Non-enzymatic synthesis of C-methylated fluostatins: discovery and reaction mechanism.

Two C-methylated fluostatins (FSTs) B3 (1) and B4 (2) were synthesized from flavin-mediated nonenzymatic epoxide ring-opening reactions of FST C. The structures of 1 and 2 were elucidated by HRESIMS, NMR, and ECD spectroscopic analyses. A subsequent 13C labeling study demonstrated that the C-methyl groups of 1 and 2 were derived from DMSO and enabled the mechanistic proposal of a nonenzymatic C-methylation.

Defensive Specialized Metabolites from the Latex of Euphorbia jolkinii.

Plant latex is sequestered in laticiferous structures and exuded immediately from damaged plant tissues. The primary function of plant latex is related to defense responses to their natural enemies. Euphorbia jolkinii Boiss. is a perennial herbaceous plant that greatly threaten the biodiversity and ecological integrity of northwest Yunnan, China. Nine triterpenes (1-9), four non-protein amino acids (10-13) and three glycosides (14-16) including a new isopentenyl disaccharide (14), were isolated and identified from the latex of E. jolkinii. Their structures were established on the basis of comprehensive spectroscopic data analyses. Bioassay revealed that meta-tyrosine (10) showed significant phytotoxic activity, inhibiting root and shoot growth of Zea mays, Medicago sativa, Brassica campestris, and Arabidopsis thaliana, with EC50 values ranging from 4.41 ± 1.08 to 37.60 ± 3.59 µg/mL. Interestingly, meta-tyrosine inhibited the root growth of Oryza sativa, but promoted their shoot growth at the concentrations below 20 µg/mL. meta-Tyrosine was found to be the predominant constituent in polar part of the latex extract from both stems and roots of E. jolkinii, but undetectable in the rhizosphere soil. In addition, some triterpenes showed antibacterial and nematicidal effects. The results suggested that meta-tyrosine and triterpenes in the latex might function as defensive substances for E. jolkinii against other organisms.

Unraveling the iterative type I polyketide synthases hidden in Streptomyces.

Type I polyketide synthases (T1PKSs) are one of the most extensively studied PKSs, which can act either iteratively or via an assembly-line mechanism. Domains in the T1PKSs can readily be predicted by computational tools based on their highly conserved sequences. However, to distinguish between iterative and noniterative at the module level remains an overwhelming challenge, which may account for the seemingly biased distribution of T1PKSs in fungi and bacteria: small iterative monomodular T1PKSs that are responsible for the enormously diverse fungal natural products exist almost exclusively in fungi. Here we report the discovery of iterative T1PKSs that are unexpectedly both abundant and widespread in Streptomyces Seven of 11 systematically selected T1PKS monomodules from monomodular T1PKS biosynthetic gene clusters (BGCs) were experimentally confirmed to be iteratively acting, synthesizing diverse branched/nonbranched linear intermediates, and two of them produced bioactive allenic polyketides and citreodiols as end products, respectively. This study indicates the huge potential of iterative T1PKS BGCs from streptomycetes in the discovery of novel polyketides.

Ene Reductase Enabled Intramolecular ß-C-H Functionalization of Substituted Cyclohexanones for Efficient Synthesis of Bridged Bicyclic Nitrogen Scaffolds.

Herein we report that ene reductases (EREDs) can facilitate an unprecedented intramolecular ß-C-H functionalization reaction for the synthesis of bridged bicyclic nitrogen heterocycles containing the 6-azabicyclo[3.2.1]octane scaffold. To streamline the synthesis of these privileged motifs, we developed a gram-scale one-pot chemoenzymatic cascade by combining iridium photocatalysis with EREDs, using readily available N-phenylglycines and cyclohexenones that can be obtained from biomass. Further derivatization using enzymatic or chemical methods can convert 6-azabicyclo[3.2.1]octan-3-one into 6-azabicyclo[3.2.1]octan-3α-ols, which can be potentially utilized for the synthesis of azaprophen and its analogues for drug discovery. Mechanistic studies revealed the reaction requires oxygen, presumably to produce oxidized flavin, which can selectively dehydrogenate the 3-substituted cyclohexanone derivatives to form the α,ß-unsaturated ketone, which subsequently undergoes spontaneous intramolecular aza-Michael addition under basic conditions.

Metabolic engineering of oleaginous yeast Rhodotorula toruloides for overproduction of triacetic acid lactone.

