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1.
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
2.
J Microbiol Biotechnol ; 29(7): 1144-1154, 2019 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-31288301

RESUMO

There have been several studies regarding lichen-associated bacteria obtained from diverse environments. Our screening process identified 49 bacterial species in two lichens from the Himalayas: 17 species of Actinobacteria, 19 species of Firmicutes, and 13 species of Proteobacteria. We discovered five types of strong antimicrobial agent-producing bacteria. Although some strains exhibited weak antimicrobial activity, NP088, NP131, NP132, NP134, and NP160 exhibited strong antimicrobial activity against all multidrug-resistant strains. Polyketide synthase (PKS) fingerprinting revealed results for 69 of 148 strains; these had similar genes, such as fatty acid-related PKS, adenylation domain genes, PfaA, and PksD. Although the association between antimicrobial activity and the PKS fingerprinting results is poorly resolved, NP160 had six types of PKS fingerprinting genes, as well as strong antimicrobial activity. Therefore, we sequenced the draft genome of strain NP160, and predicted its secondary metabolism using antiSMASH version 4.2. NP160 had 46 clusters and was predicted to produce similar secondary metabolites with similarities of 5-100%. Although NP160 had 100% similarity with the alkylresorcinol biosynthetic gene cluster, our results showed low similarity with existing members of this biosynthetic gene cluster, and most have not yet been revealed. In conclusion, we expect that lichen-associated bacteria from the Himalayas can produce new secondary metabolites, and we found several secondary metabolite-related biosynthetic gene clusters to support this hypothesis.


Assuntos
Anti-Infecciosos/metabolismo , Genoma Bacteriano/genética , Líquens/microbiologia , Streptomyces/genética , Streptomyces/metabolismo , Anti-Infecciosos/farmacologia , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Sequência de Bases , Impressões Digitais de DNA , DNA Bacteriano/genética , Testes de Sensibilidade Microbiana , Família Multigênica , Filogenia , Policetídeo Sintases/genética , RNA Ribossômico 16S/genética , Metabolismo Secundário/genética , Análise de Sequência de DNA
3.
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
4.
Proc Natl Acad Sci U S A ; 116(12): 5499-5504, 2019 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-30842286

RESUMO

Fungi have the potential to produce a large repertoire of bioactive molecules, many of which can affect the growth and development of plants. Genomic survey of sequenced biofertilizer fungi showed many secondary metabolite gene clusters are anchored by iterative polyketide synthases (IPKSs), which are multidomain enzymes noted for generating diverse small molecules. Focusing on the biofertilizer Trichoderma harzianum t-22, we identified and characterized a cryptic IPKS-containing cluster that synthesizes tricholignan A, a redox-active ortho-hydroquinone. Tricholignan A is shown to reduce Fe(III) and may play a role in promoting plant growth under iron-deficient conditions. The construction of tricholignan by a pair of collaborating IPKSs was investigated using heterologous reconstitution and biochemical studies. A regioselective methylation step is shown to be a key step in formation of the ortho-hydroquinone. The responsible methyltransferase (MT) is fused with an N-terminal pseudo-acyl carrier protein (ψACP), in which the apo state of the ACP is essential for methylation of the growing polyketide chain. The ψACP is proposed to bind to the IPKS and enable the trans MT to access the growing polyketide. Our studies show that a genome-driven approach to discovering bioactive natural products from biofertilizer fungi can lead to unique compounds and biosynthetic knowledge.


Assuntos
Arabidopsis/metabolismo , Ferro/metabolismo , Policetídeos/metabolismo , Trichoderma/genética , Arabidopsis/enzimologia , Redes e Vias Metabólicas/genética , Metilação , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Família Multigênica/genética , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo , Trichoderma/enzimologia , Trichoderma/metabolismo
5.
Curr Microbiol ; 76(3): 376-381, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30710153

RESUMO

4-Methyl-5-pentylbenzene-1,3-diol (MPBD), a product of the polyketide synthase SteelyA, is a signaling molecule that regulates Dictyostelium discoideum development. During early development, MPBD controls chemotactic cell aggregation by regulating the expression of genes in the cAMP signaling pathway; however, during culmination at late development, it induces spore maturation. In the present study, we analyzed the effects of MPBD, its derivatives, and a putative MPBD-derived metabolite on developmental defects in the MPBD-less stlA null mutant. Using structure-activity relationship studies, it was observed that in MPBD, the functional groups that were essential for induction of spore maturation were different from those essential for induction of cell aggregation. Dictyoquinone, a putative MPBD metabolite rescued the aggregation defect in stlA null mutant in early development, but not the spore maturation defect at the later stage. Our data suggest that MPBD regulates chemotactic cell aggregation and spore maturation via different mechanisms.


