Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 5.158
Filtrar
1.
Microb Cell Fact ; 18(1): 163, 2019 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-31581944

RESUMO

BACKGROUND: Sustainable production of microbial fatty acids derivatives has the potential to replace petroleum based equivalents in the chemical, cosmetic and pharmaceutical industry. Most fatty acid sources for production oleochemicals are currently plant derived. However, utilization of these crops are associated with land use change and food competition. Microbial oils could be an alternative source of fatty acids, which circumvents the issue with agricultural competition. RESULTS: In this study, we generated a chimeric microbial production system that features aspects of both prokaryotic and eukaryotic fatty acid biosynthetic pathways targeted towards the generation of long chain fatty acids. We redirected the type-II fatty acid biosynthetic pathway of Escherichia coli BL21 (DE3) strain by incorporating two homologues of the beta-ketoacyl-[acyl carrier protein] synthase I and II from the chloroplastic fatty acid biosynthetic pathway of Arabidopsis thaliana. The microbial clones harboring the heterologous pathway yielded 292 mg/g and 220 mg/g DCW for KAS I and KAS II harboring plasmids respectively. Surprisingly, beta-ketoacyl synthases KASI/II isolated from A. thaliana showed compatibility with the FAB pathway in E. coli. CONCLUSION: The efficiency of the heterologous plant enzymes supersedes the overexpression of the native enzyme in the E. coli production system, which leads to cell death in fabF overexpression and fabB deletion mutants. The utilization of our plasmid based system would allow generation of plant like fatty acids in E. coli and their subsequent chemical or enzymatic conversion to high end oleochemical products.


Assuntos
Arabidopsis/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Ácido Graxo Sintases/metabolismo , Ácidos Graxos/biossíntese , Engenharia Metabólica , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/síntese química , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/genética , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/metabolismo , Arabidopsis/enzimologia , Proteínas de Arabidopsis/síntese química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Vias Biossintéticas , Escherichia coli/enzimologia , Proteínas de Escherichia coli/genética , Ácido Graxo Sintases/genética , Ácidos Graxos/química , Isoenzimas/síntese química , Isoenzimas/genética , Isoenzimas/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo
2.
World J Microbiol Biotechnol ; 35(8): 128, 2019 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-31375920

RESUMO

Large patch disease, caused by Rhizoctonia solani AG2-2, is the most devastating disease in Zoysiagrass (Zoysia japonica). Current large patch disease control strategies rely primarily upon the use of chemical pesticides. Streptomyces sp. S8 is known to possess exceptional antagonistic properties that could potentially suppress the large patch pathogen found at turfgrass plantations. This study aims to demonstrate the feasibility of using the strain as a biological control mechanism. Sequencing of the S8 strain genome revealed a valinomycin biosynthesis gene cluster. This cluster is composed of the vlm1 and vlm2 genes, which are known to produce antifungal compounds. In order to verify this finding for the large patch pathogen, a valinomycin biosynthesis knockout mutant was created via the CRISPR/Cas9 system. The mutant lost antifungal activity against the large patch pathogen. Consequently, it is anticipated that eco-friendly microbial preparations derived from the S8 strain can be utilized to biologically control large patch disease.


Assuntos
Antifúngicos/metabolismo , Antifúngicos/farmacologia , Rhizoctonia/efeitos dos fármacos , Streptomyces/metabolismo , Valinomicina/metabolismo , Valinomicina/farmacologia , Vias Biossintéticas/genética , Técnicas de Inativação de Genes , Genoma Bacteriano , Família Multigênica , Controle Biológico de Vetores/métodos , Doenças das Plantas/microbiologia , Doenças das Plantas/prevenção & controle , Poaceae/microbiologia , Rhizoctonia/crescimento & desenvolvimento , Análise de Sequência de DNA , Streptomyces/genética
3.
World J Microbiol Biotechnol ; 35(9): 133, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31432254

RESUMO

There is a significant increase in the discovery of new antimicrobial compounds in recent past to combat drug resistant pathogens. Members of the genus Bacillus and related genera have been screened extensively due to their ability to produce wide range of antimicrobial compounds. In this study, we have isolated and characterized a new antimicrobial peptide from a marine bacterium identified as Virgibacillus species. The low molecular mass and stability of the antimicrobial substance pointed towards the bacteriocinogenic nature of the compound. The RAST analysis of genome sequence showed presence of a putative bacteriocin biosynthetic cluster containing genes necessary for synthesis of a lanthipeptide. Translated amino acid sequence of mature C-terminal propeptide showed identity with salivaricin A (52.2%) and lacticin A (33.3%). Accordingly, the mass (2417 Da) obtained by MALDI analysis was in agreement with posttranslational modifications of the leader peptide to yield three methyl lanthionine rings and a disulfide bond between two free cysteine residues. The lanthipeptide was named as virgicin, which selectively inhibited the growth of Gram-positive bacteria and biofilm formation by Enterococcus faecalis. Inhibition of biofilm formation by E. faecalis was also observed in in vitro model experiments using hydroxyapatite discs. Thus, virgicin appears to be a promising new bacteriocin to control oral biofilm formation by selective pathogens.


