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1.
C R Biol ; 342(5-6): 142-153, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31447175

RESUMO

Ranbir Basmati is one of the traditional Basmati varieties of India and of the most popular traditional Basmati variety grown in Jammu's region (State of Jammu & Kashmir). It is a tall and short-duration variety with strong aroma and excellent cooking quality. However, it is susceptible to bacterial blight (BB) disease caused by Xanthomonas oryzae pv oryzae (Xoo) and prone to lodging. In this study, semi-dwarf (sd1) and BB resistance genes (Xa21 and xa13) were introgressed into Ranbir Basmati using marker-assisted backcross breeding (MABB) scheme. A high-yielding PAU148 carrying Xa21, xa13 and sd1 genes was used as a donor parent. On each generation target, genes were selected, while polymorphic SSR markers were used to select plants having maximum recovery of the recurrent genome. The maximum genome recovery of Ranbir Basmati in BC2F2 was 86.9% in introgressed line SBTIL121. The genotypes carrying resistant genes exhibited very high levels of tolerance against BB disease along with good Basmati rice grain quality traits. The agronomic traits of introgressed lines evaluated in the field and the laboratory showed that most of the agro-morphological traits were similar or superior to Ranbir Basmati. The identified lines can be further evaluated and released as Improved Ranbir Basmati variety.


Assuntos
Cruzamentos Genéticos , Melhoramento Genético/métodos , Oryza/genética , Controle Biológico de Vetores/métodos , Doenças das Plantas/microbiologia , Doenças das Plantas/prevenção & controle , Seleção Genética/genética , Aspergillus oryzae , Cruzamento , Culinária , DNA de Plantas/genética , DNA de Plantas/isolamento & purificação , Resistência à Doença , Marcadores Genéticos , Genoma de Planta/genética , Índia
2.
Biotechnol J ; 14(11): e1900125, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31271264

RESUMO

For the industrial production of recombinant proteins in mammalian cell lines, a high rate of gene expression is desired. Therefore, strong viral promoters are commonly used. However, these have several drawbacks as they override cellular responses, are not integrated into the cellular network, and thus can induce stress and potentially epigenetic silencing. Endogenous promoters potentially have the advantage of a better response to cellular state and thus a lower stress level by uncontrolled overexpression of the transgene. Such fine-tuning is typically achieved by endogenous enhancers and other regulatory elements, which are difficult to identify purely based on the genomic sequence. Here, Chinese hamster ovary (CHO) endogenous promoters and enhancers are identified using histone marks and chromatin states, ranked based on expression level and tested for normalized promoter strength. Successive truncation of these promoters at the 5'- and 3'-end as well as the combination with enhancers are identified in the vicinity of the promoter sequence further enhance promoter activity up to threefold. In an initial screen within stable cell lines, the strongest CHO promoter appears to be more stable than the human cytomegalovirus promoter with enhancer, making it a promising candidate for recombinant protein production and cell engineering applications. A deeper understanding of promoter functionality and response elements will be required to take full advantage of such promoters for cell engineering, in particular, for multigene network engineering applications.


Assuntos
Células CHO , Expressão Gênica , Melhoramento Genético/métodos , Regiões Promotoras Genéticas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Animais , Técnicas de Cultura de Células , Engenharia Celular , Clonagem Molecular , Simulação por Computador , Cricetinae , Cricetulus , Epigênese Genética , Escherichia coli/genética , Humanos , Técnicas In Vitro , Transgenes/genética
3.
Front Med ; 13(2): 131-137, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30159670

RESUMO

The inhibitory environment that surrounds the lesion site and the lack of intrinsic regenerative capacity of the adult mammalian central nervous system (CNS) impede the regrowth of injured axons and thereby the reestablishment of neural circuits required for functional recovery after spinal cord injuries (SCI). To circumvent these barriers, biomaterial scaffolds are applied to bridge the lesion gaps for the regrowing axons to follow, and, often by combining stem cell transplantation, to enable the local environment in the growth-supportive direction. Manipulations, such as the modulation of PTEN/mTOR pathways, can also enhance intrinsic CNS axon regrowth after injury. Given the complex pathophysiology of SCI, combining biomaterial scaffolds and genetic manipulation may provide synergistic effects and promote maximal axonal regrowth. Future directions will primarily focus on the translatability of these approaches and promote therapeutic avenues toward the functional rehabilitation of patients with SCIs.


