Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 7.421
Filtrar
1.
An Acad Bras Cienc ; 91(suppl 3): e20190568, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31576934

RESUMO

Early humans have domesticated plant and animal species based on ancient empirical concepts (Darwin 1868, 1876). In 1886, Mendel established a new paradigm of hereditary laws (Mendel 1866, 1870, 1950) based on genotypic and phenotypic traits of cross-compatible species, establishing a complex breeding technology that is currently utilized for the development of most food and livestock-derived products. Recently, studies on deciphering the double-helical structure (Watson and Crick 1953a, b) and how to restrict DNA (Arber 2012) have established the foundation of recombinant DNA technology. A new era is paving the way for genetic manipulation of important traits among all the kingdom's organisms, allowing for the development of innovative and widely utilized products for the agricultural, industrial and pharmaceutical production sectors (Mc Elroy 2003, 2004, ISAAA 2016).


Assuntos
Biodiversidade , Conservação dos Recursos Naturais/métodos , Engenharia Genética/métodos , Criação de Animais Domésticos , Animais , Biotecnologia
2.
Mol Plant Microbe Interact ; 32(9): 1095-1109, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31365325

RESUMO

Verticillium longisporum is a vascular fungal pathogen leading to severe crop loss, particular in oilseed rape. Transcription factors (TF) are highly suited for genetic engineering of pathogen-resistant crops, as they control sets of functionally associated genes. Applying the AtTORF-Ex (Arabidopsis thaliana transcription factor open reading frame expression) collection, a simple and robust screen of TF-overexpressing plants was established displaying reduced fungal colonization. Distinct members of the large ethylene response factor (ERF) family, namely ERF96 and the six highly related subgroup IXb members ERF102 to ERF107, were identified. Whereas overexpression of these ERF significantly reduces fungal propagation, single loss-of-function approaches did not reveal altered susceptibility. Hence, this gain-of-function approach is particularly suited to identify redundant family members. Expression analyses disclosed distinct ERF gene activation patterns in roots and leaves, suggesting functional differences. Transcriptome studies performed on chemically induced ERF106 expression revealed an enrichment of genes involved in the biosynthesis of antimicrobial indole glucosinolates (IG), such as CYP81F2 (CYTOCHROME P450-MONOOXYGENASE 81F2), which is directly regulated by IXb-ERF via two GCC-like cis-elements. The impact of IG in restricting fungal propagation was further supported as the cyp81f2 mutant displayed significantly enhanced susceptibility. Taken together, this proof-of-concept approach provides a novel strategy to identify candidate TF that are valuable genetic resources for engineering or breeding pathogen-resistant crop plants.


Assuntos
Cruzamento , Resistência à Doença , Engenharia Genética , Fatores de Transcrição , Verticillium , Brassica rapa/microbiologia , Resistência à Doença/genética , Mutação com Ganho de Função , Regulação da Expressão Gênica de Plantas , Engenharia Genética/métodos , Fatores de Transcrição/genética
3.
BMC Plant Biol ; 19(1): 246, 2019 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-31182023

RESUMO

BACKGROUND: Rapid-cycling Brassica rapa (RCBr), also known as Wisconsin Fast Plants, are small robust plants with a short lifecycle that are widely used in biology teaching. RCBr have been used for decades but there are no published reports of RCBr genetic transformation. Agrobacterium-mediated vacuum infiltration has been used to transform pakchoi (Brassica rapa ssp. chinensis) and may be suitable for RCBr transformation. The floral dip transformation method, an improved version of vacuum infiltration, could make the procedure easier. RESULTS: Based on previous findings from Arabidopsis and pakchoi, plants of three different ages were inoculated with Agrobacterium. Kanamycin selection was suboptimal with RCBr; a GFP screen was used to identify candidate transformants. RCBr floral bud dissection showed that only buds with a diameter less than 1 mm carried unsealed carpels, a key point of successful floral dip transformation. Plants across a wide range of inflorescence maturities but containing these immature buds were successfully transformed, at an overall rate of 0.1% (one per 1000 T1 seeds). Transformation was successful using either vacuum infiltration or the floral dip method, as confirmed by PCR and Southern blot. CONCLUSION: A genetic transformation system for RCBr was established in this study. This will promote development of new biology teaching tools as well as basic biology research on Brassica rapa.