The plant-sourced polyketide triacetic acid lactone (TAL) has been recognized as a promising platform chemical for the biorefinery industry. However, its practical application was rather limited due to low natural abundance and inefficient cell factories for biosynthesis. Here, we report the metabolic engineering of oleaginous yeast Rhodotorula toruloides for TAL overproduction. We first introduced a 2-pyrone synthase gene from Gerbera hybrida (GhPS) into R. toruloides and investigated the effects of different carbon sources on TAL production. We then systematically employed a variety of metabolic engineering strategies to increase the flux of acetyl-CoA by enhancing its biosynthetic pathways and disrupting its competing pathways. We found that overexpression of ATP-citrate lyase (ACL1) improved TAL production by 45% compared to the GhPS overexpressing strain, and additional overexpression of acetyl-CoA carboxylase (ACC1) further increased TAL production by 29%. Finally, we characterized the resulting strain I12-ACL1-ACC1 using fed-batch bioreactor fermentation in glucose or oilcane juice medium with acetate supplementation and achieved a titer of 28 or 23 g/L TAL, respectively. This study demonstrates that R. toruloides is a promising host for the production of TAL and other acetyl-CoA-derived polyketides from low-cost carbon sources.

Inactivation of Flavoenzyme-Encoding Gene flsO1 in Fluostatin Biosynthesis Leads to Diversified Angucyclinone Derivatives.

Inactivation of the flavoenzyme-encoding gene flsO1 in fluostatin biosynthesis led to the isolation of four new angucyclinone derivatives (11, 12, 14, and 15), among which fluostarenes A (14) and B (15) featured the unprecedented 6/6/5/6/6 pentacyclic skeleton with fusion of a benzo[b]fluorene and a six-membered lactone ring. Both 14 and 15 were putatively generated via quinone methide-mediated nonenzymatic reactions. Fluostarene B (15) exhibited cytotoxicity against several cancer cell lines with IC50 values ranging from 7 to 10 µM. Fluostarenes A (14), B (15), and PK1 (16) showed α-glucosidase inhibition activity with IC50 of 0.89, 1.58, and 0.13 µM, respectively. Successful complementation of the ΔflsO1 mutant with alpK from kinamycin biosynthesis suggests that FlsO1 should function equivalently to AlpK as a putative C-5 hydroxylase.

Discovery of an Unexpected 1,4-Oxazepine-Linked seco-Fluostatin Heterodimer by Inactivation of the Oxidoreductase-Encoding Gene flsP.

Fluostatins belong to the atypical angucyclinone aromatic polyketides featuring a distinctive tetracyclic benzo[a]fluorene skeleton. To understand the formation of the heavily oxidized A-ring in fluostatins, a flavin adenine dinucleotide-binding oxidoreductase-encoding gene flsP was inactivated, leading to the production of an unprecedented 1,4-oxazepine-linked seco-fluostatin heterodimer difluostatin I (7) and five new fluostatin-related derivatives, fluostatins T-X (8-12). Their structures were elucidated by mass spectrometry, nuclear magnetic resonance, X-ray diffraction analysis, and biosynthetic considerations. Difluostatin I (7) represents the first example with an A-ring-cleaved 3',4'-seco-fluostatin skeleton. The absolute configuration of fluostatin T (8) was determined by X-ray diffraction analysis. Fluostatin W (11) contains an uncommon isoxazolinone ring. These findings highlight the structural diversity of fluostatins.

Leucoflavonine, a new bioactive racemic flavoalkaloid from the leaves of Leucosceptrum canum.

A new flavoalkaloid racemate, leucoflavonine (1), together with its flavonoid precursor pectolinarigenin (2), was isolated from the leaves of Leucosceptrum canum collected from Tibet. Its structure was established by comprehensive spectroscopic analysis. Chrial separation of the enantiomers of 1 was achieved, and their absolute configurations were determined as S-(+)- and R-(-)-leucoflavonines ((+)-1a and (-)-1b) by comparison of their computational and experimental optical rotations. Biological assays indicated that both (+)-1a and (-)-1b exhibited inhibitory activity against acetylchlorinesterase (AChE) in vitro (IC50 = 68.0 ±â€¯8.6 and 18.3 ±â€¯1.8 µM, respectively). Moreover, (-)-1b displayed cytotoxicity against human hepatoma cells HepG2 (IC50 = 52.9 ±â€¯3.6 µM), and inhibited the production of interleukelin-2 (IL-2) in Jurkat cells (IC50 = 16.5 ±â€¯0.9 µM), while (+)-1a showed no obvious activity in these assays.

Discovery of Stealthin Derivatives and Implication of the Amidotransferase FlsN3 in the Biosynthesis of Nitrogen-Containing Fluostatins.