Assuntos
Quimiotaxia/fisiologia , Dictyostelium/fisiologia , Resorcinóis/metabolismo , Esporos de Protozoários/crescimento & desenvolvimento , Benzoquinonas/farmacologia , Quimiotaxia/efeitos dos fármacos , Dictyostelium/genética , Dictyostelium/crescimento & desenvolvimento , Dictyostelium/metabolismo , Expressão Gênica/efeitos dos fármacos , Mutação , Policetídeo Sintases/genética , Proteínas de Protozoários/genética , Resorcinóis/química , Resorcinóis/farmacologia , Esporos de Protozoários/genética , Esporos de Protozoários/metabolismo , Esporos de Protozoários/fisiologia , Relação Estrutura-Atividade
6.
BMC Genomics ; 20(1): 129, 2019 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-30755157

RESUMO

BACKGROUND: A previous laboratory study involving wild type, mutant and devR/dosR complemented strains of Mycobacterium tuberculosis reported the attenuation phenotype of complemented strain, Comp1. This phenotype was intriguing since the parental strain H37Rv, devR mutant (Mut1) and additional complemented strains, Comp9 and Comp11, were virulent in the guinea pig model. RESULTS: Towards deciphering the mechanism underlying the attenuation of Comp1, a whole genome sequencing approach was undertaken. Eight Single Nucleotide Polymorphisms (SNPs) unique to the Comp1 strain were identified. Of these, 5 SNPs were non-synonymous and included a G➞A mutation resulting in a W1591Stop mutation in ppsD gene of the phthiocerol dimycocerosate (PDIM) biosynthetic cluster. Targeted sequence analysis confirmed this mutation in only Comp1 strain and not in wild type (H37Rv), devR knockout (Mut1) or other complemented (Comp9 and Comp11) bacteria. Differential expression of the PDIM locus in Comp1 bacteria was observed which was associated with a partial deficiency of PDIM, an increased sensitivity to detergent and a compromised ability to infect human THP-1 cells. CONCLUSIONS: It is proposed that a spontaneous mutation in the ppsD gene of Comp1 underlies down-modulation of the PDIM locus which is associated with defects in permeability and infectivity as well as virulence attenuation in guinea pigs. Our study demonstrates the value of whole genome sequencing for resolving unexplainable bacterial phenotypes and recommends the assessment of PDIM status while assessing virulence properties of laboratory-manipulated strains of M. tuberculosis.


Assuntos
Códon sem Sentido , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidade , Policetídeo Sintases/genética , Tuberculose/microbiologia , Animais , Proteínas de Bactérias/genética , Parede Celular/química , Modelos Animais de Doenças , Regulação Bacteriana da Expressão Gênica , Cobaias , Humanos , Lipídeos/biossíntese , Lipídeos/genética , Mycobacterium tuberculosis/classificação , Polimorfismo de Nucleotídeo Único , Células THP-1 , Virulência/genética , Sequenciamento Completo do Genoma
7.
Genome Biol Evol ; 11(3): 890-905, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30793159

RESUMO

White grain disorder is a recently emerged wheat disease in Australia, caused by Eutiarosporella darliae, E. pseudodarliae, and E. tritici-australis. The disease cycle of these pathogens and the molecular basis of their interaction with wheat are poorly understood. To address this knowledge gap, we undertook a comparative genomics analysis focused on the secondary metabolite gene repertoire among these three species. This analysis revealed a diverse array of secondary metabolite gene clusters in these pathogens, including modular polyketide synthase genes. These genes have only been previously associated with bacteria and this is the first report of such genes in fungi. Subsequent phylogenetic analyses provided strong evidence that the modular PKS genes were horizontally acquired from a bacterial or a protist species. We also uncovered a secondary metabolite gene cluster with three polyketide/nonribosomal peptide synthase genes (Hybrid-1, -2, and -3) in E. darliae and E. pseudodarliae. In contrast, only remnant and partial genes homologous to this cluster were identified in E. tritici-australis, suggesting loss of this cluster. Homologues of Hybrid-2 in other fungi have been proposed to facilitate disease in woody plants, suggesting a possible alternative host range for E. darliae and E. pseudodarliae. Subsequent assays confirmed that E. darliae and E. pseudodarliae were both pathogenic on woody plants, but E. tritici-australis was not, implicating woody plants as potential host reservoirs for the fungi. Combined, these data have advanced our understanding of the lifestyle and potential host-range of these recently emerged wheat pathogens and shed new light on fungal secondary metabolism.


