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1.
Gene ; 718: 144073, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31446096

RESUMO

Cell morphology of the oleaginous fungus, Aspergillus oryzae BCC7051, was genetically engineered by disruption of non-essential genes involved in cell wall biosynthesis. Comparative phenotypic analysis of two disruptant strains defective either in α-1,3-glucan synthase 1 (ΔAoAgs1) or chitin synthase B (ΔAoChsB), and the wild type showed that the ΔAoAgs1 strain had no alterations in colonial growth and sporulation when grown on agar medium whereas the ΔAoChsB disruptant showed growth retardation and lower sporulation. However, tiny and loose pellets were found in the ΔAoAgs1 culture grown in liquid medium, where fungal pellet size was decreased by 35-50% of the wild type size. Further investigation of the ΔAoAgs1 mutant grown under stress-induced conditions, including high salt concentration, ionic strength and osmolarity, showed that its growth and development remained similar to that of the wild type. When cultivating the ΔAoAgs1 strain in a stirred-tank bioreactor, lipid production in terms of titer and productivity was significantly improved. As compared to the wild type, an increase of triacylglycerol and ergosterol contents with a proportional decrease in steryl ester content was observed in the ΔAoAgs1 strain. These results suggest that the morphologically engineered strain of A. oryzae is a robust cell chassis useful for exploitation in further production development of functional lipids with industrial significance.


Assuntos
Aspergillus oryzae/metabolismo , Ergosterol/biossíntese , Engenharia Metabólica , Microrganismos Geneticamente Modificados/metabolismo , Triglicerídeos/biossíntese , Aspergillus oryzae/genética , Quitina Sintase/genética , Quitina Sintase/metabolismo , Ergosterol/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Deleção de Genes , Genes Fúngicos , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Microrganismos Geneticamente Modificados/genética , Triglicerídeos/genética
2.
Malar J ; 18(1): 215, 2019 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-31238932

RESUMO

BACKGROUND: Reverse genetics approaches have become powerful tools to dissect the biology of malaria parasites. In a previous study, development of an in vitro drug selection method for generating transgenic parasite of Plasmodium berghei was reported. Using this method, two novel and independent selection markers using the P. berghei heat shock protein 70 promoter was previously established. While the approach permits the easy and flexible genetic manipulation of P. berghei, shortcomings include a low variety in promoter options to drive marker gene expression and increased complexity of the selection procedure. In this study, addressing these issues was attempted. METHODS: To secure a variety of promoters, the use of a P. berghei elongation factor-1α promoter for marker gene expression was attempted. To simplify the procedure of in vitro selection, the establishment of a two cell-cycle culture method and its application for drug selection were attempted. RESULTS: The P. berghei elongation factor-1α (pbef-1α) promoter, which is commonly used to drive marker gene expression, was successfully applied as an alternative promoter model for marker gene expression, using the parasite's codon-optimized marker sequence. To simplify the in vitro selection method, a two cell-cycle culture method in which the merozoite was released by filtration of the culture containing matured schizont-infected erythrocytes was also developed and successfully applied for drug selection. CONCLUSION: The pbef-1α promoter was successfully applied in an in vitro selection system. The in vitro selection procedure also could be simplified for practical use using a two cell-cycle culture method. These improvements provide a more versatile platform for the genetic manipulation of P. berghei.


Assuntos
Técnicas de Cultura de Células/métodos , Plasmodium berghei/genética , Animais , Antimaláricos/farmacologia , Feminino , Malária/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos ICR , Microrganismos Geneticamente Modificados/efeitos dos fármacos , Microrganismos Geneticamente Modificados/genética , Plasmodium berghei/efeitos dos fármacos
3.
Microb Cell Fact ; 18(1): 95, 2019 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-31138208

