RESUMEN

Hyaluronic acid (HA) is an exopolysaccharide extracted from several sources such as rooster combs, umbilical cords and microorganisms. A system that controls temperature, agitation and aeration of bacterial cultures could make the HA production autonomous. Therefore, HA of microbial origin is set to take over alternative methods of production. Furthermore, the use of different nutrient sources in the culture medium and the purification stage applied in the process can cause physicochemical alterations on the bioproduct. For instance, structural modifications that change the molecular weight of HA may alter its elastic and viscoelastic properties. As a result, HA synthesized by microbes has applications in pharmacology, biotechnology, and tissue engineering. Our aim here, is to show the vast range of applications by compiling articles and patents on the culture media or genetic modifications of microorganisms that synthesize HA.

Asunto(s)

Ácido Hialurónico , Biotecnología , Medios de Cultivo , Ácido Hialurónico/biosíntesis , Ácido Hialurónico/aislamiento & purificación , Microorganismos Modificados Genéticamente

RESUMEN

Genomic islands (GIs) are horizontally transferred elements that shape bacterial genomes and contributes to the adaptation to different environments. Some GIs encode an integrase and a recombination directionality factor (RDF), which are the molecular GI-encoded machinery that promotes the island excision from the chromosome, the first step for the spread of GIs by horizontal transfer. Although less studied, this process can also play a role in the virulence of bacterial pathogens. While the excision of GIs is thought to be similar to that observed in bacteriophages, this mechanism has been only studied in a few families of islands. Here, we aimed to gain a better understanding of the factors involved in the excision of ROD21 a pathogenicity island of the food-borne pathogen Salmonella enterica serovar Enteritidis and the most studied member of the recently described Enterobacteriaceae-associated ROD21-like family of GIs. Using bioinformatic and experimental approaches, we characterized the conserved gene SEN1998, showing that it encodes a protein with the features of an RDF that binds to the regulatory regions involved in the excision of ROD21. While deletion or overexpression of SEN1998 did not alter the expression of the integrase-encoding gene SEN1970, a slight but significant trend was observed in the excision of the island. Surprisingly, we found that the expression of both genes, SEN1998 and SEN1970, were negatively correlated to the excision of ROD21 which showed a growth phase-dependent pattern. Our findings contribute to the growing body of knowledge regarding the excision of GIs, providing insights about ROD21 and the recently described EARL family of genomic islands.

Asunto(s)

Biología Computacional/métodos , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Genes Bacterianos , Islas Genómicas/genética , Salmonella enteritidis/genética , Transducción de Señal/genética , Secuencia de Aminoácidos , Cromosomas Bacterianos/genética , Cromosomas Bacterianos/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Expresión Génica , Regulación Bacteriana de la Expresión Génica , Integrasas/genética , Integrasas/metabolismo , Microorganismos Modificados Genéticamente , Mutación , Filogenia , Unión Proteica , Salmonella enteritidis/metabolismo , Salmonella enteritidis/patogenicidad , Virulencia/genética

RESUMEN

Optogenetic switches allow light-controlled gene expression with reversible and spatiotemporal resolution. In Saccharomyces cerevisiae, optogenetic tools hold great potential for a variety of metabolic engineering and biotechnology applications. In this work, we report on the modular optimization of the fungal light-oxygen-voltage (FUN-LOV) system, an optogenetic switch based on photoreceptors from the fungus Neurospora crassa. We also describe new switch variants obtained by replacing the Gal4 DNA-binding domain (DBD) of FUN-LOV with nine different DBDs from yeast transcription factors of the zinc cluster family. Among the tested modules, the variant carrying the Hap1p DBD, which we call "HAP-LOV", displayed higher levels of luciferase expression upon induction compared to FUN-LOV. Further, the combination of the Hap1p DBD with either p65 or VP16 activation domains also resulted in higher levels of reporter expression compared to the original switch. Finally, we assessed the effects of the plasmid copy number and promoter strength controlling the expression of the FUN-LOV and HAP-LOV components, and observed that when low-copy plasmids and strong promoters were used, a stronger response was achieved in both systems. Altogether, we describe a new set of blue-light optogenetic switches carrying different protein modules, which expands the available suite of optogenetic tools in yeast and can additionally be applied to other systems.