Assuntos
Ascomicetos/genética , Evolução Biológica , Policetídeo Sintases/genética , Triticum/microbiologia , Ascomicetos/metabolismo , Ascomicetos/patogenicidade , Duplicação Gênica , Transferência Genética Horizontal , Genoma Fúngico , Doenças das Plantas , Metabolismo Secundário
8.
J Ind Microbiol Biotechnol ; 46(3-4): 469-475, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30610412

RESUMO

The software antiSMASH examines microbial genome data to identify and analyze biosynthetic gene clusters for a wide range of natural products. So far, type II polyketide synthase (PKS) gene clusters could only be identified, but no detailed predictions for type II PKS gene clusters could be provided. In this study, an antiSMASH module for analyzing type II PKS gene clusters has been developed. The module detects genes/proteins in the type II PKS gene cluster involved with polyketide biosynthesis and is able to make predictions about the aromatic polyketide product. Predictions include the putative starter unit, the number of malonyl elongations during polyketide biosynthesis, the putative class and the molecular weight of the product. Furthermore, putative cyclization patterns are predicted. The accuracy of the predictions generated with the new PKSII antiSMASH module was evaluated using a leave-one-out cross validation. The prediction module is available in antiSMASH version 5 at https://antismash.secondarymetabolites.org .


Assuntos
Família Multigênica , Policetídeo Sintases/genética , Software , Bactérias/genética , Bactérias/metabolismo , Vias Biossintéticas/genética , Fungos/genética , Fungos/metabolismo , Genoma Bacteriano , Genoma Fúngico , Policetídeo Sintases/metabolismo , Policetídeos/química
9.
Nat Plants ; 5(2): 225-237, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30692678

RESUMO

Lignin is the main cause of lignocellulosic biomass recalcitrance to industrial enzymatic hydrolysis. By partially replacing the traditional lignin monomers by alternative ones, lignin extractability can be enhanced. To design a lignin that is easier to degrade under alkaline conditions, curcumin (diferuloylmethane) was produced in the model plant Arabidopsis thaliana via simultaneous expression of the turmeric (Curcuma longa) genes DIKETIDE-CoA SYNTHASE (DCS) and CURCUMIN SYNTHASE 2 (CURS2). The transgenic plants produced a plethora of curcumin- and phenylpentanoid-derived compounds with no negative impact on growth. Catalytic hydrogenolysis gave evidence that both curcumin and phenylpentanoids were incorporated into the lignifying cell wall, thereby significantly increasing saccharification efficiency after alkaline pretreatment of the transgenic lines by 14-24% as compared with the wild type. These results demonstrate that non-native monomers can be synthesized and incorporated into the lignin polymer in plants to enhance their biomass processing efficiency.


Assuntos
Arabidopsis/metabolismo , Curcumina/metabolismo , Lignina/metabolismo , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/metabolismo , Arabidopsis/genética , Biomassa , Parede Celular/genética , Parede Celular/metabolismo , Celulose/metabolismo , Curcuma/genética , Glucose/metabolismo , Ligases/genética , Ligases/metabolismo , Lignina/genética , Proteínas de Plantas/metabolismo , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo , Temperatura Ambiente
10.
Nat Chem Biol ; 15(2): 111-114, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30598544

RESUMO

Here we report a transcription factor decoy strategy for targeted activation of eight large silent polyketide synthase and non-ribosomal peptide synthetase gene clusters, ranging from 50 to 134 kilobases (kb) in multiple streptomycetes, and characterization of a novel oxazole family compound produced by a 98-kb biosynthetic gene cluster. Owing to its simplicity and ease of use, this strategy can be scaled up readily for discovery of natural products in streptomycetes.


