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1.
Sci Total Environ ; 717: 137184, 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32084685

RESUMO

Gambierdiscus spp. can produce the polyketide compound, ciguatoxin (CTX), and are hence responsible for ciguatera fish poisoning (CFP). Studying the molecular mechanism that regulates CTX production is crucial for understanding the environmental trigger of CTX as well as for better informing fishery management. Commonly, polyketide synthases are important for polyketide synthesis; however, no gene has been confirmatively assigned to CTX production. Here, suppression subtractive hybridization (SSH) and transcriptome sequencing (RNA-Seq) were used to compare a CTX-producing strain with a non-CTX-producing strain. Using both methods, a total of 52 polyketide synthase (PKS) genes were identified to be up-regulated in the CTX-producing G. balechii, including transcripts encoding single-domain PKSs as well as transcripts encoding multi-domain PKSs. Using reverse transcription quantitative PCR, the expression of these genes in the CTX-producing strain and in nitrogen-limited cultures of the strain was further documented. These data suggest that PKSs are likely involved in polyketide synthesis and potentially in CTX synthesis in this dinoflagellate species. Our study provides the candidate biomarkers for the detection of CTXs or CFP in waters or any other organisms as well as a valuable genomic resource for the research on Gambierdiscus and other dinoflagellates.


Assuntos
Dinoflagelados , Animais , Intoxicação por Ciguatera , Ciguatoxinas , Policetídeo Sintases , Transcriptoma
2.
PLoS One ; 15(1): e0228217, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31990962

RESUMO

Escherichia coli (E. coli) from the B2 phylogenetic group is implicated in colorectal cancer (CRC) as it possesses a genomic island, termed polyketide synthetase (pks), which codes for the synthesis of colibactin, a genotoxin that induces DNA damage, cell cycle arrest, mutations and chromosomal instability in eukaryotic cells. The aim of this study was to detect and compare the prevalence of E. coli expressing pks (pks+ E. coli) in CRC patients and healthy controls followed by investigating the virulence triggered by pks+ E. coli using an in-vitro model. Mucosal colon tissues were collected and processed to determine the presence of pks+ E. coli. Thereafter, primary colon epithelial (PCE) and colorectal carcinoma (HCT116) cell lines were used to detect cytopathic response to the isolated pks+ E. coli strains. Our results showed 16.7% and 4.3% of CRC and healthy controls, respectively were pks+ E. coli. Further, PCE displayed syncytia and cell swelling and HCT116 cells, megalocytosis, in response to treatment with the isolated pks+ E. coli strains. In conclusion, pks+ E. coli was more often isolated from tissue of CRC patients compared to healthy individuals, and our in-vitro assays suggest these isolated strains may be involved in the initiation and development of CRC.


Assuntos
Centros Médicos Acadêmicos/estatística & dados numéricos , Neoplasias Colorretais/epidemiologia , Neoplasias Colorretais/microbiologia , Escherichia coli/enzimologia , Escherichia coli/fisiologia , Policetídeo Sintases/metabolismo , Idoso , Feminino , Humanos , Malásia/epidemiologia , Masculino , Pessoa de Meia-Idade , Prevalência
3.
Arch Microbiol ; 202(4): 905-920, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31897537

RESUMO

The development of drug-resistant bacteria and the necessity for unique antimicrobial agents, directed to the search of new habitats to screen the production of anti-infective substances. Culture-dependent studies of heterotrophic bacteria from the intertidal macroalgae thriving along the Southern coast of India resulted in the isolation of 148 strains, which were assayed for antibacterial activities against wide spectrum of pathogens including drug-resistant pathogens, methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecalis (VRE). Two of the most active strains with a zone of inhibition ≥ 30 mm on spot over lawn assay, belonging to the phyla Firmicutes and Gamma-proteobacteria, isolated from a  Rhodophycean marine macroalga, Hypnea valentiae, were selected for bioprospecting studies. They were further characterized as Shewanella algae MTCC 12715 and Bacillus amyloliquefaciens MTCC 12716, based on integrated phenotypic and genotypic analysis. The bacterial extracts exhibited significant antibacterial activities against MRSA and VRE with minimum inhibitory concentrations of 6.25-12.5 µg/mL. Time kill kinetic profiles of these bacteria revealed rapid bactericidal activity against both MRSA and E. coli, showing a ≥ 3log10 decline in viable cell count compared to the initial. In BacLight™ live/dead staining technique, the propidium iodide uptake results appropriately attributed that the components in the B. amyloliquefaciens extract might compromise the integrity of the cytoplasmic membrane of the pathogenic bacteria. Type-1 pks gene (MH157093) of S. algae and hybrid nrps/pks gene (MH157092) of B. amyloliquefaciens could be amplified. Antibacterial activity study combined with the results of amplified genes coding for polyketide synthase and nonribosomal peptide synthetase showed that these marine symbiotic bacteria had a promising broad-spectrum activity, and therefore, could be used against the emerging dilemma of antibiotic-resistant bacterial infections.


