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l-Isovaline biosynthesis by TqaLFM- ti from Tolypocladium inflatum was demonstrated in vitro . The biochemical analysis of the α-ketoglutarate-dependent oxygenase TqaL- ti revealed that it produces (2 S ,3 S )-3-ethyl-3-methylaziridine-2-carboxylic acid from l-isoleucine, thus exhibiting a stereoselectivity different from those of the reported homologues. Remarkably, a single mutation on I295 in TqaL- ti completely exchanged its stereoselectivity to produce the C-3 stereoisomer. TqaFM- ti generates d-isovaline from (2 S ,3 R )-aziridine-2-carboxylic acid, suggesting that the stereochemistry of the TqaL product defines that of isovaline.

Cordycicadins A-D ( 1 - 4 ) are four novel polyketides that were isolated from the liquid fermentation of the insect-pathogenic fungus Cordyceps cicadae JXCH1. The structures were determined by a combination of spectroscopic analysis, single-crystal X-ray diffraction, and computational methods. Compounds 1 , 3 , and 4 harbor an unusual exocyclic enol ether bridge that connects the separated ring systems. Hypothetical biosynthetic pathways for 1 - 4 were proposed. Cordycicadins A ( 1 ) and B ( 2 ) showed antifeedant activity against silkworm larvae ( Bombyx mori ) with EC 50 values of 65.4 and 57.0 μg/cm 2 , respectively.

Tabtoxin is a β-lactam ring-containing phytotoxin produced by a plant pathogenic Pseudomonas species. Here, we describe the early stages of tabtoxin biosynthesis, involving a C -methylation reaction catalyzed by the S -adenosyl-l-methionine-dependent methyltransferase TblA as the initial step for the β-lactam construction. Gene deletion and in vitro biochemical assays demonstrated that the Gcn5-related N -acetyltransferase domain of TblD catalyzes the acetylation of the α-amino group of 5-methyl-l-lysine. This establishment of the early reaction steps lays the foundation for characterizing unique β-lactam biosynthesis.

The biosynthetic gene cluster of atlantinone B ( 10 ) was discovered in Penicillium chrysogenum MT-40. A multifunctional cytochrome P450 (AtlD) encoded by the cluster is responsible for the formation of the unique lactone-bridged ring and the 16β-hydroxyl of atlantinone B, and a new terpene cyclase (AtlC) can unprecedentedly accept the demethylated substrate epoxyfarnesyl-DMOA ( 4a ) to generate three bicyclic meroterpenoids ( 5a - 5c ). This study paves the way for combinatorial synthesis of structurally diverse meroterpenoids for drug discovery.

The Fe- and 2-oxoglutarate-dependent oxygenase SptF is a promising powerful biocatalys with unusual catalytic versatility and promiscuity. The site-specific random substitution of N150, I63, and N65, which are involved in substrate interactions, generated three compounds that were not produced by the SptF wild type. The substrate binding mode was dramatically altered by the introduction of only one or two substitutions. These results provide insights into the engineering of Fe- and 2-oxoglutarate-dependent oxygenases for chemoenzymatic syntheses of bioactive compounds.

Pelletierine, a proposed building block of Lycopodium alkaloids (LAs), was demonstrated to be synthesized via the non-enzymatic Mannich-like condensation of Δ 1 -piperideine and 3-oxoglutaric acid produced by two new type III PKSs (HsPKS4 and PcPKS1) characterized from Huperzia serrata and Phlegmariurus cryptomerianus , respectively. The findings provide new insights for further understanding the biosynthesis of LAs such as huperzine A.

The biosynthetic gene cluster of the fungal meroterpenoid emeridone F ( 8 ) was discovered in the genome of Aspergillus sp. TJ23, and its late-stage biosynthesis was elucidated by characterizing two α-ketoglutarate (αKG)-dependent dioxygenases, SptF and SptN. SptF catalyzes oxidative rearrangement followed by epoxidation, whereas SptN serves as the C-9 hydroxylase. Our study provides insight into the biosynthetic mechanisms of other andiconin ( 1 )-derived natural products, exemplifying the important roles of αKG-dependent enzymes in the structural complexifications.

Tenebrathin ( 1 ), a new C-5-substituted γ-pyrone with a nitroaryl side chain, was isolated from the rare actinomycete Streptoalloteichus tenebrarius NBRC 16177. The chemical structure of 1 was elucidated by a spectroscopic analysis using the crystalline sponge method of crystallization-free X-ray crystallography. The biosynthetic origin of the unusual C-5-substituted γ-pyrone in 1 was revealed by a 13 C-labeling experiment. Compound 1 exhibited moderate cytotoxicity against several cancer cell lines and likely targets some protein kinases.