Diazobenzofluorene-containing atypical angucyclines exhibit promising biological activities. Here we report the inactivation of an amidotransferase-encoding gene flsN3 in Micromonospora rosaria SCSIO N160, a producer of fluostatins. Bioinformatics analysis indicated that FlsN3 was involved in the diazo formation. Chemical investigation of the flsN3-inactivation mutant resulted in the isolation of a variety of angucycline aromatic polyketides, including four racemic aminobenzo[b]fluorenes stealthins D⁻G (9⁻12) harboring a stealthin C-like core skeleton with an acetone or butanone-like side chain. Their structures were elucidated on the basis of nuclear magnetic resonance (NMR) spectroscopic data and X-ray diffraction analysis. A plausible mechanism for the formation of stealthins D⁻G (9⁻12) was proposed. These results suggested a functional role of FlsN3 in the formation/modification of N⁻N bond-containing fluostatins.

Biologically active dichapetalins from Dichapetalum gelonioides.

A phytochemical investigation of the toxic tropical plant Dichapetalum gelonioides led to the isolation and identification of 14 new dichapetalins (1-14) and the known dichapetalins A (15) and K (16). The structures of the new compounds were determined by analyses of their NMR, MS, electronic circular dichroism, and X-ray diffraction data. The esterification at C-25 by 4-hydroxyphenylpropanoic acid and the hydroxylation at C-2' are unique in this unusual class of natural products. In addition to the known cytotoxicity, an array of biological activities, including antifeedant, nematicidal, antifungal, and NO and AChE inhibitory activities, were observed for this class of compounds. These findings suggested that dichapetalin hybrid triterpenoids as a class have broad biologically active cellular functions including defense against insect herbivores and pathogens.

Non-modular fatty acid synthases yield distinct N-terminal acylation in ribosomal peptides.

Recent efforts in genome mining of ribosomally synthesized and post-translationally modified peptides (RiPPs) have expanded the diversity of post-translational modification chemistries. However, RiPPs are rarely reported as hybrid molecules incorporating biosynthetic machinery from other natural product families. Here we report lipoavitides, a class of RiPP/fatty-acid hybrid lipopeptides that display a unique, putatively membrane-targeting 4-hydroxy-2,4-dimethylpentanoyl (HMP)-modified N terminus. The HMP is formed via condensation of isobutyryl-coenzyme A (isobutyryl-CoA) and methylmalonyl-CoA catalysed by a 3-ketoacyl-(acyl carrier protein) synthase III enzyme, followed by successive tailoring reactions in the fatty acid biosynthetic pathway. The HMP and RiPP substructures are then connected by an acyltransferase exhibiting promiscuous activity towards the fatty acyl and RiPP substrates. Overall, the discovery of lipoavitides contributes a prototype of RiPP/fatty-acid hybrids and provides possible enzymatic tools for lipopeptide bioengineering.

Investigation of the Biosynthetic Mechanism of Bipentaromycin Featuring an Unprecedented Cyclic Head-to-Tail Dimeric Scaffold.

Bipentaromycins are heterodimeric aromatic polyketides featuring two distinctive 5/6/6/6/5 pentacyclic ring systems and exhibit antibacterial activities. However, their overall biosynthetic mechanism, particularly the mechanism for early-stage modifications, such as hydrogenation and methylation, and late-stage dimerization, remains unknown. Herein, by integrating heterologous expression, isotope labeling, gene knockout and complementation, and computational modeling, we determined the biosynthetic origin of the skeleton, identified the enzymes involved in stereo-/regioselective hydrogenation and methylation, and provided new mechanistic insights into the dimerization. This work not only deciphers the biosynthetic mechanism of bipentaromycins but also provides new strategies for creating biologically active dimeric pharmacophores for drug discovery and development.

Automated, self-resistance gene-guided, and high-throughput genome mining of bioactive natural products from Streptomyces.

Natural products (NPs) produced by bacteria, fungi and plants are a major source of drug leads. Streptomyces species are particularly important in this regard as they produce numerous natural products with prominent bioactivities. Here we report a fully a utomated, s calable and high-throughput platform for discovery of bioactive n atural p roducts in S treptomyces (FAST-NPS). This platform comprises computational prediction and prioritization of target biosynthetic gene clusters (BGCs) guided by self-resistance genes, highly efficient and automated direct cloning and heterologous expression of BGCs, followed by high-throughput fermentation and product extraction from Streptomyces strains. As a proof of concept, we applied this platform to clone 105 BGCs ranging from 10 to 100 kb that contain potential self-resistance genes from 11 Streptomyces strains with a success rate of 95%. Heterologous expression of all successfully cloned BGCs in Streptomyces lividans TK24 led to the discovery of 23 natural products from 12 BGCs. We selected 5 of these 12 BGCs for further characterization and found each of them could produce at least one natural product with antibacterial and/or anti-tumor activity, which resulted in a total of 8 bioactive natural products. Overall, this work would greatly accelerate the discovery of bioactive natural products for biomedical and biotechnological applications.

