Flavin-Dependent Monooxygenase-Mediated 1,2-Oxazine Construction via Meisenheimer Rearrangement in the Biosynthesis of Paeciloxazine.

The [1,2]-Meisenheimer rearrangement is well known as the [1,2]-migration of an O-substituted hydroxylamine from a tertiary amine N-oxide, and it is frequently employed in organic synthesis to enforce adjacent carbon oxidation or install a 1,2-oxazine core, which is a prevalent structural feature and pharmacophore of many bioactive natural products. Although the [1,2]-Meisenheimer rearrangement was proposed to occur in the biosynthesis of a number of 1,2-oxazine-containing natural products, it has never been proved biosynthetically. Here, we identified the biosynthetic gene cluster of an insecticidal natural product, paeciloxazine (1), from Penicillium janthinellum and characterized a flavin-dependent monooxygenase, PaxA, as the first example that mediates the formation of a 1,2-oxazine moiety via Meisenheimer rearrangement. In vitro biochemical assays, site-directed mutations, docking and molecular dynamics simulations, and density functional theory calculations support the mechanism that PaxA first catalyzes N-oxidation to form an N-oxide intermediate, which undergoes [1,2]-Meisenheimer rearrangement with the assistance of an amino acid with proton transfer property. This study expands the repertoire of rearrangement reactions during the biosynthesis of natural products and provides a new strategy for discovering natural products with N-O tethers by genome mining.

Highly Oxygenated Caryophyllene-Type Sesquiterpenes from a Plant-Associated Fungus, Pestalotiopsis hainanensis, and Their Biosynthetic Gene Cluster.

Seven new ß-caryophyllene derivatives, pestalotiphains A-G (1-7), along with six known analogues (8-13), were isolated from the plant-associated Pestalotiopsis hainanensis. Compound 1 represents the first example of a caryophyllene-adenine hybrid, and 2 contains a novel oxatricyclo[4.3.1.0] system. Their structures and absolute configurations were assigned by interpretation of a combination of spectroscopic data and electronic circular dichroism calculations. Compound 8 exhibited moderate inhibition of HL-60 and THP-1 cell lines (IC50, 6.2 and 2.0 µM, respectively). A candidate biosynthetic gene cluster responsible for these compounds was uncovered by bioinformatics analyses and confirmed by a biochemical approach.

Chrysoxanthones A⁻C, Three New Xanthone⁻Chromanone Heterdimers from Sponge-Associated Penicillium chrysogenum HLS111 Treated with Histone Deacetylase Inhibitor.

By treating with histone-deacetylase inhibitor valproate sodium, three new heterdimeric tetrahydroxanthone⁻chromanone lactones chrysoxanthones A⁻C (1⁻3), along with 17 known compounds were isolated from a sponge-associated Penicillium chrysogenum HLS111. The planar structures of chrysoxanthones A⁻C were elucidated by means of spectroscopic analyses, including MS, 1D, and 2D NMR. Their absolute configurations were established by electronic circular dichroism (ECD) calculations. Chrysoxanthones A⁻C exhibited moderate antibacterial activities against Bacillus subtilis with minimum inhibitory concentration (MIC) values of 5⁻10 µg/mL.

Diphenyl Ethers from a Marine-Derived Aspergillus sydowii.

Six new diphenyl ethers (1⁻6) along with eleven known analogs were isolated from the ethyl acetate extract of a marine-derived Aspergillus sydowii guided by LC-UV-MS. Their structures were unambiguously characterized by HRESIMS, NMR, as well as chemical derivatization. Compounds 1 and 2 are rare diphenyl ether glycosides containing d-ribose. The absolute configuration of the sugar moieties in compounds 1⁻3 was determined by a LC-MS method. All the compounds were evaluated for their cytotoxicities against eight cancer cell lines, including 4T1, U937, PC3, HL-60, HT-29, A549, NCI-H460, and K562, and compounds 1, 5, 6, and 8⁻11 were found to exhibit selective cytotoxicity against different cancer cell lines.

A Cascade of Redox Reactions Generates Complexity in the Biosynthesis of the Protein Phosphatase-2 Inhibitor Rubratoxin†A.

Redox modifications are key complexity-generating steps in the biosynthesis of natural products. The unique structure of rubratoxin A (1), many of which arise through redox modifications, make it a nanomolar inhibitor of protein phosphatase 2A (PP2A). We identified the biosynthetic pathway of 1 and completely mapped the enzymatic sequence of redox reactions starting from the nonadride 5. Six redox enzymes are involved, including four α-ketoglutarate- and iron(II)-dependent dioxygenases that hydroxylate four sp3 carbons; one flavin-dependent dehydrogenase that is involved in formation of the unsaturated lactone; and the ferric-reductase-like enzyme RbtH, which regioselectively reduces one of the maleic anhydride moieties in rubratoxin B to the γ-hydroxybutenolide that is critical for PP2A inhibition. RbtH is proposed to perform sequential single-electron reductions of the maleic anhydride using electrons derived from NADH and transferred through a ferredoxin and ferredoxin reductase pair.

Heterotrophic nitrification and aerobic denitrification by a novel Halomonas campisalis.

A novel halophilic strain that could carry out heterotrophic nitrification and aerobic denitrification was isolated and named as Halomonas campisalis ha3. It removed inorganic nitrogen compounds (e.g. NO3 (-), NO2 (-) and NH4 (+)) simultaneously, and grew well in the medium containing up to 20 % (w/v) NaCl. PCR revealed four genes in the genome of ha3 related to aerobic denitrification: napA, nirS, norB and nosZ. The optimal conditions for aerobic denitrification were pH 9.0, at 37 °C, with 4 % (w/v) NaCl and sodium succinate as carbon source. The nitrogen removal rate was 87.5 mg NO3 (-)-N l(-1 )h(-1). Therefore, this strain is a potential aerobic denitrifier for the treatment of saline wastewater.

Phaeosphspirone (1/1'), a pair of unique polyketide enantiomers with an unusual 6/5/5/6 tetracyclic ring from the desert plant endophytic fungus Phaeosphaeriaceae sp.

Phaeosphspirone, an undescribed polyketide with a unique 6/5/5/6-fused tetracyclic system, and two known analogues, herbarin and O-methylherbarin, were purified from the endophytic fungus Phaeosphaeriaceae sp. isolated from the desert plant Bassia dasyphylla. The connectivity and relative configuration of phaeosphspirone was elucidated by comprehensive HR-ESI-MS and NMR analysis together with a computer-assisted structure elucidation (CASE) method. A pair of enantiomers existing in phaeosphspirone were separated by HPLC chromatography after reacting with chiral reagents, from which the absolute configuration of phaeosphspirone was simultaneously determined based on Mosher's rule. This tandem strategy provides a useful approach for the separation and stereochemical determination of enantiomers possessing secondary hydroxyl groups. The structural feature of phaeosphspirone, herbarin and O-methylherbarin together with gene cluster analysis suggested their polyketide biosynthetic origin. Herbarin and O-methylherbarin exhibited moderate cytotoxicity against three cancer cell lines.

Gene mining and efficient biosynthesis of a fungal peptidyl alkaloid.

Objective: Peptidyl alkaloids, a series of important natural products can be assembled by fungal non-ribosomal peptide synthetases (NRPSs). However, many of the NRPSs associated gene clusters are silent under laboratory conditions, and the traditional chemical separation yields are low. In this study, we aim to discovery and efficiently prepare fungal peptidyl alkaloids assembled by fungal NRPSs. Methods: Bioinformatics analysis of gene cluster containing NRPSs from the genome of Penicillium thymicola, and heterologous expression of the putative gene cluster in Aspergillus nidulans were performed. Isolation, structural identification, and biological evaluation of the product from heterologous expression were carried out. Results: The putative tri-modular NRPS AncA was heterologous-expressed in A. nidulans to give anacine (1) with high yield, which showed moderate and selective cytotoxic activity against A549 cell line. Conclusion: Heterologous expression in A. nidulans is an efficient strategy for mining fungal peptidyl alkaloids.

Genome mining combined metabolic shunting and OSMAC strategy of an endophytic fungus leads to the production of diverse natural products.

Endophytic fungi are promising producers of bioactive small molecules. Bioinformatic analysis of the genome of an endophytic fungus Penicillium dangeardii revealed 43 biosynthetic gene clusters, exhibited its strong ability to produce numbers of secondary metabolites. However, this strain mainly produce rubratoxins alone with high yield in varied culture conditions, suggested most gene clusters are silent. Efforts for mining the cryptic gene clusters in P. dangeardii, including epigenetic regulation and one-strain-many-compounds (OSMAC) approach were failed probably due to the high yield of rubratoxins. A metabolic shunting strategy by deleting the key gene for rubratoxins biosynthesis combining with optimization of culture condition successfully activated multiple silent genes encoding for other polyketide synthases (PKSs), and led to the trace compounds detectable. As a result, a total of 23 new compounds including azaphilone monomers, dimers, trimers with unprecedented polycyclic bridged heterocycle and spiral structures, as well as siderophores were identified. Some compounds showed significant cytotoxicities, anti-inflammatory or antioxidant activities. The attractive dual PKSs gene clusters for azaphilones biosynthesis were mined by bioinformatic analysis and overexpression of a pathway specific transcription factor. Our work therefor provides an efficient approach to mine the chemical diversity of endophytic fungi.

Complexity and Diversity Generation in the Biosynthesis of Fumiquinazoline-Related Peptidyl Alkaloids.

Fumiquinazolines are multicyclic peptidyl alkaloids where FAD-dependent oxidases are main tailing redox enzymes in their biosynthesis. Here, we characterized the use of an α-KG/Fe(II)-dependent dioxygenase (α-KGD) as a new strategy in Nature to increase structural complexity in fumiquinazolines biosynthesis by elucidating the concise three enzymes biosynthetic pathway of heptacyclical alanditrypinone (1). Further genome mining led to the discovery of additional gene cluster with α-KGD and trimodular NRPS as partner, which generates diverse fumiquinazolines.

Six new monacolin analogs from red yeast rice.

Six novel monacolin analogs, monacolins V1-V6 (1-6), together with seven known ones (7-13), were isolated from the ethyl acetate extract of red yeast rice. Their structures and absolute configurations were determined by spectroscopic methods, especially 2D NMR (1H-1HCOSY, HSQC, HMBC, and NOESY/ROESY) and CD spectroscopic analyses as well as chemical derivation. Monacolins V2 (2) and V3 (3) represent the first examples of monacolins with 3-hydroxybutyrate substitute. The anti-inflammatory inhibitory activities against the lipopolysaccharide (LPS) induced NO production in BV-2 cells as well as antioxidant activities against rat liver microsomal lipid peroxidation were evaluated.

Epigenetic modification in histone deacetylase deletion strain of Calcarisporium arbuscula leads to diverse diterpenoids.

Epigenetic modifications have been proved to be a powerful way to activate silent gene clusters and lead to diverse secondary metabolites in fungi. Previously, inactivation of a histone H3 deacetylase in Calcarisporium arbuscula had led to pleiotropic activation and overexpression of more than 75% of the biosynthetic genes and isolation of ten compounds. Further investigation of the crude extract of C. arbuscula ΔhdaA strain resulted in the isolation of twelve new diterpenoids including three cassanes (1-3), one cleistanthane (4), six pimaranes (5-10), and two isopimaranes (11 and 12) along with two know cleistanthane analogues. Their structures were elucidated by extensive NMR spectroscopic data analysis. Compounds 2 and 4 showed potent inhibitory effects on the expression of MMP1 and MMP2 (matrix metalloproteinases family) in human breast cancer (MCF-7) cells.

PCR-Based Seamless Genome Editing with High Efficiency and Fidelity in Escherichia coli.

Efficiency and fidelity are the key obstacles for genome editing toolboxes. In the present study, a PCR-based tandem repeat assisted genome editing (TRAGE) method with high efficiency and fidelity was developed. The design of TRAGE is based on the mechanism of repair of spontaneous double-strand breakage (DSB) via replication fork reactivation. First, cat-sacB cassette flanked by tandem repeat sequence was integrated into target site in chromosome assisted by Red enzymes. Then, for the excision of the cat-sacB cassette, only subculturing is needed. The developed method was successfully applied for seamlessly deleting, substituting and inserting targeted genes using PCR products. The effects of different manipulations including sucrose addition time, subculture times in LB with sucrose and stages of inoculation on the efficiency were investigated. With our recommended procedure, seamless excision of cat-sacB cassette can be realized in 48 h efficiently. We believe that the developed method has great potential for seamless genome editing in E. coli.

Ionic liquid pretreatment to increase succinic acid production from lignocellulosic biomass.

In this study, pinewood and corn stover pretreated with the ionic liquid (IL) 1-allyl-3-methylimidazolium chloride (AmimCl) were used as a feedstock for succinic acid production. Results reveal that 5% (v/v) AmimCl inhibited bacterial growth, whereas 0.01% (v/v) AmimCl inhibited succinic acid production. AmimCl was effective in extracting cellulose from pinewood and in degrading pinewood into a uniform pulp, as revealed by scanning electron microscopy (SEM). The rate of enzymatic hydrolysis of pinewood extract reached 72.16%. The combinations of AmimCl pretreatment with steam explosion or with hot compressed water were effective in treating corn stover, whereas AmimCl treatment alone did not result in a significant improvement. Pinewood extract produced 20.7g/L succinic acid with an average yield of 0.37g per gram of biomass. Workflow calculations indicated pine wood pretreated with IL has a theoretical yield of succinic acid of 57.1%. IL pretreatment led to increase in succinic acid yields.

Construction of reductive pathway in Saccharomyces cerevisiae for effective succinic acid fermentation at low pH value.

Succinic acid is an important precursor for the synthesis of high-value-added products. Saccharomyces cerevisiae is a suitable platform for succinic acid production because of its high tolerance towards acidity. In this study, a modified pathway for succinate production was established and investigated in S. cerevisiae. The engineered strain could produce up to 6.17±0.34g/L of succinate through the constructed pathway. The succinate titer was further improved to 8.09±0.28g/L by the deletion of GPD1 and even higher to 9.98±0.23g/L with a yield of 0.32mol/mol glucose through regulation of biotin and urea levels. Under optimal supplemental CO2 conditions in a bioreactor, the engineered strain produced 12.97±0.42g/L succinate with a yield of 0.21mol/mol glucose at pH 3.8. These results demonstrated that the proposed engineering strategy was efficient for succinic acid production at low pH value.

Performance of a haloalkaliphilic bioreactor and bacterial community shifts under different COD/SO4²â» ratios and hydraulic retention times.

Sulfur dioxide from flue gas was converted into sulfate after the absorption of alkaline solutions. Haloalkaliphilic microorganisms have been used in reducing sulfate to decrease expenses and avoid sulfide inhibition. The effects of different COD/SO4(2-) ratios and hydraulic retention times (HRTs) on the sulfate removal efficiency and bacterial community were investigated in model experiments. Ethanol showed better performance as an electron donor than lactate. The optimum COD/SO4(2-) ratio and HRT were 4.0 and 18 h, respectively, with respective sulfate removal efficiency and rate of 97.8 ± 1.11% and 6.26 ± 0.0710 g/Ld. Sulfide concentrations reached 1,603 ± 3.38 mg/L. Based on denaturing gradient gel electrophoresis analysis of 16S rDNA, the major sulfate-reducing bacterium (SRB) was Desulfonatronovibrio sp., which was only detected at a COD/SO4(2-) ratio of 4.0 using ethanol as an electron donor. Different HRTs had no significant effect on the band corresponding to this species. PCR results show that methane-producing archaea (MPA) were from the acetoclastic methanogenic family Methanosarcinaceae. Quantitative real-time PCR did not demonstrate any significant competition between SRB and MPA. The findings of this study indicate that sulfate reduction, nitrate reduction, and sulfide oxidization may occur in the same bioreactor.

Performance of a haloalkaliphilic bioreactor under different NO3(-)/SO4(2-) ratios.

Effects of NO3(-)/SO4(2-) ratio on denitrification and sulfate removal efficiency were investigated in model experiments applying haloalkaliphilic bioreactor. The reduction of both substrates performed well at different NO3(-)/SO4(2-) ratios ranging from 17.6 to l.5. The removal rates of nitrate and sulfate were 6 and 1.39kgm(-3)d(-1), respectively, at NO3(-)/SO4(2-) ratio 3.0, while sulfide concentration reached up to 703gm(-3). The major sulfate-reducing and denitrifying bacteria were Desulfonatronovibrio sp. and Halomonas campisalis, respectively. Decrease in NO3(-)/SO4(2-) ratio led to obvious changes in bacterial community. Although the sulfate reducers became dominant, the population of denitrifying ones also increased as it was demonstrated by analysis of PCR-amplified 16S rDNA fragments, which suggested that SRB and DB coexisted well in bioreactor.

Novel membrane-based biotechnological alternative process for succinic acid production and chemical synthesis of bio-based poly (butylene succinate).

Succinic acid was produced in a novel membrane-based fermentation and separation integrated system. With this integrated system, product inhibition was alleviated by removing acids and replenishing fresh broth. High cell density maintain for a longer time from 75 to 130h and succinic acid concentration increased from 53 to 73g/L. In the developed separation process, succinic acid was crystallized at a recovery of 85-90%. The purity of the obtained succinic acid crystals reached 99.4% as found by HPLC and (1)H NMR analysis. A crystallization experiment indicated that among by-products glucose had a negative effect on succinic acid crystallization. Poly (butylene succinate) (PBS) was synthesized using the purified succinic acid and (1)H NMR analysis confirmed that the composition of the synthesized PBS is in agreement with that from petro-based succinic acid.

Analysis of mixtures of fatty acids and fatty alcohols in fermentation broth.

Microbial production of fatty acids and fatty alcohols has attracted increasing concerns because of energy crisis and environmental impact of fossil fuels. Therefore, simple and efficient methods for the extraction and quantification of these compounds become necessary. In this study, a high-performance liquid chromatography-refractive index detection (HPLC-RID) method was developed for the simultaneous quantification of fatty acids and fatty alcohols in these samples. The optimum chromatographic conditions are C18 column eluted with methanol:water:acetic acid (90:9.9:0.1, v/v/v); column temperature, 26°C; flow rate, 1.0mL/min. Calibration curves of all selected analytes showed good linearity (r(2)≥0.9989). The intra-day and inter-day relative standard deviations (RSDs) of the 10 compounds were less than 4.46% and 5.38%, respectively, which indicated that the method had good repeatability and precision. Besides, a method for simultaneous extraction of fatty acids and fatty alcohols from fermentation broth was optimized by orthogonal design. The optimal extraction conditions were as follows: solvent, ethyl acetate; solvent to sample ratio, 0.5:1; rotation speed, 2min at 260rpm; extraction temperature, 10°C. This study provides simple and fast methods to simultaneously extract and quantify fatty acids and fatty alcohols for the first time. It will be useful for the study of microbial production of these products.

[Construction and fermentation control of reductive TCA pathway for malic acid production in Saccharomyces cerevisiae].

Malic acid is widely used in food, and chemical industries. Through overexpressing pyruvate carboxylase and malate dehydrogenase in pdc1-deficient Saccharomyces cerevisiae, malic acid was successfully produced through the reductive TCA pathway. No malic acid was detected in wild type Saccharomyces cerevisiae, however, 45 mmol/L malic acid was produced in engineered strain, and the concentration of byproduct ethanol also reduced by 18%. The production of malic acid enhanced 6% by increasing the concentration of Ca2+. In addition, the final concentration reached 52.5 mmol/L malic acid by addition of biotin. The increasing is almost 16% higher than that of the original strain.