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1.
Ecotoxicol Environ Saf ; 203: 110984, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32888605

RESUMO

The use of water for drinking and agriculture requires knowledge of its toxicity. In this study, we compared the use of genetically modified bioluminescent (GMB) bacteria whose luminescence increases in the presence of toxicants and Chinese Hamster Ovary (CHO) cells for the characterization of the toxicity of water samples collected from a lake and streams, hydroponic and aquaponic farms, and a wastewater treatment plant. GMB bacteria were used to probe genotoxicity, cytotoxicity and reactive oxygen species-induced effects in the whole water samples. Unlike GMB bacteria, the use of CHO cells requires XAD resin-based pre-concentration of toxic material present in water samples for the subsequent cytotoxicity assay. In addition to the examination of the toxicity of the water from the different sources, the GMB bacteria were also used to test the XAD extracts diluted to the concentrations causing 50% growth inhibition of the CHO cells. The two biomonitoring tools provided different results when they were used to test the above-mentioned diluted XAD extracts. A pre-concentration procedure based on adsorption by XAD resins with subsequent elution was not sufficient to represent the material responsible for the toxicity of the whole water samples toward the GMB bacteria. Therefore, the use of XAD resin extracts may lead to major underestimates of the toxicity of water samples. Although the toxicity findings obtained using the GMB bacteria and CHO cells may not correlate with each another, the GMB bacteria assay did provide a mechanism-specific biomonitoring tool to probe the toxicity of water samples without a need for the pre-concentration step.


Assuntos
Bactérias , Monitoramento Biológico/métodos , Água Potável/análise , Lagos/análise , Rios , Águas Residuárias/análise , Animais , Bactérias/genética , Células CHO , Cricetulus , Hidroponia , Luminescência , Microrganismos Geneticamente Modificados/genética , Eliminação de Resíduos Líquidos
2.
Ecotoxicol Environ Saf ; 204: 111060, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32768747

RESUMO

OBJECTIVE: Serratia sp. S2 is a wild strain with chromium resistance and reduction ability. Chromium(VI) metabolic-protein-coding gene ChrA and ChrT were cloned from Serratia sp. S2, and ligated with prokaryotic expression vectors pET-28a (+) and transformed into E. coli BL21 to construct ChrA, ChrT and ChrAT engineered bacteria. By studying the characteristics of Cr(VI) metabolism in engineered bacteria, the function and mechanism of the sole expression and coexpression of ChrA and ChrT genes were studied. METHODS: Using Serratia sp. S2 genome as template, ChrA and ChrT genes were amplified by PCR, and prokaryotic expression vectors was ligated to form the recombinant plasmid pET-28a (+)-ChrA, pET-28a (+)-ChrT and pET-28a (+)-ChrAT, and transformed into E. coli BL21 to construct ChrA, ChrT, ChrAT engineered bacteria. The growth curve, tolerance, and reduction of Cr(VI), the distribution of intracellular and extracellular Cr, activity of chromium reductase and intracellular oxidative stress in engineered bacteria were measured to explore the metabolic characteristics of Cr(VI) in ChrA, ChrT, ChrAT engineered bacteria. RESULTS: ChrA, ChrT and ChrAT engineered bacteria were successfully constructed by gene recombination technology. The tolerance to Cr(VI) was Serratia sp. S2 > ChrAT ≈ ChrA > ChrT > Control (P < 0.05), and the reduction ability to Cr(VI) was Serratia sp. S2 > ChrAT ≈ ChrT > ChrA (P < 0.05). The chromium distribution experiments confirmed that Cr(VI) and Cr(III) were the main valence states. Effect of electron donors on chromium reductase activity was NADPH > NADH > non-NAD(P)H (P < 0.05). The activity of chromium reductase increased significantly with NAD(P)H (P < 0.05). The Glutathione and NPSH (Non-protein Sulfhydryl) levels of ChrA, ChrAT engineered bacteria increased significantly (P < 0.05) under the condition of Cr(VI), but there was no significant difference in the indexes of ChrT engineered bacteria (P > 0.05). CONCLUSION: ChrAT engineered bacteria possesses resistance and reduction abilities of Cr(VI). ChrA protein endows the strain with the ability to resist Cr(VI). ChrT protein reduces Cr(VI) to Cr(III) by using NAD(P)H as electronic donor. The reduction process promotes the production of GSH, GSSG and NPSH to maintain the intracellular reduction state, which further improves the Cr(VI) tolerance and reduction ability of ChrAT engineered bacteria.