Assuntos
Bactérias/efeitos dos fármacos , Firmicutes/química , Gammaproteobacteria/química , Alga Marinha/microbiologia , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Bacillus amyloliquefaciens/enzimologia , Bacillus amyloliquefaciens/genética , Descoberta de Drogas , Escherichia coli/efeitos dos fármacos , Firmicutes/enzimologia , Firmicutes/genética , Gammaproteobacteria/enzimologia , Gammaproteobacteria/genética , Índia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Peptídeo Sintases/genética , Policetídeo Sintases/genética , Estudos Prospectivos , Enterococos Resistentes à Vancomicina/efeitos dos fármacos
4.
Nat Commun ; 11(1): 80, 2020 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-31900404

RESUMO

To harness the synthetic power of modular polyketide synthases (PKSs), many aspects of their biochemistry must be elucidated. A robust platform to study these megadalton assembly lines has not yet been described. Here, we in vitro reconstitute the venemycin PKS, a short assembly line that generates an aromatic product. Incubating its polypeptides, VemG and VemH, with 3,5-dihydroxybenzoic acid, ATP, malonate, coenzyme A, and the malonyl-CoA ligase MatB, venemycin production can be monitored by HPLC and NMR. Multi-milligram quantities of venemycin are isolable from dialysis-based reactors without chromatography, and the enzymes can be recycled. Assembly line engineering is performed using pikromycin modules, with synthases designed using the updated module boundaries outperforming those using the traditional module boundaries by over an order of magnitude. Using combinations of VemG, VemH, and their engineered derivatives, as well as the alternate starter unit 3-hydroxybenzoic acid, a combinatorial library of six polyketide products is readily accessed.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Policetídeo Sintases/química , Policetídeo Sintases/genética , Streptomyces/enzimologia , Proteínas de Bactérias/metabolismo , Macrolídeos/química , Policetídeo Sintases/metabolismo , Policetídeos/química , Engenharia de Proteínas , Streptomyces/química , Streptomyces/genética , Especificidade por Substrato
5.
Chem Commun (Camb) ; 56(5): 822-825, 2020 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-31848534

RESUMO

Produced by a newly isolated Streptomycetes strain, meijiemycin is a gigantic linear polyene-polyol that exhibits structural features not seen in other members of the polyene-polyol family. We propose a biosynthetic mechanism and demonstrate that meijiemycin inhibits hyphal growth by inducing the aggregation of ergosterol and restructuring of the fungal plasma membrane.


Assuntos
Antifúngicos/farmacologia , Álcoois Graxos/farmacologia , Polienos/farmacologia , Antifúngicos/isolamento & purificação , Antifúngicos/metabolismo , Candida albicans/efeitos dos fármacos , Descoberta de Drogas , Álcoois Graxos/isolamento & purificação , Álcoois Graxos/metabolismo , Genes Bacterianos , Genômica , Testes de Sensibilidade Microbiana , Família Multigênica , Polienos/isolamento & purificação , Polienos/metabolismo , Policetídeo Sintases/genética , Streptomyces/química
6.
Proc Natl Acad Sci U S A ; 117(2): 1174-1180, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31882449

