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Microbial Cell Factories Mar 2024Natural tetramates are a family of hybrid polyketides bearing tetramic acid (pyrrolidine-2,4-dione) moiety exhibiting a broad range of bioactivities. Biosynthesis of...
BACKGROUND
Natural tetramates are a family of hybrid polyketides bearing tetramic acid (pyrrolidine-2,4-dione) moiety exhibiting a broad range of bioactivities. Biosynthesis of tetramates in microorganisms is normally directed by hybrid polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) machineries, which form the tetramic acid ring by recruiting trans- or cis-acting thioesterase-like Dieckmann cyclase in bacteria. There are a group of tetramates with unique skeleton of 3-(2H-pyran-2-ylidene)pyrrolidine-2,4-dione, which remain to be investigated for their biosynthetic logics.
RESULTS
Herein, the tetramate type compounds bripiodionen (BPD) and its new analog, featuring the rare skeleton of 3-(2H-pyran-2-ylidene)pyrrolidine-2,4-dione, were discovered from the sponge symbiotic bacterial Streptomyces reniochalinae LHW50302. Gene deletion and mutant complementation revealed the production of BPDs being correlated with a PKS-NRPS biosynthetic gene cluster (BGC), in which a Dieckmann cyclase gene bpdE was identified by sit-directed mutations. According to bioinformatic analysis, the tetramic acid moiety of BPDs should be formed on an atypical NRPS module constituted by two discrete proteins, including the C (condensation)-A (adenylation)-T (thiolation) domains of BpdC and the A-T domains of BpdD. Further site-directed mutagenetic analysis confirmed the natural silence of the A domain in BpdC and the functional necessities of the two T domains, therefore suggesting that an unusual aminoacyl transthiolation should occur between the T domains of two NRPS subunits. Additionally, characterization of a LuxR type regulator gene led to seven- to eight-fold increasement of BPDs production. The study presents the first biosynthesis case of the natural molecule with 3-(2H-pyran-2-ylidene)pyrrolidine-2,4-dione skeleton. Genomic mining using BpdD as probe reveals that the aminoacyl transthiolation between separate NRPS subunits should occur in a certain population of NRPSs in nature.
Topics: Biosynthetic Pathways; Polyketide Synthases; Bacteria; Pyrans; Skeleton; Peptide Synthases; Pyrrolidinones
PubMed: 38515152
DOI: 10.1186/s12934-024-02364-7 -
Science (New York, N.Y.) Mar 2024Bacterial multimodular polyketide synthases (PKSs) are giant enzymes that generate a wide range of therapeutically important but synthetically challenging natural...
Bacterial multimodular polyketide synthases (PKSs) are giant enzymes that generate a wide range of therapeutically important but synthetically challenging natural products. Diversification of polyketide structures can be achieved by engineering these enzymes. However, notwithstanding successes made with textbook -acyltransferase (-AT) PKSs, tailoring such large assembly lines remains challenging. Unlike textbook PKSs, -AT PKSs feature an extraordinary diversity of PKS modules and commonly evolve to form hybrid PKSs. In this study, we analyzed amino acid coevolution to identify a common module site that yields functional PKSs. We used this site to insert and delete diverse PKS parts and create 22 engineered -AT PKSs from various pathways and in two bacterial producers. The high success rates of our engineering approach highlight the broader applicability to generate complex designer polyketides.
Topics: Acyltransferases; Polyketide Synthases; Polyketides; Directed Molecular Evolution; Bacterial Proteins; Serratia; Amino Acid Motifs; Recombinant Fusion Proteins
PubMed: 38513027
DOI: 10.1126/science.adj7621 -
Heliyon Mar 2024is an actinorhizal plant that coexists with the nitrogen-fixing actinomycete via nodules. It produces a variety of polyketides, including flavonoids, stilbenoids, and...
is an actinorhizal plant that coexists with the nitrogen-fixing actinomycete via nodules. It produces a variety of polyketides, including flavonoids, stilbenoids, and diarylheptanoids. These compounds have beneficial biological activities. Plant polyketides are produced by type III polyketide synthases (PKSIII). In this study, three PKSIIIs (AsPKSIII1, AsPKSIII2, and AsPKSIII3) predicted from next-generation sequencing analysis of seedling RNA were amplified and cloned. Phylogenetic tree analysis classified AsPKSIII2 and AsPKSIII3 into the chalcone synthase (CHS) group, whereas AsPKSIII1 was not classified into this group. We attempted to produce polyketides by adding cinnamic acid analogs to the culture medium of in which the respective PKSIII gene and the acetyl-CoA carboxylase (ACC) and 4-coumarate: CoA ligase (4CL) genes were simultaneously recombined. AsPKSIII1 is an enzyme that condensed only one molecule of malonyl-CoA to cinnamoyl-CoAs. In contrast, AsPKSIII2 and AsPKSIII3 produced chalcones as shown in a phylogenetic tree analysis, but also produced triketide pyrone. The ratio of these products differed between the two enzymes. We determined the gene and amino acid sequences as well as the substrate specificities of the two enzymes involved in flavonoid production and one enzyme potentially involved in diarylheptanoid production in .
