Unusual chemistries in fungal meroterpenoid biosynthesis.
Matsuda Yudai,Awakawa Takayoshi,Mori Takahiro,Abe Ikuro
Current opinion in chemical biology
Meroterpenoids are polyketide and terpenoid hybrid natural products with remarkable biological activities. Recent progress in fungal meroterpenoid biosynthesis has revealed several unusual enzyme reactions and novel enzymes, including unique terpene cyclization reactions by a novel family of membrane-bound terpene cyclases and post-cyclization modification reactions by oxygenases, such as non-heme iron-dependent dioxygenases, flavin adenine dinucleotide-dependent monooxygenases, and cytochrome P450 monooxygenases. They contribute to the structural diversification and increase in complexity of fungal meroterpenoids. Structure-function studies of these enzymes provide strategies for engineering the biosynthetic machinery to create novel molecular scaffolds for drug discovery.
10.1016/j.cbpa.2015.11.001
Global analysis of biosynthetic gene clusters reveals vast potential of secondary metabolite production in Penicillium species.
Nielsen Jens Christian,Grijseels Sietske,Prigent Sylvain,Ji Boyang,Dainat Jacques,Nielsen Kristian Fog,Frisvad Jens Christian,Workman Mhairi,Nielsen Jens
Nature microbiology
Filamentous fungi produce a wide range of bioactive compounds with important pharmaceutical applications, such as antibiotic penicillins and cholesterol-lowering statins. However, less attention has been paid to fungal secondary metabolites compared to those from bacteria. In this study, we sequenced the genomes of 9 Penicillium species and, together with 15 published genomes, we investigated the secondary metabolism of Penicillium and identified an immense, unexploited potential for producing secondary metabolites by this genus. A total of 1,317 putative biosynthetic gene clusters (BGCs) were identified, and polyketide synthase and non-ribosomal peptide synthetase based BGCs were grouped into gene cluster families and mapped to known pathways. The grouping of BGCs allowed us to study the evolutionary trajectory of pathways based on 6-methylsalicylic acid (6-MSA) synthases. Finally, we cross-referenced the predicted pathways with published data on the production of secondary metabolites and experimentally validated the production of antibiotic yanuthones in Penicillia and identified a previously undescribed compound from the yanuthone pathway. This study is the first genus-wide analysis of the genomic diversity of Penicillia and highlights the potential of these species as a source of new antibiotics and other pharmaceuticals.
10.1038/nmicrobiol.2017.44
Biosynthesis and applications of prenylquinones.
Kawamukai Makoto
Bioscience, biotechnology, and biochemistry
Prenylquinones are isoprenoid compounds with a characteristic quinone structure and isoprenyl tail that are ubiquitous in almost all living organisms. There are four major prenylquinone classes: ubiquinone (UQ), menaquinone (MK), plastoquinone (PQ), and rhodoquinone (RQ). The quinone structure and isoprenyl tail length differ among organisms. UQ, PQ, and RQ contain benzoquinone, while MK contains naphthoquinone. UQ, MK, and RQ are involved in oxidative phosphorylation, while PQ functions in photosynthetic electron transfer. Some organisms possess two types of prenylquinones; Escherichia coli has UQ and MK, and Caenorhabditis elegans has UQ and RQ. Crystal structures of most of the enzymes involved in MK synthesis have been solved. Studies on the biosynthesis and functions of quinones have advanced recently, including for phylloquinone (PhQ), which has a phytyl moiety instead of an isoprenyl tail. Herein, the synthesis and applications of prenylquinones are reviewed.
10.1080/09168451.2018.1433020
Molecular basis of dimer formation during the biosynthesis of benzofluorene-containing atypical angucyclines.