Assuntos
Proteínas de Bactérias/genética , Compostos de Cromo/metabolismo , Poluentes Ambientais/metabolismo , Genes Bacterianos , Proteínas de Membrana/genética , Microrganismos Geneticamente Modificados/genética , Serratia/genética , Biodegradação Ambiental , Escherichia coli/genética , Escherichia coli/metabolismo , Microrganismos Geneticamente Modificados/metabolismo , Modelos Teóricos , Oxirredução , Oxirredutases/metabolismo , Serratia/metabolismo
3.
Int J Food Microbiol ; 331: 108749, 2020 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-32622259

RESUMO

As genetically modified microorganisms (GMM), commonly used by the food and feed industry to produce additives, enzymes and flavourings, are frequently harbouring antimicrobial resistance (AMR) genes as selection markers, health and environmental concerns were consequently raised. For this reason, the interest of the competent authorities to control such microbial fermentation products has strongly increased, especially since several recent accidental contaminations of unauthorized GMM, or associated recombinant DNA, in bacterial fermentation products intended for the European food and feed chain. However, no global screening strategy is currently available in enforcement laboratories to assess the presence of GMM harbouring AMR genes and/or the presence of full-length AMR genes. Moreover, the confidentiality of the related GMM dossiers strongly hampers the development of methods to perform such control. To overcome this issue, an analysis of related publicly available patents was performed in this study to identify all reported AMR genes. On this basis, the aminoglycoside adenyltransferase (aadD) gene, conferring a resistance to both kanamycin and neomycin, was identified as a key target to cover a large spectrum of GM bacteria. A real-time PCR method to screen for its potential presence as well as a nested-PCR method associated with a sequencing analysis to assess its full-length were developed to target this aadD gene. The performance of these new methods were successfully evaluated in terms of specificity, sensitivity and applicability, allowing their easy implementation in enforcement laboratories. Moreover, the integration of these newly developed methods to our very recently proposed strategy, initially targeting GMM carrying a chloramphenicol resistance gene, allows to drastically increase the detection spectrum of GM bacteria producing fermentation food and feed products. The data generated by the proposed strategy represents therefore a crucial support for the competent authorities, especially to evaluate potential risks for the food and feed safety.


Assuntos
Bactérias/genética , Farmacorresistência Bacteriana/genética , Microbiologia de Alimentos , Microrganismos Geneticamente Modificados/genética , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Fermentação , Microrganismos Geneticamente Modificados/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real
4.
Mol Cell ; 78(4): 614-623, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32442504

RESUMO

Synthetic biology has promised and delivered on an impressive array of applications based on genetically modified microorganisms. While novel biotechnology undoubtedly offers benefits, like all new technology, precautions should be considered during implementation to reduce the risk of both known and unknown adverse effects. To achieve containment of transgenic microorganisms, confidence to a near-scientific certainty that they cannot transfer their transgenic genes to other organisms, and that they cannot survive to propagate in unintended environments, is a priority. Here, we present an in-depth summary of biological containment systems for micro-organisms published to date, including the production of a genetic firewall through genome recoding and physical containment of microbes using auxotrophies, regulation of essential genes, and expression of toxic genes. The level of containment required to consider a transgenic organism suitable for deployment is discussed, as well as standards of practice for developing new containment systems.


Assuntos
Biotecnologia/métodos , Genes Essenciais , Engenharia Genética/métodos , Microbiota/genética , Microrganismos Geneticamente Modificados/genética , Biologia Sintética/métodos , Humanos , Microrganismos Geneticamente Modificados/crescimento & desenvolvimento
5.
Artigo em Inglês | MEDLINE | ID: mdl-32276152

RESUMO

Due to the rapid decline in oceanic fish stock, ω-3 fatty acid (C18:3) has attracted serious attention and, hence, the identification of genotypes with high ω-3 content has become the main objective of Brassica napus (rapeseed) breeding. A candidate genes association study permitted us to delineate a genomic region linked to ω-3 content, offering a detailed understanding of the complex genetic mechanism of fatty acid biosynthesis in B. napus. Herein, the candidate genes association study, conducted on 324 genetically diverse rapeseed accessions, detected 114 single nucleotide polymorphisms (SNPs) associated with ω-3 fatty acid. Furthermore, these loci were functionally characterized in Saccharomyces cerevisiae. These associated loci were selected based on their contribution to a high C18:3 ratio, and the selected candidate loci were validated using allele-specific SNPs markers in an inbred population through polymerase chain reaction (PCR). These findings may contribute to improving the fatty acid composition by marker-based breeding and will facilitate the development of rapeseed varieties with high ω-3 content.


Assuntos
Brassica napus/genética , Ácidos Graxos Ômega-3/genética , Genes de Plantas , Proteínas de Plantas/genética , Polimorfismo de Nucleotídeo Único , Sequência de Aminoácidos , Brassica napus/metabolismo , Ácidos Graxos Ômega-3/metabolismo , Marcadores Genéticos , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/metabolismo , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência
6.
Appl Environ Microbiol ; 86(8)2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32033952