Assuntos
Agrobacterium/fisiologia , Brassica rapa/genética , Brassica rapa/microbiologia , Engenharia Genética/métodos , Transformação Genética , Southern Blotting , Flores/genética , Reação em Cadeia da Polimerase
4.
BMC Plant Biol ; 19(1): 237, 2019 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-31170915

RESUMO

BACKGROUND: The Agrobacterium-mediated transient transformation is a versatile and indispensable way of rapid analyzing gene function in plants. Despite this transient expression system has been successfully applied in a number of plant species, it is poorly developed in Caragana intermedia. RESULTS: In this study, we established an Agrobacterium-mediated transient expression system in C. intermedia leaves and optimized the effect of different Agrobacterial strains, several surfactants and the concentration of Silwet L-77, which would affect transient expression efficiency. Among the 5 Agrobacterial strains examined, GV3101 produced the highest GUS expression level. Besides, higher level of transient expression was observed in plants infiltrated with Silwet L-77 than with Triton X-100 or Tween-20. Silwet L-77 at a concentration of 0.001% greatly improved the level of GUS transient expression. Real-time PCR showed that expression of CiDREB1C was highly up-regulated in transiently expressed plants and reached the highest level at the 2nd day after infiltration. Based on this optimized transient transformation method, we characterized CiDREB1C function in response to drought, salt and ABA treatment. The results showed that transiently expressed CiDREB1C in C. intermedia leaves could enhance the survival rate and chlorophyll content, and reduce the lodging rate compared with the control seedlings under drought, salt and ABA treatments. Furthermore, the rate of leaf shedding of CiDREB1C transient expression seedlings was lower than that of the control under ABA treatment. CONCLUSIONS: The optimized transient expression condition in C. intermedia leaves were infiltrated with Agrobacterial strains GV3101 plus Silwet L-77 at a concentration of 0.001% added into the infiltration medium. Transiently expressed CiDREB1C enhanced drought, salt and ABA stress tolerance, indicated that it was a suitable and effective tool to determine gene function involved in abiotic stress response in C. intermedia.


Assuntos
Agrobacterium/genética , Caragana/fisiologia , Regulação da Expressão Gênica de Plantas , Engenharia Genética/métodos , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/fisiologia , Fatores de Transcrição/genética , Proteínas de Arabidopsis , Caragana/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Fatores de Transcrição/metabolismo
5.
Nat Commun ; 10(1): 2883, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253768

RESUMO

A substantial number of mouse genes, about 25%, are embryonically lethal when knocked out. Using current genetic tools, such as the CRISPR-Cas9 system, it is difficult-or even impossible-to produce viable mice with heritable embryonically lethal mutations. Here, we establish a one-step method for microinjection of CRISPR reagents into one blastomere of two-cell embryos to generate viable chimeric founder mice with a heritable embryonically lethal mutation, of either Virma or Dpm1. By examining founder mice, we identify a phenotype and role of Virma in regulating kidney metabolism in adult mice. Additionally, we generate knockout mice with a heritable postnatally lethal mutation, of either Slc17a5 or Ctla-4, and study its function in vivo. This one-step method provides a convenient system that rapidly generates knockout mice possessing lethal phenotypes. This allows relatively easy in vivo study of the associated genes' functions.