Asunto(s)

Proteínas Fúngicas , Microorganismos Modificados Genéticamente , Neurospora crassa/genética , Optogenética , Fotorreceptores Microbianos , Saccharomyces cerevisiae , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/genética , Microorganismos Modificados Genéticamente/genética , Microorganismos Modificados Genéticamente/metabolismo , Neurospora crassa/metabolismo , Fotorreceptores Microbianos/biosíntesis , Fotorreceptores Microbianos/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo

RESUMEN

Visceral leishmaniosis is one of the most important zoonotic diseases on the planet and dogs are the main reservoir of canine visceral leishmaniosis (CVL) in endemic areas. They play an important role in human infection because in dogs the disease appears long time after infection, and they can move uncontrollably, contributing to disperse the parasite. To take the decision to treat the animals or for euthanasia, in an elimination programme, in order to reduce the parasitic load, it is necessary to diagnose correctly, having more effective tools. Our group has developed a new recombinant antigen-based kinesin-related gene of Leishmania braziliensis (Lbk39), which shows 59% amino acid identity to the L. infantum homologue. The Lbk39 gene was synthesized, inserted into the pLEXSY-sat2 vector and transfected into L. tarentolae cells by electroporation. The recombinant protein was secreted in the culture with a C-terminal histidine marker, purified, generating a product at 337.68 µg mL-1. A total of 152 sera from dog's endemic and non-endemic areas were used, being 78 positives and 75 negatives. The antigen Lbk39 showed 100% sensitivity and 96.1% specificity. We compared this antigen with other antigens such as total extract of the parasite, TRDPP, and our data indicate that Lbk39 has potential application in the diagnosis of CVL through antibody detection.

Asunto(s)

Enfermedades de los Perros/diagnóstico , Leishmania braziliensis/genética , Leishmaniasis Visceral/veterinaria , Proteínas Protozoarias/uso terapéutico , Animales , Enfermedades de los Perros/parasitología , Perros , Leishmania/genética , Leishmaniasis Visceral/diagnóstico , Leishmaniasis Visceral/parasitología , Microorganismos Modificados Genéticamente/genética , Proteínas Recombinantes/uso terapéutico

RESUMEN

Intestinal fibrosis associated with Crohn's disease (CD), which a common and serious complication of inflammatory bowel diseases. In this context, heat shock proteins (HSPs) might serve as an alternative treatment because these antigens play important roles in the regulation of effector T cells. We thus evaluated the anti-inflammatory and antifibrotic capacities of an invasive and Hsp65-producing strain-Lactococcus lactis NCDO2118 FnBPA+ (pXYCYT:Hsp65)-in chronic intestinal inflammation to assess its potential as an alternative therapeutic strategy against fibrotic CD. Experimental colitis was induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS) in BALB/c mice, and the mice were treated orally with L. lactis NCDO2118 FnBPA+ (pXYCYT:Hsp65) via intragastric gavage. The oral administration of this strain significantly attenuated the severity of inflammation and intestinal fibrosis in mice (p < 0.05). These results are mainly justified by reductions in the levels of the pro-fibrotic cytokines IL-13 and TGF-ß and increases in the concentration of the regulatory cytokine IL-10. The L. lactis NCDO2118 FnBPA+ (pXYCYT:Hsp65) strain contributed to reductions in the severity of inflammatory damage in chronic experimental CD, and these findings confirm the effectiveness of this new antifibrotic strategy based on the delivery of therapeutic proteins to inside cells of the host intestinal mucosa.