RESUMO

BACKGROUND: Glycyrrhetinic acid (GA) is the most important ingredient in licorice due to its outstanding anti-inflammatory activity and wide application in the medicine and cosmetics industries. Contemporary industrial production of GA by acid hydrolysis of glycyrrhizin which was extracted from Glycyrrhiza plants, is not environment-friendly and devastates farmland since the Glycyrrhiza rhizomes grow up to 10 m underground. RESULTS: In this study, GA was produced through metabolically engineering Saccharomyces cerevisiae by introducing the entire heterogeneous biosynthetic pathway of GA. Codon optimized CYP88D6 and CYP72A154, combined with ß-AS (ß-amyrin synthase encoding gene) and the NADPH-cytochrome P450 reductase gene of Arabidopsis thaliana were introduced into S. cerevisiae. The resulting strain (Y1) produced 2.5 mg/L of ß-amyrin and 14 µg/L of GA. The cytochrome b5 from G. uralensis (GuCYB5) was identified and the introduction of this novel GuCYB5 increased the efficiency of GA production by eightfold. The joint utilization of the GuCYB5 gene along with 10 known MVA pathway genes from S. cerevisiae were overexpressed in a stable chromosome integration to achieve higher GA production. Using the combined strategy, GA concentration improved by 40-fold during batch fermentation. The production was further improved to 8.78 mg/L in fed-batch fermentation, which was increased by a factor of nearly 630. CONCLUSIONS: This study first investigated the influence of carbon flux in the upstream module and the introduction of a newly identified GuCYB5 on GA production. The newly identified GuCYB5 was highly effective in improving GA production. An integrated strategy including enzyme discovery, pathway optimization, and fusion protein construction was provided in improving GA production, achieving a 630 fold increase in GA production. The metabolically engineered yeast cell factories provide an alternative approach to glycyrrhetinic acid production, replacing the traditional method of plant extraction.


Assuntos
Vias Biossintéticas/genética , Ácido Glicirretínico/metabolismo , Engenharia Metabólica/métodos , Microrganismos Geneticamente Modificados , Saccharomyces cerevisiae , Citocromos b5/genética , Fermentação , Transferases Intramoleculares/genética , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/metabolismo , NADPH-Ferri-Hemoproteína Redutase/genética , Proteínas Recombinantes de Fusão/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
4.
Sci Total Environ ; 659: 540-547, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-31096383

RESUMO

Mercury is a potentially toxic trace metal that poses threats to aquatic life and to humans. In this study, a mercury-binding peptide was displayed on the surface of Escherichia coli cells using an N-terminal region ice nucleation protein anchor. The surface-engineered E. coli facilitated selective adsorption of mercury ions (Hg2+) from a solution containing various metal ions. The Hg2+ adsorption capacity of the surface-engineered cell was four-fold higher than that of the original E. coli cells. Approximately 95% of Hg2+ was removed from solution by these whole-cell sorbents. The transformed strains were fed to Carassius auratus, so that the bacteria could colonize fish intestine. Engineered bacteria-fed C. auratus showed significantly less (51.1%) accumulation of total mercury when compared with the group that had not been fed engineered bacteria. The surface-engineered E. coli effectively protected fish against the toxicity of Hg2+ in aquatic environments by adsorbing more Hg2+. Furthermore, the surface-engineered E. coli mitigated microbial diversity changes in the intestine caused by Hg2+ exposure, thereby protecting the intestinal microbial community. This strategy is a novel approach for controlling Hg2+ contamination in fish.


Assuntos
Membrana Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , Carpa Dourada/metabolismo , Mercúrio/metabolismo , Peptídeos/metabolismo , Poluentes Químicos da Água/metabolismo , Animais , Animais Geneticamente Modificados/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Engenharia Genética , Mucosa Intestinal/metabolismo , Microrganismos Geneticamente Modificados/genética , Peptídeos/genética
5.
Nat Commun ; 10(1): 2142, 2019 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-31086174

RESUMO

Metabolic engineers endeavor to create a bio-based manufacturing industry using microbes to produce fuels, chemicals, and medicines. Plant natural products (PNPs) are historically challenging to produce and are ubiquitous in medicines, flavors, and fragrances. Engineering PNP pathways into new hosts requires finding or modifying a suitable host to accommodate the pathway, planning and implementing a biosynthetic route to the compound, and discovering or engineering enzymes for missing steps. In this review, we describe recent developments in metabolic engineering at the level of host, pathway, and enzyme, and discuss how the field is approaching ever more complex biosynthetic opportunities.