Assuntos
Peptídeo Sintases/genética , Policetídeo Sintases/genética , Fatores de Transcrição/biossíntese , Regulação da Expressão Gênica/genética , Família Multigênica/fisiologia , Peptídeo Sintases/fisiologia , Policetídeo Sintases/fisiologia , Streptomycetaceae/metabolismo
11.
Org Biomol Chem ; 17(2): 374-379, 2019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-30556556

RESUMO

Menisporopsin A is a bioactive macrocyclic polylactone produced by the fungus Menisporopsis theobromae BCC 4162. A scheme for the biosynthesis of this compound has been proposed, in which reducing (R) and non-reducing (NR) polyketide synthases (PKSs) would catalyze the formation of each menisporopsin A subunit, while an additional non-ribosomal peptide synthetase (NRPS)-like enzyme would be required to perform multiple esterification and cyclolactonization reactions. Transcriptome analysis of M. theobromae identified an R-PKS gene, men1, and an NR-PKS gene, men2, which both exhibited highest expression levels during the menisporopsin A production phase. These were cloned into separate vectors for heterologous expression in Aspergillus oryzae NSAR1. Unexpectedly, coexpression of the two PKSs alone was sufficient to catalyze the formation of the macrocyclic polylactone, ascotrichalactone A, a structural derivative of menisporopsin A. The unanticipated esterification and cyclolactonization activities could reside in the unusual thioesterase domain of the NR-PKS, which is similar to that of the NRPS catalyzing elongation and cyclization of trilactone in enterobactin biosynthesis and that of modular PKSs catalyzing macrodiolide formation in elaiophylin and conglobatin biosyntheses.


Assuntos
Ascomicetos/metabolismo , Aspergillus oryzae/metabolismo , Vias Biossintéticas , Proteínas Fúngicas/metabolismo , Macrolídeos/metabolismo , Policetídeo Sintases/metabolismo , Ascomicetos/genética , Aspergillus oryzae/genética , Clonagem Molecular , Proteínas Fúngicas/genética , Expressão Gênica , Genes Fúngicos , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismo , Policetídeo Sintases/genética , Transcriptoma
12.
Pol J Microbiol ; 67(4): 441-454, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30550230

RESUMO

Paeonia ostii is known for its excellent medicinal values as Chinese traditional plant. To date, the diversity of culturable endophytes associated with P. ostii is in its initial phase of exploration. In this study, 56 endophytic bacteria and 51 endophytic fungi were isolated from P. ostii roots in China. Subsequent characterization of 56 bacterial strains by 16S rDNA gene sequence analysis revealed that nine families and 13 different genera were represented. All the fungal strains were classed into six families and 12 genera based on ITS gene sequence. The biosynthetic potential of all the endophytes was further investigated by the detection of putative polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) genes. The PCR screens were successful in targeting thirteen bacterial PKS, five bacterial NRPS, ten fungal PKS and nine fungal NRPS gene fragments. Bioinformatic analysis of these detected endophyte gene fragments facilitated inference of the potential bioactivity of endophyte bioactive products, suggesting that the isolated endophytes are capable of producing a plethora of secondary metabolites. These results suggest that endophytes isolated from P. ostii had abundant population diversity and biosynthetic potential, which further proved that endophytes are valuable reservoirs of novel bioactive compounds.Paeonia ostii is known for its excellent medicinal values as Chinese traditional plant. To date, the diversity of culturable endophytes associated with P. ostii is in its initial phase of exploration. In this study, 56 endophytic bacteria and 51 endophytic fungi were isolated from P. ostii roots in China. Subsequent characterization of 56 bacterial strains by 16S rDNA gene sequence analysis revealed that nine families and 13 different genera were represented. All the fungal strains were classed into six families and 12 genera based on ITS gene sequence. The biosynthetic potential of all the endophytes was further investigated by the detection of putative polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) genes. The PCR screens were successful in targeting thirteen bacterial PKS, five bacterial NRPS, ten fungal PKS and nine fungal NRPS gene fragments. Bioinformatic analysis of these detected endophyte gene fragments facilitated inference of the potential bioactivity of endophyte bioactive products, suggesting that the isolated endophytes are capable of producing a plethora of secondary metabolites. These results suggest that endophytes isolated from P. ostii had abundant population diversity and biosynthetic potential, which further proved that endophytes are valuable reservoirs of novel bioactive compounds.