Asunto(s)

Proteínas Bacterianas/farmacología , Chaperonina 60/farmacología , Colitis/tratamiento farmacológico , Lactococcus lactis/genética , Animales , Proteínas Bacterianas/administración & dosificación , Chaperonina 60/administración & dosificación , Colitis/inducido químicamente , Colitis/patología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Fibrosis/tratamiento farmacológico , Fibrosis/patología , Inmunoglobulina A/metabolismo , Ratones Endogámicos BALB C , Microorganismos Modificados Genéticamente , Ácido Trinitrobencenosulfónico/toxicidad

RESUMEN

Engineering resource allocation in biological systems is an ongoing challenge. Organisms allocate resources for ensuring survival, reducing the productivity of synthetic biology functions. Here we present a new approach for engineering the resource allocation of Escherichia coli by rationally modifying its transcriptional regulatory network. Our method (ReProMin) identifies the minimal set of genetic interventions that maximizes the savings in cell resources. To this end, we categorized transcription factors according to the essentiality of its targets and we used proteomic data to rank them. We designed the combinatorial removal of transcription factors that maximize the release of resources. Our resulting strain containing only three mutations, theoretically releasing 0.5% of its proteome, had higher proteome budget, increased production of an engineered metabolic pathway and showed that the regulatory interventions are highly specific. This approach shows that combining proteomic and regulatory data is an effective way of optimizing strains using conventional molecular methods.

Asunto(s)

Escherichia coli/genética , Escherichia coli/metabolismo , Ingeniería Genética/métodos , Proteoma/metabolismo , Biología Computacional/métodos , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Redes Reguladoras de Genes , Microorganismos Modificados Genéticamente , Mutación , Proteoma/genética , Análisis de Secuencia de ARN , Factores de Transcripción/genética , Factores de Transcripción/metabolismo

RESUMEN

For long time, studies on ectomycorrhiza (ECM) have been limited by inefficient expression of fluorescent proteins (FPs) in the fungal partner. To convert this situation, we have evaluated the basic requirements of FP expression in the model ECM homobasidiomycete Laccaria bicolor and established eGFP and mCherry as functional FP markers. Comparison of intron-containing and intronless FP-expression cassettes confirmed that intron-processing is indispensable for efficient FP expression in Laccaria. Nuclear FP localization was obtained via in-frame fusion of FPs between the intron-containing genomic gene sequences of Laccaria histone H2B, while cytosolic FP expression was produced by incorporating the intron-containing 5' fragment of the glyceraldehyde-3-phosphate dehydrogenase encoding gene. In addition, we have characterized the consensus Kozak sequence of strongly expressed genes in Laccaria and demonstrated its boosting effect on transgene mRNA accumulation. Based on these results, an Agrobacterium-mediated transformation compatible plasmid set was designed for easy use of FPs in Laccaria. The four cloning plasmids presented here allow fast and highly flexible construction of C-terminal in-frame fusions between the sequences of interest and the two FPs, expressed either from the endogenous gene promoter, allowing thus evaluation of the native regulation modes of the gene under study, or alternatively, from the constitutive Agaricus bisporus gpdII promoter for enhanced cellular protein localization assays. The molecular tools described here for cell-biological studies in Laccaria can also be exploited in studies of other biotrophic or saprotrophic basidiomycete species susceptible to genetic transformation.

Asunto(s)

Proteínas Fluorescentes Verdes/genética , Laccaria/genética , Proteínas Luminiscentes/genética , Plásmidos/genética , Proteínas Recombinantes de Fusión/genética , Agrobacterium/genética , Basidiomycota/genética , Núcleo Celular/genética , Citosol/metabolismo , Regulación Fúngica de la Expresión Génica , Proteínas Fluorescentes Verdes/metabolismo , Histonas/genética , Laccaria/metabolismo , Proteínas Luminiscentes/metabolismo , Microorganismos Modificados Genéticamente , Microscopía Fluorescente , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/metabolismo , Transformación Genética , Proteína Fluorescente Roja

