosynthesis of the remaining amide bond present in l ‐ACL. The results are consistent with the adenylation domain of CapU as the essential catalytic component for l ‐Lys activation and thioesterification of the adjacent thiolation domain. However, in contrast to expectations, lactamization does not require any additional domains or proteins and is likely a nonenzymatic event. The results set the stage for examining whether a similar NRPS‐mediated mechanism is employed in the biosynthesis of other l ‐ACL‐containing natural products and, just as intriguingly, how spontaneous lactamization is avoided in the numerous NRPS‐derived peptides that contain an unmodified l ‐Lys residue.     

New Insights into the Glycosylation Steps in the Biosynthesis of Sch47554 and Sch47555

Sch47554 and Sch47555 are antifungal compounds from Streptomyces sp. SCC‐2136. The availability of the biosynthetic gene cluster made it possible to track genes that encode biosynthetic enzymes responsible for the structural features of these two angucyclines. Sugar moieties play important roles in the biological activities of many natural products. An investigation into glycosyltransferases (GTs) might potentially help to diversify pharmaceutically significant drugs through combinatorial biosynthesis. Sequence analysis indicates that SchS7 is a putative C‐GT, whereas SchS9 and SchS10 are proposed to be O‐GTs. In this study, the roles of these three GTs in the biosynthesis of Sch47554 and Sch47555 are characterized. Coexpression of the aglycone and sugar biosynthetic genes with schS7 in Streptomyces lividans K4 resulted in the production of C‐glycosylated rabelomycin, which revealed that SchS7 attached a d ‐amicetose moiety to the aglycone core structure at the C‐9 position. Gene inactivation studies revealed that subsequent glycosylation steps took place in a sequential manner, in which SchS9 first attached either an l ‐aculose or l ‐amicetose moiety to 4′‐OH of the C‐glycosylated aglycone, then SchS10 transferred an l ‐aculose moiety to 3‐OH of the angucycline core.     

Discovery of the Tiancilactone Antibiotics by Genome Mining of Atypical Bacterial Type II Diterpene Synthases

Although genome mining has advanced the identification, discovery, and study of microbial natural products, the discovery of bacterial diterpenoids continues to lag behind. Herein, we report the identification of 66 putative producers of novel bacterial diterpenoids, and the discovery of the tiancilactone (TNL) family of antibiotics, by genome mining of type II diterpene synthases that do not possess the canonical DXDD motif. The TNLs, which are broad‐spectrum antibiotics with moderate activities, are produced by both Streptomyces sp. CB03234 and Streptomyces sp. CB03238 and feature a highly functionalized diterpenoid skeleton that is further decorated with chloroanthranilate and γ‐butyrolactone moieties. Genetic manipulation of the tnl gene cluster resulted in TNL congeners, which provided insights into their biosynthesis and structure–activity relationships. This work highlights the biosynthetic potential that bacteria possess to produce diterpenoids and should inspire continued efforts to discover terpenoid natural products from bacteria.     

Genome Mining of the Biosynthetic Gene Cluster of the Polyene Macrolide Antibiotic Tetramycin and Characterization of a P450 Monooxygenase Involved in the Hydroxylation of the Tetramycin B Polyol Segment

A polyene macrolide antibiotic tetramycin biosynthetic gene cluster was identified by genome mining and isolated from Streptomyces hygrospinosus var. beijingensis. Genetic and in silico analyses gave insights into the mechanism of biosynthesis of tetramycin, and a model of the tetramycin biosynthetic pathway is proposed. Inactivation of a cytochrome P450 monooxygenase gene, tetrK, resulted in the production of a tetramycin B precursor: tetramycin A, which lacks a hydroxy group in its polyol region. TetrK was subsequently overexpressed heterologously in E. coli with a His₆ tag, and purified TetrK efficiently hydroxylated tetramycin A to afford tetramycin B. Kinetic studies revealed no inhibition of TetrK by substrate or product. Surprisingly, sequence‐alignment analysis showed that TetrK, as a hydroxylase, has much higher homology with epoxidase PimD than with hydroxylases NysL and AmphL. The 3D structure of TetrK was then constructed by homology modeling with PimD as reference. Although TetrK and PimD catalyzed different chemical reactions, homology modeling indicated that they might share the same catalytic sites, despite also possessing some different sites correlated with substrate binding and substrate specificity. These findings offer good prospects for the production of improved antifungal polyene analogues.     

Discovery and Biosynthesis of Ureidopeptide Natural Products Macrocyclized via Indole N-acylation in Marine Microbulbifer spp. Bacteria

Commensal bacteria associated with marine invertebrates are underappreciated sources of chemically novel natural products. Using mass spectrometry, we had previously detected the presence of peptidic natural products in obligate marine bacteria of the genus Microbulbifer cultured from marine sponges. In this report, the isolation and structural characterization of a panel of ureidohexapeptide natural products, termed the bulbiferamides, from Microbulbifer strains is reported wherein the tryptophan side chain indole participates in a macrocyclizing peptide bond formation. Genome sequencing identifies biosynthetic gene clusters encoding production of the bulbiferamides and implicates the involvement of a thioesterase in the indolic macrocycle formation. The structural diversity and widespread presence of bulbiferamides in commensal microbiomes of marine invertebrates point toward a possible ecological role for these natural products.     

Bioengineering of Anti-Inflammatory Natural Products

Inflammatory processes occur as a generic response of the immune system and can be triggered by various factors, such as infection with pathogenic microorganisms or damaged tissue. Due to the complexity of the inflammation process and its role in common diseases like asthma, cancer, skin disorders or Alzheimer's disease, anti-inflammatory drugs are of high pharmaceutical interest. Nature is a rich source for compounds with anti-inflammatory properties. Several studies have focused on the structural optimization of natural products to improve their pharmacological properties. As derivatization through total synthesis is often laborious with low yields and limited stereoselectivity, the use of biosynthetic, enzyme-driven reactions is an attractive alternative for synthesizing and modifying complex bioactive molecules. In this minireview, we present an outline of the biotechnological methods used to derivatize anti-inflammatory natural products, including precursor-directed biosynthesis, mutasynthesis, combinatorial biosynthesis, as well as whole-cell and in vitro biotransformation.     

Genome Mining of Streptomyces sp. Tü 6176: Characterization of the Nataxazole Biosynthesis Pathway

Streptomyces sp. Tü 6176 produces the cytotoxic benzoxazole nataxazole. Bioinformatic analysis of the genome of this organism predicts the presence of 38 putative secondary‐metabolite biosynthesis gene clusters, including those involved in the biosynthesis of AJI9561 and its derivative nataxazole, the antibiotic hygromycin B, and ionophores enterobactin and coelibactin. The nataxazole biosynthesis gene cluster was identified and characterized: it lacks the O‐methyltransferase gene required to convert AJI9561 into nataxazole. This O‐methyltransferase activity might act as a resistance mechanism, as AJI9561 shows antibiotic activity whereas nataxazole is inactive. Moreover, heterologous expression of the nataxazole biosynthesis gene cluster in S. lividans JT46 resulted in the production of AJI9561. Nataxazole biosynthesis requires the shikimate pathway to generate 3‐hydroxyanthranilate and an iterative type I PKS to generate 6‐methylsalicylate. Production of nataxazole was improved up to fourfold by disrupting one regulatory gene in the cluster. An additional benzoxazole, 5‐hydroxynataxazole is produced by Streptomyces sp. Tü 6176. 5‐Hydroxynataxazole derives from nataxazole by the activity of an as yet unidentified oxygenase; this implies cross‐talk between the nataxazole biosynthesis pathway and an unknown pathway.     

具有抗菌活性的杂环天然产物的研究进展

综述了近十年来国内外报道的部分杂环类天然产物抗菌活性物质的提取分离方法及抗菌活性研究,这些物质主要包括生物碱类、黄酮类、内酯类、多糖类和香豆素类等。     

Reevaluation of the violacein biosynthetic pathway and its relationship to indolocarbazole biosynthesis

The biosynthetic pathways for violacein and for indolocarbazoles (rebeccamycin, staurosporine) include a decarboxylative fusion of two tryptophan units. However, in the case of violacein, one of the tryptophans experiences an unusual 1-->2 shift of the indole ring. The violacein biosynthetic gene cluster was previously reported to consist of four genes, vioABCD. Here we studied the violacein pathway through expression of vio genes in Escherichia coli and Streptomyces albus. A pair of genes (vioAB), responsible for the earliest steps in violacein biosynthesis, was functionally equivalent to the homologous pair in the indolocarbazole pathway (rebOD), directing the formation of chromopyrrolic acid. However, chromopyrrolic acid appeared to be a shunt product, not a violacein intermediate. In addition to vioABCD, a fifth gene (vioE) was essential for violacein biosynthesis, specifically for production of the characteristic 1-->2 shift of the indole ring. We also report new findings on the roles played by the VioC and VioD oxygenases, and on the origin of violacein derivatives of the chromoviridans type.     

Characterization of BlsM, a nucleotide hydrolase involved in cytosine production for the biosynthesis of blasticidin S

Biosynthesis of the antifungal agent blasticidin S in Streptomyces griseochromogenes requires the formation of free cytosine. The blsM gene in the blasticidin S gene cluster is predicted to encode a protein that has sequence homology with several nucleoside transferases. In vitro analysis of recombinant BlsM revealed that the enzyme functions as a nucleotide hydrolase and catalyzes the formation of free cytosine by using cytidine 5'-monophosphate (CMP) as the preferred substrate. Cytosine production was significantly lower with CDP, CTP, and dCMP as alternate substrates. BlsM was also observed to have low-level cytidine deaminase activity, converting cytidine and deoxycytidine to uridine and deoxyuridine, respectively. Point mutations were introduced in blsM at putative catalytic residues to generate three mutant enzymes, BlsM Ser98Asp, Glu104Ala, and Glu104Asp. All three mutants lost CMP hydrolysis activity, but the Ser98Asp mutant showed a modest increase in cytidine deaminase activity.     