Assuntos
Materiais Biocompatíveis , Melhoramento Genético/métodos , Regeneração Nervosa , Traumatismos da Medula Espinal/fisiopatologia , Animais , Axônios/fisiologia , Humanos , PTEN Fosfo-Hidrolase/metabolismo , Recuperação de Função Fisiológica , Engenharia Tecidual/métodos , Tecidos Suporte
4.
Reprod Fertil Dev ; 32(2): 40-49, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32188556

RESUMO

Traditionally, genetic engineering in the pig was a challenging task. Genetic engineering of somatic cells followed by somatic cell nuclear transfer (SCNT) could produce genetically engineered (GE) pigs carrying site-specific modifications. However, due to difficulties in engineering the genome of somatic cells and developmental defects associated with SCNT, a limited number of GE pig models were reported. Recent developments in genome-editing tools, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) 9 system, have markedly changed the effort and time required to produce GE pig models. The frequency of genetic engineering in somatic cells is now practical. In addition, SCNT is no longer essential in producing GE pigs carrying site-specific modifications, because direct injection of genome-editing systems into developing embryos introduces targeted modifications. To date, the CRISPR/Cas9 system is the most convenient, cost-effective, timely and commonly used genome-editing technology. Several applicable biomedical and agricultural pig models have been generated using the CRISPR/Cas9 system. Although the efficiency of genetic engineering has been markedly enhanced with the use of genome-editing systems, improvements are still needed to optimally use the emerging technology. Current and future advances in genome-editing strategies will have a monumental effect on pig models used in agriculture and biomedicine.


Assuntos
Agricultura/tendências , Pesquisa Biomédica/tendências , Sistemas CRISPR-Cas/genética , Edição de Genes/veterinária , Engenharia Genética/veterinária , Suínos/genética , Agricultura/métodos , Animais , Animais Geneticamente Modificados , Pesquisa Biomédica/métodos , Edição de Genes/métodos , Engenharia Genética/métodos , Engenharia Genética/tendências , Melhoramento Genético/métodos
5.
Biochem Biophys Res Commun ; 497(1): 160-166, 2018 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-29428735

RESUMO

Germin-like proteins (GLPs) are ubiquitous water-soluble glycoproteins that are located in the extracellular matrix. These proteins have been reported to play vital roles in diverse biological processes. In the present study, a GLP in soybean (Glycine max L. Merr.), GmGLP10, was characterized. Sequence analysis revealed that the GmGLP10 gene (GenBank Accession Number EU916258) encodes a 213-amino acid (aa) protein, which contains a N-terminal signal peptide at 1-22 aa and is highly homologous to the members of the GER2 subfamily. GmGLP10 was highly expressed in the leaves, but very faint in the roots. The expression of GmGLP10 was induced by methyl jasmonate (MeJA), ethylene (ET), salicylic acid (SA), oxalate acid (OA), and the infection of Sclerotinia sclerotiorum. Overexpression of GmGLP10 in transgenic tobacco significantly enhanced tolerance to OA and S. sclerotiorum infection. Moreover, higher levels of H2O2 and the upregulated expression of a set of plant defense-related genes and HR (hypersensitive response)-associated genes were detected in the transgenic plants. These results suggest that GmGLP10 functions as a positive regulator of resistance to S. sclerotiorum.