Ascochlorin is a medicinally important fungal meroterpenoid. Its biosynthetic pathway in Fusarium sp. was identified, and the stereoselective epoxidation of the farnesyl group by the multidomain, soluble P450 monooxygenase AscE and the subsequent formation of the unique timethylcyclohexanone ring by the membrane-bound cyclase AscF were investigated. Precursor-directed biosynthesis generated novel bromo-substituted derivatives, which exhibited potent cytotoxic activities. This study paves the way for the future metabolic engineering of medicinally important meroterpenoids for drug discovery.

A pair of enantiomeric norsesquiterpenoids, (+)- and (-)-preuisolactone A (1) [(+)-1 and (-)-1)] featuring an unprecedented tricyclo[4.4.0 1,6 .0 2,8 ]decane carbon scaffold were isolated from Preussia isomera. Their structures were determined by spectroscopic and computed methods and X-ray crystallography. Compounds (+)-1 and (-)-1 are two rare naturally occurring sesquiterpenoidal enantiomers. A plausible biosynthetic pathway for 1 is proposed. Additionally, (±)-1 exhibited remarkable antibacterial activity against Micrococcus luteus with an MIC value of 10.2 μM.

The biosynthetic gene cluster of the fungal meroterpenoid chrodrimanin B (4) was discovered in Penicillium verruculosum TPU1311, and the complete biosynthetic pathway of 4 has been elucidated by heterologous reconstitution of its biosynthesis in Aspergillus oryzae, as well as by in vitro characterizations of selected enzymes. The present study has identified the polyketide synthase that produces 6-hydroxymellein (3) and also provided a biosynthetic platform of chrodrimanins for further bioengineering.

By the genome-mining approach, a chimeric enzyme of prenyltransferase-diterpene synthase was discovered from Penicillium chrysogenum MT-12. Since its product exhibited broadened NMR signals, the structural determination by only the NMR analysis was difficult, but the crystalline sponge method successfully revealed the structure with a 6-5-5-5 fused ring system. This demonstrated that the collaboration between the genome-mining and crystalline sponge method has the potential to facilitate rapid inquiries into the unexplored chemical space of small molecules.

Two novel macrolactams, dracolactams A and B, were identified from a combined-culture of Micromonospora species and a mycolic-acid containing bacterium (MACB). Their structures and stereochemistries were completely assigned, based on spectroscopic analyses and chemical derivatization. Both dracolactams were probably generated from a common macrolactam precursor produced by the Micromonospora species. In this combined-culture system, MACB is likely to activate cryptic oxidase genes in the Micromonospora species and induce the downstream polyene macrolactam cyclization.

Daurichromenic acid is a meroterpenoid with various pharmacological activities that is biosynthesized from grifolic acid in Rhododendron dauricum. Heterologous expression of grifolic acid synthases from Stachybotrys bisbyi and a daurichromenic acid synthase from R. dauricum in Aspergillus oryzae mediated three-step combinatorial biosynthesis of (+)-daurichromenic acid through enantioselective 6-endo-trig cyclization. Additional introduction of a halogenase from Fusarium sp. into the strain resulted in the biosynthesis of (+)-5-chlorodaurichromenic acid, which exceeds the antibacterial activity of the original compounds.

A biosynthetic gene cluster similar to that for andrastin A (1) was discovered in Emericella variecolor NBRC 32302. Ctr-P450, a cytochrome P450 uniquely present in the cluster, was coexpressed with the andrastin A biosynthetic genes, leading to the production of the antifeedant agent citreohybridonol (4), along with four new andrastin derivatives. The results revealed the unusual multifunctionality of Ctr-P450 and indicated that this approach can be applied for further natural product diversification.

The search for a new sesterterpene synthase in the genome of Emericella variecolor, which reportedly produces diverse sesterterpenoids, is described. One gene product (a chimeric protein with prenyltransferase and terpene cyclase domains) led to the synthesis of a novel tricyclic sesterterpene, stellata-2,6,19-triene (1), from DMAPP and IPP, and the hydrocarbon was further transformed into stellatic acid (2) by cytochrome P450 monooxygenase encoded by the gene adjacent to the sesterterpene synthase gene.