RESUMEN

Microbial production of carotenoids has mainly focused towards a few products, such as ß-carotene, lycopene and astaxanthin. However, other less explored carotenoids, like violaxanthin, have also shown unique properties and promissory applications. Violaxanthin is a plant-derived epoxidated carotenoid with strong antioxidant activity and a key precursor of valuable compounds, such as fucoxanthin and ß-damascenone. In this study, we report for the first time the heterologous production of epoxycarotenoids in yeast. We engineered the yeast Saccharomyces cerevisiae following multi-level strategies for the efficient accumulation of violaxanthin. Starting from a ß-carotenogenic yeast strain, we first evaluated the performance of several ß-carotene hydroxylases (CrtZ), and zeaxanthin epoxidases (ZEP) from different species, together with their respective N-terminal truncated variants. The combined expression of CrtZ from Pantoea ananatis and truncated ZEP of Haematococcus lacustris showed the best performance and led to a yield of 1.6 mg/gDCW of violaxanthin. Further improvement of the epoxidase activity was achieved by promoting the transfer of reducing equivalents to ZEP by expressing several redox partner systems. The co-expression of the plant truncated ferredoxin-3, and truncated root ferredoxin oxidoreductase-1 resulted in a 2.2-fold increase in violaxanthin yield (3.2 mg/gDCW). Finally, increasing gene copy number of carotenogenic genes enabled reaching a final production of 7.3 mg/gDCW in shake flask cultures and batch bioreactors, which is the highest yield of microbially produced violaxanthin reported to date.

Asunto(s)

Ingeniería Metabólica , Microorganismos Modificados Genéticamente , Saccharomyces cerevisiae , Microorganismos Modificados Genéticamente/genética , Microorganismos Modificados Genéticamente/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Xantófilas/metabolismo

RESUMEN

Under stressful conditions some microorganisms adopt a quiescent stage characterized by a reversible non or slow proliferative condition that allows their survival. This adaptation was only recently discovered in Leishmania. We developed an in vitro model and a biosensor to track quiescence at population and single cell levels. The biosensor is a GFP reporter gene integrated within the 18S rDNA locus, which allows monitoring the expression of 18S rRNA (rGFP expression). We showed that rGFP expression decreased significantly and rapidly during the transition from extracellular promastigotes to intracellular amastigotes and that it was coupled in vitro with a decrease in replication as measured by BrdU incorporation. rGFP expression was useful to track the reversibility of quiescence in live cells and showed for the first time the heterogeneity of physiological stages among the population of amastigotes in which shallow and deep quiescent stages may coexist. We also validated the use of rGFP expression as a biosensor in animal models of latent infection. Our models and biosensor should allow further characterization of quiescence at metabolic and molecular level.

Asunto(s)

ADN Protozoario , ADN Ribosómico , Sitios Genéticos , Proteínas Fluorescentes Verdes , Leishmania braziliensis , Leishmania mexicana , Microorganismos Modificados Genéticamente , Animales , ADN Protozoario/genética , ADN Protozoario/metabolismo , ADN Ribosómico/genética , ADN Ribosómico/metabolismo , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Leishmania braziliensis/citología , Leishmania braziliensis/crecimiento & desarrollo , Leishmania braziliensis/metabolismo , Leishmania mexicana/citología , Leishmania mexicana/genética , Leishmania mexicana/metabolismo , Ratones

RESUMEN

The live attenuated mycobacterial strain BCG, in use as vaccine against tuberculosis, is considered the gold standard for primary therapy of carcinoma in situ of the bladder. Despite its limitations, to date it has not been surpassed by any other treatment. Our group has developed a recombinant BCG strain expressing the detoxified S1 pertussis toxin (rBCG-S1PT) that proved more effective than wild type BCG (WT-BCG) in increasing survival time in an experimental mouse model of bladder cancer, due to the well-known adjuvant properties of pertussis toxin. Here, we investigated the capacity of rBCG-S1PT to stimulate human immune responses, in comparison to WT-BCG, using an in vitro stimulation assay based on human whole blood cells that allows for a comprehensive evaluation of leukocyte activation. Blood leukocytes stimulated with rBCG-S1PT produced increased levels of IL-6, IL-8, and IL-10 as compared to WT-BCG, but comparable levels of IL-1ß, IL-2, IFN-γ, and TNF-α. Stimulation of blood cells with the recombinant BCG strain also enhanced the expression of CD25 and CD69 on human CD4+ T cells. PBMC stimulated with rBCG-S1PT induced higher cytotoxicity to MB49 bladder cancer cells than WT-BCG-stimulated PBMC. These results suggest that the rBCG-S1PT strain is able to activate an immune response in human leukocytes that is higher than that induced by WT-BCG for parameters linked to better prognosis in bladder cancer (regulation of immune and early inflammatory responses), while fully comparable to WT-BCG for classical inflammatory parameters. This establishes rBCG-S1PT as a new highly effective candidate as immunotherapeutic agent against bladder cancer.