Assuntos
Ascomicetos/fisiologia , Glicoproteínas/metabolismo , Doenças das Plantas/microbiologia , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/microbiologia , Plantas Geneticamente Modificadas/fisiologia , Tabaco/metabolismo , Tabaco/microbiologia , Resistência à Doença/fisiologia , Melhoramento Genético/métodos , Glicoproteínas/genética , Doenças das Plantas/prevenção & controle , Proteínas de Plantas/genética , Regulação para Cima
6.
Biochem Biophys Res Commun ; 497(1): 25-31, 2018 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-29407173

RESUMO

Scopolia lurida, a medicinal plant native to the Tibetan Plateau, is among the most effective producers of pharmaceutical tropane alkaloids (TAs). The hyoscyamine 6ß-hydroxylase genes of Hyoscyamus niger (HnH6H) and S. lurida (SlH6H) were cloned and respectively overexpressed in hairy root cultures of S. lurida, to compare their effects on promoting the production of TAs, especially the high-value scopolamine. Root cultures with SlH6H/HnH6H overexpression were confirmed by PCR and real-time quantitative PCR, suggesting that the enzymatic steps defined by H6H were strongly elevated at the transcriptional level. Tropane alkaloids, including hyoscyamine, anisodamine and scopolamine, were analyzed by HPLC. Scopolamine and anisodamine contents were remarkably elevated in the root cultures overexpressing SlH6H/HnH6H, whereas that of hyoscyamine was more or less reduced, when compared with those of the control. These results also indicated that SlH6H and HnH6H promoted anisodamine production at similar levels in S. lurida root cultures. More importantly, HnH6H-overexpressing root cultures had more scopolamine in them that did SlH6H-overexpressing root cultures. This study not only provides a feasible way of overexpressing H6H to produce high-value scopolamine in engineered root cultures of S. lurida but also found that HnH6H was better than SlH6H for engineering scopolamine production.


Assuntos
Engenharia Metabólica/métodos , Oxigenases de Função Mista/genética , Raízes de Plantas/fisiologia , Plantas Geneticamente Modificadas/fisiologia , Escopolamina/metabolismo , Scopolia/fisiologia , Ativação Enzimática , Estabilidade Enzimática , Melhoramento Genético/métodos , Oxigenases de Função Mista/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Escopolamina/isolamento & purificação
7.
Biochem Biophys Res Commun ; 495(1): 1221-1226, 2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29175328

RESUMO

Producing virus at high yield is critically important for development of whole virion inactivated vaccines or live attenuated vaccines. Most dengue virus (DENV) clinical isolates, however, replicate at low levels in cultured cells, which limits their use for vaccine development. The present study examined differences between low-replicating DENV clinical isolates and high-replicating laboratory strains with the aim of engineering high-yield DENV clinical isolates. Construction of a series of recombinant chimeric viruses derived from a high-replicating laboratory DENV type 4 (DENV-4) H241 strain and a clinical isolate revealed that the NS3-NS4B region of H241 conferred a replication advantage in cultured cells. Furthermore, northern blot analysis revealed that this advantage was due to more efficient synthesis of viral RNA. Importantly, replacement of the NS3-NS4B region of H241 did not increase virulence in mice, suggesting that viral production can be increased safely. This study provided information that will facilitate engineering of safe and high-yield viruses that can be used for vaccine development.


Assuntos
Técnicas de Cultura Celular por Lotes/métodos , Vírus da Dengue/crescimento & desenvolvimento , Vírus da Dengue/genética , Melhoramento Genético/métodos , Carga Viral/genética , Proteínas não Estruturais Virais/metabolismo , Virulência/fisiologia , Recombinação Genética/genética , Carga Viral/fisiologia , Proteínas não Estruturais Virais/genética
8.
J Ind Microbiol Biotechnol ; 45(7): 589-598, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29255989

RESUMO

Escherichia coli KO11 is a popular ethanologenic strain, but is more sensitive to ethanol than other producers. Here, an ethanol-tolerant mutant EM was isolated from ultraviolet mutagenesis library of KO11. Comparative genomic analysis added by piecewise knockout strategy and complementation assay revealed EKO11_3023 (espA) within the 36.6-kb deletion from KO11 was the only locus responsible for ethanol sensitivity. Interestingly, when espA was deleted in strain W (the parent strain of KO11), ethanol tolerance was dramatically elevated to the level of espA-free hosts [e.g., MG1655 and BL21(DE3)]. And overexpression of espA in strains MG1655 and BL21(DE3) led to significantly enhanced ethanol sensitivity. In addition to ethanol, deletion of espA also improved cell tolerance to other short-chain (C2-C4) alcohols, including methanol, isopropanol, n-butanol, isobutanol and 2-butanol. Therefore, espA was responsible for short-chain alcohol sensitivity of W-strains compared to other cells, which provides a potential engineering target for alcohols production.