RESUMO

Blakeslea trispora is an industrial fungal species used for large-scale production of carotenoids. However, B. trispora light-regulated physiological processes, such as carotenoid biosynthesis and phototropism, are not fully understood. In this study, we isolated and characterized three photoreceptor genes, btwc-1a, btwc-1b, and btwc-1c, in B. trispora Bioinformatics analyses of these genes and their protein sequences revealed that the functional domains (PAS/LOV [Per-ARNT-Sim/light-oxygen-voltage] domain and zinc finger structure) of the proteins have significant homology to those of other fungal blue-light regulator proteins expressed by Mucor circinelloides and Neurospora crassa The photoreceptor proteins were synthesized by heterologous expression in Escherichia coli The chromogenic groups consisting of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) were detected to accompany BTWC-1 proteins by using high-performance liquid chromatography (HPLC) and fluorescence spectrometry, demonstrating that the proteins may be photosensitive. The absorbance changes of the purified BTWC-1 proteins seen under dark and light conditions indicated that they were light responsive and underwent a characteristic photocycle by light induction. Site-directed mutagenesis of the cysteine residual (Cys) in BTWC-1 did not affect the normal expression of the protein in E. coli but did lead to the loss of photocycle response, indicating that Cys represents a flavin-binding domain for photon detection. We then analyzed the functions of BTWC-1 proteins by complementing btwc-1a, btwc-1b, and btwc-1c into the counterpart knockout strains of M. circinelloides for each mcwc-1 gene. Transformation of the btwc-1a complement into mcwc-1a knockout strains restored the positive phototropism, while the addition of btwc-1c complement remedied the deficiency of carotene biosynthesis in the mcwc-1c knockout strains under conditions of illumination. These results indicate that btwc-1a and btwc-1c are involved in phototropism and light-inducible carotenogenesis. Thus, btwc-1 genes share a conserved flavin-binding domain and act as photoreceptors for control of different light transduction pathways in B. trispora IMPORTANCE Studies have confirmed that light-regulated carotenogenesis is prevalent in filamentous fungi, especially in mucorales. However, few investigations have been done to understand photoinduced synthesis of carotenoids and related mechanisms in B. trispora, a well-known industrial microbial strains. In the present study, three photoreceptor genes in B. trispora were cloned, expressed, and characterized by bioinformatics and photoreception analyses, and then in vivo functional analyses of these genes were constructed in M. circinelloides The results of this study will lead to a better understanding of photoreception and light-regulated carotenoid synthesis and other physiological responses in B. trispora.


Assuntos
Proteínas Fúngicas/genética , Mucorales/genética , Fotorreceptores Microbianos/genética , Sequência de Aminoácidos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/metabolismo , Mucorales/metabolismo , Fotorreceptores Microbianos/química , Fotorreceptores Microbianos/metabolismo , Alinhamento de Sequência
7.
Microb Cell Fact ; 19(1): 45, 2020 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-32093734

RESUMO

BACKGROUND: Bacillus licheniformis 2709 is extensively applied as a host for the high-level production of heterologous proteins, but Bacillus cells often possess unfavorable wild-type properties, such as production of viscous materials and foam during fermentation, which seriously influenced the application in industrial fermentation. How to develop it from a soil bacterium to a super-secreting cell factory harboring less undomesticated properties always plays vital role in industrial production. Besides, the optimal expression pattern of the inducible enzymes like alkaline protease has not been optimized by comparing the transcriptional efficiency of different plasmids and genomic integration sites in B. licheniformis. RESULT: Bacillus licheniformis 2709 was genetically modified by disrupting the native lchAC genes related to foaming and the eps cluster encoding the extracellular mucopolysaccharide via a markerless genome-editing method. We further optimized the expression of the alkaline protease gene (aprE) by screening the most efficient expression system among different modular plasmids and genomic loci. The results indicated that genomic expression of aprE was superior to plasmid expression and finally the transcriptional level of aprE greatly increased 1.67-fold through host optimization and chromosomal integration in the vicinity of the origin of replication, while the enzyme activity significantly improved 62.19% compared with the wild-type alkaline protease-producing strain B. licheniformis. CONCLUSION: We successfully engineered an AprE high-yielding strain free of undesirable properties and its fermentation traits could be applied to bulk-production by host genetic modification and expression optimization. In summary, host optimization is an enabling technology for improving enzyme production by eliminating the harmful traits of the host and optimizing expression patterns. We believe that these strategies can be applied to improve heterologous protein expression in other Bacillus species.


Assuntos
Bacillus licheniformis/metabolismo , Proteínas de Bactérias/biossíntese , Endopeptidases/biossíntese , Bacillus licheniformis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Fermentação , Engenharia Genética , Microbiologia Industrial , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/metabolismo , Plasmídeos/genética
8.
Microb Cell Fact ; 19(1): 46, 2020 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-32093713

RESUMO

BACKGROUND: Escherichia coli is an important strain for L-threonine production. Genetic switch is a ubiquitous regulatory tool for gene expression in prokaryotic cells. To sense and regulate intracellular or extracellular chemicals, bacteria evolve a variety of transcription factors. The key enzymes required for L-threonine biosynthesis in E. coli are encoded by the thr operon. The thr operon could coordinate expression of these genes when L-threonine is in short supply in the cell. RESULTS: The thrL leader regulatory elements were applied to regulate the expression of genes iclR, arcA, cpxR, gadE, fadR and pykF, while the threonine-activating promoters PcysH, PcysJ and PcysD were applied to regulate the expression of gene aspC, resulting in the increase of L-threonine production in an L-threonine producing E. coli strain TWF001. Firstly, different parts of the regulator thrL were inserted in the iclR regulator region in TWF001, and the best resulting strain TWF063 produced 16.34 g L-threonine from 40 g glucose after 30 h cultivation. Secondly, the gene aspC following different threonine-activating promoters was inserted into the chromosome of TWF063, and the best resulting strain TWF066 produced 17.56 g L-threonine from 40 g glucose after 30 h cultivation. Thirdly, the effect of expression regulation of arcA, cpxR, gadE, pykF and fadR was individually investigated on L-threonine production in TWF001. Finally, using TWF066 as the starting strain, the expression of genes arcA, cpxR, gadE, pykF and fadR was regulated individually or in combination to obtain the best strain for L-threonine production. The resulting strain TWF083, in which the expression of seven genes (iclR, aspC, arcA, cpxR, gadE, pykF, fadR and aspC) was regulated, produced 18.76 g L-threonine from 30 g glucose, 26.50 g L-threonine from 40 g glucose, or 26.93 g L-threonine from 50 g glucose after 30 h cultivation. In 48 h fed-batch fermentation, TWF083 could produce 116.62 g/L L-threonine with a yield of 0.486 g/g glucose and productivity of 2.43 g/L/h. CONCLUSION: The genetic engineering through the expression regulation of key genes is a better strategy than simple deletion of these genes to improve L-threonine production in E. coli. This strategy has little effect on the intracellular metabolism in the early stage of the growth but could increase L-threonine biosynthesis in the late stage.


Assuntos
Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Treonina/biossíntese , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Fermentação , Genes Bacterianos , Engenharia Genética , Microbiologia Industrial , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/metabolismo
9.
Microb Cell Fact ; 19(1): 49, 2020 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-32103761

RESUMO

BACKGROUND: The GRAS and oleaginous yeast Yarrowia lipolytica (Y. lipolytica) is an attractive cell factory for the production of chemicals and biofuels. The production of many natural products of commercial interest have been investigated in this cell factory by introducing heterologous biosynthetic pathways and by modifying the endogenous pathways. However, since natural products anabolism involves long pathways and complex regulation, re-channelling carbon into the product of target compounds is still a cumbersome work, and often resulting in low production performance. RESULTS: In this work, the carotenogenic genes contained carB and bi-functional carRP from Mucor circinelloides and carotenoid cleavage dioxygenase 1 (CCD1) from Petunia hybrida were introduced to Y. lipolytica and led to the low production of ß-ionone of 3.5 mg/L. To further improve the ß-ionone synthesis, we implemented a modular engineering strategy for the construction and optimization of a biosynthetic pathway for the overproduction of ß-ionone in Y. lipolytica. The strategy involved the enhancement of the cytosolic acetyl-CoA supply and the increase of MVA pathway flux, yielding a ß-ionone titer of 358 mg/L in shake-flask fermentation and approximately 1 g/L (~ 280-fold higher than the baseline strain) in fed-batch fermentation. CONCLUSIONS: An efficient ß-ionone producing GRAS Y. lipolytica platform was constructed by combining integrated overexpressed of heterologous and native genes. A modular engineering strategy involved the optimization pathway and fermentation condition was investigated in the engineered strain and the highest ß-ionone titer reported to date by a cell factory was achieved. This effective strategy can be adapted to enhance the biosynthesis of other terpenoids in Y. lipolytica.


Assuntos
Engenharia Metabólica , Norisoprenoides/metabolismo , Yarrowia/metabolismo , Acetilcoenzima A/metabolismo , Fermentação , Microbiologia Industrial , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/metabolismo , Oxigênio/metabolismo , Yarrowia/genética
10.
Infect Immun ; 88(5)2020 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-32094255

RESUMO

Efficient delivery of antigenic cargo to trigger protective immune responses is critical to the success of vaccination. Genetically engineered microorganisms, including virus, bacteria, and protozoa, can be modified to carry and deliver heterologous antigens to the host immune system. The biological vectors can induce a broad range of immune responses and enhance heterologous antigen-specific immunological outcomes. The protozoan genus Eimeria is widespread in domestic animals, causing serious coccidiosis. Eimeria parasites with strong immunogenicity are potent coccidiosis vaccine candidates and offer a valuable model of live vaccines against infectious diseases in animals. Eimeria parasites can also function as a vaccine vector. Herein, we review recent advances in design and application of recombinant Eimeria as a vaccine vector, which has been a topic of ongoing research in our laboratory. By recapitulating the establishment of an Eimeria transfection platform and its application, it will help lay the foundation for the future development of effective parasite-based vaccine delivery vectors and beyond.


Assuntos
Eimeria/genética , Vacinas Protozoárias/genética , Vacinas Sintéticas/genética , Animais , Antígenos de Protozoários/genética , Coccidiose/imunologia , Humanos , Microrganismos Geneticamente Modificados/genética , Vacinação/métodos , Vacinas Atenuadas/genética
11.
Appl Environ Microbiol ; 86(6)2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-31924615

RESUMO

The mevalonate pathway is a well-known metabolic route that provides biosynthetic precursors for myriad isoprenoids. An unexpected variety of the pathway has been discovered from recent studies on microorganisms, mainly on archaea. The most recently discovered example, called the "archaeal" mevalonate pathway, is a modified version of the canonical eukaryotic mevalonate pathway and was elucidated in our previous study using the hyperthermophilic archaeon Aeropyrum pernix This pathway comprises four known enzymes that can produce mevalonate 5-phosphate from acetyl coenzyme A, two recently discovered enzymes designated phosphomevalonate dehydratase and anhydromevalonate phosphate decarboxylase, and two more known enzymes, i.e., isopentenyl phosphate kinase and isopentenyl pyrophosphate:dimethylallyl pyrophosphate isomerase. To show its wide distribution in archaea and to confirm if its enzyme configuration is identical among species, the putative genes of a lower portion of the pathway-from mevalonate to isopentenyl pyrophosphate-were isolated from the methanogenic archaeon Methanosarcina mazei, which is taxonomically distant from A. pernix, and were introduced into an engineered Escherichia coli strain that produces lycopene, a red carotenoid pigment. Lycopene production, as a measure of isoprenoid productivity, was enhanced when the cells were grown semianaerobically with the supplementation of mevalonolactone, which demonstrates that the archaeal pathway can function in bacterial cells to convert mevalonate into isopentenyl pyrophosphate. Gene deletion and complementation analysis using the carotenogenic E. coli strain suggests that both phosphomevalonate dehydratase and anhydromevalonate phosphate decarboxylase from M. mazei are required for the enhancement of lycopene production.IMPORTANCE Two enzymes that have recently been identified from the hyperthermophilic archaeon A. pernix as components of the archaeal mevalonate pathway do not require ATP for their reactions. This pathway, therefore, might consume less energy than other mevalonate pathways to produce precursors for isoprenoids. Thus, the pathway might be applicable to metabolic engineering and production of valuable isoprenoids that have application as pharmaceuticals. The archaeal mevalonate pathway was successfully reconstructed in E. coli cells by introducing several genes from the methanogenic or hyperthermophilic archaeon, which demonstrated that the pathway requires the same components even in distantly related archaeal species and can function in bacterial cells.


Assuntos
Escherichia coli/metabolismo , Methanosarcina/metabolismo , Ácido Mevalônico/metabolismo , Escherichia coli/genética , Redes e Vias Metabólicas , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/metabolismo
12.
Appl Environ Microbiol ; 86(6)2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-31900306

RESUMO

The Gram-positive soil bacterium Arthrobacter sp. strain TS-15 (DSM 32400), which is capable of metabolizing ephedrine as a sole source of carbon and energy, was isolated. According to 16S rRNA gene sequences and comparative genomic analysis, Arthrobacter sp. TS-15 is closely related to Arthrobacter aurescens Distinct from all known physiological paths, ephedrine metabolism by Arthrobacter sp. TS-15 is initiated by the selective oxidation of the hydroxyl function at the α-C atom, yielding methcathinone as the primary degradation product. Rational genome mining revealed a gene cluster potentially encoding the novel pathway. Two genes from the cluster, which encoded putative short-chain dehydrogenases, were cloned and expressed in Escherichia coli The obtained enzymes were strictly NAD+ dependent and catalyzed the oxidation of ephedrine to methcathinone. Pseudoephedrine dehydrogenase (PseDH) selectively converted (S,S)-(+)-pseudoephedrine and (S,R)-(+)-ephedrine to (S)- and (R)-methcathinone, respectively. Ephedrine dehydrogenase (EDH) exhibited strict selectivity for the oxidation of the diastereomers (R,S)-(-)-ephedrine and (R,R)-(-)-pseudoephedrine.IMPORTANCE Arthrobacter sp. TS-15 is a newly isolated bacterium with the unique ability to degrade ephedrine isomers. The initiating steps of the novel metabolic pathway are described. Arthrobacter sp. TS-15 and its isolated ephedrine-oxidizing enzymes have potential for use in decontamination and synthetic applications.


Assuntos
Arthrobacter/metabolismo , Efedrina/metabolismo , Regulação Bacteriana da Expressão Gênica , Pseudoefedrina/metabolismo , Arthrobacter/classificação , Biodegradação Ambiental , Efedrina/química , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Bacterianos , Micrococcaceae , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/metabolismo , Família Multigênica , Pseudoefedrina/química , Estereoisomerismo
13.
Sci Rep ; 10(1): 1383, 2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-31992813

RESUMO

Small heat shock proteins (sHSPs) have been thought to function as chaperones, protecting their targets from denaturation and aggregation when organisms are subjected to various biotic and abiotic stresses. We previously reported an sHSP from Oryza sativa (OsHSP20) that homodimerizes and forms granules within the cytoplasm but its function was unclear. We now show that OsHSP20 transcripts were significantly up-regulated by heat shock and high salinity but not by drought. A recombinant protein was purified and shown to inhibit the thermal aggregation of the mitochondrial malate dehydrogenase (MDH) enzyme in vitro, and this molecular chaperone activity suggested that OsHSP20 might be involved in stress resistance. Heterologous expression of OsHSP20 in Escherichia coli or Pichia pastoris cells enhanced heat and salt stress tolerance when compared with the control cultures. Transgenic rice plants constitutively overexpressing OsHSP20 and exposed to heat and salt treatments had longer roots and higher germination rates than those of control plants. A series of assays using its truncated mutants showed that its N-terminal arm plus the ACD domain was crucial for its homodimerization, molecular chaperone activity in vitro, and stress tolerance in vivo. The results supported the viewpoint that OsHSP20 could confer heat and salt tolerance by its molecular chaperone activity in different organisms and also provided a more thorough characterization of HSP20-mediated stress tolerance in O. sativa.