Asunto(s)

Linfocitos T CD4-Positivos/inmunología , Inmunidad Celular , Microorganismos Modificados Genéticamente/inmunología , Mycobacterium bovis/inmunología , Neoplasias de la Vejiga Urinaria/terapia , Adulto , Anciano , Animales , Linfocitos T CD4-Positivos/patología , Línea Celular Tumoral , Citocinas/inmunología , Femenino , Humanos , Masculino , Ratones , Microorganismos Modificados Genéticamente/genética , Persona de Mediana Edad , Mycobacterium bovis/genética , Toxina del Pertussis/genética , Toxina del Pertussis/inmunología , Neoplasias de la Vejiga Urinaria/inmunología , Neoplasias de la Vejiga Urinaria/patología

RESUMEN

Trypanosoma cruzi is under the attack of reactive species produced by its mammalian and insect hosts. To survive, it must repair its damaged DNA. We have shown that a base excision DNA repair (BER)-specific parasite TcAP1 endonuclease is involved in the resistance to H2 O2 . However, a putative TcAP1 negative dominant form impairing TcAP1 activity in vitro did not show any in vivo effect. Here, we show that a negative dominant form of the human APE1 apurinic/apyrimidinic (AP) endonuclease (hAPE1DN) induces a decrease in epimastigote and metacyclic trypomastigote viability when parasites were exposed to H2 O2 . Those results confirm that TcAP1 AP endonuclease activity plays an important role in epimastigote and in infective metacyclic trypomastigote oxidative DNA damage resistance leading to parasite persistence in the insect and mammalian hosts. All along its biological cycle and in its different cellular forms, T. cruzi, the etiological parasite agent of Chagas' disease, is under the attack of reactive species produced by its mammalian and insect hosts. To survive, T. cruzi must repair their oxidative damaged DNA. We have previously shown that a specific parasite TcAP1 AP endonuclease of the BER is involved in the T. cruzi resistance to oxidative DNA damage. We have also demonstrated that epimastigotes and cell-derived trypomastigotes parasite forms expressing a putative TcAP1 negative dominant form (that impairs the TcAP1 activity in vitro), did not show any in vivo effect in parasite viability when exposed to oxidative stress. In this work, we show the expression of a negative dominant form of the human APE1 AP endonuclease fused to a green fluorescent protein (GFP; hAPE1DN-GFP) in T. cruzi epimastigotes. The fusion protein is found both in the nucleus and cytoplasm of noninfective epimastigotes but only in the nucleus in metacyclic and cell-derived trypomastigote infective forms. Contrarily to the TcAP1 negative dominant form, the ectopic expression of hAPE1DN-GFP induces a decrease in epimastigote and metacyclic trypomastigote viability when parasites were exposed to increasing H2 O2 concentrations. No such effect was evident in expressing hAPE1DN-GFP cell-derived trypomastigotes. Although the viability of both wild-type infective trypomastigote forms diminishes when parasites are submitted to acute oxidative stress, the metacyclic forms are more resistant to H2 O2 exposure than cell-derived trypomastigotes.Those results confirm that the BER pathway and particularly the AP endonuclease activity play an important role in epimastigote and metacyclic trypomastigote oxidative DNA damage resistance leading to parasite survival and persistence inside the mammalian and insect host cells.