Assuntos
Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Etanol/metabolismo , Etanol/farmacologia , Regulação Bacteriana da Expressão Gênica/fisiologia , Biologia Sintética/métodos , Evolução Molecular Direcionada/métodos , Resistência Microbiana a Medicamentos/genética , Escherichia coli/metabolismo , Melhoramento Genético/métodos
9.
Biochem Biophys Res Commun ; 495(1): 686-692, 2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29113801

RESUMO

Although Escherichia coli has been engineered to perform N-glycosylation of recombinant proteins, an optimal glycosylating strain has not been created. By inserting a codon optimised Campylobacter oligosaccharyltransferase onto the E. coli chromosome, we created a glycoprotein platform strain, where the target glycoprotein, sugar synthesis and glycosyltransferase enzymes, can be inserted using expression vectors to produce the desired homogenous glycoform. To assess the functionality and glycoprotein producing capacity of the chromosomally based OST, a combined Western blot and parallel reaction monitoring mass spectrometry approach was applied, with absolute quantification of glycoprotein. We demonstrated that chromosomal oligosaccharyltransferase remained functional and facilitated N-glycosylation. Although the engineered strain produced less total recombinant protein, the glycosylation efficiency increased by 85%, and total glycoprotein production was enhanced by 17%.


Assuntos
Proteínas de Bactérias/genética , Escherichia coli/fisiologia , Edição de Genes/métodos , Genoma Bacteriano/genética , Glicoproteínas/biossíntese , Hexosiltransferases/genética , Proteínas de Membrana/genética , Engenharia Metabólica/métodos , Proteínas de Bactérias/metabolismo , Melhoramento Genético/métodos , Glicoproteínas/genética , Glicosilação , Hexosiltransferases/metabolismo , Proteínas de Membrana/metabolismo
10.
Biochem Biophys Res Commun ; 495(2): 1851-1857, 2018 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-29233696

RESUMO

Reactive oxygen species (ROS) are a key factor in abiotic stresses; excess ROS is harmful to plants. Glutathione reductase (GR) plays an important role in scavenging ROS in plants. Here, a GR gene, named SpGR, was cloned from Stipa purpurea and characterized. The full-length open reading frame was 1497 bp, encoding 498 amino acids. Subcellular localization analysis indicated that SpGR was localized to both the plasma membrane and nucleus. The expression of SpGR was induced by cold, salt, and drought stresses. Functional analysis indicated that ectopic expression of SpGR in Arabidopsis thaliana resulted in greater tolerance to salt stress than that of wild-type plants, but no difference under cold or drought treatments. The results of GR activity and GSSG and GSH content analyses suggested that, under salt stress, transgenic plants produced more GR to reduce GSSG to GSH for scavenging ROS than wild-type plants. Therefore, SpGR may be a candidate gene for plants to resist abiotic stress.


Assuntos
Arabidopsis/fisiologia , Glutationa Redutase/química , Glutationa Redutase/metabolismo , Plantas Geneticamente Modificadas/fisiologia , Poaceae/enzimologia , Espécies Reativas de Oxigênio/metabolismo , Plantas Tolerantes a Sal/genética , Clonagem Molecular/métodos , Ativação Enzimática , Melhoramento Genético/métodos , Glutationa Redutase/genética , Poaceae/genética
11.
Biotechnol Bioeng ; 114(12): 2848-2856, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28926673

RESUMO

One of the key quality attributes of monoclonal antibodies is the glycan pattern and distribution. Two terminal galactose residues typically represent a small fraction of the total glycans from antibodies. However, antibodies with defined glycosylation properties including enhanced galactosylation have been shown to exhibit altered properties for these important biomedical modalities. In this study, the disruption of two α-2,3 sialyltransferases (ST3GAL4 and ST3GAL6) from Chinese Hamster Ovary (CHO) cells was combined with protein engineering of the Fc region to generate an IgG containing 80% bigalactosylated and fucosylated (G2F) glycoforms. Expression of the same single amino acid mutant (F241A) IgG in CHO cells with a triple gene knockout of fucosyltransferase (FUT8) plus ST3GAL4 and ST3GAL6 lowered the galactosylation glycoprofile to 65% bigalactosylated G2 glycans. However, overexpression of IgGs with four amino acid substitutions recovered the G2 glycoform composition approximately 80%. Combining genome and protein engineering in CHO cells will provide a new antibody production platform that enables biotechnologists to generate glycoforms standards for specific biomedical and biotechnology applications.


Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/genética , Técnicas de Química Combinatória/métodos , Galactose/metabolismo , Imunoglobulina G/metabolismo , Engenharia de Proteínas/métodos , Animais , Células CHO , Mapeamento Cromossômico/métodos , Cricetulus , Melhoramento Genético/métodos , Glicosilação , Imunoglobulina G/genética
12.
Biotechnol Bioeng ; 114(12): 2837-2847, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28926680

RESUMO

There has been much effort exerted to reduce one carbon (C1) gas emission to address climate change. As one promising way to more conveniently utilize C1 gas, several technologies have been developed to convert C1 gas into useful chemicals such as formic acid (FA). In this study, systems metabolic engineering was utilized to engineer Mannheimia succiniciproducens to efficiently utilize FA. 13 C isotope analysis of M. succiniciproducens showed that FA could be utilized through formate dehydrogenase (FDH) reaction and/or the reverse reaction of pyruvate formate lyase (PFL). However, the naturally favored forward reaction of PFL was found to lower the SA yield from FA. In addition, FA assimilation via FDH was found to be more efficient than the reverse reaction of PFL. Thus, the M. succiniciproducens LPK7 strain, which lacks in pfl, ldh, pta, and ack genes, was selected as a base strain. In silico metabolic analysis confirmed that utilization of FA would be beneficial for the enhanced production of SA and suggested FDH as an amplification target. To find a suitable FDH, four different FDHs from M. succiniciproducens, Methylobacterium extorquens, and Candida boidinii were amplified in LPK7 strain to enhance FA assimilation. High-inoculum density cultivation using 13 C labeled sodium formate was performed to evaluate FA assimilation efficiency. Fed-batch fermentations of the LPK7 (pMS3-fdh2 meq) strain was carried out using glucose, sucrose, or glycerol as a primary carbon source and FA as a secondary carbon source. As a result, this strain produced 76.11 g/L SA with the yield and productivity of 1.28 mol/mol and 4.08 g/L/h, respectively, using sucrose and FA as dual carbon sources. The strategy employed here will be similarly applicable in developing microorganisms to utilize FA and to produce valuable chemicals and materials from FA.


Assuntos
Formiato Desidrogenases/genética , Formiatos/metabolismo , Melhoramento Genético/métodos , Mannheimia/fisiologia , Engenharia Metabólica/métodos , Análise do Fluxo Metabólico/métodos , Ácido Succínico/metabolismo , Simulação por Computador , Mannheimia/classificação , Modelos Biológicos , Especificidade da Espécie , Especificidade por Substrato , Ácido Succínico/isolamento & purificação , Regulação para Cima/genética
13.
Biochem Biophys Res Commun ; 491(3): 642-648, 2017 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-28757414

RESUMO

MYB transcription factors are a large family of proteins involved in plant development and responses to stress. In this study, the wheat salinity-induced R2R3-MYB transcription factor TaSIM was functionally characterized, with a focus on its role in salt stress tolerance. TaSIM protein enters the nucleus and binds to the MYB-binding site II motif. Expression analysis revealed that TaSIM was induced by drought, high salinity, low temperature, and abscisic acid treatment. Overexpression of TaSIM improved salt stress tolerance in transgenic plants. Furthermore, the transcript levels of genes involved in abscisic acid (ABA)-dependent (RD22) and ABA-independent (RD29A) signaling were higher in TaSIM-overexpressing plants than in the wild type. These results suggest that TaSIM positively modulates salt stress tolerance and has potential applications in molecular breeding to enhance salt tolerance in crops.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/fisiologia , Melhoramento Genético/métodos , Tolerância ao Sal/fisiologia , Estresse Fisiológico/fisiologia , Fatores de Transcrição/genética , Triticum/genética , Plantas Geneticamente Modificadas/fisiologia
14.
Biotechnol Bioeng ; 114(12): 2828-2836, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28842980

RESUMO

Numerous high-value proteins are secreted into the Escherichia coli periplasm by the General Secretory (Sec) pathway, but Sec-based production chassis cannot handle many potential target proteins. The Tat pathway offers a promising alternative because it transports fully folded proteins; however, yields have been too low for commercial use. To facilitate Tat export, we have engineered the TatExpress series of super-secreting strains by introducing the strong inducible bacterial promoter, ptac, upstream of the chromosomal tatABCD operon, to drive its expression in E. coli strains commonly used by industry (e.g., W3110 and BL21). This modification significantly improves the Tat-dependent secretion of human growth hormone (hGH) into the bacterial periplasm, to the extent that secreted hGH is the dominant periplasmic protein after only 1 hr induction. TatExpress strains accumulate in excess of 30 mg L-1 periplasmic recombinant hGH, even in shake flask cultures. A second target protein, an scFv, is also shown to be exported at much higher rates in TatExpress strains.


Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Produtos do Gene tat/genética , Melhoramento Genético/métodos , Hormônio do Crescimento/biossíntese , Periplasma/metabolismo , Via Secretória/genética , Hormônio do Crescimento/genética , Hormônio do Crescimento/isolamento & purificação , Humanos , Redes e Vias Metabólicas/genética , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação
15.
Biotechnol Bioeng ; 114(12): 2739-2752, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28842992

RESUMO

The exciting promises of functional metagenomics for the efficient discovery of novel biomolecules from nature are often hindered by factors associated with expression hosts. Aiming to shift functional metagenomics to a host independent innovative system, we here report on the cloning, heterologous expression, and reconstitution of an RNA polymerase (RNAP) from the thermophilic Geobacillus sp. GHH01 and in vitro transcription thereafter. The five genes coding for RNAP subunits, a house keeping sigma factor and two transcription elongation factors were cloned and over expressed as His6 -tagged and/ or tag-free proteins. Purified subunits were reconstituted into a functional polymerase through either the classical method of denaturation and subsequent renaturation or through a new resource and time efficient thermo-reconstitution method which takes advantage of the subunits' temperature stability. Additionally, all subunits were cloned into a single vector system for a co-expression and in vivo reconstitution to the RNAP core enzyme. Both the core and holoenzyme form of the RNAP exhibited a robust transcription activity and were stable up to a temperature of 55°C close to their fullest activity. The Geobacillus RNAP showed a remarkable in vitro transcription profile recognizing DNA template sequences of diverse bacteria and archaea as well as metagenomic samples. Coupled with a subsequent in vitro translation step, this recombinant transcription system could allow a new, clone-free, and functional metagenomic screening approach.


Assuntos
RNA Polimerases Dirigidas por DNA/genética , Melhoramento Genético/métodos , Geobacillus/genética , Metagenoma/genética , RNA/biossíntese , Proteínas Recombinantes/genética , Regulação Bacteriana da Expressão Gênica/genética , Regulação Enzimológica da Expressão Gênica/genética , RNA/genética , Proteínas Recombinantes/metabolismo
16.
Biotechnol Bioeng ; 114(12): 2896-2906, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28832943