Assuntos
Escherichia coli/metabolismo , Proteínas de Choque Térmico HSP20 , Microrganismos Geneticamente Modificados/metabolismo , Oryza/genética , Pichia/metabolismo , Proteínas de Plantas , Multimerização Proteica , Tolerância ao Sal , Escherichia coli/genética , Proteínas de Choque Térmico HSP20/biossíntese , Proteínas de Choque Térmico HSP20/genética , Microrganismos Geneticamente Modificados/genética , Pichia/genética , Proteínas de Plantas/biossíntese , Proteínas de Plantas/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Estresse Salino/genética
14.
Appl Environ Microbiol ; 86(7)2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-31980430

RESUMO

Unspecific peroxygenases (UPOs) constitute a new family of fungal heme-thiolate enzymes in which there is high biotechnological interest. Although several thousand genes encoding hypothetical UPO-type proteins have been identified in sequenced fungal genomes and other databases, only a few UPO enzymes have been experimentally characterized to date. Therefore, gene screening and heterologous expression from genetic databases are a priority in the search for ad hoc UPOs for oxyfunctionalization reactions of interest. Very recently, Escherichia coli production of a previously described basidiomycete UPO (as a soluble and active enzyme) has been reported. Here, we explored this convenient heterologous expression system to obtain the protein products from available putative UPO genes. In this way, two UPOs from the ascomycetes Collariella virescens (syn., Chaetomium virescens) and Daldinia caldariorum were successfully obtained, purified, and characterized. Comparison of their kinetic constants for oxidation of model substrates revealed 10- to 20-fold-higher catalytic efficiency of the latter enzyme in oxidizing simple aromatic compounds (such as veratryl alcohol, naphthalene, and benzyl alcohol). Homology molecular models of these enzymes showed three conserved and two differing residues in the distal side of the heme (the latter representing two different positions of a phenylalanine residue). Interestingly, replacement of the C. virescens UPO Phe88 by the homologous residue in the D. caldariorum UPO resulted in an F88L variant with 5- to 21-fold-higher efficiency in oxidizing these aromatic compounds.IMPORTANCE UPOs catalyze regio- and stereoselective oxygenations of both aromatic and aliphatic compounds. Similar reactions were previously described for cytochrome P450 monooxygenases, but UPOs have the noteworthy biotechnological advantage of being stable enzymes requiring only H2O2 to be activated. Both characteristics are related to the extracellular nature of UPOs as secreted proteins. In the present study, the limited repertoire of UPO enzymes available for organic synthesis and other applications is expanded with the description of two new ascomycete UPOs obtained by Escherichia coli expression of the corresponding genes as soluble and active enzymes. Moreover, directed mutagenesis in E. coli, together with enzyme molecular modeling, provided relevant structure-function information on aromatic substrate oxidation by these two new biocatalysts.


Assuntos
Chaetomium/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/genética , Oxigenases de Função Mista/genética , Xylariales/genética , Chaetomium/metabolismo , Escherichia coli/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/metabolismo , Oxigenases de Função Mista/metabolismo , Xylariales/metabolismo
15.
Res Vet Sci ; 128: 1-8, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31706217

RESUMO

The aim of this work was to identify the molecular characteristics of a chymotrypsin-like enzyme from Trichinella spiralis (Tschy) and its facilitation of larval penetration into enteral epithelial cells (EECs). The complete Tschy cDNA sequence was cloned and expressed in Escherichia coli BL21. RT-PCR, IIFA and western blotting showed that Tschy was expressed at the T. spiralis muscle larvae (ML), intestinal infective L1 larvae (IL1), adult worms (AW) and embryo stages and was primarily located in the stichosome of this parasite. The results of ELISA, IIFA and Far-western assays showed that there was a specific binding between rTschy and EECs, and the binding was dependent on the dose of both rTschy and EEC proteins. Confocal microscopy demonstrated that the binding was located in the EEC cytoplasm. rTschy facilitated T. spiralis larval penetration of EECs, and anti-rTschy antibodies impeded the larval intrusion of EECs. These results demonstrate that Tschy facilitated the larval intrusion of the host's enteral epithelium and could be a candidate molecular target for vaccine against the enteral invasive phase of T. spiralis.


Assuntos
Quimotripsina/genética , Expressão Gênica , Proteínas de Helminto/genética , Interações Hospedeiro-Parasita/fisiologia , Trichinella spiralis/fisiologia , Animais , Quimotripsina/metabolismo , Embrião não Mamífero/enzimologia , Embrião não Mamífero/fisiologia , Células Epiteliais/parasitologia , Escherichia coli/genética , Proteínas de Helminto/metabolismo , Intestino Delgado/parasitologia , Larva/enzimologia , Larva/genética , Larva/crescimento & desenvolvimento , Larva/fisiologia , Microrganismos Geneticamente Modificados/genética , Trichinella spiralis/enzimologia , Trichinella spiralis/genética , Trichinella spiralis/crescimento & desenvolvimento , Vacinas/análise
16.
PLoS One ; 14(12): e0226884, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31860644