Assuntos
Produtos Biológicos/metabolismo , Engenharia Metabólica/métodos , Microrganismos Geneticamente Modificados/metabolismo , Plantas/metabolismo , Vias Biossintéticas/genética , Enzimas/genética , Enzimas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Engenharia Metabólica/tendências , Microrganismos Geneticamente Modificados/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Biologia Sintética/métodos , Biologia Sintética/tendências
6.
Metab Eng ; 53: 24-34, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30605774

RESUMO

The microbial membrane serves as a biological barrier that separates the interior of cells from the external environment, thus playing an important role in tolerance to stress conditions during industrial bioprocessing. Accordingly, engineering or regulation of membrane functions provides a great opportunity to improve the robustness of industrial strains and may enable increased titers, yield, and production of the targeted metabolites. In this review, we summarize the recent progress in metabolic engineering strategies to enhance membrane integrity, regulate membrane fluidity, and tune membrane permeability.


Assuntos
Membrana Celular , Microbiologia Industrial/métodos , Engenharia Metabólica/métodos , Microrganismos Geneticamente Modificados , Estresse Fisiológico , Membrana Celular/genética , Membrana Celular/metabolismo , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/metabolismo
7.
J Ind Microbiol Biotechnol ; 46(2): 221-230, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30600411

RESUMO

L-Serine is widely used in pharmaceutical, food and cosmetic industries, and the direct fermentation to produce L-serine from cheap carbon sources such as glycerol is greatly desired. The production of L-serine by engineered Escherichia coli from glycerol has not been achieved so far. In this study, E. coli was engineered to efficiently produce L-serine from glycerol. To this end, three L-serine deaminase genes were deleted in turn, and all of the deletions caused the maximal accumulation of L-serine at 0.06 g/L. Furthermore, removal of feedback inhibition by L-serine resulted in a titer of 1.1 g/L. Additionally, adaptive laboratory evolution was employed to improve glycerol utilization in combination with the overexpression of the cysteine/acetyl serine transporter gene eamA, leading to 2.36 g/L L-serine (23.6% of the theoretical yield). In 5-L bioreactor, L-serine titer could reach up to 7.53 g/L from glycerol, demonstrating the potential of the established strain and bioprocess.


Assuntos
Escherichia coli/metabolismo , Glicerol/metabolismo , Serina/biossíntese , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Reatores Biológicos , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fermentação , Regulação Bacteriana da Expressão Gênica , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/metabolismo
8.
J Ind Microbiol Biotechnol ; 46(2): 231-239, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30604237

RESUMO

Salt stress can trigger several physiological responses in microorganisms such as the increasing accumulation of unsaturated fatty acid, which was biosynthesized by delta-9 fatty acid desaturases (D9D) at the first step. In the present study, two D9D genes, designated AoD9D1 and AoD9D2, were isolated from Aspergillus oryzae. The expression analysis showed that AoD9D1 and AoD9D2 were upregulated under salt stress. To investigate the function of AoD9D, transgenic Saccharomyces cerevisiae strains that heterologously expressed AoD9D were exposed to salinity condition. These transgenic strains exhibited greater tolerance to salt stress than wild-type strains, and the heterologous expression of AoD9D increased the content in unsaturated fatty acids as compared to control cells. Moreover, AoD9D1 and AoD9D2 both contained fatty acid desaturase (FAD) and cytochrome b5-like Heme/Steroid-binding domains (Cyt-b5). S. cerevisiae separately transformed with the gene fragments coding for the FAD and Cyt-b5 domains in the AoD9D1 protein grew better and accumulated a higher concentration of unsaturated FAs than the control. Altogether, the heterologous expression of AoD9D enhanced the tolerance of transgenic S. cerevisiae to high salinity stress with increased accumulation of unsaturated fatty acid. The results provide some practical basis for the successful development of salt-tolerant fermentation microorganisms.


Assuntos
Ácidos Graxos Insaturados/metabolismo , Saccharomyces cerevisiae/metabolismo , Tolerância ao Sal/genética , Estearoil-CoA Dessaturase/metabolismo , Sequência de Aminoácidos , Aspergillus oryzae/genética , Aspergillus oryzae/metabolismo , Citocromos b5/genética , Citocromos b5/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Deleção de Genes , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/metabolismo , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/genética , Estearoil-CoA Dessaturase/genética
9.
J Ind Microbiol Biotechnol ; 46(2): 125-132, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30623269

RESUMO

Typical N-acetyl-D-neuraminic acid (Neu5Ac) production uses N-acetyl-D-glucosamine (GlcNAc) and excess pyruvate as substrates in the enzymatic or whole-cell biocatalysis process. In a previous study, a Neu5Ac-producing biocatalytic process via engineered Escherichia coli SA-05/pDTrc-AB/pCDF-pck-ppsA was constructed without exogenous pyruvate. In this study, glycerol was found to be a good energy source compared with glucose for the catalytic system with resting cells, and Neu5Ac production increased to 13.97 ± 0.27 g L-1. In addition, a two-stage pH shift strategy was carried out, and the Neu5Ac yield was improved to 14.61 ± 0.31 g L-1. The GlcNAc concentration for Neu5Ac production was optimized. Finally, an integrated strategy was developed for Neu5Ac production, and the Neu5Ac yield reached as high as 18.17 ± 0.27 g L-1. These results provide a new biocatalysis technology for Neu5Ac production without exogenous pyruvate.


Assuntos
Escherichia coli/genética , Microrganismos Geneticamente Modificados/genética , Ácido N-Acetilneuramínico/biossíntese , Acetilglucosamina/metabolismo , Técnicas de Cultura Celular por Lotes , Biocatálise , Escherichia coli/metabolismo , Glicerol/metabolismo , Concentração de Íons de Hidrogênio , Engenharia Metabólica , Ácido Pirúvico/metabolismo
10.
BMC Vet Res ; 15(1): 6, 2019 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-30606265

RESUMO

BACKGROUND: Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia and represents a major burden to the livestock industry. Virulence can largely be attributed to the secretion of a series of haemolytic toxins, which are highly immunogenic. A. pleuropneumoniae also encodes a cytoplasmic N-glycosylation system, which involves the modification of high molecular weight adhesins with glucose residues. Central to this process is the soluble N-glycosyl transferase, ngt, which is encoded in an operon with a subsequent glycosyl transferase, agt. Plasmid-borne recombinant expression of these genes in E. coli results in the production of a glucose polymer on peptides containing the appropriate acceptor sequon, NX(S/T). However to date, there is little evidence to suggest that such a glucose polymer is formed on its target peptides in A. pleuropneumoniae. Both the toxins and glycosylation system represent potential targets for the basis of a vaccine against A. pleuropneumoniae infection. RESULTS: In this study, we developed cytoplasmic glycoengineering to construct glycoconjugate vaccine candidates composed of soluble toxin fragments modified by glucose. We transferred ngt and agt to the chromosome of Escherichia coli in order to generate a native-like operon for glycoengineering. A single chromosomal copy of ngt and agt resulted in the glucosylation of toxin fragments by a short glycan, rather than a polymer. CONCLUSIONS: A vaccine candidate that combines toxin fragment with a conserved glycan offers a novel approach to generating epitopes important for both colonisation and disease progression.


Assuntos
Infecções por Actinobacillus/veterinária , Actinobacillus pleuropneumoniae/imunologia , Toxinas Bacterianas/imunologia , Vacinas Bacterianas/imunologia , Infecções por Actinobacillus/imunologia , Infecções por Actinobacillus/prevenção & controle , Animais , Escherichia coli/genética , Engenharia Genética/métodos , Engenharia Genética/veterinária , Glicoconjugados/genética , Glicoconjugados/imunologia , Microrganismos Geneticamente Modificados/genética , Pleuropneumonia/imunologia , Pleuropneumonia/prevenção & controle , Pleuropneumonia/veterinária , Suínos , Doenças dos Suínos/imunologia , Doenças dos Suínos/microbiologia , Doenças dos Suínos/prevenção & controle , Vacinas Conjugadas/imunologia
11.
Protist ; 170(1): 52-63, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30576875