PubMed: 38509908
DOI: 10.1016/j.heliyon.2024.e27698 -
Heliyon Mar 2024Latest studies indicated that agro-food wastes are considered renewable sources of bioactive compounds. This investigation aimed to utilize natural extracts of citrus...
Latest studies indicated that agro-food wastes are considered renewable sources of bioactive compounds. This investigation aimed to utilize natural extracts of citrus peels as antimicrobial and anti-aflatoxigenic agents for food safety. The bioactivity of two citrus peels was assessed by total phenolic, flavonoids, and antioxidant activity. Nanoemulsions were manufactured using high-speed homogenization. The mean particle size of the nanoemulsions ranged from 29.41 to 66.41 nm with a polydispersity index of 0.11-0.16. The zeta potential values ranged from -14.27 to -26.74 mV, indicating stability between 81.44% and 99.26%. The orange peel extract showed the highest contents of total phenolic and flavonoids compared to the other extracts and nanoemulsions (39.54 mg GAE/g and 79.54 mg CE/100 g, respectively), which agreed with its potential antioxidant activity performed by DPPH free radical-scavenging and ABTS assays. Chlorogenic, caffeic, ferulic, and catechin were the dominant phenolic acids in the extracts and nanoemulsions, while quercitrin, rutin, and hesperidin were the most abundant flavonoids. Limonene was the major volatile component in both oils; however, it was reduced dramatically from 92.52% to 76.62% in orange peel oil and from 91.79 to 79.12% in tangerine peel oil. Consistent with the differences in phenolics, flavonoids, and volatiles between orange and tangerine peel extracts, the antibacterial properties of orange extracts had more potential than tangerine ones. Gram-positive bacteria were more affected by all the examined extracts than Gram-negative ones. The antifungal activity of orange extract and nanoemulsion on seven fungal strains from spp had more potential than tangerine extracts. Additionally, using a simulated media, the orange peel extract and its nanoemulsion had a more anti-aflatoxigenic influence. Molecular docking confirmed the high inhibitory action of flavonoids, especially hesperidin, on the polyketide synthase (-9.3 kcal/mol) and cytochrome P450 monooxygenase (-10.1 kcal/mol) key enzymes of the aflatoxin biosynthetic mechanism.
PubMed: 38509881
DOI: 10.1016/j.heliyon.2024.e27737 -
Plant Physiology Jun 2024Polyploid hybrid rice (Oryza sativa) has great potential for increasing yields. However, hybrid rice depends on male fertility and its regulation, which is less well...
Polyploid hybrid rice (Oryza sativa) has great potential for increasing yields. However, hybrid rice depends on male fertility and its regulation, which is less well studied in polyploid rice than in diploid rice. We previously identified an MYB transcription factor, MORE FLORET1 (MOF1), whose mutation causes male sterility in neo-tetraploid rice. MOF1 expression in anthers peaks at anther Stage 7 (S7) and progressively decreases to low levels at S10. However, it remains unclear how the dynamics of MOF1 expression contribute to male fertility. Here, we carefully examined anther development in both diploid and tetraploid mof1 rice mutants, as well as lines ectopically expressing MOF1 in a temporal manner. MOF1 mutations caused delayed degeneration of the tapetum and middle layer of anthers and aberrant pollen wall organization. Ectopic MOF1 expression at later stages of anther development led to retarded cytoplasmic reorganization of tapetal cells. In both cases, pollen grains were aborted and seed production was abolished, indicating that precise control of MOF1 expression is essential for male reproduction. We demonstrated that 5 key tapetal genes, CYP703A3 (CYTOCHROME P450 HYDROXYLASE 703A3), OsABCG26 (O. sativa ATP BINDING CASSETTE G26), PTC1 (PERSISTENT TAPETAL CELL1), PKS2 (POLYKETIDE SYNTHASE 2), and OsABCG15 (O. sativa ATP BINDING CASSETTE G15), exhibit expression patterns opposite to those of MOF1 and are negatively regulated by MOF1. Moreover, DNA affinity purification sequencing (DAP-seq), luciferase activity assays, and electrophoretic mobility shift assays indicated that MOF1 binds directly to the PKS2 promoter for transcriptional repression. Our results provide a mechanistic basis for the regulation of male reproduction by MOF1 in both diploid and tetraploid rice. This study will facilitate the development of polyploid male sterile lines, which are useful for breeding of polyploid hybrid rice.