RESUMO

Plasmodium vivax is the leading cause of malaria outside Africa and represents a significant health and economic burden on affected countries. A major obstacle for P. vivax eradication is the dormant hypnozoite liver stage that causes relapse infections and the limited antimalarial drugs that clear this stage. Advances in studying the hypnozoite and other unique biological aspects of this parasite are hampered by the lack of a continuous in vitro laboratory culture system and poor availability of molecular tools for genetic manipulation. In this study, we aim to develop molecular tools that can be used for genetic manipulation of P. vivax. A putative P. vivax centromere sequence (PvCEN) was cloned and episomal centromere based plasmids expressing a GFP marker were constructed. Centromere activity was evaluated using a rodent malaria parasite Plasmodium yoelii. A plasmid carrying PvCEN was stably maintained in asexual-stage parasites in the absence of drug pressure, and approximately 45% of the parasites retained the plasmid four weeks later. The same retention rate was observed in parasites possessing a native P. yoelii centromere (PyCEN)-based control plasmid. The segregation efficiency of the plasmid per nuclear division was > 99% in PvCEN parasites, compared to ~90% in a control parasite harboring a plasmid without a centromere. In addition, we observed a clear GFP signal in both oocysts and salivary gland sporozoites isolated from mosquitoes. In blood-stage parasites after liver stage development, GFP positivity in PvCEN parasites was comparable to control PyCEN parasites. Thus, PvCEN plasmids were maintained throughout the parasite life cycle. We also validated several P. vivax promoter activities and showed that hsp70 promoter (~1 kb) was active throughout the parasite life cycle. This is the first data for the functional characterization of a P. vivax centromere that can be used in future P. vivax biological research.


Assuntos
Centrômero/genética , Plasmodium vivax/genética , Plasmodium yoelii/genética , Regiões Promotoras Genéticas/genética , Animais , Segregação de Cromossomos/genética , Culicidae/parasitologia , Feminino , Proteínas de Fluorescência Verde/metabolismo , Humanos , Malária Vivax/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos ICR , Microrganismos Geneticamente Modificados/genética , Plasmídeos/genética , Plasmodium yoelii/crescimento & desenvolvimento , Glândulas Salivares/parasitologia , Esporozoítos/metabolismo , Tetra-Hidrofolato Desidrogenase/genética
17.
Malar J ; 18(1): 426, 2019 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-31849326

RESUMO

BACKGROUND: The circumsporozoite protein (CSP) of Plasmodium is a key surface antigen that induces antibodies and T-cells, conferring immune protection in animal models and humans. However, much of the work on CSP and immunity has been developed based on studies using rodent or non-human primate CSP antigens, which may not be entirely translatable to CSP expressed by human malaria parasites, especially considering the host specificity of the different species. METHODS: Using a genetically engineered strain of Plasmodium berghei that expresses luciferase, GFP and the Plasmodium falciparum orthologue of CSP, the effect of laboratory preparation, mosquito treatment and mouse factors on sporozoite infectivity was assessed using an in vivo bioluminescence assay on mice. This assay was compared with a PCR-based protection assay using an already described monoclonal antibody that can provide sterile protection against sporozoite challenge. RESULTS: Bioluminescence assay demonstrated similar detection levels of the quantity and kinetics of liver-stage infection, compared to PCR-based detection. This assay was used to evaluate treatment of sporozoite and delivery method on mouse infectivity, as well as the effects of age, sex and strain of mice. Finally, this assay was used to test the protective capacity of monoclonal antibody AB317; results strongly recapitulate the findings of previous work on this antibody. CONCLUSIONS: The PbGFP-Luc line and in vivo bioluminescence imaging provide highly sensitive read-outs of liver-stage infection in mice, and this method can be useful to reliably evaluate potency of pre-erythrocytic interventions.


Assuntos
Malária/imunologia , Plasmodium berghei/fisiologia , Animais , Anopheles/parasitologia , Feminino , Ensaios de Triagem em Larga Escala , Fígado/parasitologia , Luciferases/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/fisiologia , Plasmodium berghei/genética , Plasmodium falciparum/genética , Proteínas de Protozoários/metabolismo , Esporozoítos/crescimento & desenvolvimento
18.
Microb Cell Fact ; 18(1): 189, 2019 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-31690310