RESUMO

Volvox carteri is an excellent model for investigating the evolution of multicellularity and cell differentiation, and the rate of future progress with this system will depend on improved molecular genetic tools. Several selectable markers for nuclear transformation of V. carteri have been developed, including the nitrate reductase (nitA) gene, but it would be useful to have additional markers to multiplex transgenes in this species. To further facilitate molecular genetic analyses of V. carteri, we developed two new selectable markers that provide rapid, easily selected, and stable resistance to the antibiotics hygromycin and blasticidin. We generated constructs with Volvox-specific regulatory sequences and codon-optimized hygromycin (VcHyg) and blasticidin (VcBlast) resistance genes from Coccidioides posadasii and Bacillus cereus, respectively. With these constructs, transformants were obtained via biolistic bombardment at rates of 0.5-13 per million target cells bombarded. Antibiotic-resistant survivors were readily isolated 7days post bombardment. VcHyg and VcBlast transgenes and transcripts were detected in transformants. Co-transformation rates using the VcHyg or VcBlast markers with unselected genes were comparable to those obtained with nitA. These results indicate that the pVcHyg and pVcBlast plasmids are highly efficient and convenient for transforming and co-transforming a broad range of V. carteri strains.


Assuntos
Antibacterianos/farmacologia , Cinamatos/farmacologia , Resistência Microbiana a Medicamentos/genética , Higromicina B/análogos & derivados , Transformação Genética/genética , Volvox/genética , Bacillus cereus/genética , Coccidioides/genética , Genes Bacterianos/genética , Genes Fúngicos/genética , Marcadores Genéticos/genética , Higromicina B/farmacologia , Microrganismos Geneticamente Modificados/genética , Nucleosídeos/farmacologia , Transformação Genética/efeitos dos fármacos , Volvox/efeitos dos fármacos
12.
Biotechnol Appl Biochem ; 66(2): 153-162, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30571850

RESUMO

Renewable energy resources are considered to be promising for the development of a sustainable circular economy. Among various alternatives, the microbial route for various biofuels production is quite lucrative. Use of cellulose and lignocellulose for methane, H2 , organic acids, ethanol, and cellulase has been explored a lot in the past few decades. The major leftover or a coproduct of these processes belongs to lignin-an aromatic cross-link polymer and one of the most abundant complex compounds on earth. A successful bioconversion route of lignin into high-value products is highly desirable for biorefinery perspective. It requires a complex set of enzymes/catalysts to decompose lignin through depolymerization and oxygen removal leading to its monomers that can be metabolized by engineered organisms to synthesize muconic acids, polyhydroxyalkanoates (PHAs), methane, and other high-value products. This article will focus on the opportunities and challenges in the bioconversion of lignin and its derivatives into PHAs.


Assuntos
Lignina/metabolismo , Metano/metabolismo , Microrganismos Geneticamente Modificados/metabolismo , Poli-Hidroxialcanoatos/biossíntese , Ácido Sórbico/análogos & derivados , Microrganismos Geneticamente Modificados/genética , Ácido Sórbico/metabolismo
13.
Int J Mol Sci ; 20(1)2018 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-30583567

RESUMO

Natural rubber is a kind of indispensable biopolymers with great use and strategic importance in human society. However, its production relies almost exclusively on rubber-producing plants Hevea brasiliensis, which have high requirements for growth conditions, and the mechanism of natural rubber biosynthesis remains largely unknown. In the past two decades, details of the rubber chain polymerization and proteins involved in natural rubber biosynthesis have been investigated intensively. Meanwhile, omics and other advanced biotechnologies bring new insight into rubber production and development of new rubber-producing plants. This review summarizes the achievements of the past two decades in understanding the biosynthesis of natural rubber, especially the massive information obtained from the omics analyses. Possibilities of natural rubber biosynthesis in vitro or in genetically engineered microorganisms are also discussed.


