Polyketide Cyclase Research Articles

Seasonal activities of the phyllosphere microbiome of perennial crops

Understanding the interactions between plants and microorganisms can inform microbiome management to enhance crop productivity and resilience to stress. Here, we apply a genome-centric approach to identify ecologically important leaf microbiome members on replicated plots of field-grown switchgrass and miscanthus, and to quantify their activities over two growing seasons for switchgrass. We use metagenome and metatranscriptome sequencing and curate 40 medium- and high-quality metagenome-assembled-genomes (MAGs). We find that classes represented by these MAGs (Actinomycetia, Alpha- and Gamma- Proteobacteria, and Bacteroidota) are active in the late season, and upregulate transcripts for short-chain dehydrogenase, molybdopterin oxidoreductase, and polyketide cyclase. Stress-associated pathways are expressed for most MAGs, suggesting engagement with the host environment. We also detect seasonally activated biosynthetic pathways for terpenes and various non-ribosomal peptide pathways that are poorly annotated. Our findings support that leaf-associated bacterial populations are seasonally dynamic and responsive to host cues.

Spatial Genomic Resource Reveals Molecular Insights into Key Bioactive-Metabolite Biosynthesis in Endangered Angelica glauca Edgew.

Angelica glauca Edgew, which is an endangered medicinal and aromatic herb, is a rich source of numerous industrially important bioactive metabolites, including terpenoids, phenolics, and phthalides. Nevertheless, genomic interventions for the sustainable utilization and restoration of its genetic resources are greatly offset due to the scarcity of the genomic resources and key regulators of the underlying specialized metabolism. To unravel the global atlas of the specialized metabolism, the first spatial transcriptome sequencing of the leaf, stem, and root generated 109 million high-quality paired-end reads, assembled de novo into 81,162 unigenes, which exhibit a 61.53% significant homology with the six public protein databases. The organ-specific clustering grouped 1136 differentially expressed unigenes into four subclusters differentially enriched in the leaf, stem, and root tissues. The prediction of the transcriptional-interactome network by integrating enriched gene ontology (GO) and the KEGG metabolic pathways identified the key regulatory unigenes that correspond to terpenoid, flavonoid, and carotenoid biosynthesis in the leaf tissue, followed by the stem and root tissues. Furthermore, the stem and root-specific significant enrichments of phenylalanine ammonia lyase (PAL), cinnamate-4-hydroxylase (C4H), and caffeic acid 3-O-methyltransferase (COMT) indicate that phenylalanine mediated the ferulic acid biosynthesis in the stem and root. However, the root-specific expressions of NADPH-dependent alkenal/one oxidoreductase (NADPH-AOR), S-adenosyl-L-methionine-dependent methyltransferases (SDMs), polyketide cyclase (PKC), and CYP72A15 suggest the “root” as the primary site of phthalide biosynthesis. Additionally, the GC-MS and UPLC analyses corresponded to the organ-specific gene expressions, with higher contents of limonene and phthalide compounds in the roots, while there was a higher accumulation of ferulic acid in the stem, followed by in the root and leaf tissues. The first comprehensive genomic resource with an array of candidate genes of the key metabolic pathways can be potentially utilized for the targeted upscaling of aromatic and pharmaceutically important bioactive metabolites. This will also expedite genomic-assisted conservation and breeding strategies for the revival of the endangered A. glauca.

Crystal structure of the polyketide cyclase from Mycobacterium tuberculosis.

About 40% of proteins are classified as conserved hypothetical proteins in Mycobacterium tuberculosis (TB). Identification and characterization of these proteins are beneficial to understand the pathogenesis of TB and exploiting novel drugs for TB treatments. The polyketide cyclase, a protein from M. tuberculosis ( MtPC) has been annotated as a hypothetical protein in Uniprot database. Sequence analysis shows that the MtPC belongs to the NTF2-like superfamily proteins with diverse functions. Here, we determined the crystal structure of MtPC at a resolution of 2.4 Å and measured backbone relaxation parameters for the MtPC protein. MtPC exists as a dimer in solution, and each subunit contains a six-stranded mixed β-sheet and three α helixes which are arranged in the order α1-α2-β1-β2-α3-β3-β4-β5-β6. The NMR dynamics analysis showed that the overall structure of MtPC is highly rigid on ps-ns time scales. Furthermore, we predicted the potential function of MtPC based on the crystal structure. Our results lay the basis for further exploiting and mechanistically understanding the biological functions of MtPC.

1H, 13C, 15N backbone and side-chain chemical shift assignments of the polyketide cyclase from Mycobacterium tuberculosis.

Polyketide cyclase from Mycobacterium tuberculosis (MtPC) is related to the formation of sterol derivatives, which may play a role in immune escape in the initial stage of macrophage infection by Mycobacterium tuberculosis. However, the structure and specific functions of MtPC are still unknown. Here we report the backbone and side-chain NMR resonance assignments for the MtPC. Most resonances were assigned and the secondary structure was predicted according to the assigned backbone resonances by TALOS-N and PECAN. These NMR assignments represent a first step towards researching the structure and function of MtPC.

Quantitative Proteomics Analysis Reveals the Function of the Putative Ester Cyclase UvEC1 in the Pathogenicity of the Rice False Smut Fungus Ustilaginoidea virens.

Rice false smut is a fungal disease distributed worldwide and caused by Ustilaginoidea virens. In this study, we identified a putative ester cyclase (named as UvEC1) as being significantly upregulated during U. virens infection. UvEC1 contained a SnoaL-like polyketide cyclase domain, but the functions of ketone cyclases such as SnoaL in plant fungal pathogens remain unclear. Deletion of UvEC1 caused defects in vegetative growth and conidiation. UvEC1 was also required for response to hyperosmotic and oxidative stresses and for maintenance of cell wall integrity. Importantly, ΔUvEC1 mutants exhibited reduced virulence. We performed a tandem mass tag (TMT)-based quantitative proteomic analysis to identify differentially accumulating proteins (DAPs) between the ΔUvEC1-1 mutant and the wild-type isolate HWD-2. Proteomics data revealed that UvEC1 has a variety of effects on metabolism, protein localization, catalytic activity, binding, toxin biosynthesis and the spliceosome. Taken together, our findings suggest that UvEC1 is critical for the development and virulence of U. virens.

Differential transcriptome and metabolome analysis of Plumbago zeylanica L. reveal putative genes involved in plumbagin biosynthesis

Plumbagin is a pharmacologically active naphthoquinone present in the Plumbago zeylanica L. having important medicinal properties. The root of P. zeylanica is rich and primary tissue of the plumbagin biosynthesis and accumulation. The complete biosynthetic pathway of plumbagin in plant is still obscure. The present study attempts to understand the plumbagin biosynthetic pathway with the help of differential transcriptome and metabolome analysis of P. zeylanica leaf and root. The transcriptome data showed co-expression of Aldo-keto reductase (PzAKR), Polyketide cyclase (Pzcyclase) and Cytochrome P450 (PzCYPs) transcripts along with the Polyketide synthase (PzPKS) transcripts. Their higher expression in root as compared to leaf supports their possible involvement in plumbagin biosynthesis. The metabolome data of leaf and root revealed naphthalene derivative isoshinanolone that could be potential precursor of plumbagin. Pathway elucidation and transcriptome data of P. zeylanica, will enable and accelerate research on naphthoquinone biosynthesis in plants.

Structural characterization of three noncanonical NTF2-like superfamily proteins: implications for polyketide biosynthesis.

Proteins belonging to the NTF2-like superfamily are present in the biosynthetic pathways of numerous polyketide natural products, such as anthracyclins and benzoisochromanequinones. Some have been found to be bona fide polyketide cyclases, but many of them have roles that are currently unknown. Here, the X-ray crystal structures of three NTF2-like proteins of unknown function are reported: those of ActVI-ORFA from Streptomyces coelicolor A3(2) and its homologs Caci_6494, a protein from an uncharacterized biosynthetic cluster in Catenulispora acidiphila, and Aln2 from Streptomyces sp. CM020, a protein in the biosynthetic pathway of alnumycin. The presence of a solvent-accessible cavity and the conservation of the His/Asp dyad that is characteristic of many polyketide cyclases suggest a potential enzymatic role for these enzymes in polyketide biosynthesis.