Assuntos
Bactérias/classificação , Endófitos/classificação , Fungos/classificação , Variação Genética , Paeonia/microbiologia , Bactérias/isolamento & purificação , China , DNA Bacteriano/genética , DNA Fúngico/genética , DNA Espaçador Ribossômico/genética , Endófitos/metabolismo , Fungos/isolamento & purificação , Peptídeo Sintases/genética , Filogenia , Raízes de Plantas/microbiologia , Plantas Medicinais/microbiologia , Policetídeo Sintases/genética , Reação em Cadeia da Polimerase , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
13.
Pol J Microbiol ; 67(4): 501-516, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30550237

RESUMO

A collection of heterotrophic bacteria consisting of 167 strains was obtained from microbial communities of biofilms formed on solid substrates in the littoral zone of Lake Baikal. Based on the analysis of 16S rRNA gene fragments, the isolates were classified to four phyla: Proteobacteria , Firmicutes , Actinobacteria , and Bacteroidetes . To assess their biotechnological potential, bacteria were screened for the presence of PKS (polyketide synthase) and NRPS (non-ribosomal peptide synthetases) genes. PKS genes were detected in 41 strains (25%) and NRPS genes in 73 (43%) strains by PCR analysis. The occurrence of PKS genes in members of the phylum Firmicutes (the genera Bacillus and Paenibacillus ) was 34% and NRPS genes were found in 78%. In Proteobacteria , PKS and NRPS genes were found in 20% and 32%, and in 22% and 22% of Actinobacteria , respectively. For further analysis of PKS and NRPS genes, six Bacillus and Paenibacillus strains with antagonistic activity were selected and underwent phylogenetic analysis of 16S rRNA genes. The identification of PKS and NRPS genes in the strains investigated was demonstrated among the homologues the genes involved in the biosynthesis of antibiotics (bacillaene, difficidine, erythromycin, bacitracin, tridecaptin, and fusaricidin), biosurfactants (iturin, bacillomycin, plipastatin, fengycin, and surfactin) and antitumor agents (epothilone, calyculin, and briostatin). Bacillus spp. 9A and 2A strains showed the highest diversity of PKS and NRPS genes. Bacillus and Paenibacillus strains isolated from epilithic biofilms in Lake Baikal are potential producers of antimicrobial compounds and may be of practical interest for biotechnological purposes.A collection of heterotrophic bacteria consisting of 167 strains was obtained from microbial communities of biofilms formed on solid substrates in the littoral zone of Lake Baikal. Based on the analysis of 16S rRNA gene fragments, the isolates were classified to four phyla: Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes. To assess their biotechnological potential, bacteria were screened for the presence of PKS (polyketide synthase) and NRPS (non-ribosomal peptide synthetases) genes. PKS genes were detected in 41 strains (25%) and NRPS genes in 73 (43%) strains by PCR analysis. The occurrence of PKS genes in members of the phylum Firmicutes (the genera Bacillus and Paenibacillus) was 34% and NRPS genes were found in 78%. In Proteobacteria, PKS and NRPS genes were found in 20% and 32%, and in 22% and 22% of Actinobacteria, respectively. For further analysis of PKS and NRPS genes, six Bacillus and Paenibacillus strains with antagonistic activity were selected and underwent phylogenetic analysis of 16S rRNA genes. The identification of PKS and NRPS genes in the strains investigated was demonstrated among the homologues the genes involved in the biosynthesis of antibiotics (bacillaene, difficidine, erythromycin, bacitracin, tridecaptin, and fusaricidin), biosurfactants (iturin, bacillomycin, plipastatin, fengycin, and surfactin) and antitumor agents (epothilone, calyculin, and briostatin). Bacillus spp. 9A and 2A strains showed the highest diversity of PKS and NRPS genes. Bacillus and Paenibacillus strains isolated from epilithic biofilms in Lake Baikal are potential producers of antimicrobial compounds and may be of practical interest for biotechnological purposes.


Assuntos
Biofilmes , Variação Genética , Genoma Bacteriano , Lagos/microbiologia , Microbiota/genética , Peptídeo Sintases/genética , Policetídeo Sintases/genética , Actinobacteria/enzimologia , Actinobacteria/genética , Bacteroidetes/enzimologia , Bacteroidetes/genética , Firmicutes/enzimologia , Firmicutes/genética , Processos Heterotróficos , Proteobactérias/enzimologia , Proteobactérias/genética , RNA Ribossômico 16S/genética , Metabolismo Secundário
14.
Nat Commun ; 9(1): 5281, 2018 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-30538251

RESUMO

Tropinone is the first intermediate in the biosynthesis of the pharmacologically important tropane alkaloids that possesses the 8-azabicyclo[3.2.1]octane core bicyclic structure that defines this alkaloid class. Chemical synthesis of tropinone was achieved in 1901 but the mechanism of tropinone biosynthesis has remained elusive. In this study, we identify a root-expressed type III polyketide synthase from Atropa belladonna (AbPYKS) that catalyzes the formation of 4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoic acid. This catalysis proceeds through a non-canonical mechanism that directly utilizes an unconjugated N-methyl-Δ1-pyrrolinium cation as the starter substrate for two rounds of malonyl-Coenzyme A mediated decarboxylative condensation. Subsequent formation of tropinone from 4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoic acid is achieved through cytochrome P450-mediated catalysis by AbCYP82M3. Silencing of AbPYKS and AbCYP82M3 reduces tropane levels in A. belladonna. This study reveals the mechanism of tropinone biosynthesis, explains the in planta co-occurrence of pyrrolidines and tropanes, and demonstrates the feasibility of tropane engineering in a non-tropane producing plant.


