Publications by authors named "Aili Fan"

26 Publications

  • Page 1 of 1

Enhancing robustness of activated sludge with Aspergillus tubingensis as a protective backbone structure under high-salinity stress.

J Environ Manage 2021 Nov 19;297:113302. Epub 2021 Jul 19.

State Key Laboratory of Chemical Resource Engineering, Beijing Key Laboratory of Bioprocess, Beijing, 100029, People's Republic of China; Beijing Advanced Innovation Center for Soft Matter Science and Engineering, Beijing, 100029, People's Republic of China; Beijing University of Chemical Technology, Beijing, 100029, People's Republic of China. Electronic address:

High salt seriously destroys the stable interactions among key functional species of activated sludge, which in turn limits the performance of high-salinity wastewater biological treatment. In this study, pelletized Aspergillus tubingensis (AT) was used as a protective backbone structure for activated sludge under high-salinity stress, and a superior salt-tolerant AT-based aerobic granular sludge (AT-AGS) was developed. Results showed that the COD and NH-N removal efficiencies of salt-domesticated AT-AGS were 11.83% and 7.18% higher than those of salt-domesticated flocculent activated sludge (FAS) at 50 gNaCl/L salinity. Compared to the salt-domesticated FAS, salt-domesticated AT-AGS showed stronger biomass retention capacity (with a MLVSS concentration of 7.92 g/L) and higher metabolic activity (with a dehydrogenase activity of 48.06 mgTF/gVSS·h). AT modified the extracellular polymeric substances pattern of microbes, and the total extracellular polysaccharide content of AT-AGS (80.7 mg/gVSS) was nearly twice than that of FAS (46.3 mg/gVSS) after salt-domestication, which demonstrated that extracellular polysaccharide played a key role in keeping the system stable. The high-throughput sequencing analysis illustrated that AT contributed to maintain the microbial richness and diversity of AT-AGS in high-salt environment, and Marinobacterium (with a relative abundance of 32.04%) became the most predominant genus in salt-tolerant AT-AGS. This study provided a novel insight into enhancing the robustness of activated sludge under high-salinity stress.
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http://dx.doi.org/10.1016/j.jenvman.2021.113302DOI Listing
November 2021

Epigenetic Manipulation to Trigger Production of Guaiane-Type Sesquiterpenes from a Marine-Derived sp. Fungus with Antineuroinflammatory Effects.

J Nat Prod 2021 07 23;84(7):1993-2003. Epub 2021 Jun 23.

State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191, P.R. China.

Epigenetic manipulation of a deep-sea sediment-derived sp. fungus using suberoylanilide hydroxamic acid (SAHA) induction resulted in the activation of a terpene-related biosynthetic gene cluster, and nine new guaiane-type sesquiterpenes, spiromaterpenes A-I (-), were isolated. Their structures were determined using various spectroscopic techniques, in association with the modified Mosher's method, computed electronic circular dichroism (ECD) spectra, and chemical conversion for configurational assignments. Compounds - exhibited significant effects against the NO production on lipopolysaccharide (LPS)-induced microglia cells BV2, and the preliminary SAR analyses demonstrated that a 2(),11-diol unit is favorable. The most active abolished LPS-induced NF-κB translocation from the cytosol to the nucleus in BV-2 microglial cells, accompanied by the marked reduction of the transcription levels of pro-inflammatory cytokines, including IL-1β, IL-6, and TNF-α dose-dependently in both LPS-induced BV-2 and BV-2 cells, as well as the protein and mRNA levels of iNOS and COX-2. This study complements the gap in knowledge regarding the anti-neuroinflammatory activity of guaiane-type sesquiterpenoids at the cellular level and suggests that is promising for further optimization as a multifunctional agent for antineuroinflammation.
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http://dx.doi.org/10.1021/acs.jnatprod.1c00293DOI Listing
July 2021

Discovery of cyclohexadepsipeptides with anti-Zika virus activities and biosynthesis of the nonproteinogenic building block (3S)-methyl-l-proline.

J Biol Chem 2021 07 23;297(1):100822. Epub 2021 May 23.

State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing, China; Institute of Ocean Research, Peking University, Beijing, China. Electronic address:

The fungal cyclohexadepsipeptides destruxins (DTXs), isaridins (ISDs), and isariins (ISRs) are nonribosomal peptides whose structures include a 19-membered ring composed of five amino acid residues and one α- or β-hydroxy acid residue. These cyclohexadepsipeptides contain unusual nonproteinogenic amino acid-building blocks and possess a range of antiviral, antibacterial, and other activities. The biosynthetic gene clusters for ISDs and ISRs have not been identified, and the biosynthesis of the nonproteinogenic (3S)-methyl-l-proline residue, which is found in DTXs, ISDs, and many other natural products, lacks full characterization. In an ongoing effort to identify compounds that can inhibit the Zika virus (ZIKV), we examined the extract of marine-derived fungus Beauveria felina SX-6-22 and discovered 30 DTXs, ISDs, and ISRs (1-30) including seven new compounds (1-7). The anti-ZIKV assays showed that 9-12 and 16-18 possess inhibitory activities against ZIKV RNA replication and NS5 (nonstructural protein 5) production in ZIKV-infected A549 cells. We sequenced the genome of B. felina SX-6-22 and identified three biosynthetic gene clusters detx, isd and isr, which are responsible for the biosynthesis of DTXs, ISDs, and ISRs, respectively. Comparative analyses of the three gene clusters clarified the biosynthetic relationships among these cyclohexadepsipeptides. Finally, we characterized the entire biosynthesis of nonproteinogenic building block (3S)-methyl-l-proline. The Δ-pyrroline-5-carboxylate reductases (P5CRs), also used in the biosynthesis of l-proline, were demonstrated to catalyze the final reduction step in (3S)-methyl-l-proline formation, suggesting potential cross talk between primary and secondary metabolisms. These results provide opportunities for biosynthetic pathway engineering to generate new anti-ZIKV cyclohexadepsipeptides.
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http://dx.doi.org/10.1016/j.jbc.2021.100822DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8233208PMC
July 2021

Ustethylin Biosynthesis Implies Phenethyl Derivative Formation in .

Org Lett 2020 10 2;22(20):7837-7841. Epub 2020 Oct 2.

Institut für Pharmazeutische Biologie und Biotechnologie, Fachbereich Pharmazie, Philipps-Universität Marburg, Robert-Koch Straße 4, 35037 Marburg, Germany.

A highly oxygenated phenethyl derivative ustethylin A was isolated from . Gene deletion, isotope labeling, and heterologous expression proved that the phenethyl core structure is assembled from malonyl-CoA by a polyketide synthase harboring a methyltransferase domain. Propionate was converted via acetyl-CoA to malonyl-CoA and incorporated into the molecule. Modifications on the core structure by three different oxidoreductases and one -methyltransferase lead to the final product, ustethylin A.
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http://dx.doi.org/10.1021/acs.orglett.0c02719DOI Listing
October 2020

Oxepinamide F biosynthesis involves enzymatic D-aminoacyl epimerization, 3H-oxepin formation, and hydroxylation induced double bond migration.

Nat Commun 2020 10 1;11(1):4914. Epub 2020 Oct 1.

Institut für Pharmazeutische Biologie und Biotechnologie, Fachbereich Pharmazie, Philipps-Universität Marburg, Robert-Koch Straße 4, 35037, Marburg, Germany.

Oxepinamides are derivatives of anthranilyl-containing tripeptides and share an oxepin ring and a fused pyrimidinone moiety. To the best of our knowledge, no studies have been reported on the elucidation of an oxepinamide biosynthetic pathway and conversion of a quinazolinone to a pyrimidinone-fused 1H-oxepin framework by a cytochrome P450 enzyme in fungal natural product biosynthesis. Here we report the isolation of oxepinamide F from Aspergillus ustus and identification of its biosynthetic pathway by gene deletion, heterologous expression, feeding experiments, and enzyme assays. The nonribosomal peptide synthase (NRPS) OpaA assembles the quinazolinone core with D-Phe incorporation. The cytochrome P450 enzyme OpaB catalyzes alone the oxepin ring formation. The flavoenzyme OpaC installs subsequently one hydroxyl group at the oxepin ring, accompanied by double bond migration. The epimerase OpaE changes the D-Phe residue back to L-form, which is essential for the final methylation by OpaF.
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http://dx.doi.org/10.1038/s41467-020-18713-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7530659PMC
October 2020

Enhanced resistance of Trichoderma harzianum LZDX-32-08 to hygromycin B induced by sea salt.

Biotechnol Lett 2021 Jan 26;43(1):213-222. Epub 2020 Aug 26.

Beijing Key Laboratory of Bioprocess, College of Life Science and Technology, Beijing University of Chemical Technology, North Third Ring Road 15, Chaoyang District, Beijing, 100029, People's Republic of China.

Objectives: To determine the effect of sea salt on the resistance of Trichoderma harzianum LZDX-32-08 to hygromycin B and speculate the possible mechanisms involved via transcriptome analysis.

Results: Sea salt addition in media to simulate marine environment significantly increased the tolerance of marine-derived fungus Trichoderma harzianum LZDX-32-08 to hygromycin B from 40 to 500 μg/ml. Meanwhile, sea salt addition also elicited the hygromycin B resistance of 5 other marine or terrestrial fungi. Transcriptomic analyses of T. harzianum cultivated on PDA, PDA supplemented with sea salt and PDA with both sea salt and hygromycin B revealed that genes coding for P-type ATPases, multidrug resistance related transporters and acetyltransferases were up-regulated, while genes coding for Ca/H antiporter and 1,3-glucosidase were down-regulated, indicating probable increased efflux and inactivation of hygromycin B as well as enhanced biofilm formation, which could jointly contribute to the drug resistance.

Conclusions: Marine environment or high ion concentration in the environment could be an importance inducer for antifungal resistance. Possible mechanisms and related key genes were proposed for understanding the molecular basis and overcoming this resistance.
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http://dx.doi.org/10.1007/s10529-020-02994-yDOI Listing
January 2021

Enhancing robustness of aerobic granule sludge under low C/N ratios with addition of kitchen wastewater.

J Environ Manage 2020 Jul 13;265:110503. Epub 2020 Apr 13.

State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing, 100029, PR China. Electronic address:

Aerobic granular sludge (AGS) is one of the most promising biotechnologies for wastewater treatment. However, the instability of AGS at low carbon to nitrogen (C/N) ratios limited its application. In this study, kitchen wastewater addition in the influent was found to improve the morphology, characteristics, and treatment performance of AGS at low C/N ratios of 10, 5 and 2, which strongly reduced the negative impact of low C/N ratios on the biomass concentration, settleability, EPS secretion, stability and performance of AGS. At C/N ratio of 2, sludge disintegration was observed in RA with synthetic wastewater as influent, while the sludge in RB was able to keep a compact microbial structure with particle size of 1.0-1.5 mm. When C/N ratio decreased from 20 to 2 (phase 1 to 4), the MLSS, SVI and EPS secretion in RB were negatively affected at the beginning of each phase, but recovered to 4800 mg L, 60 mL g, and 86 mg/g SS at the end of phase 4 (C/N ratio of 2), which were 1.3, 0.6 and 1.3 times of those in RA, respectively. Meanwhile, the removal efficiencies of COD, TN, TP and NH-N in RB were 90%, 73%, 53%, and 99% at the end of phase 4, which were 1.1, 1.2, 2.2 and 2.4 times of those in RA, respectively. Thus, high-performance AGS with enhanced robustness and high abundance of HN-AD functional bacteria Paracoccus was obtained. These findings provided a promising and cost-effective method to improve the long-term stability and performance of AGS dealing with wastewater of low C/N ratio.
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http://dx.doi.org/10.1016/j.jenvman.2020.110503DOI Listing
July 2020

New perspectives on the treatment of mycobacterial infections using antibiotics.

Appl Microbiol Biotechnol 2020 May 17;104(10):4197-4209. Epub 2020 Mar 17.

State Key Laboratory of Chemical Resources Engineering, Beijing University of Chemical Technology, Beijing, 100029, People's Republic of China.

More than 100 years have passed since the discovery of Mycobacterium tuberculosis, in 1882, as the pathogen that causes tuberculosis (TB). However, globally, TB is still one of the leading causes of death by infectious diseases. In 2018, approximately 10.0 million people were diagnosed with TB owing to the development of advanced strategies by M. tuberculosis to resist antibiotics, including the development of a dormant state. The World Health Organization (WHO) and the Sustainable Development Goals (SDGs) are dedicated to ending TB by 2030. However, the development of strategies to discover new TB drugs and new therapies is crucial for the achievement of this goal. Unfortunately, the rapid occurrence of multidrug-resistant strains of M. tuberculosis has worsened the current situation, thereby warranting prioritized discovery of new anti-TB drugs and the development of new treatment regimens in academia and the pharmaceutical industry. In this mini review, we provide a brief overview of the current research and development pipeline for new anti-TB drugs and present our perspective of TB drug innovation. The data presented herein may enable the introduction of more effective medicines and therapeutic regimens into the market.Key Points• The Updated Global New TB Drug Pipelines are briefly summarized.• Novel strategies for the discovery of new TB drugs, including novel sources, bioinformatics, and synthetic biology strategies, are discussed.• New therapeutic options, including living therapeutics and phage therapy, are proposed.
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http://dx.doi.org/10.1007/s00253-020-10513-2DOI Listing
May 2020

Reinvestigation of the substrate specificity of a reverse prenyltransferase NotF from Aspergillus sp. MF297-2.

Arch Microbiol 2020 Aug 17;202(6):1419-1424. Epub 2020 Mar 17.

Beijing Key Laboratory of Bioprocess, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, 100029, China.

NotF from Aspergillus sp. MF297-2 and BrePT from Aspergillus versicolor catalyze a reverse C2-prenylation of brevianamide F in the biosynthetic pathway of brevianamides and notoamides. NotF was reported to use only brevianamide F as substrate while BrePT demonstrated broad substrate promiscuity. With high identity at amino acid level, it is interesting to reinvestigate the catalytic activities of these two prenyltransferases in vitro toward 14 cyclodipeptides. Product identification of the in vitro assays by MS proved that NotF and BrePT share similar catalytic ability and substrate promiscuity.
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http://dx.doi.org/10.1007/s00203-020-01854-7DOI Listing
August 2020

High-strength anaerobic digestion wastewater treatment by aerobic granular sludge in a step-by-step strategy.

J Environ Manage 2020 May 20;262:110245. Epub 2020 Feb 20.