Assuntos
Genoma de Planta/fisiologia , Hevea/fisiologia , Proteínas de Plantas/metabolismo , Borracha/metabolismo , Biotecnologia , Engenharia Genética , Genoma de Planta/genética , Hevea/genética , Humanos , Técnicas In Vitro , Microrganismos Geneticamente Modificados/genética , Proteínas de Plantas/genética , Proteoma , Transcriptoma
14.
Proc Natl Acad Sci U S A ; 115(40): E9271-E9279, 2018 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-30224468

RESUMO

Gaseous one-carbon (C1) compounds or formic acid (FA) converted from CO2 can be an attractive raw material for bio-based chemicals. Here, we report the development of Escherichia coli strains assimilating FA and CO2 through the reconstructed tetrahydrofolate (THF) cycle and reverse glycine cleavage (gcv) pathway. The Methylobacterium extorquens formate-THF ligase, methenyl-THF cyclohydrolase, and methylene-THF dehydrogenase genes were expressed to allow FA assimilation. The gcv reaction was reversed by knocking out the repressor gene (gcvR) and overexpressing the gcvTHP genes. This engineered strain synthesized 96% and 86% of proteinogenic glycine and serine, respectively, from FA and CO2 in a glucose-containing medium. Native serine deaminase converted serine to pyruvate, showing 4.5% of pyruvate-forming flux comes from FA and CO2 The pyruvate-forming flux from FA and CO2 could be increased to 14.9% by knocking out gcvR, pflB, and serA, chromosomally expressing gcvTHP under trc, and overexpressing the reconstructed THF cycle, gcvTHP, and lpd genes in one vector. To reduce glucose usage required for energy and redox generation, the Candida boidinii formate dehydrogenase (Fdh) gene was expressed. The resulting strain showed specific glucose, FA, and CO2 consumption rates of 370.2, 145.6, and 14.9 mg⋅g dry cell weight (DCW)-1⋅h-1, respectively. The C1 assimilation pathway consumed 21.3 wt% of FA. Furthermore, cells sustained slight growth using only FA and CO2 after glucose depletion, suggesting that combined use of the C1 assimilation pathway and C. boidinii Fdh will be useful for eventually developing a strain capable of utilizing FA and CO2 without an additional carbon source such as glucose.


Assuntos
Proteínas de Bactérias , Dióxido de Carbono/metabolismo , Escherichia coli , Formiato-Tetra-Hidrofolato Ligase , Formiatos/metabolismo , Methylobacterium extorquens/genética , Microrganismos Geneticamente Modificados , Proteínas de Bactérias/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Formiato-Tetra-Hidrofolato Ligase/genética , Formiato-Tetra-Hidrofolato Ligase/metabolismo , Técnicas de Silenciamento de Genes , Methylobacterium extorquens/enzimologia , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/metabolismo
15.
Vet Res ; 49(1): 87, 2018 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-30189894

RESUMO

Trichinellosis is a worldwide important food-borne zoonosis caused mainly by ingesting raw or undercooked pork infected with Trichinella spiralis larvae. The development of vaccine is needed for preventing swine from Trichinella infection to ensure pork safety. Previous studies showed that T. spiralis serine protease 1.2 (TsSP1.2) is a vaccine candidate against Trichinella infection. In this study, the complete TsSP1.2 cDNA sequences were cloned into pcDNA3.1, and the rTsSP1.2 DNA was transformed into attenuated Salmonella typhimurium strain ΔcyaSL1344. Oral vaccination of mice with Salmonella-delivered rTsSP1.2 DNA vaccine induced an obvious intestinal mucosal IgA response and a systemic Th1/Th2 immune response; the vaccinated mice showed a 33.45% reduction of intestinal adult worms and 71.84% reduction of muscle larvae after T. spiralis larval challenge. The protection might be due to the rTsSP1.2-induced production of specific anti-TsSP1.2 sIgA, IgG, IgG1/IgG2a, and secretion of IFN-γ, IL-4 and IL-10, which protected intestinal mucosa from the parasite invasion, inhibited worm development and reduced female fecundity. The results indicate that the attenuated Salmonella-delivered rTsSP1.2 DNA vaccine offers a prospective strategy for the prevention and control of animal Trichinella infection.