Asunto(s)

Daño del ADN , Reparación del ADN , Estrés Oxidativo , Proteínas Protozoarias/metabolismo , Trypanosoma cruzi/enzimología , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , Genes Dominantes , Humanos , Peróxido de Hidrógeno/farmacología , Microorganismos Modificados Genéticamente , Proteínas Protozoarias/genética , Trypanosoma cruzi/genética

RESUMEN

Escherichia coli strains W3110 and BL21 were engineered for the production of plasmid DNA (pDNA) under aerobic and transitions to microaerobic conditions. The gene coding for recombinase A (recA) was deleted in both strains. In addition, the Vitreoscilla hemoglobin (VHb) gene (vgb) was chromosomally inserted and constitutively expressed in each E. coli recA mutant and wild type. The recA inactivation increased the supercoiled pDNA fraction (SCF) in both strains, while VHb expression improved the pDNA production in W3110, but not in BL21. Therefore, a codon-optimized version of vgb was inserted in strain BL21recA-, which, together with W3110recA-vgb+, was tested in cultures with shifts from aerobic to oxygen-limited regimes. VHb expression lowered the accumulation of fermentative by-products in both strains. VHb-expressing cells displayed higher oxidative activity as indicated by the Redox Sensor Green fluorescence, which was more intense in BL21 than in W3110. Furthermore, VHb expression did not change pDNA production in W3110, but decreased it in BL21. These results are useful for understanding the physiological effects of VHb expression in two industrially relevant E. coli strains, and for the selection of a host for pDNA production.

Asunto(s)

Escherichia coli/metabolismo , Microorganismos Modificados Genéticamente/metabolismo , Plásmidos/biosíntesis , Aerobiosis , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Cromosomas Bacterianos/genética , Cromosomas Bacterianos/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Eliminación de Gen , Microorganismos Modificados Genéticamente/genética , Plásmidos/genética , Rec A Recombinasas/genética , Rec A Recombinasas/metabolismo , Hemoglobinas Truncadas/biosíntesis , Hemoglobinas Truncadas/genética

RESUMEN

Cyanobacteria have been considered as promising candidates for sustainable bioproduction from inexpensive raw materials, as they grow on light, carbon dioxide, and minimal inorganic nutrients. In this study, we present a genome-scale metabolic network model for Synechocystis sp. PCC 6803 and study the optimal design of the strain for ethanol production by using a mixed integer linear problem reformulation of a bilevel programming problem that identifies gene knockouts which lead to coupling between growth and product synthesis. Five mutants were found, where the in silico model predicts coupling between biomass growth and ethanol production in photoautotrophic conditions. The best mutant gives an in silico ethanol production of 1.054 mmol·gDW -1 ·h -1 .

Asunto(s)

Biocombustibles , Etanol/metabolismo , Synechocystis/genética , Synechocystis/metabolismo , Técnicas de Inactivación de Genes , Microbiología Industrial , Redes y Vías Metabólicas , Microorganismos Modificados Genéticamente , Modelos Biológicos

RESUMEN

A synthetic plasmid consisting of the minimal elements for replication control of the R1 replicon and kanamycin resistance marker, which was named pminiR1, was developed. pminiR1 production was tested at 30 °C under aerobic and microaerobic conditions in Escherichia coli W3110 recA- (W1). The plasmid DNA yields from biomass (YpDNA/X) were only 0.06 ± 0.02 and 0.22 ± 0.11 mg/g under aerobic and microaerobic conditions, respectively. As an option to increase YpDNA/X values, pminiR1 was introduced in an engineered E. coli strain expressing the Vitreoscilla hemoglobin inserted in chromosome (W12). The YpDNA/X values using strain W12 increased to 0.85 ± 0.05 and 1.53 ± 0.14 mg/g under aerobic and microaerobic conditions, respectively. pminiR1 production in both strains was compared with that of pUC57Kan at 37 °C under aerobic and microaerobic conditions. The YpDNA/X values for pminiR1 using strain W12 were 6.25 ± 0.16 and 9.27 ± 0.95 mg/g under aerobic and microaerobic conditions, respectively. Such yields were similar to those obtained for plasmid pUC57Kan using strain W12 (6.9 ± 0.64 and 10.85 ± 1.06 mg/g for aerobic and microaerobic cultures, respectively). Therefore, the synthetic minimal plasmid based on the R1 replicon is a valuable alternative to pUC plasmids for biotechnological applications.