The soil-derived bacterium, Streptomyces sp. CJ-5, was cocultured with the mycolic acid-containing bacterium Tsukamurella pulmonis TP-B0596. The combined culture method significantly enhanced the production of the secondary metabolites in Streptomyces sp. CJ-5, leading to the isolation of three novel butanolide chojalactones A-C (1-3), with unusual γ-butyrolactone scaffolds. The complete structures, including the absolute configurations of 1-3, were determined based on spectroscopic data and total syntheses. In methylthiazole tetrazolium (MTT) assays, 1 and 2 showed moderate cytotoxicity against P388 cells.

Lipodiscamides A-C, three new lipodepsipeptides, were characterized from the marine sponge Discodermia kiiensis. These structurally rare cyclic lipodepsipeptides were found to possess an unprecedented dilactone macrocycle and, thus, represent a new family of lipopeptides. They are the only lipopeptides bearing 4S-hydroxy-trans-2-enoate, and noncanonical amino acids, l-3-ureidoalanine (Uda), E-dehydronorvaline (Denor), and d-citrulline (Cit). MTT assays against P388 and HeLa cells revealed the moderate cytotoxicity of all three compounds.

We herein report comparative analysis of two versions of the biosynthetic gene clusters of antimycins, a natural product family possessing up to 44 distinct entities. The biosynthetic pathway of antimycins is amenable to the high structural variation of the substrates, supported by successes in heterologous expression of the ant cluster and in fluorine incorporation. The latter facilitated the investigation of the structure-activity relationship into the usually invariable 3-formamidosalicylic acid moiety of the molecules.

FtmPT1 from Aspergillus fumigatus is a fungal indole prenyltransferase (PT) that normally catalyzes the regiospecific prenylation of brevianamide F (cyclo-L-Trp-L-Pro) at the C-2 position of the indole ring with dimethylallyl diphosphate (DMAPP). Interestingly, FtmPT1 exhibited remarkable substrate tolerance and accepted (E)-4-(1H-indol-3-yl)but-3-en-2-one (1) as a substrate to produce an unnatural novel α-prenylindolylbutenone (1a). This is the first demonstration of the prenylation of a nonaromatic carbon of the acceptor substrate by a fungal indole PT.

A C(19) hexaketide stilbene and a C(21) heptaketide chalcone were synthesized by Aloe arborescens octaketide synthase (OKS), a plant-specific type III polyketide synthase (PKS). Remarkably, the C(21) chalcone-forming activity was dramatically increased in a structure-guided OKS N222G mutant that produces a C(20) decaketide SEK15 from 10 molecules of malonyl-CoA. The findings suggested further strategies for production of unnatural polyketides by combination of the precursor-directed biosynthesis and the structure-guided engineering of type III PKS.

[Structure: see text] Benzalacetone synthase from Rheum palmatum efficiently catalyzed condensation of N-methylanthraniloyl-CoA (or anthraniloyl-CoA) with malonyl-CoA (or methylmalonyl-CoA) to produce 4-hydroxy-2(1H)-quinolones, a novel alkaloidal scaffold produced by a type III polyketide synthase (PKS). Manipulation of the functionally divergent type III PKSs by a nonphysiological substrate thus provides an efficient method for production of pharmaceutically important quinolone alkaloids.

[reaction: see text]. Chalcone synthase (CHS) is a plant-specific type III polyketide synthase catalyzing condensation of 4-coumaroyl-CoA with three molecules of malonyl-CoA. Surprisingly, it was demonstrated that S338V mutant of Scutellaria baicalensis CHS produced octaketides SEK4/SEK4b from eight molecules of malonyl-CoA. Further, the octaketides-forming activity was dramatically increased in a CHS triple mutant (T197G/G256L/S338T). The functional conversion is based on the simple steric modulation of a chemically inert residue lining the active-site cavity.

[reaction: see text] Substrate specificities of plant polyketide synthases (PKSs) were investigated using analogues of malonyl-CoA, the extension unit of the polyketide chain elongation reactions. When incubated with methylmalonyl-CoA and 4-coumaroyl-CoA, plant PKSs (chalcone synthase from Scutellaria baicalensis, stilbene synthase from Arachis hypogaea, and benzalacetone synthase from Rheum palmatum) afforded an unnatural C(6)-C(5) aromatic polyketide, 1-(4-hydroxyphenyl)pent-1-en-3-one, formed by one-step decarboxylative condensation of the two substrates. In contrast, succinyl-CoA was not accepted as a substrate by the enzymes.