Assuntos
Proteínas de Helminto/imunologia , Imunidade nas Mucosas , Serina Proteases/imunologia , Trichinella spiralis/imunologia , Vacinas de DNA/administração & dosagem , Administração Oral , Animais , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Microrganismos Geneticamente Modificados/genética , Salmonella typhimurium/genética , Vacinação , Vacinas Atenuadas/administração & dosagem
16.
Proc Natl Acad Sci U S A ; 115(36): E8509-E8517, 2018 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-30061389

RESUMO

Re-engineering of complex biological systems (CBS) is an important goal for applications in synthetic biology. Efforts have been made to simplify CBS by refactoring a large number of genes with rearranged polycistrons and synthetic regulatory circuits. Here, a posttranslational protein-splicing strategy derived from RNA viruses was exploited to minimize gene numbers of the classic nitrogenase system, where the expression stoichiometry is particularly important. Operon-based nif genes from Klebsiella oxytoca were regrouped into giant genes either by fusing genes together or by expressing polyproteins that are subsequently cleaved with Tobacco Etch Virus protease. After several rounds of selection based on protein expression levels and tolerance toward a remnant C-terminal ENLYFQ-tail, a system with only five giant genes showed optimal nitrogenase activity and supported diazotrophic growth of Escherichia coli This study provides an approach for efficient translation from an operon-based system into a polyprotein-based assembly that has the potential for portable and stoichiometric expression of the complex nitrogenase system in eukaryotic organisms.


Assuntos
Proteínas de Bactérias , Escherichia coli , Klebsiella oxytoca/genética , Microrganismos Geneticamente Modificados , Fixação de Nitrogênio , Óperon , Poliproteínas , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Endopeptidases/genética , Endopeptidases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/metabolismo , Poliproteínas/biossíntese , Poliproteínas/genética
17.
Int J Mol Sci ; 19(8)2018 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-30071625

RESUMO

Ipomoea pes-caprae L. is an extremophile halophyte with strong adaptability to seawater and drought. It is widely used in the ecological restoration of coastal areas or degraded islands in tropical and subtropical regions. In this study, a new abscisic acid, stressandripening (ASR) gene, IpASR, was reported, and is mainly associated with biological functions involved in salt and drought tolerance. Sequence analysis of IpASR showed that this protein contains an ABA/WDS (abscisic acid/water deficit stress) domain, which is a common feature of all plant ASR members. Overexpression of IpASR improved Escherichia coli growth performance compared with the control under abiotic stress treatment. The transgenic overexpressing IpASR Arabidopsis showed higher tolerance to salt and drought stress than the wild type and lower accumulation of hydrogen peroxide (H2O2) and superoxide (O2-) accompanied by increased antioxidant enzyme activity in vivo. IpASR exhibits transcription factor's activity. Therefore, the overexpression of IpASR in Arabidopsis is supposed to influence the expression of some genes involved in anti-oxidative and abiotic stresses. The results indicate that IpASR is involved in the plant response to salt and drought and probably acts as a reactive oxygen species scavenger or transcription factor, and therefore influences physiological processes associated with various abiotic stresses in plants.


Assuntos
Arabidopsis , Escherichia coli , Ipomoea/genética , Microrganismos Geneticamente Modificados , Proteínas de Plantas , Plantas Geneticamente Modificadas , Salinidade , Plantas Tolerantes a Sal , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Desidratação/genética , Desidratação/metabolismo , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/crescimento & desenvolvimento , Proteínas de Plantas/biossíntese , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Tolerantes a Sal/genética , Plantas Tolerantes a Sal/crescimento & desenvolvimento
18.
J Microbiol Biotechnol ; 28(8): 1225-1232, 2018 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-29996621

RESUMO

Phenylethanoids, including 2-phenylethanol, tyrosol, and salidroside are a group of phenolic compounds with a C6-C2 carbon skeleton synthesized by plants. Phenylethanoids display a variety of biological activities, including antibacterial, anticancer, anti-inflammatory, neuroprotective, and anti-asthmatic activities. Recently, successful microbial synthesis of phenylethanoids through metabolic engineering and synthetic biology approaches has been reported and could allow phenylethanoid production from alternative microbial sources. Here, we review the recent achievements in the synthesis of phenylethanoids by microorganisms. The work done so far will contribute to the production of diverse phenylethanoids using various microbial systems and facilitate exploration of further diverse biological activities of phenylethanoids.