Hydrosoluble phylloplane components of Theobroma cacao modulate the metabolism of Moniliophthora perniciosa spores during germination

The surface of plants forms a defense barrier that directly inhibits the first point of contact of microorganisms with the host. To understand this defense mechanism in Moniliophthora perniciosa interaction with Theobroma cacao cv Catongo, the aim of this study was to compare the changes in protein expression in basidiospores of the fungus M. perniciosa in response the leaf water washes (LWW) of two contrasting cocoa varieties for resistance to witches’ broom disease. A total of 8.1 × 108 basidiospores were used for each treatment containing washed leaf material. Germinated basidiospores in the absence of LWW were used as control. The proteomic analysis was performed by the 2D-PAGE technique combined with mass spectrometry (MS). Protein extraction was based on the SDS-dense method followed by sonication for cell disruption and phenol extraction. Sixty-four proteins had accumulation of variation when compared to the control (no LWW). Proteins were identified associated with energy (ATP synthase) and protein (BiP) metabolism, whose accumulation was reduced by basidiospores germinated in leaf wash from Catongo cocoa. The reduction in ATP synthase of the basidiospores germinated the Catongo LWW suggests a shift from aerobic to fermentative metabolism of the fungus in response to components of the LWW. Furthermore, proteins involved in virulence were identified along with fungal resistance to polyketide cyclase, glycoside hydrolase, multidrug transporter protein (SFM) and proteins related to oxidative stress and fermentation, such as catalase A and alcohol dehydrogenase (ADH). The data showed an effect of cocoa phylloplane substances on the germination of fungal basidiospores through differences in protein expression patterns in the presence of LWW of the CCN51 and Catongo genotypes. These results may reveal mechanisms of resistance, host susceptibility and pathogen virulence.

Solution NMR structure of CGL2373, a polyketide cyclase-like protein from Corynebacterium glutamicum.

Protein CGL2373 from Corynebacterium glutamicum was previously proposed to be a member of the polyketide_cyc2 family, based on amino-acid sequence and secondary structure features derived from NMR chemical shift assignments. We report here the solution NMR structure of CGL2373, which contains three α-helices and one antiparallel β-sheet and adopts a helix-grip fold. This structure shows moderate similarities to the representative polyketide cyclases, TcmN, WhiE, and ZhuI. Nevertheless, unlike the structures of these homologs, CGL2373 structure looks like a half-open shell with a much larger pocket, and key residues in the representative polyketide cyclases for binding substrate and catalyzing aromatic ring formation are replaced with different residues in CGL2373. Also, the gene cluster where the CGL2373-encoding gene is located in C. glutamicum contains additional genes encoding nucleoside diphosphate kinase, folylpolyglutamate synthase, and valine-tRNA ligase, different from the typical gene cluster encoding polyketide cyclase in Streptomyces. Thus, although CGL2373 is structurally a polyketide cyclase-like protein, the function of CGL2373 may differ from the known polyketide cyclases and needs to be further investigated. The solution structure of CGL2373 lays a foundation for in silico ligand screening and binding site identifying in future functional study.

Exploration of Nicotine Metabolism in Paenarthrobacter nicotinovorans pAO1 by Microbial Proteomics.

Proteomics, or the large-scale study of proteins, is a post-genomics field that, together with transcriptomics and metabolomics, has moved the study of bacteria to a new era based on system-wide understanding of bacterial metabolic and regulatory networks. The study of bacterial proteins or microbial proteomics has found a wide array of applications in many fields of microbiology, from food, clinical, and industrial microbiology to microbial ecology and physiology. The current chapter makes a brief technical introduction into the available approaches for the large-scale study of bacterial proteins using mass-spectrometry. Furthermore, the advantages and disadvantages of using bacteria for proteomics studies are indicated as well as several example studies where MS-based bacterial proteomics had a fundamental role in deciphering the scientific question. Finally, the proteomics study of nicotine catabolism in Paenarthrobacter nicotinovorans pAO1 using nanoLC-MS/MS is given as an in-depth example for possible applications of microbial proteomics.The nicotine degradation pathway functioning in Paenarthrobacter nicotinovorans is encoded by the catabolic megaplasmid pAO1 that contains about 40 nicotine-related genes making out the nic-gens cluster. Despite the promising biotechnological potential for the production of green-chemicals, only half of the nic-genes have been experimentally linked to nicotine. In an attempt to systematically identify all the proteins involved in nicotine degradation, a gel-based proteomics approach was used to identify a total of 801 proteins when Paenarthrobacter nicotinovorans was grown on three carbon sources: citrate, nicotine and nicotine and citrate. The differences in protein abundance showed that the bacterium is able to switch between deamination and demethylation in the lower nicotine pathway based on the available C source. Several pAO1 putative genes including a hypothetical polyketide cyclase have been shown to have a nicotine-dependent expression and we hypothesize that the polyketide cyclase would hydrolyze the N1-C6 bond from the pyridine ring with the formation of alpha-keto-glutaramate. Two chromosomal proteins, a malate dehydrogenase, and a D-3-phosphoglycerate dehydrogenase were shown to be strongly upregulated when nicotine was the sole carbon source and could be related to the production of the alpha-keto-glutaramate by the polyketide cyclase.

Proteomics based analysis of the nicotine catabolism in Paenarthrobacter nicotinovorans pAO1

Paenarthrobacter nicotinovorans is a nicotine-degrading microorganism that shows a promising biotechnological potential for the production of compounds with industrial and pharmaceutical importance. Its ability to use nicotine was linked to the presence of the catabolic megaplasmid pAO1. Although extensive work has been performed on the molecular biology of nicotine degradation in this bacterium, only half of the genes putatively involved have been experimentally linked to nicotine. In the current approach, we used nanoLC–MS/MS to identify a total of 801 proteins grouped in 511 non-redundant protein clusters when P. nicotinovorans was grown on citrate, nicotine and nicotine and citrate as the only carbon sources. The differences in protein abundance showed that deamination is preferred when citrate is present. Several putative genes from the pAO1 megaplasmid have been shown to have a nicotine-dependent expression, including a hypothetical polyketide cyclase. We hypothesize that the enzyme would hydrolyze the N1-C6 bond from the pyridine ring with the formation of α-keto- glutaramate. Two chromosomally-encoded proteins, a malate dehydrogenase, and a D-3-phosphoglycerate dehydrogenase were shown to be strongly up-regulated when nicotine was the sole carbon source and could be related to the production the α-keto-glutarate. The data have been deposited to the ProteomeXchange with identifier PXD008756.

Comparative Metatranscriptomics of Wheat Rhizosphere Microbiomes in Disease Suppressive and Non-suppressive Soils for Rhizoctonia solani AG8.

The soilborne fungus Rhizoctonia solani anastomosis group (AG) 8 is a major pathogen of grain crops resulting in substantial production losses. In the absence of resistant cultivars of wheat or barley, a sustainable and enduring method for disease control may lie in the enhancement of biological disease suppression. Evidence of effective biological control of R. solani AG8 through disease suppression has been well documented at our study site in Avon, South Australia. A comparative metatranscriptomic approach was applied to assess the taxonomic and functional characteristics of the rhizosphere microbiome of wheat plants grown in adjacent fields which are suppressive and non-suppressive to the plant pathogen R. solani AG8. Analysis of 12 rhizosphere metatranscriptomes (six per field) was undertaken using two bioinformatic approaches involving unassembled and assembled reads. Differential expression analysis showed the dominant taxa in the rhizosphere based on mRNA annotation were Arthrobacter spp. and Pseudomonas spp. for non-suppressive samples and Stenotrophomonas spp. and Buttiauxella spp. for the suppressive samples. The assembled metatranscriptome analysis identified more differentially expressed genes than the unassembled analysis in the comparison of suppressive and non-suppressive samples. Suppressive samples showed greater expression of a polyketide cyclase, a terpenoid biosynthesis backbone gene (dxs) and many cold shock proteins (csp). Non-suppressive samples were characterised by greater expression of antibiotic genes such as non-heme chloroperoxidase (cpo) which is involved in pyrrolnitrin synthesis, and phenazine biosynthesis family protein F (phzF) and its transcriptional activator protein (phzR). A large number of genes involved in detoxifying reactive oxygen species (ROS) and superoxide radicals (sod, cat, ahp, bcp, gpx1, trx) were also expressed in the non-suppressive rhizosphere samples most likely in response to the infection of wheat roots by R. solani AG8. Together these results provide new insight into microbial gene expression in the rhizosphere of wheat in soils suppressive and non-suppressive to R. solani AG8. The approach taken and the genes involved in these functions provide direction for future studies to determine more precisely the molecular interplay of plant-microbe-pathogen interactions with the ultimate goal of the development of management options that promote beneficial rhizosphere microflora to reduce R. solani AG8 infection of crops.

Chemical shift assignments of polyketide cyclase_like protein CGL2373 from Corynebacterium glutamicum.

Protein CGL2373 from Corynebacterium glutamicum, which is 155 amino acids long and 17.7kDa, is a member of the polyketide_cyc2 family. As a potential polyketide cyclase, it may play an important role in the biosynthesis of aromatic polyketides that are the source of many bioactive molecules. Here we report the complete 1H, 13C and 15N chemical shift assignments of CGL2373, which lays a foundation for further structural and functional research.

Structural basis for olivetolic acid formation by a polyketide cyclase from Cannabis sativa.

Structural data reported in this paper are available in the Protein Data Bank under the accession numbers 5B08 for the OAC apo, 5B09 for the OAC-OA binary complex and 5B0A, 5B0B, 5B0C, 5B0D, 5B0E, 5B0F and 5B0G for the OAC His5Q, Ile7F, Tyr27F, Tyr27W, Val59M, Tyr72F and His78S mutant enzymes, respectively.

Expression, purification and crystallization of a plant polyketide cyclase from Cannabis sativa.

Plant polyketides are a structurally diverse family of natural products. In the biosynthesis of plant polyketides, the construction of the carbocyclic scaffold is a key step in diversifying the polyketide structure. Olivetolic acid cyclase (OAC) from Cannabis sativa L. is the only known plant polyketide cyclase that catalyzes the C2-C7 intramolecular aldol cyclization of linear pentyl tetra-β-ketide-CoA to generate olivetolic acid in the biosynthesis of cannabinoids. The enzyme is also thought to belong to the dimeric α+β barrel (DABB) protein family. However, because of a lack of functional analysis of other plant DABB proteins and low sequence identity with the functionally distinct bacterial DABB proteins, the catalytic mechanism of OAC has remained unclear. To clarify the intimate catalytic mechanism of OAC, the enzyme was overexpressed in Escherichia coli and crystallized using the vapour-diffusion method. The crystals diffracted X-rays to 1.40 Å resolution and belonged to space group P3121 or P3221, with unit-cell parameters a = b = 47.3, c = 176.0 Å. Further crystallographic analysis will provide valuable insights into the structure-function relationship and catalytic mechanism of OAC.

HIGH CHLOROPHYLL FLUORESCENCE145 Binds to and Stabilizes the psaA 5' UTR via a Newly Defined Repeat Motif in Embryophyta.

The seedling-lethal Arabidopsis thaliana high chlorophyll fluorescence145 (hcf145) mutation leads to reduced stability of the plastid tricistronic psaA-psaB-rps14 mRNA and photosystem I (PSI) deficiency. Here, we genetically mapped the HCF145 gene, which encodes a plant-specific, chloroplast-localized, modular protein containing two homologous domains related to the polyketide cyclase family comprising 37 annotated Arabidopsis proteins of unknown function. Two further highly conserved and previously uncharacterized tandem repeat motifs at the C terminus, herein designated the transcript binding motif repeat (TMR) domains, confer sequence-specific RNA binding capability to HCF145. Homologous TMR motifs are often found as multiple repeats in quite diverse proteins of green and red algae and in the cyanobacterium Microcoleus sp PCC 7113 with unknown function. HCF145 represents the only TMR protein found in vascular plants. Detailed analysis of hcf145 mutants in Arabidopsis and Physcomitrella patens as well as in vivo and in vitro RNA binding assays indicate that HCF145 has been recruited in embryophyta for the stabilization of the psaA-psaB-rps14 mRNA via specific binding to its 5' untranslated region. The polyketide cyclase-related motifs support association of the TMRs to the psaA RNA, presumably pointing to a regulatory role in adjusting PSI levels according to the requirements of the plant cell.