Assuntos
Sistemas CRISPR-Cas , Embrião de Mamíferos/fisiologia , Animais , Desenvolvimento Embrionário , Feminino , Edição de Genes/métodos , Engenharia Genética/métodos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Knockout , Mutação , RNA Guia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
6.
Nat Commun ; 10(1): 2673, 2019 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-31209208

RESUMO

Alternative splicing performs a central role in expanding genomic coding capacity and proteomic diversity. However, programming of splicing patterns in engineered biological systems remains underused. Synthetic approaches thus far have predominantly focused on controlling expression of a single protein through alternative splicing. Here, we describe a modular and extensible platform for regulating four programmable exons that undergo a mutually exclusive alternative splicing event to generate multiple functionally-distinct proteins. We present an intron framework that enforces the mutual exclusivity of two internal exons and demonstrate a graded series of consensus sequence elements of varying strengths that set the ratio of two mutually exclusive isoforms. We apply this framework to program the DNA-binding domains of modular transcription factors to differentially control downstream gene activation. This splicing platform advances an approach for generating diverse isoforms and can ultimately be applied to program modular proteins and increase coding capacity of synthetic biological systems.


Assuntos
Processamento Alternativo/genética , Regulação da Expressão Gênica/genética , Engenharia Genética/métodos , RNA/genética , Fatores de Transcrição/genética , Motivos de Aminoácidos/genética , Animais , Linhagem Celular , Biologia Computacional , Sequência Consenso/genética , Éxons/genética , Biblioteca Gênica , Genes Reporter/genética , Humanos , Íntrons/genética , Mutagênese Sítio-Dirigida/métodos , Domínios Proteicos/genética , Isoformas de Proteínas/genética , RNA/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética
7.
World J Microbiol Biotechnol ; 35(6): 79, 2019 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-31134410

RESUMO

The methylotrophic yeast Pichia pastoris is widely used in recombinant expression of eukaryotic proteins owing to the ability of post-translational modification, tightly regulated promoters, and high cell density fermentation. However, episomal plasmids for heterologous gene expression and the CRISPR/Cas9 system for genome editing have not been well developed in P. pastoris. In the present study, a panel of episomal plasmids containing various autonomously replicating sequences (ARSs) were constructed and their performance in transformation efficiency, copy numbers, and propagation stability were systematically compared. Among the five ARSs with different origins, panARS isolated from Kluyveromyces lactis was determined to have the best performance and used to develop an efficient CRISPR/Cas9 based genome editing system. Compared with a previously reported system using the endogenous and most commonly used ARS (PARS1), the CRISPR/Cas9 genome editing efficiency was increased for more than tenfold. Owing to the higher plasmid stability with panARS, efficient CRISPR/Cas9-mediated genome editing with a type III promoter (i.e. SER promoter) to drive the expression of the single guide RNA (sgRNA) was achieved for the first time. The constructed episomal plasmids and developed CRISPR/Cas9 system will be important synthetic biology tools for both fundamental studies and industrial applications of P. pastoris.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Engenharia Genética/métodos , Pichia/genética , Plasmídeos/genética , Transformação Genética , Replicação do DNA , Escherichia coli/genética , Dosagem de Genes , Regulação Fúngica da Expressão Gênica , Técnicas de Inativação de Genes , Vetores Genéticos , Instabilidade Genômica , Microbiologia Industrial , Kluyveromyces/genética , Regiões Promotoras Genéticas , RNA Guia , Biologia Sintética
8.
Nat Commun ; 10(1): 2212, 2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-31101808

RESUMO

In mammalian cells, double-stranded DNA breaks (DSBs) are preferentially repaired through end-joining processes that generally lead to mixtures of insertions and deletions (indels) or other rearrangements at the cleavage site. In the presence of homologous DNA, homology-directed repair (HDR) can generate specific mutations, albeit typically with modest efficiency and a low ratio of HDR products:indels. Here, we develop hRad51 mutants fused to Cas9(D10A) nickase (RDN) that mediate HDR while minimizing indels. We use RDN to install disease-associated point mutations in HEK293T cells with comparable or better efficiency than Cas9 nuclease and a 2.7-to-53-fold higher ratio of desired HDR product:undesired byproducts. Across five different human cell types, RDN variants generally result in higher HDR:indel ratios and lower off-target activity than Cas9 nuclease, although HDR efficiencies remain strongly site- and cell type-dependent. RDN variants provide precision editing options in cell types amenable to HDR, especially when byproducts of DSBs must be minimized.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Engenharia Genética/métodos , Rad51 Recombinase/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Reparo de DNA por Recombinação , Proteína 9 Associada à CRISPR/genética , Quebras de DNA de Cadeia Dupla , Edição de Genes/métodos , Células HEK293 , Células HeLa , Humanos , Células-Tronco Pluripotentes Induzidas , Células K562 , Rad51 Recombinase/genética , Proteínas Recombinantes de Fusão/genética , Transfecção/métodos
9.
Nat Med ; 25(5): 730-733, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31068712

RESUMO

A 15-year-old patient with cystic fibrosis with a disseminated Mycobacterium abscessus infection was treated with a three-phage cocktail following bilateral lung transplantation. Effective lytic phage derivatives that efficiently kill the infectious M. abscessus strain were developed by genome engineering and forward genetics. Intravenous phage treatment was well tolerated and associated with objective clinical improvement, including sternal wound closure, improved liver function, and substantial resolution of infected skin nodules.


Assuntos
Infecções por Micobactéria não Tuberculosa/terapia , Mycobacterium abscessus , Terapia por Fagos/métodos , Adolescente , Fibrose Cística/microbiologia , Farmacorresistência Bacteriana , Feminino , Engenharia Genética/métodos , Humanos , Infecções por Micobactéria não Tuberculosa/microbiologia , Mycobacterium abscessus/efeitos dos fármacos
10.
Plant Cell Physiol ; 60(6): 1197-1204, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-31076774

RESUMO

The seed oil quality of Brassica oilseed species has been improved in the last few decades, using conventional breeding approaches. Modern biotechnology has enabled the significant development of new seed lipid traits in many oil crops. Alternation of seed lipid component with gene knockout by RNAi gene silencing, artificial microRNA or gene editing within the crop is relative straightforward. Introducing a new pathway from an exogenous source via biotechnology enables the creation of a new trait, where the biosynthetic pathway for such a new trait is not available in the host crop. This review updates the recent development of new seed lipid traits in six major Brassica species and highlights the capability of biotechnology to improve the composition of important fatty acids for both industrial and nutritional purposes.


Assuntos
Brassica/genética , Engenharia Genética , Característica Quantitativa Herdável , Óleo de Brassica napus/metabolismo , Sementes/metabolismo , Brassica/metabolismo , Edição de Genes , Engenharia Genética/métodos
11.
J Microbiol ; 57(8): 637-643, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31079333

RESUMO

Bacteria sense and respond to the environment, communicate, and continuously interact with their surroundings, including host bodies. For more than a century, engineers have been trying to harness the natural ability of bacteria as live biotherapeutics for the treatment of diseases. Recent advances in synthetic biology facilitate the enlargement of the repertoire of genetic parts, tools, and devices that serve as a framework for biotherapy. This review describes bacterial species developed for specific diseases shown in in vitro studies and clinical stages. Here, we focus on drug delivery by programing bacteria and discuss the challenges for safety and improvement.


Assuntos
Bactérias/genética , Sistemas de Liberação de Medicamentos , Engenharia Genética/métodos , Biologia Sintética/métodos , Humanos
12.
Bioelectrochemistry ; 129: 18-25, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31075535

RESUMO

Periplasmic c-type cytochromes are essential for the electron transport between the cytoplasmic membrane bound menquinol oxidase CymA and the terminal ferric iron reductase MtrABC in the outer membrane of Shewanella oneidensis cells. Either STC or FccA are necessary for periplasmic electron transfer. We followed the hypothesis that the elimination of potential competing reactions in the periplasm and the simultaneous overexpression of STC (cctA) could lead to an accelerated electron transfer to the cell surface. The genes nrfA, ccpA, napB and napA were replaced by cctA. This led to a 1.7-fold increased ferric iron reduction rate and a 23% higher current generation in a bioelectrochemical system. Moreover, the quadruple mutant had a higher periplasmic flavin content. Further deletion of fccA and its replacement by cctA resulted in a strain with ferric iron reduction rates similar to the wild type and a lower concentration of periplasmic flavin compared to the quadruple mutant. A transcriptomic analysis revealed that the quadruple mutant had a 3.7-fold higher cctA expression which could not be further increased by the replacement of fccA. This work indicates that a synthetic adaptation of Shewanella towards extracellular respiration holds potential for increased respiratory rates and consequently higher current densities.