Huang Chunshuai,Yang Chunfang,Zhang Wenjun,Zhang Liping,De Bidhan Chandra,Zhu Yiguang,Jiang Xiaodong,Fang Chunyan,Zhang Qingbo,Yuan Cheng-Shan,Liu Hung-Wen,Zhang Changsheng
Nature communications
Lomaiviticin A and difluostatin A are benzofluorene-containing aromatic polyketides in the atypical angucycline family. Although these dimeric compounds are potent antitumor agents, how nature constructs their complex structures remains poorly understood. Herein, we report the discovery of a number of fluostatin type dimeric aromatic polyketides with varied C-C and C-N coupling patterns. We also demonstrate that these dimers are not true secondary metabolites, but are instead derived from non-enzymatic deacylation of biosynthetic acyl fluostatins. The non-enzymatic deacylation proceeds via a transient quinone methide like intermediate which facilitates the subsequent C-C/C-N coupled dimerization. Characterization of this unusual property of acyl fluostatins explains how dimerization takes place, and suggests a strategy for the assembly of C-C and C-N coupled aromatic polyketide dimers. Additionally, a deacylase FlsH was identified which may help to prevent accumulation of toxic quinone methides by catalyzing hydrolysis of the acyl group.
10.1038/s41467-018-04487-z
Dissection of patulin biosynthesis, spatial control and regulation mechanism in Penicillium expansum.
Li Boqiang,Chen Yong,Zong Yuanyuan,Shang Yanjiao,Zhang Zhanquan,Xu Xiaodi,Wang Xiao,Long Manyuan,Tian Shiping
Environmental microbiology
The patulin biosynthesis is one of model pathways in an understanding of secondary metabolite biology and network novelties in fungi. However, molecular regulation mechanism of patulin biosynthesis and contribution of each gene related to the different catalytic enzymes in the biochemical steps of the pathway remain largely unknown in fungi. In this study, the genetic components of patulin biosynthetic pathway were systematically dissected in Penicillium expansum, which is an important fungal pathogen and patulin producer in harvested fruits and vegetables. Our results revealed that all the 15 genes in the cluster are involved in patulin biosynthesis. Proteins encoded by those genes are compartmentalized in various subcellular locations, including cytosol, nucleus, vacuole, endoplasmic reticulum, plasma membrane and cell wall. The subcellular localizations of some proteins, such as PatE and PatH, are required for the patulin production. Further, the functions of eight enzymes in the 10-step patulin biosynthetic pathway were verified in P. expansum. Moreover, velvet family proteins, VeA, VelB and VelC, were proved to be involved in the regulation of patulin biosynthesis, but not VosA. These findings provide a thorough understanding of the biosynthesis pathway, spatial control and regulation mechanism of patulin in fungi.
10.1111/1462-2920.14542
The Pathway Less Traveled: Engineering Biosynthesis of Nonstandard Functional Groups.
Sulzbach Morgan,Kunjapur Aditya M
Trends in biotechnology
The field of metabolic engineering has achieved biochemical routes for conversion of renewable inputs to structurally diverse chemicals, but these products contain a limited number of chemical functional groups. In this review, we provide an overview of the progression of uncommon or 'nonstandard' functional groups from the elucidation of their biosynthetic machinery to the pathway optimization framework of metabolic engineering. We highlight exemplary efforts from primarily the last 5 years for biosynthesis of aldehyde, ester, terminal alkyne, terminal alkene, fluoro, epoxide, nitro, nitroso, nitrile, and hydrazine functional groups. These representative nonstandard functional groups vary in development stage and showcase the pipeline of chemical diversity that could soon appear within customized, biologically produced molecules.
10.1016/j.tibtech.2019.12.014
Opposite facial specificity for two hydroquinone epoxidases: (3-si,4-re)-2,5-dihydroxyacetanilide epoxidase from Streptomyces LL-C10037 and (3-re,4-si)-2,5-dihydroxyacetanilide epoxidase from Streptomyces MPP 3051.