Role of genes snoaM, snoaL, and snoaE in the biosynthesis of nogalamycin in Streptomyces nogalater Lv65

Bioinformatic analysis of snoaL, snoaM, and snoaE genes from Streptomyces nogalater, a microorganism producing the anthracycline antibiotic nogalamycin, is reported. The results of phylogenetic analysis demonstrated high similarity of SnoaL, SnoaM, and SnoaE proteins to cyclases involved in the biosynthesis of various antibiotics. Insertional inactivation of polyketide cyclase genes in the chromosome of S. nogalater Lv65 was performed, and S. nogalater strains MI, LI, and EI were thus obtained. Inactivation of the above named genes did not affect the morphologic characteristics of S. nogalater. The inability of strains MI, LI, and EI to synthesize nogalamycin was demonstrated. Antibiotic synthesis was restored when extra copies of snoaL, snoaM, and snoaE genes were introduced into the above named strains. Streptomycete strains obtained in the present work, as well as nogalamycin cyclase genes, can be used in combinatorial biosynthesis of anthracyclin antibiotics.

Characterization of a novel cyclase-like gene family involved in controlling stress tolerance in rice

A novel cyclase-like gene family (CYL) encodes proteins containing cyclase domain, but their functions are largely unknown. We report the systematic identification and characterization of CYL genes in the rice genome. Five putative CYL protein sequences (OsCYL1 to 4b) were identified. These sequences and other CYL homologs were classified into four subgroups based on phylogenetic analysis. Distinct diversification of these CYL proteins exists between plants and non-plants. The CYL family has conserved exon-intron structures, and the organizations of putative motifs in plants are specifically diverse. All OsCYL genes were expressed in a wide range of tissues or organs and were responsive to at least one of the abiotic stresses and hormone treatments applied. Protein OsCYL4a is targeted to the cell membrane. The overexpression of one stress-responsive gene OsCYL4a in rice resulted in decreased tolerance to salt, drought, cold, and oxidative stress. The expression levels of some abiotic stress-responsive factors, including H2O2-accumulating negative factors DST and OsSKIPa in OsCYL4a-overexpressing plants, were reduced compared with the wild type under normal condition and drought stress. These results suggest that rice CYL family may be functionally conserved polyketide cyclase, resulting in the rapid accumulation of reactive oxygen species to decrease tolerance to abiotic stresses.

Bioconversion of deoxysugar moieties to the biosynthetic intermediates of daunorubicin in an engineered strain of Streptomyces coeruleobidus.

Daunorubicin (DNR) is a representative anthracycline with anti-tumor bioactivity. Its convergent biosynthetic pathway has promoted the research on pursuing novel anthracyclines by combinatorial biosynthesis. SnoaL is a special polyketide cyclase that catalyzes the closure of nogalonic acid methyl ester with the C9-S stereochemistry. In this study, the gene cluster of DNR was cloned, and snoaL was integrated into the DNR biosynthetic pathway for the substitution of dnrD in Streptomyces coeruleobidus DM, which resulted in the production of epi-aklaviketone. The biosynthetic pathway of NDP-4-deacetyl-L-chromose B was then expressed in the engineered strain, which led to the production of corresponding glycosylated anthracycline compounds. Finally, the bioactivities of these engineering strains were evaluated.

Structural and mechanistic studies of theorf12gene product from the clavulanic acid biosynthesis pathway

Structural and biochemical studies of the orf12 gene product (ORF12) from the clavulanic acid (CA) biosynthesis gene cluster are described. Sequence and crystallographic analyses reveal two domains: a C-terminal penicillin-binding protein (PBP)/β-lactamase-type fold with highest structural similarity to the class A β-lactamases fused to an N-terminal domain with a fold similar to steroid isomerases and polyketide cyclases. The C-terminal domain of ORF12 did not show β-lactamase or PBP activity for the substrates tested, but did show low-level esterase activity towards 3'-O-acetyl cephalosporins and a thioester substrate. Mutagenesis studies imply that Ser173, which is present in a conserved SXXK motif, acts as a nucleophile in catalysis, consistent with studies of related esterases, β-lactamases and D-Ala carboxypeptidases. Structures of wild-type ORF12 and of catalytic residue variants were obtained in complex with and in the absence of clavulanic acid. The role of ORF12 in clavulanic acid biosynthesis is unknown, but it may be involved in the epimerization of (3S,5S)-clavaminic acid to (3R,5R)-clavulanic acid.