RESUMO
Yarrowia lipolytica IMUFRJ 50682 is a Brazilian wild-type strain with potential application in bioconversion processes which can be improved through synthetic biology. In this study, we focused on a combinatorial dual cleavage CRISPR/Cas9-mediated for construction of irreversible auxotrophic mutants IMUFRJ 50682, which genomic information is not available, thought paired sgRNAs targeting upstream and downstream sites of URA3 gene. The disruption efficiency ranged from 5 to 28 % for sgRNAs combinations closer to URA3's start and stop codon and the auxotrophic mutants lost about 970 bp containing all coding sequence, validating this method for genomic edition of wild-type strains. In addition, we introduced a fluorescent phenotype and achieved cloning rates varying from 80 to 100 %. The ura3Δ strains IMUFRJ 50682 were also engineered for ß-carotene synthesis as proof of concept. Carotenoid-producing strains exhibited a similar growth profile compared to the wild-type strain and were able to synthesized 30.54-50.06â¯mg/L (up to 4.8â¯mg/g DCW) of ß-carotene in YPD and YNB flask cultures, indicating a promisor future of the auxotrophic mutants IMUFRJ 50682 as a chassis for production of novel value-added chemicals.
Assuntos
Sistemas CRISPR-Cas , Engenharia Metabólica/métodos , Yarrowia/genética , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Meios de Cultura/metabolismo , Fluorescência , Proteínas Fúngicas/genética , Marcação de Genes , Mutação , RNA Guia de Cinetoplastídeos/genética , Uracila/metabolismo , Yarrowia/crescimento & desenvolvimento , Yarrowia/metabolismo , beta Caroteno/biossíntese , beta Caroteno/genéticaRESUMO
This study aimed to evaluate the production of carotenoid pigments by Rhodotorula spp. in submerged fermentation, using residual glycerin from biodiesel production as a carbon source. Chromatographic analysis by HPLC showed that the residual glycerin used as substrate was 57.88% composed of glycerol. The best growth conditions were found in the fermentation medium composed of residual glycerin at a concentration of 30 g/L and pH 9. From all the Rhodotorula strains tested, R. minuta URM6693 was selected because of their performance and adaptation in all culture media assayed. The maximum volumetric production of carotenoids was found at 48 h (equivalent to 17.20 mg/L, for the R. minuta). The production of ß-carotene since the first 24 h of fermentation reach a final concentration of 1.021 mg/L. The yeast Rhodotorula minuta proved its capability to efficiently convert the substrate (mainly at the concentration of 50 g/L), obtaining products of biotechnological interest.
Assuntos
Glicerol/metabolismo , Rhodotorula/crescimento & desenvolvimento , beta Caroteno/biossínteseRESUMO
Carrot (Daucus carota) is one of the most important vegetable cultivated worldwide and the main source of dietary provitamin A. Contrary to other plants, almost all carrot varieties accumulate massive amounts of carotenoids in the root, resulting in a wide variety of colors, including those with purple, yellow, white, red and orange roots. During the first weeks of development the root, grown in darkness, is thin and pale and devoid of carotenoids. At the second month, the thickening of the root and the accumulation of carotenoids begins, and it reaches its highest level at 3 months of development. This normal root thickening and carotenoid accumulation can be completely altered when roots are grown in light, in which chromoplasts differentiation is redirected to chloroplasts development in accordance with an altered carotenoid profile. Here we discuss the current evidence on the biosynthesis of carotenoid in carrot roots in response to environmental cues that has contributed to our understanding of the mechanism that regulates the accumulation of carotenoids, as well as the carotenogenic gene expression and root development in D. carota.
Assuntos
Carotenoides/biossíntese , Daucus carota/metabolismo , Pigmentos Biológicos/biossíntese , beta Caroteno/biossíntese , Carotenoides/metabolismo , Daucus carota/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas , Humanos , Pigmentos Biológicos/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Plastídeos/genética , Plastídeos/metabolismo , Vitamina A/biossíntese , Vitamina A/metabolismo , beta Caroteno/genéticaRESUMO
There is an increasing demand for carotenoids, which are fundamental components of the human diet, for example as precursors of vitamin A. Carotenoids are also potent antioxidants and their health benefits are becoming increasingly evident. Protective effects against prostate cancer and age-related macular degeneration have been proposed for lycopene and lutein/zeaxanthin, respectively. Additionally, ß-carotene, astaxanthin and canthaxanthin are high-value carotenoids used by the food industry as feed supplements and colorants. The production and consumption of these carotenoids from natural sources, especially from seeds, constitutes an important step towards fortifying the diet of malnourished people in developing nations. Therefore, attempts to metabolically manipulate ß-carotene production in plants have received global attention, especially after the generation of Golden Rice (Oryza sativa). The endosperms of Golden Rice seeds synthesize and accumulate large quantities of ß-carotene (provitamin A), yielding a characteristic yellow color in the polished grains. Classical breeding efforts have also focused in the development of cultivars with elevated seed carotenoid content, with maize and other cereals leading the way. In this communication we will summarize transgenic efforts and modern breeding strategies to fortify various crop seeds with nutraceutical carotenoids.
Assuntos
Carotenoides/metabolismo , Oryza/metabolismo , Sementes/metabolismo , Vitamina A/metabolismo , Biotecnologia , Cruzamento , Carotenoides/biossíntese , Regulação da Expressão Gênica de Plantas , Humanos , Oryza/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Sementes/genética , beta Caroteno/biossíntese , beta Caroteno/metabolismoRESUMO
BACKGROUND: The effectiveness of hot air treatments in controlling decay and insects in mango fruit has been demonstrated and has usually been assessed as a function of the temperature of the heated air and the duration of the treatment. However, the contribution of the moisture content of the heated air has received little attention, especially with regard to fruit quality. In this study, mango fruits (cv. Manila) at mature-green stage were treated with moist (95% relative humidity (RH)) or dry (50% RH) hot forced air (43 °C, at 2.5 m s(-1) for 220 min) and then held at 20 °C for 9 days and evaluated periodically. RESULTS: The heating rate was higher with moist air. Treatments with moist and dry air did not cause injury to the fruit. Treatment with moist air temporarily slowed down color development, softening, weight loss and ß-carotene biosynthesis. This slowing down was clearly observed during the first 4-5 days at 20 °C. However, non-heated fruit and fruit heated with dry air showed similar quality at the end of storage. CONCLUSION: The moisture content of the heating air differentially modulated the postharvest ripening of 'Manila' mangoes. Moist air temporarily slowed down the ripening process of this mango cultivar.
Assuntos
Ar , Manipulação de Alimentos/métodos , Conservação de Alimentos/métodos , Frutas/química , Temperatura Alta , Mangifera/química , Água , Cor , Dieta , Frutas/normas , Humanos , Umidade , beta Caroteno/biossínteseRESUMO
Plant carotenoids are synthesized and accumulated in plastids through a highly regulated pathway. Lycopene ß-cyclase (LCYB) is a key enzyme involved directly in the synthesis of α-carotene and ß-carotene through the cyclization of lycopene. Carotenoids are produced in both carrot (Daucus carota) leaves and reserve roots, and high amounts of α-carotene and ß-carotene accumulate in the latter. In some plant models, the presence of different isoforms of carotenogenic genes is associated with an organ-specific function. D. carota harbors two Lcyb genes, of which DcLcyb1 is expressed in leaves and storage roots during carrot development, correlating with an increase in carotenoid levels. In this work, we show that DcLCYB1 is localized in the plastid and that it is a functional enzyme, as demonstrated by heterologous complementation in Escherichia coli and over expression and post transcriptional gene silencing in carrot. Transgenic plants with higher or reduced levels of DcLcyb1 had incremented or reduced levels of chlorophyll, total carotenoids and ß-carotene in leaves and in the storage roots, respectively. In addition, changes in the expression of DcLcyb1 are accompanied by a modulation in the expression of key endogenous carotenogenic genes. Our results indicate that DcLcyb1 does not possess an organ specific function and modulate carotenoid gene expression and accumulation in carrot leaves and storage roots.
Assuntos
Daucus carota/enzimologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Regulação da Expressão Gênica de Plantas/fisiologia , Liases Intramoleculares/biossíntese , Folhas de Planta/enzimologia , Proteínas de Plantas/biossíntese , Raízes de Plantas/enzimologia , Daucus carota/genética , Liases Intramoleculares/genética , Folhas de Planta/genética , Proteínas de Plantas/genética , Raízes de Plantas/genética , beta Caroteno/biossíntese , beta Caroteno/genéticaRESUMO
beta-carotene is a commonly used food colorant. In this work, a novel beta-carotene producing strain, Serratia marcescens RB3, was isolated and identified by physiological and biochemical tests, as well as 16S rDNA sequence analysis. The production of beta-carotene by S. marcescens RB3 was identified through HPLC analysis. The cultivation conditions for beta-carotene production by S. marcescens RB3 were optimized as 2.0 percent lactose, 2.0 percent peptone, 0.3 percent beef extract, 1.0 percent NaCl supplemented with 0.05 percent Fe2+, pH 6.0 and 30ºC. Under the optimal conditions, the yield of beta-carotene achieved 2.45 ug/mL.
Assuntos
Serratia marcescens/metabolismo , beta Caroteno/biossíntese , Cromatografia Líquida de Alta Pressão , Corantes de Alimentos , Concentração de Íons de Hidrogênio , TemperaturaRESUMO
Yeasts of the genera Rhodotorula are able to synthesize different pigments of high economic value like ß-carotene, torulene, and torularhodin, and therefore represent a biotechnologically interesting group of yeasts. However, the low production rate of pigment in these microorganisms limits its industrial application. Here we describe some strategies to obtain hyperpigmented mutants of Rhodotorula mucilaginosa by means of ultraviolet-B radiation, the procedures for total carotenoids extraction and quantification, and a method for identification of each pigment.
Assuntos
Carotenoides/biossíntese , Engenharia Metabólica/métodos , Rhodotorula/genética , Rhodotorula/metabolismo , Carotenoides/análise , Carotenoides/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Mutagênese , Mutação , beta Caroteno/análise , beta Caroteno/biossíntese , beta Caroteno/isolamento & purificaçãoRESUMO
In Dunaliella salina, we studied the early steps in the isoprenoid pathway for the biosynthesis of carotenoids and beta-carotene and the effects of metabolic inhibitors. When D. salina was grown under carotenogenic and non-carotenogenic conditions, mevinolin did not inhibit growth or the accumulation of carotenoids, beta-carotene or chlorophyll. In contrast, fosmidomycin progressively inhibited cell growth and the biosynthesis of carotenoids, beta-carotene and chlorophyll. In this work, we reported for the first time that in D. salina, beta-carotene biosynthesis does not proceed via the classical acetate/mevalonate pathway but via the novel glyceraldehyde 3-phosphate/pyruvate pathway. This favours the yield of C(5) isoprenoid units for synthesis of isopentenyl diphosphate, the precursor in the biosynthesis of C(20) compounds, including geranylgeranyl diphosphate. Consequently, this pathway promotes carotenogenesis and the biosynthesis of C(40) beta-carotene in D. salina.
Assuntos
Clorófitas/metabolismo , beta Caroteno/biossíntese , Fosfomicina/análogos & derivados , Gliceraldeído 3-Fosfato/metabolismo , Lovastatina , Redes e Vias Metabólicas , Ácido Pirúvico/metabolismoRESUMO
D. salina is one of the recognized natural sources to produce beta-carotene, and an useful model for studying the role of inhibitors and enhancers of carotenogenesis. However there is little information in D. salina regarding whether the isoprenoid substrate can be influenced by stress factors (carotenogenic) or selective inhibitors which in turn may further contribute to elucidate the early steps of carotenogenesis and biosynthesis of beta-carotene. In this study, Dunaliella salina (BC02) isolated from La Salina BC Mexico, was subjected to the method of isoprenoids-beta-carotene interference in order to promote the interruption or accumulation of the programmed biosynthesis of carotenoids. When Carotenogenic and non-carotenogenic cells of D. salina BC02 were grown under photoautotrophic growth conditions in the presence of 200 microM fosmidomycin, carotenogenesis and the synthesis of beta-carotene were interrupted after two days in cultured D. salina cells. This result is an indirect consequence of the inhibition of the synthesis of isoprenoids and activity of the recombinant DXR enzyme thereby preventing the conversion of 1-deoxy-D-xylulose 5-phosphate (DXP) to 2-C-methyl-D-erythritol (MEP) and consequently interrupts the early steps of carotenogenesis in D. salina. The effect at the level of proteins and RNA was not evident. Mevinolin treated D. salina cells exhibited carotenogenesis and beta-carotene levels very similar to those of control cell cultures indicating that mevinolin not pursued any indirect action in the biosynthesis of isoprenoids and had no effect at the level of the HMG-CoA reductase, the key enzyme of the Ac/MVA pathway.