Assuntos
Atropa belladonna/enzimologia , Atropa belladonna/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Proteínas de Plantas/metabolismo , Policetídeo Sintases/metabolismo , Tropanos/química , Tropanos/metabolismo , Atropa belladonna/genética , Ciclização , Sistema Enzimático do Citocromo P-450/genética , Estrutura Molecular , Proteínas de Plantas/genética , Raízes de Plantas/química , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Policetídeo Sintases/genética
15.
Phytochemistry ; 156: 142-150, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30296707

RESUMO

The biosynthesis of two polyketides, atranorin and fumarprotocetraric acid, produced from a lichen-forming fungus, Cladonia rangiferina (L.) F. H. Wigg. was correlated with the expression of eight fungal genes (CrPKS1, CrPKS3, CrPKS16, Catalase (CAT), Sugar Transporter (MFsug), Dioxygenase (YQE1), C2H2 Transcription factor (C2H2), Transcription Factor PacC (PacC), which are thought to be involved in polyketide biosynthesis, and one algal gene, NAD-dependent deacetylase sirtuin 2 (AsNAD)), using laser microdissection (LMD). The differential gene expression levels within the thallus tissue layers demonstrate that the most active region for potential polyketide biosynthesis within the lichen is the outer apical region proximal to the photobiont but some expression also occurs in reproductive tissue. This is the first study using laser microdissection to explore gene expression of these nine genes and their location of expression; it provides a proof-of-concept for future experiments exploring tissue-specific gene expression within lichens; and it highlights the utility of LMD for use in lichen systems.


Assuntos
Ascomicetos/enzimologia , Lasers , Líquens/microbiologia , Microdissecção , Policetídeo Sintases/química , Ascomicetos/metabolismo , Líquens/genética , Líquens/metabolismo , Estrutura Molecular , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo
16.
Proc Natl Acad Sci U S A ; 115(40): 9835-9844, 2018 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-30232266

RESUMO

Malonyl-CoA is an important central metabolite for the production of diverse valuable chemicals including natural products, but its intracellular availability is often limited due to the competition with essential cellular metabolism. Several malonyl-CoA biosensors have been developed for high-throughput screening of targets increasing the malonyl-CoA pool. However, they are limited for use only in Escherichia coli and Saccharomyces cerevisiae and require multiple signal transduction steps. Here we report development of a colorimetric malonyl-CoA biosensor applicable in three industrially important bacteria: E. coli, Pseudomonas putida, and Corynebacterium glutamicum RppA, a type III polyketide synthase producing red-colored flaviolin, was repurposed as a malonyl-CoA biosensor in E. coli Strains with enhanced malonyl-CoA accumulation were identifiable by the colorimetric screening of cells showing increased red color. Other type III polyketide synthases could also be repurposed as malonyl-CoA biosensors. For target screening, a 1,858 synthetic small regulatory RNA library was constructed and applied to find 14 knockdown gene targets that generally enhanced malonyl-CoA level in E. coli These knockdown targets were applied to produce two polyketide (6-methylsalicylic acid and aloesone) and two phenylpropanoid (resveratrol and naringenin) compounds. Knocking down these genes alone or in combination, and also in multiple different E. coli strains for two polyketide cases, allowed rapid development of engineered strains capable of enhanced production of 6-methylsalicylic acid, aloesone, resveratrol, and naringenin to 440.3, 30.9, 51.8, and 103.8 mg/L, respectively. The malonyl-CoA biosensor developed here is a simple tool generally applicable to metabolic engineering of microorganisms to achieve enhanced production of malonyl-CoA-derived chemicals.