Topics: Oryza; Tetraploidy; Plant Proteins; Flowers; Gene Expression Regulation, Plant; Diploidy; Pollen; Mutation; Genes, Plant; Transcription Factors
PubMed: 38507615
DOI: 10.1093/plphys/kiae145 -
Natural Product Reports Mar 2024Covering: up to the end of 2023Type I modular polyketide synthases construct polyketide natural products in an assembly line-like fashion, where the growing polyketide... (Review)
Review
Covering: up to the end of 2023Type I modular polyketide synthases construct polyketide natural products in an assembly line-like fashion, where the growing polyketide chain attached to an acyl carrier protein is passed from catalytic domain to catalytic domain. These enzymes have immense potential in drug development since they can be engineered to produce non-natural polyketides by strategically adding, exchanging, and deleting individual catalytic domains. In practice, however, this approach frequently results in complete failures or dramatically reduced product yields. A comprehensive understanding of modular polyketide synthase architecture is expected to resolve these issues. We summarize the three-dimensional structures and the proposed mechanisms of three full-length modular polyketide synthases, Lsd14, DEBS module 1, and PikAIII. We also describe the advantages and limitations of using X-ray crystallography, cryo-electron microscopy, and AlphaFold2 to study intact type I polyketide synthases.
PubMed: 38501175
DOI: 10.1039/d3np00060e -
BMC Plant Biology Mar 2024Plant microbiome confers versatile functional roles to enhance survival fitness as well as productivity. In the present study two pearl millet panicle microbiome member...
Comprehensive genomic analysis of Bacillus subtilis and Bacillus paralicheniformis associated with the pearl millet panicle reveals their antimicrobial potential against important plant pathogens.
BACKGROUND
Plant microbiome confers versatile functional roles to enhance survival fitness as well as productivity. In the present study two pearl millet panicle microbiome member species Bacillus subtilis PBs 12 and Bacillus paralicheniformis PBl 36 found to have beneficial traits including plant growth promotion and broad-spectrum antifungal activity towards taxonomically diverse plant pathogens. Understanding the genomes will assist in devising a bioformulation for crop protection while exploiting their beneficial functional roles.
RESULTS
Two potential firmicute species were isolated from pearl millet panicles. Morphological, biochemical, and molecular characterization revealed their identities as Bacillus subtilis PBs 12 and Bacillus paralicheniformis PBl 36. The seed priming assays revealed the ability of both species to enhance plant growth promotion and seedling vigour index. Invitro assays with PBs 12 and PBl 36 showed the antibiosis effect against taxonomically diverse plant pathogens (Magnaporthe grisea; Sclerotium rolfsii; Fusarium solani; Alternaria alternata; Ganoderma sp.) of crops and multipurpose tree species. The whole genome sequence analysis was performed to unveil the genetic potential of these bacteria for plant protection. The complete genomes of PBs 12 and PBl 36 consist of a single circular chromosome with a size of 4.02 and 4.33 Mb and 4,171 and 4,606 genes, with a G + C content of 43.68 and 45.83%, respectively. Comparative Average Nucleotide Identity (ANI) analysis revealed a close similarity of PBs 12 and PBl 36 with other beneficial strains of B. subtilis and B. paralicheniformis and found distant from B. altitudinis, B. amyloliquefaciens, and B. thuringiensis. Functional annotation revealed a majority of pathway classes of PBs 12 (30) and PBl 36 (29) involved in the biosynthesis of secondary metabolites, polyketides, and non-ribosomal peptides, followed by xenobiotic biodegradation and metabolism (21). Furthermore, 14 genomic regions of PBs 12 and 15 of PBl 36 associated with the synthesis of RiPP (Ribosomally synthesized and post-translationally modified peptides), terpenes, cyclic dipeptides (CDPs), type III polyketide synthases (T3PKSs), sactipeptides, lanthipeptides, siderophores, NRPS (Non-Ribosomal Peptide Synthetase), NRP-metallophone, etc. It was discovered that these areas contain between 25,458 and 33,000 secondary metabolite-coding MiBiG clusters which code for a wide range of products, such as antibiotics. The PCR-based screening for the presence of antimicrobial peptide (cyclic lipopeptide) genes in PBs 12 and 36 confirmed their broad-spectrum antifungal potential with the presence of spoVG, bacA, and srfAA AMP genes, which encode antimicrobial compounds such as subtilin, bacylisin, and surfactin.
CONCLUSION
The combined in vitro studies and genome analysis highlighted the antifungal potential of pearl millet panicle-associated Bacillus subtilis PBs12 and Bacillus paralicheniformis PBl36. The genetic ability to synthesize several antimicrobial compounds indicated the industrial value of PBs 12 and PBl 36, which shed light on further studies to establish their action as a biostimulant for crop protection.
Topics: Bacillus subtilis; Pennisetum; Antifungal Agents; Anti-Infective Agents; Genomics; Plants; Peptides; Bacillus
PubMed: 38500040
DOI: 10.1186/s12870-024-04881-4 -
Philosophical Transactions of the Royal... May 2024Many animals have pigments when they themselves cannot see colour. Perhaps those pigments enable the animal to avoid predators, or to attract mates. Maybe even those... (Review)
Review
Many animals have pigments when they themselves cannot see colour. Perhaps those pigments enable the animal to avoid predators, or to attract mates. Maybe even those pigmented surfaces are hosts for microbes, even when the microbes do not see colour. Do some pigments then serve as a chemical signal for a good or bad microbial substrate? Maybe pigments attract or repel various microbe types? Echinoderms serve as an important model to test the mechanisms of pigment-based microbial interactions. Echinoderms are marine benthic organisms, ranging from intertidal habitats to depths of thousands of metres and are exposed to large varieties of microbes. They are also highly pigmented, with a diverse variety of colours between and even within species. Here we focus on one type of pigment (naphthoquinones) made by polyketide synthase, modified by flavin-dependent monoxygenases, and on one type of function, microbial interaction. Recent successes in targeted gene inactivation by CRISPR/Cas9 in sea urchins supports the contention that colour is more than it seems. Here we dissect the players, and their interactions to better understand how such host factors influence a microbial colonization. This article is part of the theme issue 'Sculpting the microbiome: how host factors determine and respond to microbial colonization'.
Topics: Animals; Microbiota; Pigmentation
PubMed: 38497266
DOI: 10.1098/rstb.2023.0077 -
RSC Advances Mar 2024The programming of widely distributed iterative fungal hr-PKS is mysterious, yet it is central for generating polyketide natural product diversity by controlling the...
The programming of widely distributed iterative fungal hr-PKS is mysterious, yet it is central for generating polyketide natural product diversity by controlling the chain length, β-processing level and methylation patterns of fungal polyketides. For the iterative hr-PKS TENS, responsible for producing the pentaketide-tyrosine hybrid pretenellin A 1, the chain length programming is known to be determined by the KR domain. Structure prediction of the KR domain enabled the identification of a relevant substrate binding helix, which was the focus of swap experiments with corresponding sequences from the related hr-PKS DMBS and MILS that produce similar hexa- and heptaketides (2, 3). The investigations of chimeric TENS variants expressed in the host NSAR1 revealed the substrate binding helix as a promising target for further investigations, evidenced by observed increase of the chain length during swap experiments. Building on these findings, rational engineering of TENS was applied based on structural analysis and sequence alignment. A minimal set of four simultaneous amino acid mutations achieved the re-programming of TENS by producing hexaketides in minor amounts. To refine our understanding and minimize the number of mutations impacting polyketide chain length, we conducted an alanine scan, pinpointing crucial amino acid positions. Our findings give indications on the intrinsic programming of hr-PKS domains by minimal changes in the amino acid sequence as one influence factor for programming.
PubMed: 38495992
DOI: 10.1039/d3ra08463a -
Advanced Science (Weinheim,... Jun 2024Pigments such as anthraquinones (AQs) and melanins are antioxidants, protectants, or virulence factors. AQs from the entomopathogenic bacterium Photorhabdus laumondii...
Pigments such as anthraquinones (AQs) and melanins are antioxidants, protectants, or virulence factors. AQs from the entomopathogenic bacterium Photorhabdus laumondii are produced by a modular type II polyketide synthase system. A key enzyme involved in AQ biosynthesis is PlAntI, which catalyzes the hydrolysis of the bicyclic-intermediate-loaded acyl carrier protein, polyketide trimming, and assembly of the aromatic AQ scaffold. Here, multiple crystal structures of PlAntI in various conformations and with bound substrate surrogates or inhibitors are reported. Structure-based mutagenesis and activity assays provide experimental insights into the three sequential reaction steps to yield the natural product AQ-256. For comparison, a series of ligand-complex structures of two functionally related hydrolases involved in the biosynthesis of 1,8-dihydroxynaphthalene-melanin in pathogenic fungi is determined. These data provide fundamental insights into the mechanism of polyketide trimming that shapes pigments in pro- and eukaryotes.
Topics: Anthraquinones; Polyketides; Melanins; Polyketide Synthases; Photorhabdus; Naphthols; Pigments, Biological
PubMed: 38491909
DOI: 10.1002/advs.202400184