Assuntos
Bacteriocinas/isolamento & purificação , Bacteriocinas/farmacologia , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecalis/crescimento & desenvolvimento , Peptídeos/isolamento & purificação , Peptídeos/farmacologia , Virgibacillus/metabolismo , Bacteriocinas/química , Bacteriocinas/genética , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Vias Biossintéticas/genética , Genoma Bacteriano , Peso Molecular , Família Multigênica , Peptídeos/química , Peptídeos/genética , Água do Mar/microbiologia , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Virgibacillus/classificação , Virgibacillus/isolamento & purificação
4.
Microbiol Res ; 226: 34-40, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31284942

RESUMO

Carotenoid composition has been studied in mesophilic, nitrogen-fixing cyanobacterium Anabaena sp. PCC7120 grown photoautotrophically, under diazotrophic conditions at four different temperatures (15 °C, 23 °C, 30 °C and 37 °C). The relative accumulation of chlorophyll, carotenoids and proteins was the highest at temperature of 23 °C. At a suboptimal temperature (15 °C) ß-carotene was the dominant carotenoid compound, whereas the increase in temperature caused ketocarotenoids (echinenone, canthaxanthin, keto-myxoxanthophyll) to accumulate. A significant increase in the accumulation of phytoene synthase (CrtB) transcript was observed at both extreme growth temperatures (15 °C and 37 °C). The relative amount of ß-carotene ketolase (CrtW) transcript directly corresponded to the accumulation of its product (keto-myxoxanthophyll) with a maximum at 30 °C and a profound decrease at 37 °C, whereas the transcription level of ß-carotene ketolase (CrtO) was significantly decreased only at a suboptimal temperature (15 °C). These results show that temperature affects the functioning of the carotenoid biosynthesis pathway in Anabaena cells under photoautotrophic growth. Specifically, the balance between ß-carotene and ketocarotenoids is altered according to temperature conditions. The transcriptional regulation of genes encoding enzymes active both at the early (CrtB) and the final steps (CrtO, CrtW) of the carotenoid biosynthetic pathway may participate in the acclimation mechanism of cyanobacteria to low and high temperatures.


Assuntos
Anabaena/crescimento & desenvolvimento , Anabaena/metabolismo , Carotenoides/biossíntese , Temperatura Ambiente , Anabaena/enzimologia , Anabaena/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Vias Biossintéticas/genética , Vias Biossintéticas/fisiologia , Cantaxantina , Clorofila/metabolismo , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/genética , Geranil-Geranildifosfato Geranil-Geraniltransferase/genética , Geranil-Geranildifosfato Geranil-Geraniltransferase/metabolismo , Oxigenases/genética , Oxigenases/metabolismo , Estresse Fisiológico , beta Caroteno/biossíntese
5.
Sheng Wu Gong Cheng Xue Bao ; 35(7): 1184-1192, 2019 Jul 25.
Artigo em Chinês | MEDLINE | ID: mdl-31328475

RESUMO

Salidroside, as one of the main active ingredients of Rhodiala plant, has the effects of anti-hypoxia, anti-radiation, anti-fatigue, anti-tumor, hypoglycemia and improving immunity. With the increasing demand for salidroside and the decreasing of plant resources, microbial production of salidroside has attracted much attention due to its advantages of short period and easy controlling. At present, microbial production of salidroside is still at the basic research stage. In order to make it easier for researchers to understand the advances of microbial synthesis of salidroside, the biosynthesis pathways, uridine diphosphate glucosyltransferases, wild strain/natural enzymes and engineered strain/recombinant enzymes were reviewed.