RESUMO

In many organisms of biotechnological importance precise genome editing is limited by inherently low homologous recombination (HR) efficiencies. A number of strategies exist to increase the effectiveness of this native DNA repair pathway; however, most strategies rely on permanently disabling competing repair pathways, thus reducing an organism's capacity to repair naturally occurring double strand breaks. Here, we describe a CRISPR interference (CRISPRi) system for gene repression in the oleochemical-producing yeast Yarrowia lipolytica. By using a multiplexed sgRNA targeting strategy, we demonstrate efficient repression of eight out of nine targeted genes to enhance HR. Strains with nonhomologous end-joining repressed were shown to have increased rates of HR when transformed with a linear DNA fragment with homology to a genomic locus. With multiplexed targeting of KU70 and KU80, and enhanced repression with Mxi1 fused to deactivated Cas9 (dCas9), rates of HR as high as 90% were achieved. The developed CRISPRi system enables enhanced HR in Y. lipolytica without permanent genetic knockouts and promises to be a potent tool for other metabolic engineering, synthetic biology, and functional genomics studies.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Reparo do DNA por Junção de Extremidades/genética , Edição de Genes/métodos , Melhoramento Genético/métodos , Genoma Fúngico/genética , Proteínas Recombinantes/biossíntese , Yarrowia/genética , Regulação Fúngica da Expressão Gênica/genética , Recombinação Homóloga/genética , Proteínas Recombinantes/genética , Proteínas Repressoras/genética
17.
Biotechnol Bioeng ; 114(12): 2807-2817, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28802003

RESUMO

Lactic acid bacteria (LAB) are among the most interesting organisms for industrial processes with a long history of application as food starters and biocontrol agents, and an underexploited potential for biorefineries converting biomass into high-value compounds. Lactic acid (LA), their main fermentation product, is among the most requested chemicals owing to its broad range of applications. Notably, LA polymers, that is, polylactides, have high potential as biodegradable substitutes of fossil-derived plastics. However, LA production by LAB fermentation is currently too expensive for polylactide to be cost-competitive with traditional plastics. LAB have complex nutritional requirements and cannot ferment inexpensive substrates such as cellulose. Metabolic engineering could help reduce such nutritional requirements and enable LAB to directly ferment low-cost polysaccharides. Here, we engineered a Lactococcus lactis strain which constitutively secretes a ß-glucosidase and an endoglucanase. The recombinant strain can grow on cellooligosaccharides up to at least cellooctaose and efficiently metabolizes them to L-LA in single-step fermentation. This is the first report of a LAB able to directly metabolize cellooligosaccharides longer that cellohexaose and a significant step toward cost-sustainable consolidated bioprocessing of cellulose into optically pure LA.


Assuntos
Celulose/análogos & derivados , Dextrinas/metabolismo , Melhoramento Genético/métodos , Ácido Láctico/biossíntese , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Proteínas Recombinantes/metabolismo , Recombinação Genética/genética , Celulose/genética , Celulose/metabolismo , Dextrinas/genética , Ácido Láctico/isolamento & purificação
18.
Biotechnol Bioeng ; 114(12): 2794-2806, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28782796

RESUMO

Ansamitocin P-3 (AP-3) is a maytansinoid with its most compelling antitumor activity, however, the low production titer of AP-3 greatly restricts its wide commercial application. In this work, a combinatorial approach including random mutation and metabolic engineering was conducted to enhance AP-3 biosynthesis in Actinosynnema pretiosum. First, a mutant strain M was isolated by N-methyl-N'-nitro-N-nitrosoguanidine mutation, which could produce AP-3 almost threefold that of wild type (WT) in 48 deep-well plates. Then, by overexpressing key biosynthetic genes asmUdpg and asm13-17 in the M strain, a further 60% increase of AP-3 production in 250-ml shake flasks was achieved in the engineered strain M-asmUdpg:asm13-17 compared to the M strain, and its maximum AP-3 production reached 582.7 mg/L, which is the highest as ever reported. Both the gene transcription levels and intracellular intermediate concentrations in AP-3 biosynthesis pathway were significantly increased in the M and M-asmUdpg:asm13-17 during fermentation compared to the WT. The good fermentation performance of the engineered strain was also confirmed in a lab-scale bioreactor. This work demonstrated that combination of random mutation and metabolic engineering could promote AP-3 biosynthesis and might be helpful for increasing the production of other industrially important secondary metabolites.