Asunto(s)

Escherichia coli , Microorganismos Modificados Genéticamente , Plásmidos , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Cromosomas Bacterianos/genética , Cromosomas Bacterianos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Microorganismos Modificados Genéticamente/genética , Microorganismos Modificados Genéticamente/metabolismo , Plásmidos/biosíntesis , Plásmidos/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Hemoglobinas Truncadas/biosíntesis , Hemoglobinas Truncadas/genética

RESUMEN

The development of new heterologous hosts for polyketides production represents an excellent opportunity to expand the genomic, physiological, and biochemical backgrounds that better fit the sustainable production of these valuable molecules. Cyanobacteria are particularly attractive for the production of natural compounds because they have minimal nutritional demands and several strains have well established genetic tools. Using the model strain Synechococcus elongatus, a generic platform was developed for the heterologous production of polyketide synthase (PKS)-derived compounds. The versatility of this system is based on interchangeable modules harboring promiscuous enzymes for PKS activation and the production of PKS extender units, as well as inducible circuits for a regulated expression of the PKS biosynthetic gene cluster. To assess the capability of this platform, we expressed the mycobacterial PKS-based mycocerosic biosynthetic pathway to produce multimethyl-branched esters (MBE). This work is a foundational step forward for the production of high value polyketides in a photosynthetic microorganism.

Asunto(s)

Ingeniería Metabólica , Microorganismos Modificados Genéticamente , Policétidos/metabolismo , Synechococcus , Microorganismos Modificados Genéticamente/genética , Microorganismos Modificados Genéticamente/metabolismo , Synechococcus/genética , Synechococcus/metabolismo

RESUMEN

The equilibrium between protein synthesis and degradation is key to maintaining efficiency in different physiological processes. The proteinase inhibitor cystatin regulates protease activities in different developmental and physiological contexts. Here we describe for the first time the identification and the biological function of the cysteine protease inhibitor cystatin of Fragaria chiloensis, FchCYS1. Based on primary sequence and 3D-structural homology modelling, FchCYS1 is a type II phytocystatin with high identity to other cystatins of the Fragaria genus. Both the papain-like and the legumain-like protease inhibitory domains are indeed functional, based on in vitro assays performed with Escherichia coli protein extracts containing recombinant FchCYS1. FchCYS1 is differentially-expressed in achenes of F. chiloensis fruits, with highest expression as the fruit reaches the ripened stage, suggesting a role in preventing degradation of storage proteins that will nourish the embryo during seed germination. Furthermore, FchCYS1 responds transcriptionally to the application of salicylic acid and to mechanical injury, strongly suggesting that FchCYS1 could be involved in the response against pathogen attack. Overall these results point to a role for FchCYS1 in diverse physiological processes in F. chiloensis.

Asunto(s)

Cistatinas/metabolismo , Fragaria/metabolismo , Frutas/metabolismo , Proteínas de Plantas/metabolismo , Cistatinas/fisiología , Cisteína Endopeptidasas/metabolismo , Escherichia coli , Fragaria/fisiología , Frutas/crecimiento & desarrollo , Frutas/fisiología , Microorganismos Modificados Genéticamente , Papaína/metabolismo , Proteínas de Plantas/fisiología , Estrés Fisiológico , Transcriptoma

RESUMEN

The environmental effects of chemical fertilizers and pesticides have encouraged the quest for new strategies to increase crop productivity with minimal impacts on the natural medium. Plant growth promoting rhizobacteria (PGPR) can contribute to this endeavor by improving fitness through better nutrition acquisition and stress tolerance. Using the neutral (non PGPR) rhizobacterium Cupriavidus pinatubonensis JMP134 as the host, we engineered a regulatory forward loop that triggered the synthesis of the phytohormone indole-3-acetic acid (IAA) in a manner dependent on quorum sensing (QS) signals. Implementation of the device in JMP134 yielded synthesis of IAA in an autoregulated manner, improving the growth of the roots of inoculated Arabidopsis thaliana. These results not only demonstrated the value of the designed genetic module, but also validated C. pinatubonensis JMP134 as a suitable vehicle for agricultural applications, as it is amenable to genetic manipulations.

Asunto(s)

Arabidopsis/crecimiento & desarrollo , Arabidopsis/microbiología , Cupriavidus/metabolismo , Ácidos Indolacéticos/metabolismo , Ingeniería Metabólica/métodos , 4-Butirolactona/análogos & derivados , 4-Butirolactona/metabolismo , Cupriavidus/genética , Retroalimentación Fisiológica , Regulación Bacteriana de la Expresión Génica , Microorganismos Modificados Genéticamente , Raíces de Plantas/microbiología , Plásmidos/genética , Percepción de Quorum , Simbiosis

RESUMEN

Mezcal, a traditional beverage that originated in Mexico, is produced from species of the Agavaceae family. The esters associated with the yeasts utilized during fermentation are important for improving the organoleptic properties of the beverage. We improved the ester contents in a mezcal beverage by using the yeast Kluyveromyces marxianus, which was engineered with the ATF1 gene. ATF1 expression in the recombinant yeast significantly increased compared with that in the parental yeast, but its fermentative parameters were unchanged. Volatile-organic-compound-content analysis showed that esters had significantly increased in the mezcal produced with the engineered yeast. In a sensory-panel test, 48% of the panelists preferred the mezcal produced from the engineered yeast, 30% preferred the mezcal produced from the wild type, and 15 and 7% preferred the two mezcal types produced following the routine procedure. Correlation analysis showed that the fruitiness/sweetness description of the mezcal produced using the ATF1-engineered K. marxianus yeast correlated with the content of the esters, whose presence improved the organoleptic properties of the craft mezcal beverage.

Asunto(s)

Bebidas Alcohólicas/análisis , Asparagaceae/química , Ácidos Grasos/análisis , Kluyveromyces/genética , Proteínas/genética , Sensación , Comportamiento del Consumidor , Ésteres/análisis , Fermentación , Expresión Génica , Humanos , Kluyveromyces/metabolismo , México , Microorganismos Modificados Genéticamente/genética , Proteínas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo

RESUMEN

Pseudomonas sp. PHA- was used as host for PHA biosynthesis genes from Aeromonas sp. to produce 3HB-co-3HAMCL from glucose with no supply of co-substrates. A non-naturally-occurring PHA composed mainly of 3HB, 3HHx and 3HD (3HO, 3HDdΔ5 and 3HDd monomers were detected in smaller amounts) was obtained. The polymer was extracted using two different solvents (acetone and chloroform) and subject to the following characterization tests: FTIR, DSC, TGA and GPC. The latter suggests a block copolymer since a single and narrow elution peak was observed for each sample. The DSC results ruled out the possibility of a random copolymer and agrees with a single copolymer composed of two blocks: one with the typical composition of PHAMCL produced by Pseudomonas and another containing 3HB and 3HHx with a high 3HHx molar fraction. Thus, this study increases the perspectives of P(3HB-co-3HAMCL) production from carbohydrates as the sole carbon source.

Asunto(s)

Glucosa/metabolismo , Microorganismos Modificados Genéticamente/metabolismo , Poliésteres/metabolismo , Pseudomonas/metabolismo , Aeromonas/genética , Carbono/metabolismo , Microorganismos Modificados Genéticamente/genética , Pseudomonas/genética

RESUMEN

Biodiesels produced from transesterification of vegetable oils have a major problem in quality due to the presence of precipitates, which are mostly composed of steryl glucosides (SGs). We have recently described an enzymatic method for the efficient removal of SGs from biodiesel, based on the activity of a thermostable ß-glycosidase from Thermococcus litoralis. In the present work, we describe the development of an Escherichia coli-based expression system and a high cell density fermentation process. Strain and process engineering include the assessment of different promoters to drive the expression of a codon-optimized gene, the co-expression of molecular chaperones and the development of a high cell density fermentation process. A 200-fold increase in the production titers was achieved, which directly impacts on the costs of the industrial process for treating biodiesel.

Asunto(s)

Escherichia coli/genética , Fermentación , Glucosidasas/química , Microorganismos Modificados Genéticamente , Biocombustibles , Codón , Escherichia coli/enzimología , Ingeniería Genética , Aceites de Plantas/metabolismo , Thermococcus