Assuntos
Glucosídeos/metabolismo , Fenóis/metabolismo , Vias Biossintéticas
6.
J Agric Food Chem ; 67(29): 8235-8242, 2019 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-31260295

RESUMO

This research aims to analyze the biosynthetic pathway of endogenous semicarbazide (SEM) in shrimps using Litopenaeus vannamei as the model target. To achieve this objective, the content of SEM in L. vannamei throughout the whole growth cycle was monitored under the strict control of external environmental interference. Experimental results showed that SEM was found in the shrimp shell at all stages, with its content decreasing first and then increasing, and no SEM was detected in the shrimp muscle of each growth stage. This indicated that endogenous SEM in L. vannamei was derived from the shrimp shell. At the same time, the content of amino acids in the shrimp shell and the corresponding substances involved in the urea cycle in the entire growth cycle of shrimp were monitored. The correlation analysis between them and the changes in the SEM content in shrimp showed that arginine had the largest correlation coefficient (0.952) with the changes in the SEM content. The main substances of the urea cycle may be related to the production of SEM. In combination with the water environmental test of high ammonia nitrogen, it was presumed that the formation of endogenous SEM was related to the amidine group of arginine and amide structure of citrulline and urea. Arginine, citrulline, and urea in the urea cycle of L. vannamei eventually produced SEM via an oxaziridine intermediate under the action of hydrogen peroxide and ammonia, and a standardized reaction test was conducted to verify the hypothesis and, thus, provided a new idea for future endogenous SEM research.


Assuntos
Penaeidae/crescimento & desenvolvimento , Semicarbazidas/análise , Semicarbazidas/metabolismo , Aminoácidos/análise , Aminoácidos/metabolismo , Amônia/metabolismo , Exoesqueleto/química , Exoesqueleto/metabolismo , Animais , Vias Biossintéticas , Penaeidae/química , Penaeidae/metabolismo
7.
J Agric Food Chem ; 67(29): 8186-8190, 2019 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-31272146

RESUMO

Indole-3-acetic acid (IAA) is considered the most common and important naturally occurring auxin in plants and a major regulator of plant growth and development. In this study, an aldehyde dehydrogenase AldH from Escherichia coli was found to convert indole-3-acetylaldehyde into IAA. Then we established an artificial pathway in engineered E. coli for microbial production of IAA from glucose. The overall pathway includes the upstream pathway from glucose to L-tryptophan and the downstream pathway from L-tryptophan to IAA. To our knowledge, this is the first report on the biosynthesis of IAA directly from a renewable carbon source. The study described here shows the way for the development of a beneficial microbe for biosynthesis of auxin and promoting plant growth in the future.


Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Ácidos Indolacéticos/metabolismo , Vias Biossintéticas , Engenharia Genética , Glucose/metabolismo , Triptofano/metabolismo
8.
BMC Plant Biol ; 19(1): 301, 2019 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-31291885

RESUMO

BACKGROUND: Nothapodytes nimmoniana, a plant of pivotal medicinal significance is a source of potent anticancer monoterpene indole alkaloid (MIA) camptothecin (CPT). This compound owes its potency due to topoisomerase-I inhibitory activity. However, biosynthetic and regulatory aspects of CPT biosynthesis so far remain elusive. Production of CPT is also constrained due to unavailability of suitable in vitro experimental system. Contextually, there are two routes for the biosynthesis of MIAs: the mevalonate (MVA) pathway operating in cytosol and the methylerythritol phosphate (MEP) pathway in the plastids. Determination of relative precursor flux through either of these pathways may provide a new vista for manipulating the enhanced CPT production. RESULTS: In present study, specific enzyme inhibitors of MVA (lovastatin) and MEP pathways (fosmidomycin) were used to perturb the metabolic flux in N. nimmoniana. Interaction of both these pathways was investigated at transcriptional level by using qRT-PCR and at metabolite level by evaluating secologanin, tryptamine and CPT contents. In fosmidomycin treated plants, highly significant reduction was observed in both secologanin and CPT accumulation in the range 40-57% and 64-71.5% respectively, while 4.61-7.69% increase was observed in tryptamine content as compared to control. Lovastatin treatment showed reduction in CPT (7-11%) and secologanin (7.5%) accumulation while tryptamine registered slight increase (3.84%) in comparison to control. These inhibitor mediated changes were reflected at transcriptional level via altering expression levels of deoxy-xylulose-5-phosphate reductoisomerase (DXR) and hydroxymethylglutaryl-CoA reductase (HMG). Further, mRNA expression of four more genes downstream to DXR and HMG of MEP and MVA pathways respectively were also investigated. Expression analysis also included secologanin synthase (SLS) and strictosidine synthase (STR) of seco-iridoid pathway. Present investigation also entailed development of an efficient in vitro multiplication system as a precursor to pathway flux studies. Further, a robust Agrobacterium-mediated transformed hairy root protocol was also developed for its amenability for up-scaling as a future prospect. CONCLUSIONS: Metabolic and transcriptional changes reveal differential efficacy of cytosolic and plastidial inhibitors in context to pathway flux perturbations on seco-iridoid end-product camptothecin. MEP pathway plausibly is the major precursor contributor towards CPT production. These empirical findings allude towards developing suitable biotechnological interventions for enhanced CPT production.


Assuntos
Antineoplásicos Fitogênicos/biossíntese , Camptotecina/biossíntese , Magnoliopsida/genética , Vias Biossintéticas , Regulação da Expressão Gênica de Plantas , Magnoliopsida/metabolismo , Plantas Medicinais
9.
BMC Bioinformatics ; 20(1): 380, 2019 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-31288752

RESUMO

BACKGROUND: Alkaloids, a class of organic compounds that contain nitrogen bases, are mainly synthesized as secondary metabolites in plants and fungi, and they have a wide range of bioactivities. Although there are thousands of compounds in this class, few of their biosynthesis pathways are fully identified. In this study, we constructed a model to predict their precursors based on a novel kind of neural network called the molecular graph convolutional neural network. Molecular similarity is a crucial metric in the analysis of qualitative structure-activity relationships. However, it is sometimes difficult for current fingerprint representations to emphasize specific features for the target problems efficiently. It is advantageous to allow the model to select the appropriate features according to data-driven decisions for extracting more useful information, which influences a classification or regression problem substantially. RESULTS: In this study, we applied a neural network architecture for undirected graph representation of molecules. By encoding a molecule as an abstract graph and applying "convolution" on the graph and training the weight of the neural network framework, the neural network can optimize feature selection for the training problem. By incorporating the effects from adjacent atoms recursively, graph convolutional neural networks can extract the features of latent atoms that represent chemical features of a molecule efficiently. In order to investigate alkaloid biosynthesis, we trained the network to distinguish the precursors of 566 alkaloids, which are almost all of the alkaloids whose biosynthesis pathways are known, and showed that the model could predict starting substances with an averaged accuracy of 97.5%. CONCLUSION: We have showed that our model can predict more accurately compared to the random forest and general neural network when the variables and fingerprints are not selected, while the performance is comparable when we carefully select 507 variables from 18000 dimensions of descriptors. The prediction of pathways contributes to understanding of alkaloid synthesis mechanisms and the application of graph based neural network models to similar problems in bioinformatics would therefore be beneficial. We applied our model to evaluate the precursors of biosynthesis of 12000 alkaloids found in various organisms and found power-low-like distribution.


Assuntos
Alcaloides/classificação , Vias Biossintéticas , Redes Neurais (Computação) , Algoritmos , Alcaloides/química , Metaboloma , Modelos Teóricos
10.
Nat Commun ; 10(1): 2733, 2019 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-31227716

RESUMO

Cell wall antibiotics are crucial for combatting the emerging wave of resistant bacteria. Yet, our understanding of antibiotic action is limited, as many strains devoid of all resistance determinants display far higher antibiotic tolerance in vivo than suggested by the antibiotic-target binding affinity in vitro. To resolve this conflict, here we develop a comprehensive theory for the bacterial cell wall biosynthetic pathway and study its perturbation by antibiotics. We find that the closed-loop architecture of the lipid II cycle of wall biosynthesis features a highly asymmetric distribution of pathway intermediates, and show that antibiotic tolerance scales inversely with the abundance of the targeted pathway intermediate. We formalize this principle of minimal target exposure as intrinsic resistance mechanism and predict how cooperative drug-target interactions can mitigate resistance. The theory accurately predicts the in vivo efficacy for various cell wall antibiotics in different Gram-positive bacteria and contributes to a systems-level understanding of antibiotic action.


Assuntos
Vias Biossintéticas/efeitos dos fármacos , Parede Celular/efeitos dos fármacos , Farmacorresistência Bacteriana/efeitos dos fármacos , Bactérias Gram-Positivas/metabolismo , Infecções por Bactérias Gram-Positivas/tratamento farmacológico , Parede Celular/metabolismo , Bactérias Gram-Positivas/efeitos dos fármacos , Infecções por Bactérias Gram-Positivas/microbiologia , Modelos Biológicos , Uridina Difosfato Ácido N-Acetilmurâmico/análogos & derivados , Uridina Difosfato Ácido N-Acetilmurâmico/biossíntese
11.
BMC Plant Biol ; 19(1): 266, 2019 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-31221095