Non-modular Fatty Acid Synthases Yield Unique Acylation in Ribosomal Peptides.

Recent efforts in genome mining of ribosomally synthesized and post-translationally modified peptides (RiPPs) have expanded the diversity of post-translational modification chemistries 1, 2 . However, RiPPs are rarely reported as hybrid molecules incorporating biosynthetic machineries from other natural product families 3-8 . Here, we report lipoavitides, a class of RiPP/fatty acid hybrid lipopeptides that display a unique, membrane-targeting 4-hydroxy-2,4-dimethylpentanoyl (HMP)-modified N -terminus. The HMP is formed via condensation of isobutyryl-CoA and methylmalonyl-CoA catalyzed by a 3-ketoacyl-ACP synthase III enzyme, followed by successive tailoring reactions in the fatty acid biosynthetic pathway. The HMP and RiPP substructures are then connected by an acyltransferase exhibiting promiscuous activity towards the fatty acyl and RiPP substrates. Overall, the discovery of lipoavitides contributes a prototype of RiPP/fatty acid hybrids and provides possible enzymatic tools for lipopeptide bioengineering.

Genome mining unveils a class of ribosomal peptides with two amino termini.

The era of inexpensive genome sequencing and improved bioinformatics tools has reenergized the study of natural products, including the ribosomally synthesized and post-translationally modified peptides (RiPPs). In recent years, RiPP discovery has challenged preconceptions about the scope of post-translational modification chemistry, but genome mining of new RiPP classes remains an unsolved challenge. Here, we report a RiPP class defined by an unusual ( S )- N 2 , N 2 -dimethyl-1,2-propanediamine (Dmp)-modified C -terminus, which we term the daptides. Nearly 500 daptide biosynthetic gene clusters (BGCs) were identified by analyzing the RiPP Recognition Element (RRE), a common substrate-binding domain found in half of prokaryotic RiPP classes. A representative daptide BGC from Microbacterium paraoxydans DSM 15019 was selected for experimental characterization. Derived from a C -terminal threonine residue, the class-defining Dmp is installed over three steps by an oxidative decarboxylase, aminotransferase, and methyltransferase. Daptides uniquely harbor two positively charged termini, and thus we suspect this modification could aid in membrane targeting, as corroborated by hemolysis assays. Our studies further show that the oxidative decarboxylation step requires a functionally unannotated accessory protein. Fused to the C -terminus of the accessory protein is an RRE domain, which delivers the unmodified substrate peptide to the oxidative decarboxylase. This discovery of a class-defining post-translational modification in RiPPs may serve as a prototype for unveiling additional RiPP classes through genome mining.

Genome mining unveils a class of ribosomal peptides with two amino termini.

The era of inexpensive genome sequencing and improved bioinformatics tools has reenergized the study of natural products, including the ribosomally synthesized and post-translationally modified peptides (RiPPs). In recent years, RiPP discovery has challenged preconceptions about the scope of post-translational modification chemistry, but genome mining of new RiPP classes remains an unsolved challenge. Here, we report a RiPP class defined by an unusual (S)-N2,N2-dimethyl-1,2-propanediamine (Dmp)-modified C-terminus, which we term the daptides. Nearly 500 daptide biosynthetic gene clusters (BGCs) were identified by analyzing the RiPP Recognition Element (RRE), a common substrate-binding domain found in half of prokaryotic RiPP classes. A representative daptide BGC from Microbacterium paraoxydans DSM 15019 was selected for experimental characterization. Derived from a C-terminal threonine residue, the class-defining Dmp is installed over three steps by an oxidative decarboxylase, aminotransferase, and methyltransferase. Daptides uniquely harbor two positively charged termini, and thus we suspect this modification could aid in membrane targeting, as corroborated by hemolysis assays. Our studies further show that the oxidative decarboxylation step requires a functionally unannotated accessory protein. Fused to the C-terminus of the accessory protein is an RRE domain, which delivers the unmodified substrate peptide to the oxidative decarboxylase. This discovery of a class-defining post-translational modification in RiPPs may serve as a prototype for unveiling additional RiPP classes through genome mining.