RESUMO

Indolizidine alkaloids such as anticancer drugs vinblastine and vincristine are exceptionally attractive due to their widespread occurrence, prominent bioactivity, complex structure, and sophisticated involvement in the chemical defense for the producing organisms. However, the versatility of the indolizidine alkaloid biosynthesis remains incompletely addressed since the knowledge about such biosynthetic machineries is only limited to several representatives. Herein, we describe the biosynthetic gene cluster (BGC) for the biosynthesis of curvulamine, a skeletally unprecedented antibacterial indolizidine alkaloid from Curvularia sp. IFB-Z10. The molecular architecture of curvulamine results from the functional collaboration of a highly reducing polyketide synthase (CuaA), a pyridoxal-5'-phosphate (PLP)-dependent aminotransferase (CuaB), an NADPH-dependent dehydrogenase (CuaC), and a FAD-dependent monooxygenase (CuaD), with its transportation and abundance regulated by a major facilitator superfamily permease (CuaE) and a Zn(II)Cys6 transcription factor (CuaF), respectively. In contrast to expectations, CuaB is bifunctional and capable of catalyzing the Claisen condensation to form a new C-C bond and the α-hydroxylation of the alanine moiety in exposure to dioxygen. Inspired and guided by the distinct function of CuaB, our genome mining effort discovers bipolamines A-I (bipolamine G is more antibacterial than curvulamine), which represent a collection of previously undescribed polyketide alkaloids from a silent BGC in Bipolaris maydis ATCC48331. The work provides insight into nature's arsenal for the indolizidine-coined skeletal formation and adds evidence in support of the functional versatility of PLP-dependent enzymes in fungi.


Assuntos
Alcaloides/biossíntese , Ascomicetos/enzimologia , Ascomicetos/metabolismo , Indolizidinas/metabolismo , Policetídeo Sintases/metabolismo , Fosfato de Piridoxal/metabolismo , Alcaloides/genética , Alcaloides/isolamento & purificação , Antibacterianos/metabolismo , Ascomicetos/genética , Aspergillus oryzae/genética , Aspergillus oryzae/metabolismo , Catálise , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos/genética , Hidroxilação , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Família Multigênica , Filogenia , Policetídeo Sintases/classificação , Policetídeo Sintases/genética , Policetídeos , Fosfato de Piridoxal/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transaminases/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
7.
Chem Commun (Camb) ; 56(1): 157-160, 2019 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-31799975

RESUMO

Modular polyketide synthases (PKSs) are enzymatic assembly lines that fuse carbon fragments into complex chiral products. Here, their synthetic logic is employed to chemoenzymatically generate two-stereocenter triketides. Each of the four stereoisomers was constructed in a stereocontrolled manner using C-acylation and two PKS ketoreductases possessing opposite stereoselectivities.


Assuntos
Oxirredutases do Álcool/química , Policetídeo Sintases/química , Policetídeos/síntese química , Proteínas Fúngicas/química , Estereoisomerismo
8.
Nat Commun ; 10(1): 3918, 2019 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-31477708

RESUMO

Polyketides produced by modular type I polyketide synthases (PKSs) play eminent roles in the development of medicines. Yet, the production of structural analogs by genetic engineering poses a major challenge. We report an evolution-guided morphing of modular PKSs inspired by recombination processes that lead to structural diversity in nature. By deletion and insertion of PKS modules we interconvert the assembly lines for related antibiotic and antifungal agents, aureothin (aur) and neoaureothin (nor) (aka spectinabilin), in both directions. Mutational and functional analyses of the polyketide-tailoring cytochrome P450 monooxygenases, and PKS phylogenies give contradictory clues on potential evolutionary scenarios (generalist-to-specialist enzyme evolution vs. most parsimonious ancestor). The KS-AT linker proves to be well suited as fusion site for both excision and insertion of modules, which supports a model for alternative module boundaries in some PKS systems. This study teaches important lessons on the evolution of PKSs, which may guide future engineering approaches.