Shen B,Gould S J
Biochemistry
(3-si,4-re)-2,5-Dihydroxyacetanilide epoxidase (DHAE I), a key enzyme in the biosynthesis of the epoxysemiquinone antibiotic LL-C10037 alpha by Streptomyces LL-C10037 [Gould, S.J., & Shen, B. (1991) J. Am. Chem. Soc. 113, 684-686], and (3-re,4-si)-2,5-dihydroxyacetanilide epoxidase (DHAE II) isolated from Streptomyces MPP 3051--which yields the (3R,4S)-epoxyquinone mirror image product of DHAE I--are described. DHAE I was purified 640-fold. Gel permeation chromatography indicated an Mr of 117,000 +/- 10,000; SDS-PAGE gave a major band of 22,300 daltons, indicating that DHAE I is either a pentamer or hexamer in solution. The enzyme had a pH optimum of 6.5, a Km of 8.4 +/- 0.5 microM, and a Vmax of 3.7 +/- 0.2 mumol min-1 mg-1. DHAE II was purified 1489-fold. The enzyme was shown to be a dimer of Mr 33,000 +/- 2000, with 16,000-dalton subunits, with a pH optimum of 5.5 and a Km of 7.2 +/- 0.4 microM. Both enzymes required only O2 and substrate; flavin and nicotinamide coenzymes had little or no effect. Neither catalase nor EDTA affected the activity of either enzyme, but complete inhibition of both was obtained with 1,10-phenanthroline. The activity of the purified DHAE I could be enhanced, but only by Mn2+ (relative V = 246 at 0.04 mM), Ni2+ (relative V = 266 at 0.2 mM), or Co2+ (relative = 498 at 0.2 mM). Reconstitution from a DHAE I apoenzyme, generated by treatment with 1,10-phenanthroline followed by Sephadex G-25 chromatography, occurred only by addition of one of these three metals.(ABSTRACT TRUNCATED AT 250 WORDS)
10.1021/bi00101a004
Analysis of the cryptophycin P450 epoxidase reveals substrate tolerance and cooperativity.
Ding Yousong,Seufert Wolfgang H,Beck Zachary Q,Sherman David H
Journal of the American Chemical Society
Cryptophycins are potent anticancer agents isolated from Nostoc sp. ATCC 53789 and Nostoc sp. GSV 224. The most potent natural cryptophycin analogues retain a beta-epoxide at the C2'-C3' position of the molecule. A P450 epoxidase encoded by c rpE recently identified from the cryptophycin gene cluster was shown to install this key functional group into cryptophycin-4 (Cr-4) to produce cryptophycin-2 (Cr-2) in a regio- and stereospecific manner. Here we report a detailed characterization of the CrpE epoxidase using an engineered maltose binding protein (MBP)-CrpE fusion. The substrate tolerance of the CrpE polypeptide was investigated with a series of structurally related cryptophycin analogues generated by chemoenzymatic synthesis. The enzyme specifically installed a beta-epoxide between C2' and C3' of cyclic cryptophycin analogues. The kcat/Km values of the enzyme were determined to provide further insights into the P450 epoxidase catalytic efficiency affected by substrate structural variation. Finally, binding analysis revealed cooperativity of MBP-CrpE toward natural and unnatural desepoxy cryptophycin substrates.
10.1021/ja710520q
Tandem enzymatic oxygenations in biosynthesis of epoxyquinone pharmacophore of manumycin-type metabolites.
Rui Zhe,Sandy Moriah,Jung Brian,Zhang Wenjun
Chemistry & biology
Many natural products contain epoxyquinone pharmacophore with unknown biosynthetic mechanisms. Recent genetic analysis of the asukamycin biosynthetic gene cluster proposed enzyme candidates related to epoxyquinone formation for manumycin-type metabolites. Our biochemical studies reveal that 3-amino-4-hydroxyl benzoic acid (3,4-AHBA) precursor is activated and loaded on aryl carrier protein (AsuC12) by ATP-dependent adenylase (AsuA2). AsuE1 and AsuE3, both single-component flavin-dependent monooxygenases, catalyze the exquisite regio- and enantiospecific postpolyketide synthase (PKS) assembly oxygenations. AsuE1 installs a hydroxyl group on the 3,4-AHB ring to form a 4-hydroxyquinone moiety, which is epoxidized by AsuE3 to yield the epoxyquinone functionality. Despite being a single-component monooxygenase, AsuE1 activity is elicited by AsuE2, a pathway-specific flavin reductase. We further demonstrate that the epoxyquinone moiety is critical for anti-MRSA activity by analyzing the bioactivity of various manumycin-type metabolites produced through mutasynthesis.
10.1016/j.chembiol.2013.05.006
Biosynthesis of colabomycin E, a new manumycin-family metabolite, involves an unusual chain-length factor.
Petříčková Kateřina,Pospíšil Stanislav,Kuzma Marek,Tylová Tereza,Jágr Michal,Tomek Petr,Chroňáková Alica,Brabcová Eva,Anděra Ladislav,Krištůfek Václav,Petříček Miroslav
Chembiochem : a European journal of chemical biology
Colabomycin E is a new member of the manumycin-type metabolites produced by the strain Streptomyces aureus SOK1/5-04 and identified by genetic screening from a library of streptomycete strains. The structures of colabomycin E and accompanying congeners were resolved. The entire biosynthetic gene cluster was cloned and expressed in Streptomyces lividans. Bioinformatic analysis and mutagenic studies identified components of the biosynthetic pathway that are involved in the formation of both polyketide chains. Recombinant polyketide synthases (PKSs) assembled from the components of colabomycin E and asukamycin biosynthetic routes catalyzing the biosynthesis of "lower" carbon chains were constructed and expressed in S. aureus SOK1/5-04 ΔcolC11-14 deletion mutant. Analysis of the metabolites produced by recombinant strains provided evidence that in both biosynthetic pathways the length of the lower carbon chain is controlled by an unusual chain-length factor supporting biosynthesis either of a triketide in asukamycin or of a tetraketide in colabomycin E. Biological activity assays indicated that colabomycin E significantly inhibited IL-1β release from THP-1 cells and might thus potentially act as an anti-inflammatory agent.
10.1002/cbic.201400068
Biosynthetic origins of the epoxyquinone skeleton in epoxyquinols A and B.
Fujita Katsuki,Ishikawa Fumihiro,Kakeya Hideaki
Journal of natural products
The biosynthetic origins of epoxyquinols A (1) and B (2) produced by an unidentified fungus have attracted considerable interest because these compounds could be assembled from a biosynthetic precursor, epoxycyclohexenone aldehyde (3), via an electrocyclization/intermolecular Diels-Alder dimerization cascade reaction. Furthermore, very little is known about the biosynthetic origins of naturally occurring epoxyquinone moieties. We herein describe the incorporation of (13)C at specific positions within the structure of a shunt product, epoxycyclohexenone (4), using stable isotope feeding experiments with sodium [1-(13)C]-acetate and sodium [1,2-(13)C2]-acetate. The results of these experiments strongly suggest that the epoxyquinone skeleton is assembled by a polyketide synthase.
10.1021/np5004615
Heterologous Expression of Fluostatin Gene Cluster Leads to a Bioactive Heterodimer.
Yang Chunfang,Huang Chunshuai,Zhang Wenjun,Zhu Yiguang,Zhang Changsheng
Organic letters
The biosynthesis gene cluster (fls) for atypical angucycline fluostatins was identified from the marine derived Micromonospora rosaria SCSIO N160 and was confirmed by gene knockouts and the biochemical characterization of a bifunctional oxygenase FlsO2. The absolute configuration of the key biosynthetic intermediate prejadomycin was determined for the first time by Cu Kα X-ray analysis. Heterologous expression of the intact fls-gene cluster in Streptomyces coelicolor YF11 in the presence of 3% sea salts led to the isolation of two new compounds: fluostatin L (1) and difluostatin A (2). Difluostatin A (2), an unusual heterodimer, exhibited antibacterial activities.
10.1021/acs.orglett.5b02683
Late-Stage Terpene Cyclization by an Integral Membrane Cyclase in the Biosynthesis of Isoprenoid Epoxycyclohexenone Natural Products.