Biochemical and structural insights of multifunctional flavin-dependent monooxygenase FlsO1-catalyzed unexpected xanthone formation.

Xanthone-containing natural products display diverse pharmacological properties. The biosynthetic mechanisms of the xanthone formation have not been well documented. Here we show that the flavoprotein monooxygenase FlsO1 in the biosynthesis of fluostatins not only functionally compensates for the monooxygenase FlsO2 in converting prejadomycin to dehydrorabelomycin, but also unexpectedly converts prejadomycin to xanthone-containing products by catalyzing three successive oxidations including hydroxylation, epoxidation and Baeyer-Villiger oxidation. We also provide biochemical evidence to support the physiological role of FlsO1 as the benzo[b]-fluorene C5-hydrolase by using nenestatin C as a substrate mimic. Finally, we resolve the crystal structure of FlsO1 in complex with the cofactor flavin adenine dinucleotide close to the "in" conformation to enable the construction of reactive substrate-docking models to understand the basis of a single enzyme-catalyzed multiple oxidations. This study highlights a mechanistic perspective for the enzymatic xanthone formation in actinomycetes and sets an example for the versatile functions of flavoproteins.

Flavin-enabled reductive and oxidative epoxide ring opening reactions.

Epoxide ring opening reactions are common and important in both biological processes and synthetic applications and can be catalyzed in a non-redox manner by epoxide hydrolases or reductively by oxidoreductases. Here we report that fluostatins (FSTs), a family of atypical angucyclines with a benzofluorene core, can undergo nonenzyme-catalyzed epoxide ring opening reactions in the presence of flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (NADH). The 2,3-epoxide ring in FST C is shown to open reductively via a putative enol intermediate, or oxidatively via a peroxylated intermediate with molecular oxygen as the oxidant. These reactions lead to multiple products with different redox states that possess a single hydroxyl group at C-2, a 2,3-vicinal diol, a contracted five-membered A-ring, or an expanded seven-membered A-ring. Similar reactions also take place in both natural products and other organic compounds harboring an epoxide adjacent to a carbonyl group that is conjugated to an aromatic moiety. Our findings extend the repertoire of known flavin chemistry that may provide new and useful tools for organic synthesis.

Cas12a-assisted precise targeted cloning using in vivo Cre-lox recombination.

Direct cloning represents the most efficient strategy to access the vast number of uncharacterized natural product biosynthetic gene clusters (BGCs) for the discovery of novel bioactive compounds. However, due to their large size, repetitive nature, or high GC-content, large-scale cloning of these BGCs remains an overwhelming challenge. Here, we report a scalable direct cloning method named Cas12a-assisted precise targeted cloning using in vivo Cre-lox recombination (CAPTURE) which consists of Cas12a digestion, a DNA assembly approach termed T4 polymerase exo + fill-in DNA assembly, and Cre-lox in vivo DNA circularization. We apply this method to clone 47 BGCs ranging from 10 to 113 kb from both Actinomycetes and Bacilli with ~100% efficiency. Heterologous expression of cloned BGCs leads to the discovery of 15 previously uncharacterized natural products including six cyclic head-to-tail heterodimers with a unique 5/6/6/6/5 pentacyclic carbon skeleton, designated as bipentaromycins A-F. Four of the bipentaromycins show strong antimicrobial activity to both Gram-positive and Gram-negative bacteria such as methicillin-resistant Staphylococcus aureus, vancomycinresistant Enterococcus faecium, and bioweapon Bacillus anthracis. Due to its robustness and efficiency, our direct cloning method coupled with heterologous expression provides an effective strategy for large-scale discovery of novel natural products.

Albumycin, a new isoindolequinone from Streptomyces albus J1074 harboring the fluostatin biosynthetic gene cluster.

Heterologous expression of the fluostatin biosynthetic gene cluster from the marine-derived Micromonospora rosaria SCSIO N160 in Streptomyces albus J1074 led to the isolation of a novel isoindolequinone albumycin (1) and a known isoquinolinequinone mansouramycin A (2). The structure of 1 was elucidated on the basis of detailed 1D and 2D NMR spectroscopic analysis. Mansouramycin A (2) is active against methicillin-resistant Staphylococcus aureus ATCC 43300, with a MIC of 8 µg ml-1, while albumycin (1) displayed negligible antibacterial activities. This study represents another example of activation of secondary metabolites that are non-relevant to the heterologously introduced biosynthetic gene cluster in a bacterial host.