Assuntos
Cromonas/metabolismo , Oxigenases/metabolismo , Policetídeo Sintases/metabolismo , Policetídeos/metabolismo , Streptomyces/metabolismo , Sequência de Aminoácidos , Antibacterianos/química , Antibacterianos/metabolismo , Cromonas/química , Engenharia Genética/métodos , Modelos Químicos , Estrutura Molecular , Mutação , Filogenia , Policetídeo Sintases/classificação , Policetídeo Sintases/genética , Policetídeos/química , Streptomyces/genética
9.
mSphere ; 4(5)2019 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-31554725

RESUMO

The polyketide-derived secondary metabolite ascochitine is produced by species in the Didymellaceae family, including but not restricted to Ascochyta species pathogens of cool-season food legumes. Ascochitine is structurally similar to the well-known mycotoxin citrinin and exhibits broad-spectrum phytotoxicity and antimicrobial activities. Here, we identified a polyketide synthase (PKS) gene (denoted pksAC) responsible for ascochitine production in the filamentous fungus Ascochyta fabae Deletion of the pksAC prevented production of ascochitine and its derivative ascochital in A. fabae The putative ascochitine biosynthesis gene cluster comprises 11 genes that have undergone rearrangement and gain-and-loss events relative to the citrinin biosynthesis gene cluster in Monascus ruber Interestingly, we also identified pksAC homologs in two recently diverged species, A. lentis and A. lentis var. lathyri, that are sister taxa closely related to ascochitine producers such as A. fabae and A. viciae-villosae However, nonsense mutations have been independently introduced in coding sequences of the pksAC homologs of A. lentis and A. lentis var. lathyri that resulted in loss of ascochitine production. Despite its reported phytotoxicity, ascochitine was not a pathogenicity factor in A. fabae infection and colonization of faba bean (Vicia faba L.). Ascochitine was mainly produced from mature hyphae at the site of pycnidial formation, suggesting a possible protective role of the compound against other microbial competitors in nature. This report highlights the evolution of gene clusters harnessing the structural diversity of polyketides and a mechanism with the potential to alter secondary metabolite profiles via single nucleotide polymorphisms in closely related fungal species.IMPORTANCE Fungi produce a diverse array of secondary metabolites, many of which are of pharmacological importance whereas many others are noted for mycotoxins, such as aflatoxin and citrinin, that can threaten human and animal health. The polyketide-derived compound ascochitine, which is structurally similar to citrinin mycotoxin, has been considered to be important for pathogenicity of legume-associated Ascochyta species. Here, we identified the ascochitine polyketide synthase (PKS) gene in Ascochyta fabae and its neighboring genes that may be involved in ascochitine biosynthesis. Interestingly, the ascochitine PKS genes in other legume-associated Ascochyta species have been mutated, encoding truncated PKSs. This indicated that point mutations may have contributed to genetic diversity for secondary metabolite production in these fungi. We also demonstrated that ascochitine is not a pathogenicity factor in A. fabae The antifungal activities and production of ascochitine during sporulation suggested that it may play a role in competition with other saprobic fungi in nature.


Assuntos
Ascomicetos/genética , Variação Genética , Micotoxinas/biossíntese , Policetídeo Sintases/genética , Ascomicetos/enzimologia , Família Multigênica , Mutação Puntual , Análise de Sequência de DNA
10.
Nat Commun ; 10(1): 4036, 2019 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-31492848

RESUMO

The skeleton of tropane alkaloids is derived from ornithine-derived N-methylpyrrolinium and two malonyl-CoA units. The enzymatic mechanism that connects N-methylpyrrolinium and malonyl-CoA units remains unknown. Here, we report the characterization of three pyrrolidine ketide synthases (PYKS), AaPYKS, DsPYKS, and AbPYKS, from three different hyoscyamine- and scopolamine-producing plants. By examining the crystal structure and biochemical activity of AaPYKS, we show that the reaction mechanism involves PYKS-mediated malonyl-CoA condensation to generate a 3-oxo-glutaric acid intermediate that can undergo non-enzymatic Mannich-like condensation with N-methylpyrrolinium to yield the racemic 4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoic acid. This study therefore provides a long sought-after biosynthetic mechanism to explain condensation between N-methylpyrrolinium and acetate units and, more importantly, identifies an unusual plant type III polyketide synthase that can only catalyze one round of malonyl-CoA condensation.


Assuntos
Malonil Coenzima A/metabolismo , Proteínas de Plantas/metabolismo , Policetídeo Sintases/metabolismo , Pirróis/metabolismo , Alcaloides de Solanáceas/metabolismo , Tropanos/metabolismo , Sequência de Aminoácidos , Biocatálise , Cromatografia Líquida/métodos , Cristalografia por Raios X , Malonil Coenzima A/química , Modelos Químicos , Estrutura Molecular , Filogenia , Proteínas de Plantas/classificação , Proteínas de Plantas/genética , Policetídeo Sintases/química , Policetídeo Sintases/genética , Pirróis/química , Homologia de Sequência de Aminoácidos , Alcaloides de Solanáceas/química , Espectrometria de Massas em Tandem/métodos , Tropanos/química
11.
J Appl Microbiol ; 127(6): 1686-1697, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31529739

RESUMO

AIMS: Isolating culturable bacteria associated with sea cucumber (Apostichopus japonicus) and investigating their potential bioactivities are important approaches to discover natural marine products. METHODS AND RESULTS: A total of 161 isolates were obtained from sea cucumber collected along the Weihai coast of the North Yellow Sea, China. Identification and phylogenetic analysis of 61 isolates were conducted by 16S rRNA gene sequencing. The isolates belonged to 13 genera from 10 families in three phyla, including Firmicutes, Actinobacteria and Proteobacteria. The antimicrobial activities of all strains were determined using six indicator strains. Of the 161 isolates, 93 showed antibacterial activities against at least one of the indicator strains. The 26 strains with the strongest inhibitory effects were selected for screening the biosynthetic gene clusters of polyketide synthase (PKS-I, -II) and nonribosomal peptide synthetase (NRPS). Phylogenetic trees based on the amino acid sequences of the PKS or NRPS genes were constructed. Eleven strains with PKS genes and four strains with NRPS genes were detected. CONCLUSIONS: The data reveal the diversity of culturable bacteria associated with A. japonicus. Most strains showed broad-spectrum antimicrobial activities, and some strains with antimicrobial activities possessed PKS and NRPS genes. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggest that culturable bacteria associated with A. japonicus may act as a promising source of bioactive substances.


Assuntos
Bactérias/genética , Peptídeo Sintases/genética , Policetídeo Sintases/genética , Stichopus/microbiologia , Animais , Antibiose , Bactérias/classificação , Bactérias/isolamento & purificação , China , Oceanos e Mares , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
12.
J Microbiol Biotechnol ; 29(10): 1570-1579, 2019 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-31474098

RESUMO

The fungal products dibenzodioxocinones promise a novel class of inhibitors against cholesterol ester transfer protein (CEPT). Knowledge as to their biosynthesis is scarce. In this report, we characterized four more dibenzodioxocinones, which along with a previously described member pestalotiollide B, delimit the dominant spectrum of secondary metabolites in P. microspora. Through mRNA-seq profiling in gα1Δ, a process that halts the production of the dibenzodioxocinones, a gene cluster harboring 21 genes including a polyketide synthase, designated as pks8, was defined. Disruption of genes in the cluster led to loss of the compounds, concluding the anticipated role in the biosynthesis of the chemicals. The biosynthetic route to dibenzodioxocinones was temporarily speculated. This study reveals the genetic basis underlying the biosynthesis of dibenzodioxocinone in fungi, and may facilitate the practice for yield improvement in the drug development arena.


Assuntos
Família Multigênica , Policetídeos/metabolismo , Xylariales/genética , Vias Biossintéticas , Proteínas de Transferência de Ésteres de Colesterol/antagonistas & inibidores , Endófitos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Perfilação da Expressão Gênica , Família Multigênica/genética , Mutação , Paclitaxel/biossíntese , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo , Policetídeos/química , Xylariales/química , Xylariales/metabolismo
13.
Microbiol Res ; 229: 126312, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31434034

RESUMO

Due to emergence of drug resistant pathogens, nearly all available medicines are becoming ineffective against these life threatening pathogens so there is dire need for the discovery of compounds having unique modes of action. During our previous studies, actinomycetes designated as 196 and RI.24 were isolated, screened for bioactive compounds production and characterized using 16S rRNA gene sequencing. Colony 196 was identified as strain of Streptomyces albolongus (100% sequence similarity) and RI.24 as strain of Streptomyces enissocaesilis (100% sequence similarity). In current study, potential bioactive compounds produced by these strains were characterized. Cold extraction method was applied for taking out of bioactive compounds from actinomycetes. Minimum inhibitory concentration (MIC) determination of compounds from these strains showed activity nearly in the range of commercial antibiotics (strain 196 0.0075 mg/ml, RI.24 0.25 mg/ml and chloramphenicol 0.0075 mg/ml, ampicillin 0.025 mg/ml). Structural elucidation of these compounds was carried out using spectroscopic techniques of LC-MS/MS and 1H NMR. Compounds K-252-C-Aglycone, indolocarbazole alkaloid, decoyinine, cycloheximide were detected from strain 196 whereas daunorubicin, hygromycin B, agecorynin F, indinavir-N-glucuronide and minocycline were identified from strain RI.24.Current study reports these compounds for the first time from strains of Streptomyces albolongus and Streptomyces enissocaesilis. Present investigation also suggests that strains 196 and RI.24 contain polyketide synthase-I (PKS-I) and non-ribosomal peptide synthetase (NRPS) gene clusters which are responsible for the production of bioactive compounds. The results of this study can be used by the scientific world or pharmaceutical industries for the development of new drugs/formulations by applying more advanced techniques.


Assuntos
Antibacterianos/química , Antibacterianos/metabolismo , Microbiologia do Solo , Streptomyces/química , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Estrutura Molecular , Família Multigênica , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo , Streptomyces/genética , Streptomyces/isolamento & purificação , Streptomyces/metabolismo
14.
Microb Cell Fact ; 18(1): 137, 2019 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-31409353

RESUMO

Actinobacteria are characterized as the most prominent producer of natural products (NPs) with pharmaceutical importance. The production of NPs from these actinobacteria is associated with particular biosynthetic gene clusters (BGCs) in these microorganisms. The majority of these BGCs include polyketide synthase (PKS) or non-ribosomal peptide synthase (NRPS) or a combination of both PKS and NRPS. Macrolides compounds contain a core macro-lactone ring (aglycone) decorated with diverse functional groups in their chemical structures. The aglycon is generated by megaenzyme polyketide synthases (PKSs) from diverse acyl-CoA as precursor substrates. Further, post-PKS enzymes are responsible for allocating the structural diversity and functional characteristics for their biological activities. Macrolides are biologically important for their uses in therapeutics as antibiotics, anti-tumor agents, immunosuppressants, anti-parasites and many more. Thus, precise genetic/metabolic engineering of actinobacteria along with the application of various chemical/biological approaches have made it plausible for production of macrolides in industrial scale or generation of their novel derivatives with more effective biological properties. In this review, we have discussed versatile approaches for generating a wide range of macrolide structures by engineering the PKS and post-PKS cascades at either enzyme or cellular level in actinobacteria species, either the native or heterologous producer strains.


Assuntos
Actinobacteria/enzimologia , Actinobacteria/genética , Macrolídeos/metabolismo , Policetídeos/metabolismo , Produtos Biológicos/metabolismo , Engenharia Genética , Família Multigênica , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo
15.
World J Microbiol Biotechnol ; 35(7): 109, 2019 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-31280382

RESUMO

Echinocandin B (ECB) is an important lipohexapeptide used for chemical manufacture of the antifungal agent anidulafungin. Sterigmatocystin (ST) is a polyketide mycotoxin produced by certain species of Aspergillus such as Aspergillus delacroxii SIPIW15, which could produce both ECB and ST. However, the presence of the potent carcinogen ST will greatly affect the quality and safety of ECB production. Therefore, it is essential to eliminate the ST biosynthesis and increase ECB titers in Asp. delacroxii SIPIW15. In this study, the polyketide synthase gene (stcA) required for biosynthesis of ST and its flanking region in Asp. delacroxii SIPIW15 were cloned, sequenced and analyzed firstly. Based on Agrobacterium-mediated transformation, the ΔstcA mutant AMT-1 was obtained and its yield of ECB was increased by 40% without ST detected at the same time as compared to the original strain. The results of the fed-batch experiments showed that the ECB yield of the ΔstcA strain AMT-1 was increased to 2163 ± 31 mg/l and no ST was detected in the 50 l bioreactor. This work suggested that the ΔstcA strain AMT-1 has the potential for application in ECB production improvement, and more importantly, to eliminate ST-related environmental pollution in ECB fermentation industry.


Assuntos
Aspergillus/genética , Aspergillus/metabolismo , Equinocandinas/biossíntese , Equinocandinas/genética , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Genes Fúngicos/genética , Policetídeo Sintases/genética , Esterigmatocistina/biossíntese , Agrobacterium/genética , Anidulafungina , Antifúngicos , Sequência de Bases , Técnicas de Cultura Celular por Lotes , Reatores Biológicos , DNA Fúngico/isolamento & purificação , Fermentação , Engenharia Metabólica , Redes e Vias Metabólicas/genética , Metabolismo Secundário/genética , Transformação Genética
16.
Nat Chem Biol ; 15(8): 795-802, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31308531

RESUMO

Glycosylation is a common modification reaction in natural product biosynthesis and has been known to be a post-assembly line tailoring process in glycosylated polyketide biosynthesis. Here, we show that in pactamycin biosynthesis, glycosylation can take place on an acyl carrier protein (ACP)-bound polyketide intermediate. Using in vivo gene inactivation, chemical complementation and in vitro pathway reconstitution, we demonstrate that the 3-aminoacetophenone moiety of pactamycin is derived from 3-aminobenzoic acid by a set of discrete polyketide synthase proteins via a 3-(3-aminophenyl)3-oxopropionyl-ACP intermediate. This ACP-bound intermediate is then glycosylated by an N-glycosyltransferase, PtmJ, providing a sugar precursor for the formation of the aminocyclopentitol core structure of pactamycin. This is the first example of glycosylation of a small molecule while tethered to a carrier protein. Additionally, we demonstrate that PtmO is a hydrolase that is responsible for the release of the ACP-bound product to a free ß-ketoacid that subsequently undergoes decarboxylation.


Assuntos
Proteínas de Transporte/metabolismo , Pactamicina/biossíntese , Streptomyces/metabolismo , Proteínas de Bactérias , Proteínas de Transporte/química , Clonagem Molecular , Regulação Bacteriana da Expressão Gênica , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo , Policetídeos/química , Ligação Proteica
17.
Nat Chem Biol ; 15(8): 813-821, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31308532

RESUMO

Bacterial trans-acyltransferase polyketide synthases (trans-AT PKSs) are among the most complex known enzymes from secondary metabolism and are responsible for the biosynthesis of highly diverse bioactive polyketides. However, most of these metabolites remain uncharacterized, since trans-AT PKSs frequently occur in poorly studied microbes and feature a remarkable array of non-canonical biosynthetic components with poorly understood functions. As a consequence, genome-guided natural product identification has been challenging. To enable de novo structural predictions for trans-AT PKS-derived polyketides, we developed the trans-AT PKS polyketide predictor (TransATor). TransATor is a versatile bio- and chemoinformatics web application that suggests informative chemical structures for even highly aberrant trans-AT PKS biosynthetic gene clusters, thus permitting hypothesis-based, targeted biotechnological discovery and biosynthetic studies. We demonstrate the applicative scope in several examples, including the characterization of new variants of bioactive natural products as well as structurally new polyketides from unusual bacterial sources.


Assuntos
Bactérias/enzimologia , Policetídeo Sintases/metabolismo , Policetídeos/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Produtos Biológicos , Modelos Químicos , Filogenia , Policetídeo Sintases/genética , Policetídeos/química , Poríferos/microbiologia , Domínios Proteicos , Especificidade por Substrato
19.
J Agric Food Chem ; 67(31): 8581-8589, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31321975

RESUMO

Intermediates in aromatic amino acid biosynthesis can serve as substrates for the synthesis of bioactive compounds. In this study we used two intermediates in the shikimate pathway of Escherichia coli, chorismate and anthranilate, to synthesize three bioactive compounds: 4-hydroxycoumarin (4-HC), 2,4-dihydroxyquinoline (DHQ), and 4-hydroxy-1-methyl-2(1H)-quinolone (NMQ). We introduced genes for the synthesis of salicylic acid from chorismate to supply the substrate for 4-HC and the gene encoding N-methyltransferase for the synthesis of N-methylanthranilate from anthranilate. Polyketide synthases and coenzyme (Co)A ligases were tested to determine the optimal combination of genes for the synthesis of each compound. We also tested several constructs and identified the best one for increasing levels of endogenous substrates for chorismate, anthranilate, and malonyl-CoA. With the use of these strategies, 255.4 mg/L 4-HC, 753.7 mg/L DHQ, and 17.5 mg/L NMQ were synthesized. This work provides a basis for the synthesis of diverse coumarin and quinoline derivatives with potential medical applications.


Assuntos
4-Hidroxicumarinas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Engenharia Metabólica , Policetídeo Sintases/genética , Quinolinas/metabolismo , 4-Hidroxicumarinas/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ácido Corísmico/metabolismo , Coenzima A Ligases/genética , Coenzima A Ligases/metabolismo , Photorhabdus/enzimologia , Photorhabdus/genética , Policetídeo Sintases/metabolismo , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/genética , Quinolinas/química , ortoaminobenzoatos/metabolismo
20.
PLoS Biol ; 17(7): e3000347, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31318855

RESUMO

Polyketides are a class of specialised metabolites synthesised by both eukaryotes and prokaryotes. These chemically and structurally diverse molecules are heavily used in the clinic and include frontline antimicrobial and anticancer drugs such as erythromycin and doxorubicin. To replenish the clinicians' diminishing arsenal of bioactive molecules, a promising strategy aims at transferring polyketide biosynthetic pathways from their native producers into the biotechnologically desirable host Escherichia coli. This approach has been successful for type I modular polyketide synthases (PKSs); however, despite more than 3 decades of research, the large and important group of type II PKSs has until now been elusive in E. coli. Here, we report on a versatile polyketide biosynthesis pipeline, based on identification of E. coli-compatible type II PKSs. We successfully express 5 ketosynthase (KS) and chain length factor (CLF) pairs-e.g., from Photorhabdus luminescens TT01, Streptomyces resistomycificus, Streptoccocus sp. GMD2S, Pseudoalteromonas luteoviolacea, and Ktedonobacter racemifer-as soluble heterodimeric recombinant proteins in E. coli for the first time. We define the anthraquinone minimal PKS components and utilise this biosynthetic system to synthesise anthraquinones, dianthrones, and benzoisochromanequinones (BIQs). Furthermore, we demonstrate the tolerance and promiscuity of the anthraquinone heterologous biosynthetic pathway in E. coli to act as genetically applicable plug-and-play scaffold, showing it to function successfully when combined with enzymes from phylogenetically distant species, endophytic fungi and plants, which resulted in 2 new-to-nature compounds, neomedicamycin and neochaetomycin. This work enables plug-and-play combinatorial biosynthesis of aromatic polyketides using bacterial type II PKSs in E. coli, providing full access to its many advantages in terms of easy and fast genetic manipulation, accessibility for high-throughput robotics, and convenient biotechnological scale-up. Using the synthetic and systems biology toolbox, this plug-and-play biosynthetic platform can serve as an engine for the production of new and diversified bioactive polyketides in an automated, rapid, and versatile fashion.


Assuntos
Antraquinonas/metabolismo , Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , Hidrocarbonetos Policíclicos Aromáticos/metabolismo , Policetídeo Sintases/metabolismo , Policetídeos/metabolismo , Proteínas Recombinantes/metabolismo , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/classificação , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/genética , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/metabolismo , Antraquinonas/química , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Vias Biossintéticas , Escherichia coli/genética , Modelos Químicos , Estrutura Molecular , Filogenia , Hidrocarbonetos Policíclicos Aromáticos/química , Policetídeo Sintases/química , Policetídeo Sintases/genética , Policetídeos/química , Proteínas Recombinantes/química
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