Assuntos
Proteínas de Bactérias/metabolismo , Grupo dos Citocromos c/metabolismo , Proteínas Periplásmicas/metabolismo , Shewanella/metabolismo , Proteínas de Bactérias/genética , Grupo dos Citocromos c/genética , Transporte de Elétrons , Desenho de Equipamento , Fumaratos/metabolismo , Deleção de Genes , Dosagem de Genes , Engenharia Genética/métodos , Lactatos/metabolismo , Proteínas Periplásmicas/genética , Shewanella/genética , Transcriptoma , Regulação para Cima
13.
Braz J Med Biol Res ; 52(5): e8108, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31038578

RESUMO

Animal models of diseases are invaluable tools of modern medicine. More than forty years have passed since the first successful experiments and the spectrum of available models, as well as the list of methods for creating them, have expanded dramatically. The major step forward in creating specific disease models was the development of gene editing techniques, which allowed for targeted modification of the animal's genome. In this review, we discuss the available tools for creating transgenic animal models, such as transgenesis methods, recombinases, and nucleases, including zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and CRISPR/Cas9 systems. We then focus specifically on the models of atherosclerosis, especially mouse models that greatly contributed to improving our understanding of the disease pathogenesis and we outline their characteristics and limitations.


Assuntos
Animais Geneticamente Modificados , Aterosclerose/fisiopatologia , Modelos Animais de Doenças , Engenharia Genética/métodos , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/metabolismo , Animais , Aterosclerose/genética , Pesquisa Biomédica/métodos , Feminino , Técnicas de Transferência de Genes , Humanos , Masculino , Camundongos
14.
Gene ; 704: 49-58, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30935921

RESUMO

Synthetic biology with genetically engineered (GE) cyanobacteria has the potential to produce valuable products such as biofuels. However, it is also essential to assess the potential risks of synthetic biology technology before it can be widely used. In order to address key concerns posed by the application of synthetic biology to microorganisms, studies were designed to monitor the horizontal transfer of engineered genes from GE cyanobacteria Thermosynechococcus elongatus BP1 to Escherichia coli through co-incubation. The results of these experiments demonstrated that the genetically engineered DNA construct containing alcohol producing genes and kanamycin resistance can be horizontally transferred from GE T. elongatus BP1 to wild-type E. coli following two days of liquid co-culturing. The rapid and facile transfer of foreign genes, which include antibiotic resistance, between bacterial communities signifies the need to continue to deepen our understanding of the process of horizontal gene transfer, chromosomal integration as well as further biosafety-oriented research efforts. In the era of synthetic biology, the natural microbial process for sharing genetic material will also significantly impact risk assessments, containment approaches and further policy development.


Assuntos
Cianobactérias/genética , Escherichia coli/genética , Transferência Genética Horizontal , Clonagem Molecular , Cianobactérias/classificação , DNA Bacteriano/genética , Engenharia Genética/métodos , Técnicas Microbiológicas , Organismos Geneticamente Modificados , Synechococcus/genética , Biologia Sintética , Transformação Bacteriana/genética
15.
Mol Biotechnol ; 61(6): 461-468, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30997667