Assuntos
Actinobacteria/fisiologia , Vias Biossintéticas/genética , Melhoramento Genético/métodos , Maitansina/análogos & derivados , Engenharia Metabólica/métodos , Mutação/genética , Actinobacteria/classificação , Maitansina/biossíntese , Especificidade da Espécie , Regulação para Cima/genética
19.
Biotechnol Bioeng ; 114(12): 2883-2895, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28755474

RESUMO

Probiotics, whether taken as capsules or consumed in foods, have been regarded as safe for human use by regulatory agencies. Being living cells, they serve as "tunable" factories for the synthesis of a vast array of beneficial molecules. The idea of reprogramming probiotics to act as controllable factories, producing potential therapeutic molecules under user-specified conditions, represents a new and powerful concept in drug synthesis and delivery. Probiotics that serve as drug delivery vehicles pose several challenges, one being targeting (as seen with nanoparticle approaches). Here, we employ synthetic biology to control swimming directionality in a process referred to as "pseudotaxis." Escherichia coli, absent the motility regulator cheZ, swim sporadically, missing the traditional "run" in the run:tumble swimming paradigm. Upon introduction of cheZ in trans and its signal-generated upregulation, engineered bacteria can be "programmed" to swim toward the source of the chemical cue. Here, engineered cells that encounter sufficient levels of the small signal molecule pyocyanin, produce an engineered CheZ and swim with programmed directionality. By incorporating a degradation tag at the C-terminus of CheZ, the cells stop running when they exit spaces containing pyocyanin. That is, the engineered CheZ modified with a C-terminal extension derived from the putative DNA-binding transcriptional regulator YbaQ (RREERAAKKVA) is consumed by the ClpXP protease machine at a rate sufficient to "brake" the cells when pyocyanin levels are too low. Through this process, we demonstrate that over time, these engineered E. coli accumulate in pyocyanin-rich locales. We suggest that such approaches may find utility in engineering probiotics so that their beneficial functions can be focused in areas of principal benefit.


Assuntos
Quimiotaxia/fisiologia , Proteínas de Escherichia coli/genética , Escherichia coli/fisiologia , Redes Reguladoras de Genes/genética , Melhoramento Genético/métodos , Proteínas Quimiotáticas Aceptoras de Metil/genética , Transativadores/genética , Quimiotaxia/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Piocianina/administração & dosagem , Biologia Sintética/métodos
20.
Biotechnol Bioeng ; 114(12): 2782-2793, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28755490

RESUMO

Metabolic engineering of isopropyl alcohol (IPA)-producing Escherichia coli strains was conducted along with 13 C-metabolic flux analysis (MFA). A metabolically engineered E. coli strain expressing the adc gene derived from Clostridium acetobutylicum and the IPADH gene from C. beijerinckii did not produce IPA during its exponential growth phase in the aerobic batch culture. 13 C-MFA was carried out, and revealed a deficiency in NADPH regeneration for IPA production in growth phase. Based on these findings, we used nitrogen-starved culture conditions to reduce NADPH consumption for biomass synthesis. As a result, IPA yield was increased to 20% mol/mol glucose. 13 C-MFA revealed that the relative flux levels through the oxidative pentose phosphate (PP) pathway and the TCA cycle were elevated in nitrogen-starved condition relative to glucose uptake rate. To prevent CO2 release in the 6-phosphogluconate dehydrogenase (6PGDH) reaction, metabolism of this E. coli strain was further engineered to redirect glycolytic flux to the glucose 6-phosphate dehydrogenase (G6PDH) and Entner-Doudoroff (ED) pathway. IPA yield of 55% mol/mol glucose was achieved by combining the nitrogen-starved culture condition with the metabolic redirection. The 13 C-MFA data and intracellular NADPH levels obtained under these IPA production conditions revealed linear correlations between the specific IPA production rate and NADPH concentration, as well as between IPA yield and the pyruvate dehydrogenase (PDH) flux. Our results showed that 13 C-MFA is a helpful tool for metabolic engineering studies, and that further improvement in IPA production by E. coli may be achieved by fine-tuning the cofactor ratio and concentrations, as well as optimizing the metabolic pathways and culture conditions.


Assuntos
2-Propanol/metabolismo , Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , Engenharia Metabólica/métodos , Análise do Fluxo Metabólico/métodos , Redes e Vias Metabólicas/fisiologia , 2-Propanol/isolamento & purificação , Proteínas de Bactérias/genética , Isótopos de Carbono/farmacocinética , Escherichia coli/classificação , Escherichia coli/citologia , Melhoramento Genético/métodos , Especificidade da Espécie
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