Assuntos
Proteínas de Bactérias , Técnicas Biossensoriais/métodos , Corynebacterium glutamicum , Escherichia coli , Malonil Coenzima A/análise , Engenharia Metabólica , Policetídeo Sintases , Pseudomonas putida , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Corynebacterium glutamicum/enzimologia , Corynebacterium glutamicum/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo , Pseudomonas putida/enzimologia , Pseudomonas putida/genética
17.
J Microbiol Biotechnol ; 28(7): 1068-1077, 2018 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-29975996

RESUMO

DKxanthenes are a class of yellow secondary metabolites produced by myxobacterial genera Myxococcus and Stigmatella. We identified a putative 49.5 kb DKxanthene biosynthetic gene cluster from Myxococcus stipitatus DSM 14675 by genomic sequence and mutational analysis. The cluster was comprisedof 15 genes (MYSTI_06004-MYSTI_06018) encoding polyketide synthases, non-ribosomal peptide synthases, and proteins with unknown functions. Disruption of the genes by plasmid insertion resulted in defects in the production of yellow pigments. High-performance liquid chromatography and liquid chromatography-tandem mass spectrometry analysis indicated that the yellow pigments produced by M. stipitatus DSM 14675 might be noble DKxanthene derivatives. M. stipitatus did not require DKxanthenes for the formation of heat-resistant viable spores, unlike Myxococcus xanthus. Furthermore, DKxanthenes showed growth inhibitory activity against the fungi Aspergillus niger, Candida albicans, and Rhizopus stolonifer.


Assuntos
Vias Biossintéticas/genética , Família Multigênica/genética , Myxococcus/enzimologia , Myxococcus/genética , Myxococcus/metabolismo , Xantenos/metabolismo , Xantenos/farmacologia , Anti-Infecciosos/química , Anti-Infecciosos/metabolismo , Anti-Infecciosos/farmacologia , Aspergillus niger/efeitos dos fármacos , Aspergillus niger/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Candida albicans/efeitos dos fármacos , Candida albicans/crescimento & desenvolvimento , Carpóforos/efeitos dos fármacos , Genes Bacterianos/genética , Mutação , Myxococcus xanthus/metabolismo , Peptídeo Sintases/genética , Pigmentos Biológicos/genética , Pigmentos Biológicos/metabolismo , Plasmídeos/genética , Policetídeo Sintases/genética , Rhizopus/efeitos dos fármacos , Rhizopus/crescimento & desenvolvimento , Metabolismo Secundário/genética , Análise de Sequência , Esporos/efeitos dos fármacos , Xantenos/química
18.
Lett Appl Microbiol ; 67(3): 226-234, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29927502

RESUMO

Streptomyces cinnamonensis A495 is a variant of the monensin producer which instead of the native polyether antibiotic gives rise to antibiotic and anti-tumour shunt-product premonensin. Through the supplementation of the fermentation medium with suitable precursors, premonensin can be derivatized via the incorporation of new-to-nature extender units into the biosynthetic machinery. Polyketide extender units require activation, typically in form of coenzyme A-thioesters. These are membrane impermeable and thus in the past an artificial mimic was employed. Here, we show the use and preliminary characterization of a highly substrate promiscuous new enzyme for the endogenous thioester formation in a Streptomyces strain. These intracellularly activated alternative extender units are significantly better incorporated into premonensin than the synthetically activated counterparts. SIGNIFICANCE AND IMPACT OF THE STUDY: Polyketide natural products are of enormous relevance in medicine. The hit-rate in finding active compounds for the potential treatment of various diseases among this substance family of microbial origin is high. However, most polyketides require derivatization to render them suitable for the application. Of relevance in this field is the incorporation of artificial substances into the biogenesis of polyketides, hampered by both the microbial metabolism and the complexity of the enzymes involved. This manuscript describes the straightforward and selective biosynthetic incorporation of synthetic substances into a reduced polyketide and showcases a promising new enzyme to aid this purpose.


Assuntos
Antibacterianos/biossíntese , Proteínas de Bactérias/metabolismo , Monensin/biossíntese , Policetídeo Sintases/metabolismo , Streptomyces/metabolismo , Proteínas de Bactérias/genética , Vias Biossintéticas , Ativação Enzimática , Fermentação , Policetídeo Sintases/genética , Streptomyces/enzimologia , Streptomyces/genética
19.
Enzyme Microb Technol ; 115: 16-22, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29859598