RESUMO

Synthetic biology and genetic engineering in algae offer an unprecedented opportunity to develop species with traits that can help solve the problems associated with food and energy supply in the 21st century. In the green alga Chlamydomonas reinhardtii, foreign genes can be expressed from the chloroplast genome for molecular farming and metabolic engineering to obtain commodities and high-value molecules. To introduce these genes, selectable markers, which rely mostly on the use of antibiotics, are needed. This has risen social concern associated with the potential risk of horizontal gene transfer across life kingdoms, which has led to a quest for antibiotic-free selectable markers. Phosphorus (P) is a scarce nutrient element that most organisms can only assimilate in its most oxidized form as phosphate (Pi); however, some organisms are able to oxidize phosphite (Phi) to Pi prior to incorporation into the central metabolism of P. As an alternative to the use of the two positive selectable makers already available for chloroplast transformation in C. reinhardtii, the aadA and the aphA-6 genes, that require the use of antibiotics, we investigated if a phosphite-based selection method could be used for the direct recovery of chloroplast transformed lines in this alga. Here we show that following bombardment with a vector carrying the ptxD gene from Pseudomonas stutzeri WM88, only cells that integrate and express the gene proliferate and form colonies using Phi as the sole P source. Our results demonstrate that a selectable marker based on the assimilation of Phi can be used for chloroplasts transformation in a biotechnologically relevant organism. The portable selectable marker we have developed is, in more than 18 years, the latest addition to the markers available for selection of chloroplast transformed cells in C. reinhardtii. The ptxD gene will contribute to the repertoire of tools available for synthetic biology and genetic engineering in the chloroplast of C. reinhardtii.


Assuntos
Proteínas de Bactérias/genética , Chlamydomonas reinhardtii/genética , Cloroplastos/genética , NADH NADPH Oxirredutases/genética , Fosfitos/metabolismo , Fósforo/metabolismo , Proteínas de Algas/genética , Proteínas de Algas/metabolismo , Proteínas de Bactérias/metabolismo , Chlamydomonas reinhardtii/metabolismo , Cloroplastos/metabolismo , Engenharia Genética/métodos , Marcadores Genéticos , Vetores Genéticos/química , Vetores Genéticos/metabolismo , NADH NADPH Oxirredutases/metabolismo , Fosfitos/farmacologia , Pseudomonas stutzeri/química , Pseudomonas stutzeri/genética , Seleção Genética , Transformação Genética
16.
Genome Biol ; 20(1): 80, 2019 04 25.
Artigo em Inglês | MEDLINE | ID: mdl-31018865

RESUMO

BACKGROUND: Geminiviruses cause damaging diseases in several important crop species. However, limited progress has been made in developing crop varieties resistant to these highly diverse DNA viruses. Recently, the bacterial CRISPR/Cas9 system has been transferred to plants to target and confer immunity to geminiviruses. In this study, we use CRISPR-Cas9 interference in the staple food crop cassava with the aim of engineering resistance to African cassava mosaic virus, a member of a widespread and important family (Geminiviridae) of plant-pathogenic DNA viruses. RESULTS: Our results show that the CRISPR system fails to confer effective resistance to the virus during glasshouse inoculations. Further, we find that between 33 and 48% of edited virus genomes evolve a conserved single-nucleotide mutation that confers resistance to CRISPR-Cas9 cleavage. We also find that in the model plant Nicotiana benthamiana the replication of the novel, mutant virus is dependent on the presence of the wild-type virus. CONCLUSIONS: Our study highlights the risks associated with CRISPR-Cas9 virus immunity in eukaryotes given that the mutagenic nature of the system generates viral escapes in a short time period. Our in-depth analysis of virus populations also represents a template for future studies analyzing virus escape from anti-viral CRISPR transgenics. This is especially important for informing regulation of such actively mutagenic applications of CRISPR-Cas9 technology in agriculture.