RESUMO

BACKGROUND: Oxygen-evolving photoautotrophic organisms, like cyanobacteria, protect their photosynthetic machinery by a number of regulatory mechanisms, including alternative electron transfer pathways. Despite the importance in modulating the electron flux distribution between the photosystems, alternative electron transfer routes may compete with the solar-driven production of CO2-derived target chemicals in biotechnological systems under development. This work focused on engineered cyanobacterial Synechocystis sp. PCC 6803 strains, to explore possibilities to rescue excited electrons that would normally be lost to molecular oxygen by an alternative acceptor flavodiiron protein Flv1/3-an enzyme that is natively associated with transfer of electrons from PSI to O2, as part of an acclimation strategy towards varying environmental conditions. RESULTS: The effects of Flv1/3 inactivation by flv3 deletion were studied in respect to three alternative end-products, sucrose, polyhydroxybutyrate and glycogen, while the photosynthetic gas fluxes were monitored by Membrane Inlet Mass Spectrometry (MIMS) to acquire information on cellular carbon uptake, and the production and consumption of O2. The results demonstrated that a significant proportion of the excited electrons derived from photosynthetic water cleavage was lost to molecular oxygen via Flv1/3 in cells grown under high CO2, especially under high light intensities. In flv3 deletion strains these electrons could be re-routed to increase the relative metabolic flux towards the monitored target products, but the carbon distribution and the overall efficiency were determined by the light conditions and the genetic composition of the respective pathways. At the same time, the total photosynthetic capacity of the Δflv3 strains was systematically reduced, and accompanied by upregulation of oxidative glycolytic metabolism in respect to controls with the native Flv1/3 background. CONCLUSIONS: The observed metabolic changes and respective production profiles were proposedly linked with the lack of Flv1/3-mediated electron transfer, and the associated decrease in the intracellular ATP/NADPH ratio, which is bound to affect the metabolic carbon partitioning in the flv3-deficient cells. While the deletion of flv3 could offer a strategy for enhancing the photosynthetic production of desired chemicals in cyanobacteria under specified conditions, the engineered target pathways have to be carefully selected to align with the intracellular redox balance of the cells.


Assuntos
Proteínas de Bactérias/genética , Flavoproteínas/genética , Microrganismos Geneticamente Modificados/metabolismo , Fotossíntese , Synechocystis , Transporte de Elétrons , Genes Bacterianos/genética , Microrganismos Geneticamente Modificados/genética , Deleção de Sequência/genética , Synechocystis/genética , Synechocystis/metabolismo
19.
Microb Cell Fact ; 18(1): 196, 2019 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-31699090

RESUMO

BACKGROUND: The thermostable serine protease pernisine originates from the hyperthermophilic Archaeaon Aeropyrum pernix and has valuable industrial applications. Due to its properties, A. pernix cannot be cultivated in standard industrial fermentation facilities. Furthermore, pernisine is a demanding target for heterologous expression in mesophilic heterologous hosts due to the relatively complex processing step involved in its activation. RESULTS: We achieved production of active extracellular pernisine in a Streptomyces rimosus host through heterologous expression of the codon-optimised gene by applying step-by-step protein engineering approaches. To ensure secretion of fully active enzyme, the srT signal sequence from the S. rimosus protease was fused to pernisine. To promote correct processing and folding of pernisine, the srT functional cleavage site motif was fused directly to the core pernisine sequence, this way omitting the proregion. Comparative biochemical analysis of the wild-type and recombinant pernisine confirmed that the enzyme produced by S. rimosus retained all of the desired properties of native pernisine. Importantly, the recombinant pernisine also degraded cellular and infectious bovine prion proteins, which is one of the particular applications of this protease. CONCLUSION: Functional pernisine that retains all of the advantageous properties of the native enzyme from the thermophilic host was successfully produced in a S. rimosus heterologous host. Importantly, we achieved extracellular production of active pernisine, which significantly simplifies further downstream procedures and also omits the need for any pre-processing step for its activation. We demonstrate that S. rimosus can be used as an attractive host for industrial production of recombinant proteins that originate from thermophilic organisms.


Assuntos
Aeropyrum/enzimologia , Proteínas de Bactérias , Endopeptidases , Microrganismos Geneticamente Modificados , Proteínas Recombinantes , Streptomyces rimosus , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Endopeptidases/genética , Endopeptidases/metabolismo , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Streptomyces rimosus/genética , Streptomyces rimosus/metabolismo
20.
Microb Cell Fact ; 18(1): 179, 2019 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-31640713

RESUMO

BACKGROUND: Pseudomonas putida is a metabolically versatile, genetically accessible, and stress-robust species with outstanding potential to be used as a workhorse for industrial applications. While industry recognises the importance of robustness under micro-oxic conditions for a stable production process, the obligate aerobic nature of P. putida, attributed to its inability to produce sufficient ATP and maintain its redox balance without molecular oxygen, severely limits its use for biotechnology applications. RESULTS: Here, a combination of genome-scale metabolic modelling and comparative genomics is used to pinpoint essential [Formula: see text]-dependent processes. These explain the inability of the strain to grow under anoxic conditions: a deficient ATP generation and an inability to synthesize essential metabolites. Based on this, several P. putida recombinant strains were constructed harbouring acetate kinase from Escherichia coli for ATP production, and a class I dihydroorotate dehydrogenase and a class III anaerobic ribonucleotide triphosphate reductase from Lactobacillus lactis for the synthesis of essential metabolites. Initial computational designs were fine-tuned by means of adaptive laboratory evolution. CONCLUSIONS: We demonstrated the value of combining in silico approaches, experimental validation and adaptive laboratory evolution for microbial design by making the strictly aerobic Pseudomonas putida able to grow under micro-oxic conditions.


Assuntos
Proteínas de Bactérias/genética , Microrganismos Geneticamente Modificados , Oxigênio/metabolismo , Pseudomonas putida , Acetato Quinase/genética , Acetato Quinase/metabolismo , Anaerobiose , Proteínas de Bactérias/metabolismo , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Genômica , Lactobacillus/enzimologia , Lactobacillus/metabolismo , Engenharia Metabólica , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , Ribonucleotídeo Redutases/genética , Ribonucleotídeo Redutases/metabolismo
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