RESUMO

Two putative type III polyketide synthase genes (PKS) were identified from Sordariomycetes fungi. These two type III PKS genes from Sordaria macrospora (SmPKS) and Chaetomium thermophilum (CtPKS), shared 59.8% sequence identity. Both, full-length and truncated versions of type III PKSs were successfully cloned and overexpressed in a bacterial host, Escherichia Coli BL21 (DE3) using a N-terminus hexa-histidine tag. The full-length and the truncated construct of PKSs showed similar activity profiles, suggesting that additional amino acid residues at the C-terminal of both SmPKS and CtPKS may not be involved in catalytic functions. We demonstrate that these two recombinant polyketide synthases could efficiently synthesize tri- and tetraketide pyrones, resorcinols and resorcylic acids using various acyl-CoAs (C4-C20) as starter units. The truncated S. macrospora polyketide synthases (TrSmPKS) showed a maximum of 7.0 × 104 s-1 M-1 catalytic efficiency towards stearoyl-CoA.Whereas, truncated C. thermophilum polyketide synthases (TrCtPKS) preferred the long-chain acyl-CoA starter arachidoyl-CoA, to produce pentaketide and hexaketide resorcinols with a high catalytic efficiency of 6.2 × 104 s-1 M-1. Homology model and substrate docking analyses suggest a shorter distance between sulfur of catalytic Cys152 and thioester carbonyl group of arachidoyl-CoA as well as stronger imidazolium-thiolate ion pair distance in TrCtPKS between catalytic Cys152-His309 compared to TrSmPKS- arachidoyl CoA complex. Enhanced binding interactions of CtPKS residues forming intermolecular contacts at the active site could be attributed to its high specificity towards arachidoyl-CoA. This study reports the functional characterization of two fungal type III polyketide synthases, SmPKS and CtPKS with high catalytic efficiency from S. macrospora and C. thermophilum respectively. Furthermore, the results suggested that the both SmPKS and CtPKS could be attractive targets for protein engineering to discern the unique substrate specificity and catalytic efficiency.


Assuntos
Acil Coenzima A/metabolismo , Chaetomium/enzimologia , Policetídeo Sintases/metabolismo , Pironas/metabolismo , Sordariales/enzimologia , Catálise , Domínio Catalítico , Chaetomium/genética , Chaetomium/crescimento & desenvolvimento , Clonagem Molecular , Cinética , Modelos Moleculares , Policetídeo Sintases/genética , Sordariales/genética , Sordariales/crescimento & desenvolvimento , Especificidade por Substrato
20.
Proc Natl Acad Sci U S A ; 115(22): E4980-E4989, 2018 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-29760061

RESUMO

Glycosylation is a prominent strategy to optimize the pharmacokinetic and pharmacodynamic properties of drug-like small-molecule scaffolds by modulating their solubility, stability, bioavailability, and bioactivity. Glycosyltransferases applicable for "sugarcoating" various small-molecule acceptors have been isolated and characterized from plants and bacteria, but remained cryptic from filamentous fungi until recently, despite the frequent use of some fungi for whole-cell biocatalytic glycosylations. Here, we use bioinformatic and genomic tools combined with heterologous expression to identify a glycosyltransferase-methyltransferase (GT-MT) gene pair that encodes a methylglucosylation functional module in the ascomycetous fungus Beauveria bassiana The GT is the founding member of a family nonorthologous to characterized fungal enzymes. Using combinatorial biosynthetic and biocatalytic platforms, we reveal that this GT is a promiscuous enzyme that efficiently modifies a broad range of drug-like substrates, including polyketides, anthraquinones, flavonoids, and naphthalenes. It yields both O- and N-glucosides with remarkable regio- and stereospecificity, a spectrum not demonstrated for other characterized fungal enzymes. These glucosides are faithfully processed by the dedicated MT to afford 4-O-methylglucosides. The resulting "unnatural products" show increased solubility, while representative polyketide methylglucosides also display increased stability against glycoside hydrolysis. Upon methylglucosidation, specific polyketides were found to attain cancer cell line-specific antiproliferative or matrix attachment inhibitory activities. These findings will guide genome mining for fungal GTs with novel substrate and product specificities, and empower the efficient combinatorial biosynthesis of a broad range of natural and unnatural glycosides in total biosynthetic or biocatalytic formats.


Assuntos
Antineoplásicos , Descoberta de Drogas , Fungos , Glicosiltransferases , Metiltransferases , Animais , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cercopithecus aethiops , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fungos/enzimologia , Fungos/genética , Fungos/metabolismo , Glicosilação , Glicosiltransferases/química , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Humanos , Metiltransferases/química , Metiltransferases/genética , Metiltransferases/metabolismo , Policetídeo Sintases/química , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo , Policetídeos/metabolismo , Células Vero
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