Tang Man-Cheng,Cui Xiaoqing,He Xueqian,Ding Zhuang,Zhu Tianjiao,Tang Yi,Li Dehai
Organic letters
Macrophorins are representative examples of isoprenoid epoxycyclohexenones containing cyclized drimane moieties. We located and characterized the biosynthetic gene cluster of macrophorin from Penicillium terrestris. MacJ encoded by this cluster was characterized to be the first example of a membrane-bound type-II terpene cyclase catalyzing the cyclization of meroterpenoids via direct protonation of the terminal olefinic bond in acyclic yanuthones. The late-stage functionalization and substrate promiscuity of MacJ make it a potential biocatalyst for the synthesis of macrophorin analogues.
10.1021/acs.orglett.7b02653
Generation of complexity in fungal terpene biosynthesis: discovery of a multifunctional cytochrome P450 in the fumagillin pathway.
Journal of the American Chemical Society
Fumagillin (1), a meroterpenoid from Aspergillus fumigatus, is known for its antiangiogenic activity due to binding to human methionine aminopeptidase 2. 1 has a highly oxygenated structure containing a penta-substituted cyclohexane that is generated by oxidative cleavage of the bicyclic sesquiterpene β-trans-bergamotene. The chemical nature, order, and biochemical mechanism of all the oxygenative tailoring reactions has remained enigmatic despite the identification of the biosynthetic gene cluster and the use of targeted-gene deletion experiments. Here, we report the identification and characterization of three oxygenases from the fumagillin biosynthetic pathway, including a multifunctional cytochrome P450 monooxygenase, a hydroxylating nonheme-iron-dependent dioxygenase, and an ABM family monooxygenase for oxidative cleavage of the polyketide moiety. Most significantly, the P450 monooxygenase is shown to catalyze successive hydroxylation, bicyclic ring-opening, and two epoxidations that generate the sesquiterpenoid core skeleton of 1. We also characterized a truncated polyketide synthase with a ketoreductase function that controls the configuration at C-5 of hydroxylated intermediates.
10.1021/ja500881e
Epoxyquinone formation catalyzed by a two-component flavin-dependent monooxygenase involved in biosynthesis of the antibiotic actinorhodin.
Taguchi Takaaki,Okamoto Susumu,Hasegawa Kimiko,Ichinose Koji
Chembiochem : a European journal of chemical biology
The biosynthetic gene cluster of the aromatic polyketide antibiotic actinorhodin (ACT) in Streptomyces coelicolor A3(2) carries a pair of genes, actVA-ORF5 and actVB, that encode a two-component flavin-dependent monooxygenase (FMO). Our previous studies have demonstrated that the ActVA-ORF5/ActVB system functions as a quinone-forming C-6 oxygenase in ACT biosynthesis. Furthermore, we found that this enzyme system exhibits an additional oxygenation activity with dihydrokalafungin (DHK), a proposed intermediate in the ACT biosynthetic pathway, and generates two reaction products. These compounds were revealed to be monooxygenated derivatives of kalafungin, which is spontaneously formed through oxidative lactonization of DHK. Their absolute structures were elucidated from their NMR spectroscopic data and by computer modeling and X-ray crystallography as (5S,14R)-epoxykalafungin and (5R,14S)-epoxykalafungin, demonstrating an additional epoxyquinone-forming activity of the ActVA-ORF5/ActVB system in vitro.
10.1002/cbic.201100571
New polyoxygenated farnesylcyclohexenones, deacetoxyyanuthone A and its hydro derivative from the marine-derived fungus Penicillium sp.
Li Xifeng,Choi Hong Dae,Kang Jung Sook,Lee Chong-O,Son Byeng Wha
Journal of natural products
New polyoxygenated farnesylcyclohexenones, 7-deacetoxyyanuthone A (1) and its 2,3-hydro derivative (2), were isolated together with the known farnesylquinones (3, 4) from a marine isolate of the genus Penicillium. The structures of the new deacetoxyyanuthone A (1) and its 2,3-hydro derivative (2) were assigned by spectroscopic methods, including 2D NMR and CD for the Cotton effect of alpha-epoxyketone experiments. Compounds 1 and 3 showed moderate in vitro cytotoxicity in a panel of five human tumor cell lines, and 1 also exhibited mild in vitro antibacterial activity against methicillin-resistant and multidrug-resistant Staphylococcus aureus (MIC, 50 microg/mL).
10.1021/np030231u