Beijing Key Laboratory of Bioprocess, Beijing Advanced Innovation Center for Soft Matter Science and Engineering, Beijing University of Chemical Technology, Beijing, 100029, PR China. Electronic address:

To reduce the instability of aerobic granular sludge (AGS) caused by high-strength anaerobic digestion wastewater, a strategy of increasing proportion of anaerobic digestion wastewater step-by-step was adopted in this study. High-performance stable AGSs were successfully cultivated with sequencing batch reactors by this strategy, which could efficiently treat high-strength anaerobic digestion wastewater with an influent chemical oxygen demand (COD) up to 5090 mg⋅L. After six phases of stepwise increasing COD loads, the sludge sizes increased from 0.5 mm to 1.5 mm, with the final mixed liquor suspended solids increased to 13,814 mg⋅L, and the final sludge volume index decreased to 15 mL⋅g. The extracellular polymeric substance (EPS), which is crucial to keep the stability of AGS, increased continuously from 85.1 mg⋅g SS to 307.8 mg⋅g SS with the increase of COD loads. Moreover, the removal efficiency of COD and TN could reach 92% and 87% for real high-strength anaerobic digestion wastewater treatment. The bacterial community analysis revealed that the family Rhodocyclaceae, Flavobacteriaceae, and Xanthomonadaceae were the major microbes of AGS, and were responsible for COD and TN removal, as well as EPS secretion. These findings may provide novel information and enrich AGS treatment of high-strength real wastewater.
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http://dx.doi.org/10.1016/j.jenvman.2020.110245DOI Listing
May 2020

Retrospective cohort analysis of heart rate variability in patients with high altitude pulmonary hypertension in Tibet.

Clin Cardiol 2020 Mar 19;43(3):298-304. Epub 2019 Dec 19.

Department of High Altitude Sickness and Cardiovascular Disease, The People's Hospital of the Tibet Autonomous Region Heart Center, Tibet, Lisa, China.

Background: Studies from both humans and animals experiments have offered abundant evidence supporting that mountain sickness is associated with changes in autonomic nervous function (ANF), which can be measured by heart rate variability (HRV).

Hypothesis: We aimed to assess changes of ANF in chronic mountain disease by measuring HRV in patients with high altitude pulmonary hypertension (HAPH).

Methods: From November 2018 to March 2019, 120 patients in the cardiac care unit of the People's Hospital of Tibet Autonomous Region were selected as the observation group, and 50 patients without organic heart disease served as the control group. Pulmonary artery systolic pressure was evaluated by echocardiography in patients with HAPH, divided into three groups: mild (30-49 mm Hg), moderate (50-69 mm Hg) and severity (≥70 mm Hg) groups. A 24-hour dynamic electrocardiogram (DCG) was obtained for each patient. HRV (SDNN, SDANN, RMSSD, PNN50, and HRVTI for time domain; TP, VLF, LF, HF, and LF/HF for frequency domain) indexes were measured and compared.

Results: Compared with the control group, time domain parameters including SDNN, SDANN, RMSSD, PNN50, and HRVTI were reduced, as well as frequency domain indexes such as TP, VLF, LF, and HF. LF/HF was highest in mild HAPH group and lowest in the moderate HAPH group, and the difference between the two groups was statistically significant.

Conclusions: The HRV of patients with chronic HAPH in high altitude areas in Tibet is significantly reduced relative to healthy controls, and significantly negatively correlated with the severity of pulmonary artery hypertension.
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http://dx.doi.org/10.1002/clc.23312DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7068065PMC
March 2020

Switching a regular tryptophan C4-prenyltransferase to a reverse tryptophan-containing cyclic dipeptide C3-prenyltransferase by sequential site-directed mutagenesis.

Org Biomol Chem 2018 09;16(36):6688-6694

Institut für Pharmazeutische Biologie und Biotechnologie, Philipps-Universität Marburg, Robert-Koch-Straße 4, 35037 Marburg, Germany.

FgaPT2 from Aspergillus fumigatus catalyzes a regular C4- and its mutant K174A a reverse C3-prenylation of l-tryptophan in the presence of dimethylallyl diphosphate. FgaPT2 also uses tryptophan-containing cyclic dipeptides for C4-prenylation, while FgaPT2_K174A showed almost no activity toward these substrates. In contrast, Arg244 mutants of FgaPT2 accept very well cyclic dipeptides for regular C4-prenylation. In this study, we demonstrate that FgaPT2_K174F, which catalyzes a regular C3-prenylation on tyrosine, can also use cyclo-l-Trp-l-Ala, cyclo-l-Trp-l-Trp, cyclo-l-Trp-Gly, cyclo-l-Trp-l-Phe, cyclo-l-Trp-l-Pro, and cyclo-l-Trp-l-Tyr as substrates, but only with low activity. Combinational mutation on Lys174 and Arg244 increases significantly the acceptance of these cyclic dipeptides. With the exception of cyclo-l-Trp-l-Trp, the tested dipeptides were much better accepted by FgaPT2_K174F_R244X (X = L, N, Q, Y) than FgaPT2, with an increase of two- to six-fold activity. In comparison to FgaPT2_K174F, even two- to ten-fold conversion yields were calculated for the double mutants. Isolation and structural elucidation of the enzyme products revealed stereospecific reverse C3-prenylation on the indole ring, resulting in the formation of syn-cis configured hexahydropyrroloindole derivatives. The results presented in this study highlight the convenience of site-directed mutagenesis for creating new biocatalysts.
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http://dx.doi.org/10.1039/c8ob01735bDOI Listing
September 2018

Asperphenamate biosynthesis reveals a novel two-module NRPS system to synthesize amino acid esters in fungi.

Chem Sci 2018 Mar 24;9(9):2589-2594. Epub 2018 Jan 24.

State Key Laboratory of Mycology , Institute of Microbiology , Chinese Academy of Sciences , 100101 Beijing , China . Email:

Amino acid esters are a group of structurally diverse natural products with distinct activities. Some are synthesized through an inter-molecular esterification step catalysed by nonribosomal peptide synthetase (NRPS). In bacteria, the formation of the intra-molecular ester bond is usually catalysed by a thioesterase domain of NRPS. However, the mechanism by which fungal NRPSs perform this process remains unclear. Herein, by targeted gene disruption in and heterologous expression in , we show that two NRPSs, ApmA and ApmB, are sufficient for the synthesis of an amino acid ester, asperphenamate. Using the heterologous expression system, we identified that ApmA, with a reductase domain, rarely generates dipeptidyl alcohol. In contrast, ApmB was determined to not only catalyse inter-molecular ester bond formation but also accept the linear dipeptidyl precursor into the NRPS chain. The mechanism described here provides an approach for the synthesis of new small molecules with NRPS as the catalyst. Our study reveals for the first time a two-module NRPS system for the formation of amino acid esters in nature.
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http://dx.doi.org/10.1039/c7sc02396kDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5897882PMC
March 2018

A cryptic pigment biosynthetic pathway uncovered by heterologous expression is essential for conidial development in Pestalotiopsis fici.

Mol Microbiol 2017 Aug 6;105(3):469-483. Epub 2017 Jun 6.

State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, People's Republic of China.

Spore pigmentation is very common in the fungal kingdom. The best studied pigment in fungi is melanin which coats the surface of single cell spores. What and how pigments function in a fungal species with multiple cell conidia is poorly understood. Here, we identified and deleted a polyketide synthase (PKS) gene PfmaE and showed that it is essential for multicellular conidial pigmentation and development in a plant endophytic fungus, Pestalotiopsis fici. To further characterize the melanin pathway, we utilized an advanced Aspergillus nidulans heterologous system for the expression of the PKS PfmaE and the Pfma gene cluster. By structural elucidation of the pathway metabolite scytalone in A. nidulans, we provided chemical evidence that the Pfma cluster synthesizes DHN melanin. Combining genetic deletion and combinatorial gene expression of Pfma cluster genes, we determined that the putative reductase PfmaG and the PKS are sufficient for the synthesis of scytalone. Feeding scytalone back to the P. fici ΔPfmaE mutant restored pigmentation and multicellular adherence of the conidia. These results cement a growing understanding that pigments are essential not simply for protection of spores from biotic and abiotic stresses but also for spore structural development.
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http://dx.doi.org/10.1111/mmi.13711DOI Listing
August 2017

Deletion of a Histone Acetyltransferase Leads to the Pleiotropic Activation of Natural Products in Metarhizium robertsii.

Org Lett 2017 04 16;19(7):1686-1689. Epub 2017 Mar 16.

State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences , Beijing 100101, China.

Histone deacetylation normally decreases the gene expression in organisms. By genome-wide deletions of epigenetic regulators in entomopathogenic fungus Metarhizium robertsii, unexpected activations of orphan secondary metabolite genes have been found upon the disruption of a histone acetyltransferase (HAT) gene Hat1. This led to the characterization of 11 new natural products, including eight isocoumarin derivatives meromusides A-H and two nonribosomal peptides meromutides A and B. Therefore, disruption of HAT represents a new approach to mine chemical diversity from fungi.
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http://dx.doi.org/10.1021/acs.orglett.7b00476DOI Listing
April 2017

Mutation on Gly115 and Tyr205 of the cyclic dipeptide C2-prenyltransferase FtmPT1 increases its catalytic activity toward hydroxynaphthalenes.

Appl Microbiol Biotechnol 2017 Mar 10;101(5):1989-1998. Epub 2016 Nov 10.

Institut für Pharmazeutische Biologie und Biotechnologie, Philipps-Universität Marburg, 35037, Marburg, Germany.

The fungal cyclic dipeptide prenyltransferase FtmPT1 from Aspergillus fumigatus catalyzes a regular C2-prenylation of brevianamide F (cyclo-L-Trp-L-Pro) and is involved in the biosynthesis of a number of biologically active natural products including tryprostatins, spirotryprostatins, verruculogen, and fumitremorgins. FtmPT1, like other members of the dimethylallyltryptophan synthase superfamily, was shown to have high substrate promiscuity for tryptophan-containing cyclic dipeptides and a few other aromatic substrates. A previous study demonstrated the acceptance of 1-naphthol by FtmPT1, but with very low product yield. In this study, we report the significantly increased acceptance of 1-naphthol and other hydroxynaphthalenes by FtmPT1_G115A and six FtmPT1_Y205X single mutants as well as FtmPT1_G115A_Y205C. These results provided an example for creation of biocatalysts with improved catalytic activity by site-directed mutagenesis.
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http://dx.doi.org/10.1007/s00253-016-7966-xDOI Listing
March 2017

Saturation mutagenesis on Arg244 of the tryptophan C4-prenyltransferase FgaPT2 leads to enhanced catalytic ability and different preferences for tryptophan-containing cyclic dipeptides.

Appl Microbiol Biotechnol 2016 Jun 15;100(12):5389-99. Epub 2016 Feb 15.

Institut für Pharmazeutische Biologie und Biotechnologie, Philipps-Universität Marburg, Deutschhausstrasse 17A, 35037, Marburg, Germany.

FgaPT2 from Aspergillus fumigatus catalyzes a Friedel-Crafts alkylation at C-4 of L-tryptophan and is involved in the biosynthesis of the ergot alkaloids fumigaclavines. Several tryptophan-containing cyclic dipeptides had also been prenylated by FgaPT2, but the turnover rate (k cat) was low. Here, we report the generation of FgaPT2 mutants by saturation mutagenesis at the amino acid residue Arg244 to improve its catalytic efficiency toward cyclic dipeptides. Thirteen mutated enzymes demonstrated up to 76-fold higher turnover number toward seven cyclic dipeptides than the non-mutated FgaPT2. More importantly, the mutated enzymes exhibited different preferences toward these substrates. This study provides a convenient approach for creation of new biocatalysts for production of C4-prenylated cyclic dipeptides.
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http://dx.doi.org/10.1007/s00253-016-7365-3DOI Listing
June 2016

Impacts and perspectives of prenyltransferases of the DMATS superfamily for use in biotechnology.

Appl Microbiol Biotechnol 2015 Sep 31;99(18):7399-415. Epub 2015 Jul 31.

Institut für Pharmazeutische Biologie und Biotechnologie, Philipps-Universität Marburg, Deutschhausstrasse 17A, D-35037, Marburg, Germany.

Prenylated compounds are ubiquitously found in nature and demonstrate interesting biological and pharmacological activities. Prenyltransferases catalyze the attachment of prenyl moieties from different prenyl donors to various acceptors and contribute significantly to the structural and biological diversity of natural products. In the last decade, significant progress has been achieved for the prenyltransferases of the dimethylallyltryptophan synthase (DMATS) superfamily. More than 40 members of these soluble enzymes are identified in microorganisms and characterized biochemically. These enzymes were also successfully used for production of a large number of prenylated derivatives. N1-, C4-, C5-, C6-, and C7-prenylated tryptophan and N1-, C2-, C3-, C4-, and C7-prenylated tryptophan-containing peptides were obtained by using DMATS enzymes as biocatalysts. Tyrosine and xanthone prenyltransferases were used for production of prenylated derivatives of their analogs. More interestingly, the members of the DMATS superfamily demonstrated intriguing substrate and catalytic promiscuity and also used structurally quite different compounds as prenyl acceptors. Prenylated hydroxynaphthalenes, flavonoids, indolocarbazoles, and acylphloroglucinols, which are typical bacterial or plant metabolites, were produced by using several fungal DMATS enzymes. Furthermore, the potential usage of these enzymes was further expanded by using natural or unnatural DMAPP analogs as well as by coexpression with other genes like NRPS and by development of whole cell biocatalyst.
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http://dx.doi.org/10.1007/s00253-015-6813-9DOI Listing
September 2015

Prenyltransferases as key enzymes in primary and secondary metabolism.

Appl Microbiol Biotechnol 2015 Sep 28;99(18):7379-97. Epub 2015 Jul 28.

Institut für Pharmazeutische Biologie und Biotechnologie, Philipps-Universität Marburg, Deutschhausstrasse 17A, D-35037, Marburg, Germany.

Attachment of isoprene units to various acceptors by prenylation plays an important role in primary and secondary metabolism of living organisms. Protein prenylation belongs to posttranslational modification and is involved in cellular regulation process. Prenylated secondary metabolites usually demonstrate promising biological and pharmacological activities. Prenyl transfer reactions catalyzed by prenyltransferases represent the key steps in the biosynthesis and contribute significantly to the structural and biological diversity of these compounds. In the last decade, remarkable progress has been achieved in the biochemical, molecular, and structural biological investigations of prenyltransferases, especially on those of the members of the dimethylallyltryptophan synthase (DMATS) superfamily. Until now, more than 40 of such soluble enzymes are identified and characterized biochemically. They catalyze usually regioselective and stereoselective prenylations of a series of aromatic substances including tryptophan, tryptophan-containing peptides, and other indole derivatives as well as tyrosine or even nitrogen-free substrates. Crystal structures of a number of prenyltransferases have been solved in the past 10 years and provide a solid basis for understanding the mechanism of prenyl transfer reactions.
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http://dx.doi.org/10.1007/s00253-015-6811-yDOI Listing
September 2015

Tryptophan prenyltransferases showing higher catalytic activities for Friedel-Crafts alkylation of o- and m-tyrosines than tyrosine prenyltransferases.

Org Biomol Chem 2015 Jul;13(27):7551-7

Institut für Pharmazeutische Biologie und Biotechnologie, Philipps-Universität Marburg, Deutschhausstrasse 17A, 35037 Marburg, Germany.

Tryptophan prenyltransferases FgaPT2, 5-DMATS, 6-DMATSSv and 7-DMATS catalyse regiospecific C-prenylations on the indole ring, while tyrosine prenyltransferases SirD and TyrPT catalyse the O-prenylation of the phenolic hydroxyl group. In this study, we report the Friedel-Crafts alkylation of L-o-tyrosine by these enzymes. Surprisingly, no conversion was detected with SirD and three tryptophan prenyltransferases showed significantly higher activity than another tyrosine prenyltransferase TyrPT. C5-prenylated L-o-tyrosine was identified as a unique product of these enzymes. Using L-m-tyrosine as the prenylation substrate, product formation was only observed with the tryptophan prenyltransferases FgaPT2 and 7-DMATS. C4- and C6-prenylated derivatives were identified in the reaction mixture of FgaPT2. These results provided additional evidence for the similarities and differences between these two subgroups within the DMATS superfamily in their catalytic behaviours.
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http://dx.doi.org/10.1039/c5ob01040cDOI Listing
July 2015

Site-directed mutagenesis switching a dimethylallyl tryptophan synthase to a specific tyrosine C3-prenylating enzyme.

J Biol Chem 2015 Jan 4;290(3):1364-73. Epub 2014 Dec 4.

From the Institut für Pharmazeutische Biologie und Biotechnologie, Philipps-Universität Marburg, 35037 Marburg and

The tryptophan prenyltransferases FgaPT2 and 7-DMATS (7-dimethylallyl tryptophan synthase) from Aspergillus fumigatus catalyze C(4)- and C(7)-prenylation of the indole ring, respectively. 7-DMATS was found to accept l-tyrosine as substrate as well and converted it to an O-prenylated derivative. An acceptance of l-tyrosine by FgaPT2 was also observed in this study. Interestingly, isolation and structure elucidation revealed the identification of a C(3)-prenylated l-tyrosine as enzyme product. Molecular modeling and site-directed mutagenesis led to creation of a mutant FgaPT2_K174F, which showed much higher specificity toward l-tyrosine than l-tryptophan. Its catalytic efficiency toward l-tyrosine was found to be 4.9-fold in comparison with that of non-mutated FgaPT2, whereas the activity toward l-tryptophan was less than 0.4% of that of the wild-type. To the best of our knowledge, this is the first report on an enzymatic C-prenylation of l-tyrosine as free amino acid and altering the substrate preference of a prenyltransferase by mutagenesis.
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http://dx.doi.org/10.1074/jbc.M114.623413DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4340383PMC
January 2015

A new member of the DMATS superfamily from Aspergillus niger catalyzes prenylations of both tyrosine and tryptophan derivatives.

Appl Microbiol Biotechnol 2014 Dec 28;98(24):10119-29. Epub 2014 Jun 28.

Institut für Pharmazeutische Biologie und Biotechnologie, Philipps-Universität Marburg, Deutschhausstrasse 17A, 35037, Marburg, Germany.

A putative prenyltransferase gene of the dimethylallyltryptophan synthase (DMATS) family, An13g01840, was identified in the genome sequence of Aspergillus niger. The deduced polypeptide CAK41583 consists of 465 amino acids with a calculated molecular mass of 52.7 kDa. To evaluate gene function, the coding sequence was cloned into pET28a and overexpressed in Escherichia coli. The soluble His6-fusion protein was purified to near homogeneity on Ni-NTA agarose and used for enzyme assays with diverse aromatic substrates in the presence of dimethylallyl diphosphate. HPLC analysis revealed product formation in the incubation mixtures with L-tyrosine and five derivatives thereof. Structure elucidation of the enzyme products by NMR and MS analyses confirmed O-prenylations and proved the identification of a tyrosine O-prenyltransferase (TyrPT). As in the case of SirD from Leptosphaeria maculans, TyrPT also accepted 4-amino-L-phenylalanine for an N-prenylation and L-tryptophan for a C7-prenylation. The K M values of TyrPT for L-tyrosine, L-tryptophan, and dimethylallyl diphosphate (DMAPP) were found to be 0.24, 0.19, and 0.71 mM, respectively. The k cat of L-tyrosine and L-tryptophan reactions were determined at 0.58 and 0.0053 s(-1), respectively. The results presented in this study enhance the relationship of tyrosine O- and tryptophan C7-prenyltranferases and provide meanwhile a new enzyme for production of prenylated derivatives. In comparison to the known tyrosine prenyltransferase SirD, TyrPT showed significantly higher catalytic activity for several substrates, e.g., 4-amino-L-phenylalanine as well as 4- and 5-methyl-DL-tryptophan.
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http://dx.doi.org/10.1007/s00253-014-5872-7DOI Listing
December 2014

New insights into the biosynthesis of prenylated xanthones: Xptb from Aspergillus nidulans catalyses an O-prenylation of xanthones.

Chembiochem 2012 Dec 13;13(18):2764-71. Epub 2012 Nov 13.

Institut für Pharmazeutische Biologie und Biotechnologie, Philipps-Universität Marburg, Germany.

Gene-inactivation experiments have indicated that the putative prenyltransferase XptB from Aspergillus nidulans was likely to be responsible for the prenylation of 1,7-dihydroxy-6-methyl-8-hydroxymethylxanthone. Recently, it was suggested that this enzyme might also accept as substrate the benzophenone arugosin H, which is assumed to be a precursor of prenylated xanthones. In this study, five benzophenones and ten xanthones were incubated with purified recombinant XptB in the presence of dimethylallyl diphosphate (DMAPP). XptB accepted four xanthones as substrates, including the proposed natural substrate, and catalysed regiospecific O-prenylations at C-7 of the xanthone core. K(m) values in the range of 0.081-1.1 mM and turnover numbers (k(cat)) between 0.02 and 0.5 s(-1) were determined for the accepted xanthones. The kinetic parameters for DMAPP were found to be 0.024 mM (K(m)) and 0.13 s(-1) (k(cat)). Arugosin H was not accepted by XptB under the tested conditions. XptB was relatively specific towards its prenyl donor and did not accept geranyl or farnesyl diphosphate as substrate. Mn(2+) and Co(2+) strongly enhanced XptB activity (up to eightfold); this has not been reported before for prenyltransferases of the DMATS superfamily.
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http://dx.doi.org/10.1002/cbic.201200545DOI Listing
December 2012

Total synthesis of lamellarins D, H, and R and ningalin B.

Org Lett 2011 Jan 17;13(2):312-5. Epub 2010 Dec 17.

State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, 38 Xueyuan Road, Beijing 100191, China.

A concise total synthesis of lamellarins D (7 steps), H (7 steps), and R (5 steps) and ningalin B (5 steps) is achieved starting from the corresponding aldehydes and amines. The synthesis features three oxidative reactions as key steps in a biomimetic manner, involving an AgOAc-mediated oxidative coupling reaction to construct the pyrrole core, a Pb(OAc)(4)-induced oxidative cyclization to form the lactone, and Kita's oxidation reaction to form the pyrrole-arene C-C bond.
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http://dx.doi.org/10.1021/ol1027877DOI Listing
January 2011

One-pot AgOAc-mediated synthesis of polysubstituted pyrroles from primary amines and aldehydes: application to the total synthesis of purpurone.

Org Lett 2010 Sep;12(18):4066-9

State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, 38 Xueyuan Road, Beijing 100191, China.

A simple and efficient method for the synthesis of 1,3,4-trisubstituted or 3,4-disubstituted pyrroles has been developed. The reaction represents the first time that pyrroles are synthesized directly from readily available aldehydes and amines (anilines) as starting materials. This method has been successfully applied to the rapid synthesis of purpurone.
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http://dx.doi.org/10.1021/ol101644gDOI Listing
September 2010

[Effects of salicylic acid on chlorophyll fluorescence and xanthophyll cycle in cucumber leaves under high temperature and strong light].

Ying Yong Sheng Tai Xue Bao 2006 Mar;17(3):399-402

College of Horticulture, Northwest Sci-Tech University of Agriculture and Forestry, Yangling 712100, China.

In this study, cucumber leaves were under high temperature and strong light stress after two days of spraying with 50 to approximately 400 micromol x L(-1) salicylic acid (SA) solution. The measurements showed that SA pretreatment could inhibit the decrease of intrinsic photochemical efficiency (Fv/ /Fm), quantum yield of PSII(phiPSII), maximum fluorescence value (Fm), and photochemical quenching (qP) by 16.1% to approximately 30.2%, 11.9% to approximately 33.0%, 7.2% to approximately 41.0% and 27.2% to approximately 160.8%, respectively, and promote the increase of non-photochemical quenching (NPQ) by 13.1% to approximately 62.9%, but had little effect on initial fluorescence value (Fo). SA pretreatment could also inhibit the decrease of xanthophyll cycle pool in cucumber leaves, and increase the de-epoxidation extent of xanthophyll cycle (A + Z)/(V + A + Z),which was 29.5% and 24.6% higher than control, respectively. It was suggested that SA could protect the photosynthetic apparatus of cucumber leaves under high temperature and strong light through promoting non-radiative energy dissipation.
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March 2006
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