Assuntos
Produtos Biológicos/metabolismo , Engenharia Metabólica , Microrganismos Geneticamente Modificados/metabolismo , Álcool Feniletílico/análogos & derivados , Álcool Feniletílico/metabolismo , Bactérias/genética , Bactérias/metabolismo , Vias Biossintéticas , Fungos/genética , Fungos/metabolismo , Engenharia Metabólica/tendências , Microrganismos Geneticamente Modificados/genética , Biologia Sintética
19.
Vet Res ; 49(1): 57, 2018 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-29976253

RESUMO

The obligate intracellular pathogen Lawsonia intracellularis (LI), the etiological agent of proliferative enteropathy (PE), poses a substantial economic loss in the swine industry worldwide. In this study, we genetically engineered an O-antigen-deficient (rough) Salmonella strain secreting four selected immunogenic LI antigens, namely OptA, OptB, LfliC, and Lhly. The genes encoding these antigens were individually inserted in the expression vector plasmid pJHL65, and the resultant plasmids were transformed into the ∆asd ∆lon ∆cpxR ∆rfaL Salmonella Typhimurium (ST) strain JOL1800. The individual expression of the selected LI antigens in JOL1800 was validated by an immunoblotting assay. We observed significant (P < 0.05) induction of systemic IgG and mucosal IgA responses against each LI antigen or Salmonella outer membrane protein in mice immunized once orally with a mixture of four JOL1800-derived strains. Further, mRNA of IL-4 and IFN-γ were highly upregulated in splenic T cells re-stimulated in vitro with individual purified antigens. Subsequently, immunized mice showed significant protection against challenge with 106.9 TCID50 LI or 2 × 109 CFU of a virulent ST strain. At day 8 post-challenge, no mice in the immunized groups showed the presence of LI-specific genomic DNA (gDNA) in stool samples, while 50% of non-immunized mice were positive for LI-specific gDNA. Further, all the immunized mice survived the virulent ST challenge, compared to a 20% mortality rate observed in the control mice. Collectively, the constructed rough ST-based LI vaccine candidate efficiently elicited LI and ST-specific humoral and cell-mediated immunity and conferred proper dual protection against PE and salmonellosis.


Assuntos
Infecções por Desulfovibrionaceae/veterinária , Imunização/veterinária , Lawsonia (Bactéria)/imunologia , Antígenos O/imunologia , Salmonella typhimurium/imunologia , Doenças dos Suínos/prevenção & controle , Animais , Infecções por Desulfovibrionaceae/imunologia , Infecções por Desulfovibrionaceae/microbiologia , Infecções por Desulfovibrionaceae/prevenção & controle , Feminino , Imunogenicidade da Vacina/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/imunologia , Salmonella typhimurium/genética , Organismos Livres de Patógenos Específicos , Suínos , Doenças dos Suínos/imunologia , Doenças dos Suínos/microbiologia
20.
Bioprocess Biosyst Eng ; 41(9): 1383-1390, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29948210

RESUMO

(R)-2-hydroxy-4-phenylbutyric acid (R-HPBA) is a valuable intermediate for the synthesis of angiotensin-converting enzyme inhibitors. The asymmetric reduction of 2-oxo-4-phenylbutyric acid (OPBA) by oxidoreductases is an efficient approach for its synthesis. Here, we report a novel biocatalytic approach for asymmetric synthesis of R-HPBA using recombinant Pichia pastoris expressing the Tyr52Leu variant of D-lactate dehydrogenase (D-LDH) from Lactobacillus plantarum. The recombinant yeast cells showed impressive catalytic activity at a high concentration of NaOPBA (380 mM, 76 g/L) and achieved full conversion starting with 40 g/L NaOPBA or even at higher concentration. Under optimized reaction conditions (pH 7.5, 37 °C, and 2% glucose), a full conversion with > 95% reaction yield and ~ 100% product enantiomeric excess (ee) was achieved for the preparation of R-HPBA on a 2-g scale. The findings of this study promote both the biotransformation of R-HPBA and an extension of the application of recombinant yeast as biocatalysts.


Assuntos
Proteínas de Bactérias , L-Lactato Desidrogenase , Lactobacillus plantarum/genética , Microrganismos Geneticamente Modificados , Fenilbutiratos/metabolismo , Pichia , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , L-Lactato Desidrogenase/biossíntese , L-Lactato Desidrogenase/genética , Lactobacillus plantarum/enzimologia , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/metabolismo , Pichia/genética , Pichia/metabolismo
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