Parallel Post‐Polyketide Synthase Modification Mechanism Involved in FD‐891 Biosynthesis in Streptomyces graminofaciens A‐8890

To isolate a key polyketide biosynthetic intermediate for the 16‐membered macrolide FD‐891 ( 1 ), we inactivated two biosynthetic genes coding for post‐polyketide synthase (PKS) modification enzymes: a methyltransferase (GfsG) and a cytochrome P450 (GfsF). Consequently, FD‐892 ( 2 ), which lacks the epoxide moiety at C8–C9, the hydroxy group at C10, and the O‐methyl group at O‐25 of FD‐891, was isolated from the gfsF/gfsG double‐knockout mutant. In addition, 25‐O‐methyl‐FD‐892 ( 3 ) and 25‐O‐demethyl‐FD‐891 ( 4 ) were isolated from the gfsF and gfsG mutants, respectively. We also confirmed that GfsG efficiently catalyzes the methylation of 2 and 4 in vitro. Further, GfsF catalyzed the epoxidation of the double bond at C8‐C9 of 2 and 3 and subsequent hydroxylation at C10, to afford 4 and 1 , respectively. These results suggest that a parallel post‐PKS modification mechanism is involved in FD‐891 biosynthesis.     

Enzymatic Formation of Oxygen‐Containing Heterocycles in Natural Product Biosynthesis

Oxygen‐containing heterocycles are widely encountered in natural products that display diverse pharmacological properties and have potential benefits to human health. The formation of O‐heterocycles catalyzed by different types of enzymes in the biosynthesis of natural products not only contributes to the structural diversity of these compounds, but also enriches our understanding of nature's ability to construct complex molecules. This minireview focuses on the various modes of enzymatic O‐heterocyclization identified in natural product biosynthesis and summarizes the possible mechanisms involved in ring closure.     

Core Steps to the Azaphilone Family of Fungal Natural Products

Azaphilones are a family of polyketide-based fungal natural products that exhibit interesting and useful bioactivities. This minireview explores the literature on various characterised azaphilone biosynthetic pathways, which allows for a proposed consensus scheme for the production of the core azaphilone structure, as well as identifying early diversification steps during azaphilone biosynthesis. A consensus understanding of the core enzymatic steps towards a particular family of fungal natural products can aid in genome-mining experiments. Genome mining for novel fungal natural products is a powerful technique for both exploring chemical space and providing new insights into fungal natural product pathways.     

Biosynthesis of xyrrolin, a new cytotoxic hybrid polyketide/non-ribosomal peptide pyrroline with anticancer potential, in Xylaria sp. BCC 1067

A gene from Xylaria sp. BCC 1067, pks3, that encodes a putative 3660-residue hybrid polyketide synthase (PKS)/non-ribosomal peptide synthetase (NRPS) was characterised by targeted gene disruption in combination with comprehensive product identification. Studies of the features of a corresponding mutant, YA3, allowed us to demonstrate that pks3 is responsible for the synthesis of a new pyrroline compound, named xyrrolin, in the wild-type Xylaria sp. BCC 1067. The structure of xyrrolin was established by extensive spectroscopic and spectrometric analyses, including low- and high-resolution MS, IR, (1)H NMR, (13)C NMR, (13)C NMR with Dept135, HMQC 2D NMR, HMBC 2D NMR and COSY 2D NMR. On the basis of the Pks3 domain organisation and the chemical structure of xyrrolin, we proposed that biosynthesis of this compound requires the condensation of a tetraketide and an L-serine unit, followed by Dieckmann or reductive cyclisation and enzymatic removal of ketone residue(s). Bioassays of the pure xyrrolin further displayed cytotoxicity against an oral cavity (KB) cancer cell line.     

Natural Products as Zinc‐Dependent Histone Deacetylase Inhibitors

Zinc‐dependent histone deacetylases (HDACs), a family of hydrolases that remove acetyl groups from lysine residues, play an important role in the regulation of multiple processes, from gene expression to protein activity. The dysregulation of HDACs is associated with many diseases including cancer, neurological disorders, cellular metabolism disorders, and inflammation. Molecules that act as HDAC inhibitors (HDACi) exhibit a variety of related bioactivities. In particular, HDACi have been applied clinically for the treatment of cancers. Inhibition through competitive binding of the catalytic domain of these enzymes has been achieved by a diverse array of small‐molecule chemotypes, including a number of natural products. This review provides a systematic introduction of natural HDACi, with an emphasis on their enzyme inhibitory potency, selectivity, and biological activities, highlighting their various binding modes with HDACs.