Assuntos
Sistemas CRISPR-Cas , Geminiviridae/genética , Engenharia Genética/efeitos adversos , Interações Hospedeiro-Patógeno/genética , Manihot/genética , Engenharia Genética/métodos , Manihot/virologia , Plantas Geneticamente Modificadas/virologia
17.
J Microbiol Biotechnol ; 29(4): 507-517, 2019 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-30856706

RESUMO

Rhodotorula is a group of pigment-producing yeasts well known for its intracellular biosynthesis of carotenoids such as ß-carotene, γ-carotene, torulene and torularhodin. The great potential of carotenoids in applications in food and feed as well as in health products and cosmetics has generated a market value expected to reach over $2.0 billion by 2022. Due to growing public concern over food safety, the demand for natural carotenoids is rising, and this trend significantly encourages the use of microbial fermentation for natural carotenoid production. This review covers the biological properties of carotenoids and the most recent findings on the carotenoid biosynthetic pathway, as well as strategies for the metabolic engineering methods for the enhancement of carotenoid production by Rhodotorula. The practical approaches to improving carotenoid yields, which have been facilitated by advancements in strain work as well as the optimization of media and fermentation conditions, were summarized respectively.


Assuntos
Carotenoides/metabolismo , Rhodotorula/genética , Rhodotorula/metabolismo , Vias Biossintéticas/genética , Carotenoides/biossíntese , Carotenoides/química , Carotenoides/genética , Fermentação , Inocuidade dos Alimentos , Engenharia Genética/métodos , Engenharia Metabólica/métodos , Mutagênese
19.
BMC Res Notes ; 12(1): 144, 2019 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-30876440

RESUMO

OBJECTIVE: The treatment of plant tissue with Agrobacterium tumefaciens is often a critical first step to both stable and transient plant transformation. In both applications bacterial suspensions are oftentimes physically introduced into plant tissues using hand-driven pressure from a needleless syringe. While effective, this approach has several drawbacks that limit reproducibility. Pressure must be provided with the syringe perfectly perpendicular to the tissue surface. The researcher must also attempt to provide even and consistent pressure, both within and between experimental replicates. These factors mean that the procedures do not always translate well between research groups or biological replicates. RESULTS: We have devised a method to introduce Agrobacterium suspensions into plant leaves with greater reproducibility. Using a decommissioned dissecting microscope as an armature, a syringe body with the bacterial suspension is mounted to the nosepiece. Gentle, even pressure is applied by rotating the focus knob. The treatment force is measured using a basic kitchen scale. The development of the Standardized Pressure Agrobacterium Infiltration Device (SPAID) provides a means to deliver consistent amounts of bacterial suspensions into plant tissues with the goal of increasing reproducibility between replicates and laboratories.


Assuntos
Agrobacterium tumefaciens/genética , Arabidopsis/genética , Fragaria/genética , Folhas de Planta/genética , Tabaco/genética , Transformação Genética , Engenharia Genética/instrumentação , Engenharia Genética/métodos , Plantas Geneticamente Modificadas , Reprodutibilidade dos Testes
20.
J Biotechnol ; 295: 63-70, 2019 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-30853633

RESUMO

Engineering of Chinese Hamster Ovary cells by manipulating microRNA (miRNA) expression levels has been shown to induce advantageous, desired phenotypes. Most of these studies so far were concerned with increasing productivity or reducing growth rate (with the implied intention of thus freeing cellular resources to also increase productivity). Here we evaluated the ability of growth correlating miRNAs to increase the growth rate of CHO-K1 cells by transient overexpression or knock down, respectively. Candidates were selected based on the correlation between growth rate and miRNA expression levels as observed in previous studies. These candidates were then up- or downregulated initially by transfection of mimics or inhibitors and subsequently by transfection of plasmids bearing the corresponding miRNAs or sponges. None of the 40 selected candidates was able to induce a better growth phenotype under these conditions. Overlap between miRNAs identified to correlate to growth in published miRNA expression studies and those identified to actively increase growth rate in a functional screen is minimal, indicating that the here selected approach of traditional overexpression/knock down engineering of miRNAs may not be a suitable strategy for the purpose of increasing growth rate.


Assuntos
Reatores Biológicos , Proliferação de Células/genética , Engenharia Genética/métodos , MicroRNAs/genética , Animais , Células CHO , Cricetinae , Cricetulus , MicroRNAs/análise , MicroRNAs/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA