Enhancing the physicochemical properties and bioactivities of 2'-hydroxyflavanone through fungal biotransformation.

Flavonoids comprise a group of natural compounds with diverse bioactivities; however, their low water solubility and limited bioavailability often impede their potential health benefits for humans. In this study, five derivatives, namely 2',5'-dihydroxyflavanone (1), 2'-dihydroxyflavanone-5'-O-4″-O-methyl-ß-d-glucoside (2), 2'-dihydroxyflavanone-6-O-4″-O-methyl-ß-d-glucoside (3), 2'-dihydroxyflavanone-3'-O-4″-O-methyl-ß-d-glucoside (4) and hydroxyflavanone-2'-O-4″-O-methyl-ß-d-glucoside (5), were biosynthesized from 2'-hydroxyflavanone through microbial transformation using Beauveria bassiana ATCC 7159. Product 1 was identified as a known compound while 2-5 were structurally characterized as new structures through extensive 1D and 2D NMR analysis. The water solubility of biotransformed products 1-5 was enhanced by 30-280 times compared to the substrate 2'-hydroxyflavanone. Moreover, the antioxidant assay revealed that 1 and 2 exhibited improved 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity relative to the substrate, decreasing the logIC50 from 8.08 ± 0.11 µM to 6.19 ± 0.08 µM and 7.15 ± 0.08 µM, respectively. Compound 5 displayed significantly improved anticancer activity compared to the substrate 2'-hydroxyflavanone against Glioblastoma 33 cancer stem cells, decreasing the IC50 from 25.05 µM to 10.59 µM. Overall, fungal biotransformation represents an effective tool to modify flavonoids for enhanced water solubility and bioactivities.

Sustained silencing peanut allergy by xanthopurpurin is associated with suppression of peripheral and bone marrow IgE-producing B cell.

Introduction: Peanut allergy is an immunoglobulin E (IgE) mediated food allergy. Rubia cordifolia L. (R. cordifolia), a Chinese herbal medicine, protects against peanut-induced anaphylaxis by suppressing IgE production in vivo. This study aims to identify IgE-inhibitory compounds from the water extract of R. cordifolia and investigate the underlying mechanisms using in vitro and in vivo models. Methods: Compounds were isolated from R. cordifolia water extract and their bioactivity on IgE production was assessed using a human myeloma U266 cell line. The purified active compound, xanthopurpurin (XPP), was identified by LC-MS and NMR. Peanut-allergic C3H/HeJ mice were orally administered with or without XPP at 200µg or 400µg per mouse per day for 4 weeks. Serum peanut-specific IgE levels, symptom scores, body temperatures, and plasma histamine levels were measured at challenge. Cytokines in splenocyte cultures were determined by ELISA, and IgE + B cells were analyzed by flow cytometry. Acute and sub-chronic toxicity were evaluated. IL-4 promoter DNA methylation, RNA-Seq, and qPCR analysis were performed to determine the regulatory mechanisms of XPP. Results: XPP significantly and dose-dependently suppressed the IgE production in U266 cells. XPP significantly reduced peanut-specific IgE (>80%, p <0.01), and plasma histamine levels and protected the mice against peanut-allergic reactions in both early and late treatment experiments (p < 0.05, n=9). XPP showed a strong protective effect even 5 weeks after discontinuing the treatment. XPP significantly reduced the IL-4 level without affecting IgG or IgA and IFN-γ production. Flow cytometry data showed that XPP reduced peripheral and bone marrow IgE + B cells compared to the untreated group. XPP increased IL-4 promoter methylation. RNA-Seq and RT-PCR experiments revealed that XPP regulated the gene expression of CCND1, DUSP4, SDC1, ETS1, PTPRC, and IL6R, which are related to plasma cell IgE production. All safety testing results were in the normal range. Conclusions: XPP successfully protected peanut-allergic mice against peanut anaphylaxis by suppressing IgE production. XPP suppresses murine IgE-producing B cell numbers and inhibits IgE production and associated genes in human plasma cells. XPP may be a potential therapy for IgE-mediated food allergy.

Engineered production of bioactive polyphenolic O-glycosides.

Polyphenolic compounds (such as quercetin and resveratrol) possess potential medicinal values due to their various bioactivities, but poor water solubility hinders their health benefits to humankind. Glycosylation is a well-known post-modification method to biosynthesize natural product glycosides with improved hydrophilicity. Glycosylation has profound effects on decreasing toxicity, increasing bioavailability and stability, together with changing bioactivity of polyphenolic compounds. Therefore, polyphenolic glycosides can be used as food additives, therapeutics, and nutraceuticals. Engineered biosynthesis provides an environmentally friendly and cost-effective approach to generate polyphenolic glycosides through the use of various glycosyltransferases (GTs) and sugar biosynthetic enzymes. GTs transfer the sugar moieties from nucleotide-activated diphosphate sugar (NDP-sugar) donors to sugar acceptors such as polyphenolic compounds. In this review, we systematically review and summarize the representative polyphenolic O-glycosides with various bioactivities and their engineered biosynthesis in microbes with different biotechnological strategies. We also review the major routes towards NDP-sugar formation in microbes, which is significant for producing unusual or novel glycosides. Finally, we discuss the trends in NDP-sugar based glycosylation research to promote the development of prodrugs that positively impact human health and wellness.

Genus Tetrastigma: A review of its folk uses, phytochemistry and pharmacology.

The genus Tetrastigma belongs to the Vitaceae family and contains over 100 species. This paper reviewed folk uses, chemical constituents, pharmacological activities, and clinical applications of the medicinal plants in the genus Tetrastigma. In addition, the paper also discussed the current problems for the further studies. Up to now, more than 240 compounds were reported from the genus Tetrastigma, covering 74 flavonoids, 14 terpenoids, 19 steroids, 21 phenylpropanoids, 14 alkaloids and others constituents. Among them, flavonoids are the major and the characteristic chemical constituents in this genus. Modern pharmacological studies and clinical practice showed that the extracts and chemical constituents of Tetrastigma species possessed wide pharmacological activities including antitumor, antioxidative, hepatoprotective, antiviral, anti-inflammatory, and analgesic activities. The information summarized in this paper provides valuable clues for new drug discovery and an incentive to expand the research of genus Tetrastigma.

Identification of a novel glucuronyltransferase from Streptomyces chromofuscus ATCC 49982 for natural product glucuronidation.

Glycosylation is an effective way to increase the polarity of natural products. UDP-glucuronyltransferases (UGTs) are commonly observed and extensively studied in phase II drug metabolism. However, UGTs in microorganisms are not well studied, which hampered the utilization of this type of enzyme in microbial glucuronidation of natural products. Screening of five actinomycete strains showed that Streptomyces chromofuscus ATCC 49982 can convert diverse plant polyphenols into more polar products, which were characterized as various glucuronides based on their spectral data. Analysis of the genome of this strain revealed a putative glucuronidation gene cluster that contains a UGT gene (gcaC) and two UDP-glucuronic acid biosynthetic genes (gcaB and gcaD). The gcaC gene was cloned and heterologously expressed in Escherichia coli BL21(DE3). Incubation of the purified enzyme with resveratrol and UDP-glucuronic acid led to the production of resveratrol-4'-O-ß-D-glucuronide and resveratrol-3-O-ß-D-glucuronide, allowing GcaC to be characterized as a flexible UGT. The optimal in vitro reaction pH and temperature for GcaC are 7.5 and 30 °C, respectively. Its activity can be stimulated by Ca2+, Mg2+, and Mn2+, whereas Zn2+, Cu2+, and Fe2+ showed inhibitory effects. Furthermore, GcaC has a broad substrate specificity, which can glucuronidate various substrates besides resveratrol, including quercetin, ferulic acid, vanillic acid, curcumin, vanillin, chrysin, zearalenone, and apigenin. The titers of resveratrol-4'-O-ß-D-glucuronide and resveratrol-3-O-ß-D-glucuronide in E. coli-GcaC were 78.381 ± 0.366 mg/L and 14.991 ± 0.248 mg/L from 114.125 mg/L resveratrol within 3 h. Therefore, this work provides an effective way to produce glucuronides of resveratrol and other health-benefitting natural products. KEY POINTS: • A novel versatile microbial UDP-glucuronyltransferase was discovered and characterized from Streptomyces chromofuscus ATCC 49982. • The UDP-glucuronyltransferase was expressed in Escherichia coli and can convert resveratrol into two glucuronides both in vitro and in vivo. • The UDP-glucuronyltransferase has a highly flexible substrate specificity and is an effective tool to prepare mono- or diglucuronides of bioactive molecules.

Manipulation of two regulatory genes for efficient production of chromomycins in Streptomyces reseiscleroticus.

BACKGROUND: Regulatory genes play critical roles in natural product biosynthetic pathways. Chromomycins are promising anticancer natural products from actinomycetes. This study is aimed to create an efficient strain for production of these molecules by manipulating the regulatory genes. RESULTS: A putative but silent chromomycin biosynthetic gene cluster was discovered in Streptomyces reseiscleroticus. Heterologous expression of the ketosynthase, chain length factor, and acyl carrier protein in Streptomyces lividans confirmed that they are responsible for the assembly of a decaketide. Two regulatory genes are present in this gene cluster, including SARP-type activator SrcmRI and PadR-like repressor SrcmRII. Either overexpression of SrcmRI or disruption of SrcmRII turned on the biosynthetic pathway of chromomycins. The production titers of chromomycin A3/A2 in R5 agar in these two strains reached 8.9 ± 1.2/13.2 ± 1.6 and 49.3 ± 4.3/53.3 ± 3.6 mg/L, respectively. An engineered strain was then constructed with both SrcmRII disruption and SrcmRI overexpression, which produced chromomycins A3 and A2 in R5 agar at 69.4 ± 7.6 and 81.7 ± 7.2 mg/L, respectively. Optimization of the culture conditions further increased the titers of chromomycins A3 and A2 respectively to 145.1 ± 15.3 and 158.3 ± 15.4 mg/L in liquid fermentation. CONCLUSIONS: This work revealed the synergistic effect of manipulation of pathway repressor and activator genes in the engineering of a natural product biosynthetic pathway. The resulting engineered strain showed the highest production titers of chromomycins by a strain of Streptomyces, providing an efficient way to produce these pharmaceutically valuable molecules.

The Flavonoid 7,4'-Dihydroxyflavone Prevents Dexamethasone Paradoxical Adverse Effect on Eotaxin Production by Human Fibroblasts.

Eotaxin/CCL-11 is a major chemoattractant that contributes to eosinophilic inflammation in asthma. Glucocorticoids inhibit inflammation, but long-time exposure may cause paradoxical adverse effects by augmenting eotaxin/CCL-11production. The aim of this study was to determine if 7,4'-dihydroxyflavone (7,4'-DHF), the eotaxin/CCL11 inhibitor isolated from Glycyrrhiza uralensis, reduces in vitro eotaxin production induced by long-time dexamethasone (Dex) exposure, and if so, to elucidate the mechanisms of this inhibition. Human lung fibroblast-1 cells were used to identify the potency of 7,4'-DHF compared with other compounds from G. uralensis, to compare 7,4'-DHF with Dex on eotaxin production following 24-h short-time culture and 72-h longer-time (LT) culture, and to determine the effects of the 7,4'-DHF on Dex LT culture augmented eotaxin production and molecule mechanisms. 7,4'-DHF was the most potent eotaxin/CCL-11 inhibitor among the ten compounds and provided continued suppression. In contrast to short-time culture, Dex LT culture increased constitutively, and IL-4/TNF-α stimulated eotaxin/CCL11 production by human lung fibroblast-1 cells. This adverse effect was abrogated by 7,4'-DHF co-culture. 7,4'-DHF significantly inhibited Dex LT culture augmentation of p-STAT6 and impaired HDAC2 expression. This study demonstrated that 7,4'-DHF has the ability to consistently suppress eotaxin production and prevent Dex-paradoxical adverse effects on eotaxin production. Copyright © 2017 John Wiley & Sons, Ltd.

Engineered Biosynthesis of Medicinally Important Plant Natural Products in Microorganisms.

Plants produce structurally and functionally diverse natural products. Some of these compounds possess promising health-benefiting properties, such as resveratrol (antioxidant) curcumin (anti-inflammatory, anti-allergic and anticancer), paclitaxel (anticancer) and artemisinin (antimalarial). These compounds are produced through particular biosynthetic pathways in the plants. While supply of these medicinally important molecules relies on extraction from the producing species, recent years have seen significant advances in metabolic engineering of microorganisms for the production of plant natural products. Escherichia coli and Saccharomyces cerevisiae are the two most widely used heterologous hosts for expression of enzymes and reconstitution of plant natural product biosynthetic pathways. Total biosynthesis of many plant polyketide natural products such as curcumin and piceatannol in microorganisms has been achieved. While the late biosynthetic steps of more complex molecules such as paclitaxel and artemisinin remain to be understood, reconstitution of their partial biosynthetic pathways and microbial production of key intermediates have been successful. This review covers recent advances in understanding and engineering the biosynthesis of plant polyketides and terpenoids in microbial hosts.

Ganoderic acid C1 isolated from the anti-asthma formula, ASHMI™ suppresses TNF-α production by mouse macrophages and peripheral blood mononuclear cells from asthma patients.

Asthma is a heterogeneous airway inflammatory disease, which is associated with Th2 cytokine-driven inflammation and non-Th2, TNF-α mediated inflammation. Unlike Th2 mediated inflammation, TNF-α mediated asthma inflammation is generally insensitive to inhaled corticosteroids (ICS). ASHMITM, aqueous extract of three medicinal herbs-Ganoderma lucidum (G. lucidum), Sophora flavescens Ait (S. flavescens) and Glycyrrhiza uralensis Fischer (G. uralensis), showed a high safety profile and was clinically beneficial in asthma patients. It also suppresses both Th2 and TNF-α associated inflammation in murine asthma models. We previously determined that G. uralensis flavonoids are the key active compounds responsible for ASHMITM suppression of Th2 mediated inflammation. Until now, there are limited studies on anti-TNF-α compounds presented in ASHMITM. The objective of this study was to isolate and identify TNF-α inhibitory compounds in ASHMITM. Here we report that G. lucidum, but not the other two herbal extracts, S. flavescens or G. uralensis inhibited TNF-α production by murine macrophages; and that the methylene chloride (MC)-triterpenoid-enriched fraction, but not the polysaccharide-enriched fraction, contained the inhibitory compounds. Of the 15 triterpenoids isolated from the MC fraction, only ganoderic acid C1 (GAC1) significantly reduced TNF-α production by murine macrophages (RAW 264.7 cells) and peripheral blood mononuclear cells (PBMCs) from asthma patients. Inhibition was associated with down-regulation of NF-κB expression, and partial suppression of MAPK and AP-1 signaling pathways. Ganoderic acid C1 may have potential for treating TNF-α mediated inflammation in asthma and other inflammatory diseases.

Berberine and limonin suppress IgE production by human B cells and peripheral blood mononuclear cells from food-allergic patients.

BACKGROUND: Currently, there is no satisfactory treatment for IgE-mediated food allergy. Food Allergy Herbal Formula 2 (FAHF-2) and butanol-purified FAHF-2 (B-FAHF-2) have been shown to protect against peanut-induced anaphylaxis and inhibit IgE synthesis in a murine model. OBJECTIVE: To determine which herbs and compounds in FAHF-2 and B-FAHF-2 suppress IgE production. METHODS: The effect of FAHF-2 and B-FAHF-2 on IgE production was determined using a human B-cell line (U266). Individual compounds were isolated and identified using column chromatography, liquid chromatographic mass spectrometry, and nuclear magnetic resonance techniques. The potency of compounds on IgE suppression were investigated using U266 cells and verified using human peripheral blood mononuclear cells (n = 25) from peanut-allergic patients. Epsilon germline transcript expression was determined. Phosphorylated IκBα level was analyzed using the In-Cell Western assay. The mRNA expression of signal transducer and activator of transcription-3, T-box transcription factor TBX21, interferon-γ, forkhead box P3, GATA-binding protein 3, interleukin-10, and interleukin-5 also were analyzed using real-time polymerase chain reaction. RESULTS: FAHF-2 and B-FAHF-2 inhibited IgE production by U266 cells. B-FAHF-2 was 9 times more effective than FAHF-2. Two compounds that inhibited IgE production were isolated from Philodendron chinensis and identified as berberine and limonin. Berberine was more potent and inhibited IgE production by peripheral blood mononuclear cells by 80% at 0.62 µg/mL. Berberine significantly inhibited ε-germline transcript expression by peripheral blood mononuclear cells. Phosphorylated IκBα level was significantly suppressed and mRNA expressions of T-box transcription factor TBX21 and signal transducer and activator of transcription-3 were significantly increased by berberine. CONCLUSION: Berberine and limonin mediated IgE suppression. The mechanism by which berberine modulates ε-germline transcript expression might be through regulating the phosphorylated IκBα level and the expressions of signal transducer and activator of transcription-3 and T-box transcription factor TBX21. TRIAL REGISTRATION: Clinicaltrials.gov identifier NCT00602160.

Diversity-oriented combinatorial biosynthesis of benzenediol lactone scaffolds by subunit shuffling of fungal polyketide synthases.

Combinatorial biosynthesis aspires to exploit the promiscuity of microbial anabolic pathways to engineer the synthesis of new chemical entities. Fungal benzenediol lactone (BDL) polyketides are important pharmacophores with wide-ranging bioactivities, including heat shock response and immune system modulatory effects. Their biosynthesis on a pair of sequentially acting iterative polyketide synthases (iPKSs) offers a test case for the modularization of secondary metabolic pathways into "build-couple-pair" combinatorial synthetic schemes. Expression of random pairs of iPKS subunits from four BDL model systems in a yeast heterologous host created a diverse library of BDL congeners, including a polyketide with an unnatural skeleton and heat shock response-inducing activity. Pairwise heterocombinations of the iPKS subunits also helped to illuminate the innate, idiosyncratic programming of these enzymes. Even in combinatorial contexts, these biosynthetic programs remained largely unchanged, so that the iPKSs built their cognate biosynthons, coupled these building blocks into chimeric polyketide intermediates, and catalyzed intramolecular pairing to release macrocycles or α-pyrones. However, some heterocombinations also provoked stuttering, i.e., the relaxation of iPKSs chain length control to assemble larger homologous products. The success of such a plug and play approach to biosynthesize novel chemical diversity bodes well for bioprospecting unnatural polyketides for drug discovery.

Synthesis of two new hydroxylated derivatives of spironolactone by microbial transformation.

Spironolactone is a medicinally important molecule that is clinically used in the treatment and management of many diseases such as oedema and ascites in cirrhosis of the liver, malignant ascites, nephrotic syndrome, chronic lung disease, resistant hypertension, congestive heart failure, and primary hyperaldosteronism. Microbial transformations of spironolactone by Cunninghamella elegans ATCC 9245 was carried out. Two new hydroxylated derivatives, 12ß-hydroxy-spironolactone and 2α-hydroxy-spironolactone, were synthesized. Their structures were characterized on the basis of the spectroscopic data. The substrate can be efficiently converted into the products within 72 h after its addition to the fermentation broth of C. elegans ATCC 9245.

Functional dissection and module swapping of fungal cyclooligomer depsipeptide synthetases.

BbBSLS and BbBEAS were dissected and reconstituted in Saccharomyces cerevisiae. The intermodular linker is essential for the reconstitution of the separate modules. Module 1 can be swapped between BbBEAS and BbBSLS, while modules 2 and 3 control the product profiles. BbBSLS is a flexible enzyme that also synthesizes beauvericins.

Engineered production of fungal anticancer cyclooligomer depsipeptides in Saccharomyces cerevisiae.

Two fungal cyclooligomer depsipeptide synthetases(CODSs), BbBEAS (352 kDa) and BbBSLS (348 kDa) from Beauveria bassiana ATCC7159, were reconstituted in Saccharomyces cerevisiae BJ5464-NpgA, leading to the production of the corresponding anticancer natural products, beauvericins and bassianolide, respectively. The titers of beauvericins (33.8 ± 1.4 mg/l) and bassianolide (21.7± 0.1 mg/l) in the engineered S. cerevisiae BJ5464-NpgA strains were comparable to those in the native producer B. bassiana. Feeding D-hydroxyisovaleric acid (D-Hiv) and the corresponding L-amino acid precursors improved the production of beauvericins and bassianolide. However, the high price of D-Hiv limits its application in large-scale production of these cyclooligomer depsipeptides. Alternatively, we engineered another enzyme, ketoisovalerate reductase (KIVR) from B. bassiana, into S. cerevisiae BJ5464-NpgA for enhanced in situ synthesis of this expensive substrate. Co-expression of BbBEAS and KIVR in the yeast led to significant improvement of the production of beauvericins.The total titer of beauvericin and its congeners (beauvericins A-C) was increased to 61.7 ± 3.0 mg/l and reached 2.6-fold of that in the native producer B. bassiana ATCC7159. Supplement of L-Val at 10 mM improved the supply of ketoisovalerate, the substrate of KIVR, which consequently further increased the total titer of beauvericins to 105.8 ± 2.1 mg/l. Using this yeast system,we functionally characterized an unknown CODS from Fusarium venenatum NRRL 26139 as a beauvericin synthetase, which was named as FvBEAS. Our work thus provides a useful approach for functional reconstitution and engineering of fungal CODSs for efficient production of this family of anticancer molecules.

Specific 5-hydroxylation of piperlongumine by Beauveria bassiana ATCC 7159.

A new hydroxylated derivative was efficiently prepared by transforming the natural anti-cancer product, piperlongumine, with Beauveria bassiana ATCC 7159. Its structure was determined to be 5-hydroxylpiperlongumine on the basis of the spectroscopic data. The absolute configuration at C-5 was established as R by Mosher's method.

Specific inhibition of the halogenase for radicicol biosynthesis by bromide at the transcriptional level in Pochonia chlamydosporia.

Pochonia chlamydosporia produces radicicol (1), a potent antifungal and anticancer product. NaBr, but not NaF, NaCl or NaI, inhibited the biosynthesis of 1 in P. chlamydosporia in a dose-dependent manner, accompanied by the formation chlorine-lacking monocillins II-V (2-5), indicating that the dedicated halogenase, Rdc2 had been inhibited. RT-PCR analysis confirmed that transcription of rdc2 was selectively inhibited by Br(-), whereas the putative P450 epoxidase gene, rdc4, was not affected.

Biosynthesis of bacterial aromatic polyketides.

Aromatic polyketides represent important members of the family of polyketides, which have displayed a wide assortment of bioactive properties, such as antibacterial, antitumor, and antiviral activities. Bacterial aromatic polyketides are mainly synthesized by type II polyketide synthases (PKSs). Whereas malonyl-CoA is exclusively used as the extender unit, starter units can vary in different aromatic polyketide biosynthetic pathways, leading to a variety of polyketide backbones. Once the polyketide chains are elongated by the minimal PKSs to the full length, the immediate tailoring enzymes including ketoreductases, oxygenases and cyclases will work on the nascent chains to form aromatic structures, which will be further decorated by those late tailoring enzymes such as methyltransferases and glycosyltransferases. The mechanistic studies on the biosynthetic pathways of aromatic polyketides such as oxytetracycline and pradimicin A have been extensively carried out in recent years. Engineered biosynthesis of novel "unnatural" polyketides has been achieved in heterologous hosts such as Streptomyces coelicolor and Escherichia coli. This review covers the most recent advances in aromatic polyketide biosynthesis, which provide new enzymes or methods for building novel polyketide biosynthetic machinery.

Microbial metabolism of 1-aminoanthracene by Beauveria bassiana.

The carcinogen and mutagen, 1-aminoanthracene, was efficiently metabolized by the fungal strain Beauveria bassiana ATCC 7159 to yield three new metabolites identified as 1-acetamido-5-[(4'-O-methyl-beta-d-glucopyranosyl)oxy]anthracene, 1-acetamido-8-[(4'-O-methyl-beta-d-glucopyranosyl)oxy]anthraquinone, and 1-acetamido-6-[(4'-O-methyl-beta-d-glucopyranosyl)oxy]anthraquinone, together with 1-acetamidoanthracene and 1-acetamidoanthraquinone. Formation of these metabolites suggests that the metabolic pathways of 1-aminoanthracene in B. bassiana ATCC 7159 involve acetylation, oxidation, hydroxylation, and O-methylglucosylation.

Identifying the minimal enzymes required for anhydrotetracycline biosynthesis.

The cyclohexenone ring A of tetracyclines exhibits unique structural features not observed among other aromatic polyketides. These substitutions include the C2 primary amide, C4 dimethylamine, and the C12a tertiary alcohol. Here we report the identification and reconstitution of the minimum set of enzymes required for the biosynthesis of anhydrotetracycline (ATC, 5), the first intermediate in the tetracycline biosynthetic pathway that contains the fully functionalized ring A. Using a combination of in vivo and in vitro approaches, we confirmed OxyL, OxyQ, and OxyT to be the only enzymes required to convert 6-methylpretetramid 1 into 5. OxyL is a NADPH-dependent dioxygenase that introduces two oxygen atoms into 1 to yield the unstable intermediate 4-keto-ATC 2. The aminotransferase OxyQ catalyzes the reductive amination of C4-keto of 2, yielding 4-amino-ATC 3. Furthermore, the N, N-dimethyltransferase OxyT catalyzes the formation of 5 from 3 in a (S)-adenosylmethionine (SAM)-dependent manner. Finally, a "non-natural" anhydrotetracycline derivative was generated, demonstrating that our heterologous host/vector pair can be a useful platform toward the engineered biosynthesis of tetracycline analogues.

Cytotoxic and Antihaptotactic beauvericin analogues from precursor-directed biosynthesis with the insect pathogen Beauveria bassiana ATCC 7159.

Precursor-directed biosynthesis was used to produce analogues of the cyclic depsipeptide mycotoxin beauvericin (1) using the filamentous fungus Beauveria bassiana ATCC 7159. Feeding 30 analogues of D-2-hydroxyisovalerate and L-phenylalanine, the natural 2-hydroxycarboxylic acid and amino acid precursors of beauvericin, led to the biosynthesis of novel beauvericins. Six of these were isolated and characterized, and their cytotoxicity and directional cell migration (haptotaxis) inhibitory activity against the metastatic prostate cancer cell line PC-3M were evaluated. Replacement of one, two, or all three of the D-2-hydroxyisovalerate constituents in beauvericin (1) with 2-hydroxybutyrate moieties (beauvericins G(1-3), compounds 2-4) caused a parallel decline of cell migration inhibitory activity and cytotoxicity, suggesting a requirement for a branched side chain for both of these biological activities at the corresponding positions of beauvericins. Replacement of one, two, or all three N-methyl-L-phenylalanine residues of beauvericin with N-methyl-L-3-fluorophenylalanine moieties (beauvericins H(1-3), compounds 5-7) increased cytotoxicity without affecting antihaptotactic activity.