RESUMO

BACKGROUND: Pogostemon cablin (Blanco) Benth. (Patchouli) is an important aromatic and medicinal plant and widely used in traditional Chinese medicine as well as in the perfume industry. Patchoulol is the primary bioactive component in P. cablin, its biosynthesis has attracted widespread interests. Previous studies have surveyed the putative genes involved in patchoulol biosynthesis using next-generation sequencing method; however, technical limitations generated by short-read sequencing restrict the yield of full-length genes. Additionally, little is known about the expression pattern of genes especially patchoulol biosynthesis related genes in response to methyl jasmonate (MeJA). Our understanding of patchoulol biosynthetic pathway still remained largely incomplete to date. RESULTS: In this study, we analyzed the morphological character and volatile chemical compounds of P. cablin cv. 'Zhanxiang', and 39 volatile chemical components were detected in the patchouli leaf using GC-MS, most of which were sesquiterpenes. Furthermore, high-quality RNA isolated from leaves and stems of P. cablin were used to generate the first full-length transcriptome of P. cablin using PacBio isoform sequencing (Iso-Seq). In total, 9.7 Gb clean data and 82,335 full-length UniTransModels were captured. 102 transcripts were annotated as 16 encoding enzymes involved in patchouli alcohol biosynthesis. Accorded with the uptrend of patchoulol content, the vast majority of genes related to the patchoulol biosynthesis were up-regulated after MeJA treatment, indicating that MeJA led to an increasing synthesis of patchoulol through activating the expression level of genes involved in biosynthesis pathway of patchoulol. Moreover, expression pattern analysis also revealed that transcription factors participated in JA regulation of patchoulol biosynthesis were differentially expressed. CONCLUSIONS: The current study comprehensively reported the morphological specificity, volatile chemical compositions and transcriptome characterization of the Chinese-cultivated P. cablin cv. 'Zhanxiang', these results contribute to our better understanding of the physiological and molecular features of patchouli, especially the molecular mechanism of biosynthesis of patchoulol. Our full-length transcriptome data also provides a valuable genetic resource for further studies in patchouli.


Assuntos
Regulação da Expressão Gênica de Plantas , Pogostemon/genética , Sesquiterpenos/metabolismo , Acetatos , Vias Biossintéticas , Ciclopentanos , Perfilação da Expressão Gênica , Oxilipinas , Transcriptoma
12.
Cancer Sci ; 110(8): 2408-2420, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31215094

RESUMO

Esophageal squamous cell carcinomas (ESCCs) as well as adenocarcinomas (EACs) were developed in rat duodenal contents reflux models (reflux model). The present study aimed to shed light on the mechanism by which bile acid stimulation causes cancer onset and progression. Metabolomics analyses were performed on samples of neoplastic and nonneoplastic tissues from reflux models, and K14D, cultivated from a nonmetastatic, primary ESCC, and ESCC-DR, established from a metastatic thoracic lesion. ESCC-DRtca2M was prepared by treating ESCC-DR cells with taurocholic acid (TCA) to accelerate cancer progression. The lines were subjected to comprehensive genomic analyses. In addition, protein expression levels of glucose-6-phosphate dehydrogenase (G6PD), nuclear factor kappa B (NF-κB) (p65) and O-linked N-Acetylglucosamine (O-GlcNAc) were compared among lines. Cancers developed in the reflux models exhibited greater hexosamine biosynthesis pathway (HBP) activation compared with the nonneoplastic tissues. Expression of O-GlcNAc transferase (OGT) increased considerably in both ESCC and EAC compared with nonneoplastic squamous epithelium. Conversely, cell line-based experiments revealed the greater activation of the pentose phosphate pathway (PPP) at higher degrees of malignancy. G6PD overexpression in response to TCA exposure was observed. Both NF-κB (p65) and O-GlcNAc were expressed more highly in ESCC-DRtca2M than in the other cell lines. Moreover, ESCC-DRtca2M cells had additional chromosomal abnormalities in excess of ESCC-DR cells. Overall, glucose metabolism was upregulated in both esophageal cancer tissue and cell lines. While bile acids are not mutagenic, chronic exposure seems to trigger NF-κB(p65) activation, potentially inducing genetic mutations as well as facilitating carcinogenesis and cancer progression. Glucose metabolism was upregulated in both esophageal cancer tissue and cell lines, and the HBP was activated in the former. The cell line-based experiments demonstrated upregulation of the pentose phosphate pathway (PPP) at higher degrees of malignancy. While bile acids are not mutagenic, chronic exposure seems to trigger G6PD overexpression and NF-κB (p65) activation, potentially inducing genetic mutations as well as facilitating carcinogenesis and cancer progression.


Assuntos
Ácidos e Sais Biliares/metabolismo , Vias Biossintéticas/fisiologia , Neoplasias Esofágicas/metabolismo , Carcinoma de Células Escamosas do Esôfago/metabolismo , Hexosaminas/metabolismo , Via de Pentose Fosfato/fisiologia , Acetilglucosamina/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Linhagem Celular Tumoral , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/patologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Glucose/análogos & derivados , Glucose/metabolismo , Glucosefosfato Desidrogenase/metabolismo , Lisina/análogos & derivados , Lisina/metabolismo , Masculino , NF-kappa B/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais/fisiologia , Regulação para Cima/fisiologia
13.
J Microbiol Biotechnol ; 29(6): 923-932, 2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-31154747

RESUMO

Current strategies of strain improvement processes are mainly focused on enhancing the synthetic pathways of the products. However, excessive metabolic flux often creates metabolic imbalances, which lead to growth retardation and ultimately limit the yield of the product. To solve this problem, we applied a dynamic regulation strategy to produce L-phenylalanine (LPhe) in Escherichia coli. First, we constructed a series of Phe-induced promoters that exhibited different strengths through modification of the promoter region of tyrP. Then, two engineered promoters were separately introduced into a Phe-producing strain xllp1 to dynamically control the expression level of one pathway enzyme AroK. Batch fermentation results of the strain xllp3 showed that the titer of Phe reached 61.3 g/l at 48 h, representing a titer of 1.36- fold of the strain xllp1 (45.0 g/l). Moreover, the L-Phe yields on glucose of xllp3 (0.22 g/g) were also greatly improved, with an increase of 1.22-fold in comparison with the xllp1 (0.18 g/ g). In summary, we successfully improved the titer of Phe by using dynamic regulation of one key enzyme and this strategy can be applied for improving the performance of strains producing other aromatic amino acids and derived compounds.


Assuntos
Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Microbiologia Industrial/métodos , Engenharia Metabólica/métodos , Fenilalanina/biossíntese , Sistemas de Transporte de Aminoácidos Neutros/genética , Vias Biossintéticas/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Fermentação , Glucose/metabolismo , Análise do Fluxo Metabólico , Mutação , Fenilalanina/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Regiões Promotoras Genéticas
14.
J Microbiol Biotechnol ; 29(6): 839-844, 2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-31154751

RESUMO

Anthranilate derivatives have been used as flavoring and fragrant agents for a long time. Recently, these compounds are gaining attention due to new biological functions including antinociceptive and analgesic activities. Three anthranilate derivatives, N-methylanthranilate, methyl anthranilate, and methyl N-methylanthranilate were synthesized using metabolically engineered stains of Escherichia coli. NMT encoding N-methyltransferase from Ruta graveolens, AMAT encoding anthraniloyl-coenzyme A (CoA):methanol acyltransferase from Vitis labrusca, and pqsA encoding anthranilate coenzyme A ligase from Pseudomonas aeruginosa were cloned and E. coli strains harboring these genes were used to synthesize the three desired compounds. E. coli mutants (metJ, trpD, tyrR mutants), which provide more anthranilate and/or S-adenosyl methionine, were used to increase the production of the synthesized compounds. MS/MS analysis was used to determine the structure of the products. Approximately, 185.3 µM N-methylanthranilate and 95.2 µM methyl N-methylanthranilate were synthesized. This is the first report about the synthesis of anthranilate derivatives in E. coli.


Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , ortoaminobenzoatos/metabolismo , Vias Biossintéticas , Coenzima A Ligases/genética , Coenzima A Ligases/metabolismo , Coenzima A-Transferases/genética , Coenzima A-Transferases/metabolismo , Escherichia coli/enzimologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Engenharia Metabólica , Metiltransferases/genética , Metiltransferases/metabolismo , Mutação , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/genética , Proteínas Recombinantes/metabolismo , Ruta/enzimologia , Ruta/genética , Vitis/enzimologia , Vitis/genética , ortoaminobenzoatos/química
15.
J Agric Food Chem ; 67(26): 7266-7273, 2019 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-31244199

RESUMO

Chemical investigation of fungus Pochonia chlamydosporia strain 170, derived from rice fermentation sediment samples, afforded seven radicicol analogues, including two new compounds, monocillin VI (1) and monocillin VII (2), and five known compounds, monocillin II (3), monorden D (4), monocillin IV (5), monocillin V (6), and pochonin M (7). The structures of compounds 1-7 were established primarily by analysis of nuclear magnetic resonance data, and the absolute configurations of the secondary alcohol in compounds 1 and 2 were assigned by the modified Mosher method. All seven compounds have modest antibacterial activities, with a minimal inhibitory concentration (MIC) of 25.6 µg/mL for compounds 1 and 3-7 and 51.2 µg/mL for compound 2, on inhibition of the growth of the plant pathogen Xanthomonas campestris (the positive control ampicillin showed a MIC value of 12.8 µg/mL), indicating that the fungus has the potential to control bacterial disease. The biosynthetic gene cluster and putative biosynthetic pathways of these radicicol analogues in the P. chlamydosporia genome were proposed. These findings increase our knowledge of the chemical potential of P. chlamydosporia and may allow us to better utilize the fungus as a biological control agent.


Assuntos
Antibacterianos/química , Hypocreales/metabolismo , Macrolídeos/química , Antibacterianos/biossíntese , Antibacterianos/farmacologia , Vias Biossintéticas , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Hypocreales/química , Hypocreales/genética , Macrolídeos/metabolismo , Macrolídeos/farmacologia , Testes de Sensibilidade Microbiana , Família Multigênica , Xanthomonas campestris/efeitos dos fármacos , Xanthomonas campestris/crescimento & desenvolvimento
16.
Sheng Wu Gong Cheng Xue Bao ; 35(6): 1109-1116, 2019 Jun 25.
Artigo em Chinês | MEDLINE | ID: mdl-31232007

RESUMO

The discovery of hydroxylases in the anticancer drug taxol biosynthesis pathway is a hotspot and difficulty in current research. In this study, a new hydroxylase gene TcCYP725A22 (GenBank accession number: MF448646.1) was used to construct a sub-cellular localization vector pCAMIBA1303-TcCYP725A22-EGFP to get the transient expression in onion epidermal cells. Laser confocal microscopy revealed that the protein encoded by this gene was localized in the cell membrane. Furthermore, the recombinant plant expression plasmid pBI121-TcCYP725A22 was constructed. After transient transformation to the Taxus chinensis mediated by Agrobacterium tumefaciens LBA4404, qRT-PCR and LC-MS were utilized to analyze the effects of TcCYP725A22 overexpression on the synthesis of taxol. The results showed that, in the TcCYP725A22 overexpressed cell line, expression levels of most defined hydroxylase genes for taxol biosynthesis were increased, and the yield of taxanes were also increased. It was concluded that the hydroxylase gene TcCYP725A22 is likely involved in the biosynthetic pathway of taxol.


Assuntos
Taxus , Vias Biossintéticas , Oxigenases de Função Mista , Paclitaxel , Taxoides
17.
Nat Commun ; 10(1): 2615, 2019 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-31197154

RESUMO

Balanced expression of multiple genes is central for establishing new biosynthetic pathways or multiprotein cellular complexes. Methods for efficient combinatorial assembly of regulatory sequences (promoters) and protein coding sequences are therefore highly wanted. Here, we report a high-throughput cloning method, called COMPASS for COMbinatorial Pathway ASSembly, for the balanced expression of multiple genes in Saccharomyces cerevisiae. COMPASS employs orthogonal, plant-derived artificial transcription factors (ATFs) and homologous recombination-based cloning for the generation of thousands of individual DNA constructs in parallel. The method relies on a positive selection of correctly assembled pathway variants from both, in vivo and in vitro cloning procedures. To decrease the turnaround time in genomic engineering, COMPASS is equipped with multi-locus CRISPR/Cas9-mediated modification capacity. We demonstrate the application of COMPASS by generating cell libraries producing ß-carotene and co-producing ß-ionone and biosensor-responsive naringenin. COMPASS will have many applications in synthetic biology projects that require gene expression balancing.


Assuntos
Vias Biossintéticas/genética , Engenharia Metabólica/métodos , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Técnicas Biossensoriais/métodos , Sistemas CRISPR-Cas/genética , Clonagem Molecular/métodos , Flavanonas/biossíntese , Recombinação Homóloga/genética , Norisoprenoides/biossíntese , Regiões Promotoras Genéticas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Biologia Sintética/métodos , Fatores de Transcrição/genética , beta Caroteno/biossíntese
18.
Nat Commun ; 10(1): 2613, 2019 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-31197182

RESUMO

Kistamicin is a divergent member of the glycopeptide antibiotics, a structurally complex class of important, clinically relevant antibiotics often used as the last resort against resistant bacteria. The extensively crosslinked structure of these antibiotics that is essential for their activity makes their chemical synthesis highly challenging and limits their production to bacterial fermentation. Kistamicin contains three crosslinks, including an unusual 15-membered A-O-B ring, despite the presence of only two Cytochrome P450 Oxy enzymes thought to catalyse formation of such crosslinks within the biosynthetic gene cluster. In this study, we characterise the kistamicin cyclisation pathway, showing that the two Oxy enzymes are responsible for these crosslinks within kistamicin and that they function through interactions with the X-domain, unique to glycopeptide antibiotic biosynthesis. We also show that the kistamicin OxyC enzyme is a promiscuous biocatalyst, able to install multiple crosslinks into peptides containing phenolic amino acids.


Assuntos
Actinobacteria/metabolismo , Antibacterianos/metabolismo , Vias Biossintéticas/genética , Glicopeptídeos/biossíntese , Peptídeos/metabolismo , Actinobacteria/genética , Antibacterianos/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biocatálise , Ciclização/genética , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Glicopeptídeos/química , Família Multigênica , Peptídeos/química
19.
Aquat Toxicol ; 213: 105213, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31200332

RESUMO

Atrazine is a widely used pesticide which acts as an endocrine disruptor in various organisms. The aim of this study was to investigate adverse effects of atrazine on life parameters, oxidative stress, and ecdysteroid biosynthetic pathway in the marine copepod Tigriopus japonicus. In T. japonicus, no mortality was shown in response to atrazine up to 20 mg/L in acute toxicity assessment. In nauplii, retardation in the growth and prolonged molting and metamorphosis resulted under chronic exposure of atrazine at 20 mg/L. In addition, body sizes of T. japonicus nauplii were significantly decreased (P < 0.01 in length and P < 0.001 in width) in response to 20 mg/L of atrazine. Furthermore, atrazine induced oxidative stress by the generation of reactive oxygen species at all concentrations compared to the control in the nauplii. Also, significant increase in glutathione-S transferase activity was observed in adult T. japonicus at low concentration of atrazine. To understand effects of atrazine on ecdysteroid biosynthetic pathway-involved genes (e.g., neverland, CYP307E1, CYP306A1, CYP302A1, CYP3022A1 [CYP315A1], CYP314A1, and CYP18D1) were examined with mRNA expressions of ecdysone receptor (EcR) and ultraspiracle (USP) in response to 20 mg/L atrazine in nauplii and adults. In the nauplii, these genes were significantly downregulated (P < 0.05) in response to atrazine, compared to the control but not in the adult T. japonicus. These results suggest that atrazine can interfere in vivo life parameters by oxidative stress-induced retrogression and ecdysteroid biosynthetic pathway in this species.


Assuntos
Organismos Aquáticos/metabolismo , Atrazina/toxicidade , Vias Biossintéticas/efeitos dos fármacos , Copépodes/efeitos dos fármacos , Ecdisteroides/biossíntese , Estresse Oxidativo/efeitos dos fármacos , Animais , Organismos Aquáticos/efeitos dos fármacos , Organismos Aquáticos/genética , Atrazina/química , Vias Biossintéticas/genética , Tamanho Corporal/efeitos dos fármacos , Copépodes/genética , Copépodes/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glutationa Transferase/metabolismo , Estresse Oxidativo/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Poluentes Químicos da Água/toxicidade
20.
Food Chem ; 297: 124975, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31253324

RESUMO

This study aimed to evaluate the formation of tryptophan derivatives in the kynurenine pathway during wort fermentation using a multi-response kinetic model and an empirical modified logistic model. Saccharomyces cerevisiae NCYC 88 (ale yeast) and S. pastorianus NCYC 203 (lager yeast) were used to understand the effect of fermentation type on tryptophan derivatives. According to the modified logistic model, tryptophan concentration was critical for the maximum production rate of kynurenic acid, a neuroprotective compound. The results indicated that utilization of tryptophan and kynurenic acid formation were faster in wort fermented with S. cerevisiae than with S. pastorianus. The reaction rate constants implied that the kynurenic acid formation stage was minor compared to other enzymatic reactions leading to NAD+ synthesis. Multi-response kinetic modeling of kynurenine pathway provided insights into tryptophan derivative formation, which can facilitate improved beer fermentation processing.


Assuntos
Ácido Cinurênico/metabolismo , Cinurenina/metabolismo , Fármacos Neuroprotetores/metabolismo , Preparações de Plantas/química , Saccharomyces/metabolismo , Triticum/química , Triptofano/análogos & derivados , Cerveja/análise , Vias Biossintéticas , Fermentação , Cinética , Modelos Logísticos , Saccharomyces cerevisiae/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA