Construction of wild-type Yarrowia lipolytica IMUFRJ 50682 auxotrophic mutants using dual CRISPR/Cas9 strategy for novel biotechnological approaches.

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 30.54-50.06 mg/L (up to 4.8 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.

Reutilization of residual glycerin for the produce ß-carotene by Rhodotorula minuta.

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.

Carotenoid Biosynthesis in Daucus carota.

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.

Modern Breeding and Biotechnological Approaches to Enhance Carotenoid Accumulation in Seeds.

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.

Effect of the moisture content of forced hot air on the postharvest quality and bioactive compounds of mango fruit (Mangifera indica L. cv. Manila).

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.

Levels of lycopene ß-cyclase 1 modulate carotenoid gene expression and accumulation in Daucus carota.

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.

Optimization of beta-carotene production by a newly isolated Serratia marcescens strain

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.

Production of torularhodin, torulene, and ß-carotene by Rhodotorula yeasts.

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.

The biosynthesis and accumulation of beta-carotene in Dunaliella salina proceed via the glyceraldehyde 3-phosphate/pyruvate pathway.

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.

The carotenogenesis pathway via the isoprenoid-beta-carotene interference approach in a new strain of Dunaliella salina isolated from Baja California Mexico.

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.

Crecimiento y producción de metabolitos de la cianobacteria marina Synechococcus sp. (Chroococcales) en función de la irradiancia

Growth and metabolite production of the marine cyanobacterium Synechococcus sp. (Chroococcales) in function to irradiance. Changes in salinity, temperature and irradiance during wet and dry seasons have induced metabolic versatility in cyanobacteria from saline environments. Cyanobacteria from these environments have biotechnological potential for the production of metabolites with pharmaceutical and industrial interest. We studied the growth, dry mass and metabolite production of the cyanobacterium Synechococcus sp. MOF-03 in function of irradiance (78, 156 and 234 µmol q m-2 s-1). All batch cultures were maintained by triplicate in constant aeration, 12:12 h photoperiod, 30 ±2ºC and 35‰. Maximum values of protein, carbohydrates and lipids, of 530.19 ±11.16, 408.94 ±4.27 and 56.20 ±1.17 µg ml-1, respectively, were achieved at 78 µmol q m-2 s-1. Pigments, analyzed by HPLC, showed maximum values at 78 µmol q m-2 s-1 for chlorophyll a with 7.72 ±0.16 µg ml-1, and at 234 µmol q m-2 s-1 for ß-carotene and zeaxanthin with 0.70 ±0.01 and 0.67 ±0.05 µg ml-1. Chlorophyll a:ß-carotene ratio decreased from 17.15 to 6.91 at 78 and 234 µmol q m-2 s-1; whereas ß-carotene:zeaxanthin ratio showed no changes between 78 and 156 µmol q m-2 s-1, around 1.21, and decreased at 234 µmol q m-2 s-1, to 1.04. Also, this cyanobacterium produced the greatest cell density and dry mass at 156 µmol q m-2 s-1, with 406.13 ±21.74 x106 cell ml-1 and 1.49 ±0.11 mg ml-1, respectively. Exopolysaccharide production was stable between 156 y 234 µmol q m-2 s-1, around 110 µg ml-1. This Synechococcus strain shows a great potential for the production of enriched biomass with high commercial value metabolites. Rev. Biol. Trop. 56 (2): 421-429. Epub 2008 June 30.

Las cianobacterias de ambientes salinos presentan una versatilidad metabólica inducida por los cambios de salinidad, temperatura e irradiancia, durante los períodos de sequía y lluvias. Por ello es importante la búsqueda en estos ambientes de cianobacterias con potencial biotecnológico para la producción de metabolitos de interés farmacéutico e industrial. Se reporta el crecimiento, masa seca y producción de metabolitos de la cianobacteria Synechococcus sp. MOF-03 en función de la irradiancia (78, 156 y 234 µmol q m-2 s-1). Los cultivos discontinuos por triplicado, fueron mantenidos con aireación constante, fotoperiodo 12:12 h, 30 ±2ºC y a 35‰. Los máximos valores de proteínas, carbohidratos y lípidos de 530.19 ±11.16, 408.94 ±4.27 y 56.20 ±1.17 µg ml-1 respectivamente, fueron obtenidos a 78 µmol q m-2 s-1. Los pigmentos, analizados por HPLC, mostraron los máximos a 78 µmol q m-2 s-1 para clorofila a con 7.72 ±0.16 µg ml-1; y a 234 µmol q m-2 s-1 para ß-caroteno y zeaxantina con 0.70 ±0.01 and 0.67 ±0.05 µg ml-1. La relación clorofila a:ß-caroteno disminuyó de 17.15 hasta 6.91 a 78 y 234 µmol q m-2 s-1; mientras que la relación ß-caroteno:zeaxantina se mantuvo sin cambios entre 78 y 156 µmol q m-2 s-1, con cerca de 1.21 y disminuyó a 234 µmol q m-2 s-1 a 1.04. La cianobacteria produjo la mayor densidad celular y masa seca a 156 µmol q m-2 s-1, con 406.13 ±21.74 x106 cel ml-1 y 1.49 ±0.11 mg ml-1 respectivamente. La producción de exopolisacáridos se mantuvo alrededor de 110 µg ml-1 entre 156 y 234 µmol q m-2 s-1. Así, esta cepa de Synechococcus muestra un gran potencial para la producción de biomasa enriquecida con metabolitos de alto valor comercial.

[Growth and metabolite production of the marine cyanobacterium Synechococcus sp. (Chroococcales) in function to irradiance]. / Crecimiento y producción de metabolitos de la cianobacteria marina Synechococcus sp. (Chroococcales) en función de la irradiancia.

Changes in salinity, temperature and irradiance during wet and dry seasons have induced metabolic versatility in cyanobacteria from saline environments. Cyanobacteria from these environments have biotechnological potential for the production of metabolites with pharmaceutical and industrial interest. We studied the growth, dry mass and metabolite production of the cyanobacterium Synechococcus sp. MOF-03 in function of irradiance (78, 156 and 234 micromol q m(-2) s(-1)). All batch cultures were maintained by triplicate in constant aeration, 12:12 h photoperiod, 30 +/- 2 degrees C and 35% per hundred. Maximum values of protein, carbohydrates and lipids, of 530.19 +/- 11.16, 408.94 +/- 4.27 and 56.20 +/- 1.17 microg ml(-1), respectively, were achieved at 78 micromol q m(-2) s(-1). Pigments, analyzed by HPLC, showed maximum values at 78 micromol q m(-2) s(-1) for chlorophyll a with 7.72 +/- 0.16 microg ml(-1), and at 234 micromol q m(-2) s(-1) for beta-carotene and zeaxanthin with 0.70 +/- 0.01 and 0.67 +/- 0.05 microg ml(-1). Chlorophyll a:beta-carotene ratio decreased from 17.15 to 6.91 at 78 and 234 micromol q m(-2) s(-'1); whereas beta-carotene:zeaxanthin ratio showed no changes between 78 and 156 micromol q m(-2) s(-1), around 1.21, and decreased at 234 micromol q m(-2) s(-1), to 1.04. Also, this cyanobacterium produced the greatest cell density and dry mass at 156 micromol q m(-2) s(-1), with 406.13 +/- 21.74 x l0(6) cell ml(-1) and 1.49 +/- 0.11 mg ml(-1), respectively. Exopolysaccharide production was stable between 156 y 234 micromol q m(-2) s(-1), around 110 microg ml(-1). This Synechococcus strain shows a great potential for the production of enriched biomass with high commercial value metabolites.

The effect of temperature and irradiance on the growth and carotenogenic capacity of seven strains of Dunaliella salina (Chlorophyta) cultivated under laboratory conditions.

The carotenogenic microalga Dunaliella salina is cultivated as a natural source of beta-carotene. The 9-cis isomer of beta-carotene is found only in natural sources having commercial advantages over the all-trans isomer due to its high liposolubility and antioxidant power. High irradiance appears to stimulate specifically all-trans beta-carotene accumulation in D. salina, whereas low temperature apparently elicits a-carotene and 9-cis betacarotene production. We studied the effect of temperature and irradiance on the growth and the carotenogenesis of three Chilean (CONC-001, CONC-006 and CONC-007) and four non-Chilean (from Mexico, China, Australia and Israel) strains of D. salina cultivated under two photon flux densities (40 and 110 micromol photons x m(-2) x s(-1)) and two temperatures (15 and 26 degrees C). The Chilean strain CONC-001 and all of the non-Chilean strains exhibited the highest growth rates and the maximum cell densities, whereas the Chilean strains CONC-006 and CONC-007 showed the lowest values in both parameters. The Australian strain showed the highest accumulation of total carotenoids per unit volume (40.7 mg x L(-1)), whereas the Chilean strains CONC-006 and CONC-007, the only ones isolated from Andean environments, yielded the highest amounts of carotenoids per cell (61.1 and 92.4 pg x cell(-1), respectively). Temperature was found to be more effective than irradiance in changing the qualitative and quantitative carotenoids composition. The Chilean strains accumulated 3.5-fold more alpha-carotene than the non-Chilean strains when exposed to 15 degrees C and, unlike the non-Chilean strains, also accumulated this pigment at 26 degrees C. The 9-cis/all-trans beta-carotene ratio was > 1.0 in all treatments for all strains, and the values were not greatly influenced by either temperature or photon flux density. Physiological and biotechnological implications of these results are discussed.

Astaxanthin hyperproduction by Phaffia rhodozyma (now Xanthophyllomyces dendrorhous) with raw coconut milk as sole source of energy.

Natural carbon sources, such as those present in cane sugar molasses and grape juice, promote the synthesis of astaxanthin in different Phaffia rhodozyma yeasts. One of these, coconut milk, has a very rich nutrient composition. The aim of this work was to investigate the utility of coconut milk as sole source of energy for astaxanthin pigment production by P. rhodozyma strains. Currently, coconut pulp is widely used in industrial processes in Mexico for the production of shampoos, candies, food, etc. However, coconut milk is a waste product. We show that coconut milk enhances astaxanthin production. The fermentation yielded 850 microg/g yeast with the NRRL-10921 wild-type strain and 1850 microg/g yeast with the mutated R1 strain. Production was better than reported results employing other natural carbon sources.

Study of the expression of carotenoid biosynthesis genes in wild-type and deregulated strains of Xanthophyllomyces dendrorhous (Ex.: Phaffia rhodozyma).

The expression, at the mRNA level, of carotenoid biosynthetic genes from Xanthophyllomyces dendrorhous was studied by RT-PCR. The experimental conditions for the RT-PCR assay were standardized to quantify the relative transcript levels of idi, crtE, crtYB and crtI genes. This work attempted to correlate astaxanthin production with the transcript levels of carotenogenic genes in a wild-type strain (UCD 67-385) and two overproducer deregulated strains (atxS1 and atxS2). At 3 day cultures, the wild-type strain contained higher transcript levels from the crtE and crtYB genes on minimal medium than on rich medium. Similarly, carotenoid production was higher on minimal medium than on rich medium. However, carotenoid production in the atxS1 and atxS2 strains was not correlated with the transcript level of carotenogenic genes under the same experimental conditions. This result suggests that there is not a linear relationship between carotenogenic transcript levels and carotenoid biosynthesis.

Optimization of biomass, total carotenoids and astaxanthin production in Haematococcus pluvialis Flotow strain Steptoe (Nevada, USA) under laboratory conditions.

The microalga Haematococcus pluvialis Flotow is one of the natural sources of astaxanthin, a pigment widely used in salmon feed. This study was made to discover optimal conditions for biomass and astaxanthin production in H. pluvialis from Steptoe, Nevada (USA), cultured in batch mode. Growth was carried out under autotrophic (with NaNO3, NH4Cl and urea) and mixotrophic conditions (with 4, 8, 12 mM sodium acetate) under two photon flux densities (PFD) (35 and 85 mumol m-2 s-1). The carotenogenesis was induced by 1) addition of NaCl (0.2 and 0.8%), 2) N-deprivation and 3) high PFD (150 mumol m-2 s-1). Total carotenoids were estimated by spectrophotometry and total astaxanthin by HPLC. Ammonium chloride was the best N-source for growth (k = 0.7 div day-1, 228-258 mg l-1 and 2.0 x 10(5)-2.5 x 10(5) cells ml-1 at both PFD, respectively). With increasing acetate concentration, a slight increment in growth occurred only at 85 mumol m-2 s-1. Light was the best inductive carotenogenic factor, and the highest carotenoid production (4.9 mg l-1, 25.0 pg cell-1) was obtained in cultures pre-grown in nitrate at low light. The NaCl caused an increase in carotenoid content per cell at increasing salt concentrations, but resulted in a high cell mortality and did not produce any increment in carotenoid content per volume compared to cultures grown at 150 mumol m-2 s-1. The highest carotenoid content per cell (22 pg) and astaxanthin content per dry weight (10.3 mg g-1) (1% w/w) were obtained at 85 mumol m-2 s-1 with 0.8% NaCl.

Optimization of astaxanthin production by Phaffia rhodozyma through factorial design and response surface methodology.

Sequential methodology based on the application of three types of experimental designs was used to optimize the astaxanthin production of the mutant strain 25-2 of Phaffia rhodozyma in shake flask cultures. The first design employed was a factorial design 2(5), where the factors studied were: pH, temperature, percent of inoculum, carbon and nitrogen concentrations, each one at two levels. This design was performed in two medium types: rich YM medium and minimal medium, based on date juice (Yucca medium). With this first design the most important factors were determined (carbon concentration and temperature) that were used in the second experimental strategy: the method of steepest ascent was applied in order to rapidly approach the optimum. Finally, a second-order response surface design was applied using temperature and carbon concentration as factors. The optimal conditions stimulating the highest astaxanthin production were: 19.7 degrees C temperature; 11.25 g l(-1) carbon concentration; 6.0 pH; 5% inoculum and 0.5 g l(-1) nitrogen concentration. Under these conditions the astaxanthin production was 8100 microg l(-1), 92% higher than the production under the initial conditions.

Complementation analysis with new genetic markers in Phaffia rhodozyma.

Isolation and characterization of auxotrophic mutants from wild-type and astaxanthin mutant strains of Phaffia rhodozyma is described. Differences in survival were observed when u.v. irradiation of P. rhodozyma wild-type and astaxanthin mutant strains were incubated in the dark or exposed to photoreactivating light. Ultra-violet mutagenesis was not effective to produce auxotrophic mutants in this yeast. Auxotrophic mutants were obtained with high efficiency through a nystatin enrichment procedure after a N-methyl-N'-nitro-N-nitrosoguanidine (NGT) mutagenic treatment with a 0.12% survivor level. Stringent mutagenetic conditions were needed to obtain P. rhodozyma auxotrophs. The most frequent mutants were ade- and met- in a rather narrow auxotroph spectrum. These results may be associated with a possible diploid condition of this yeast. The high number of adenine auxotrophs obtained in relation to other auxotrophic mutants suggests the possibility of some degree of heterozygosity in the wild-type strain UCD 67-385.

Genetic transformation of astaxanthin mutants of Phaffia rhodozyma.

Stable red astaxanthin-producing transformants were obtained after genetic transformation of two Phaffia rhodozyma mutants. A yellow mutant, accumulating beta-carotene, and an albino mutant, accumulating phytoene, from P. rhodozyma were transformed using a genomic library of wild-type strain UCD 67-385 in the pBluescript vector. Hybridization assays, using the pBluescript DNA as a radioactive probe, indicate integration of vector sequences into the genome of the transformants. Transformants DNA was digested with restriction endonucleases, ligated with T4 DNA ligase and then used to transform E. coli. Ampicillin resistant plasmids, containing 0.1, 0.2, and 2.5 kb DNA inserts of P. rhodozyma, were rescued from the yeast red transformants. The molecular analysis indicate that transformation has occurred by an integration event of donor DNA into the genome of the host strains.

[Microbial sources of pigments]. / Fuentes microbianas de pigmentos.

Pigments from natural sources has been obtained since long time ago, and their interest has increased due to the toxicity problems caused by those of synthetic origin. In this way the pigments from microbial sources are a good alternative. Some of more important natural pigments, are the carotenoids, flavonoids (anthocyanins) and some tetrapirroles (chloropyls, phycobilliproteins). Another group less important are the betalains and quinones. The carotenoids are molecules formed by isoprenoids units and the most important used as colorant are the alpha and beta carotene which are precursors of vitamin A, and some xantophylls as astaxanthin. The pigment more used in the industry is the beta-carotene which is obtained from some microalgae and cyanobacteria. The astaxanthin another important carotenoid is a red pigment of great commercial value, and it is used in the pharmaceutical feed and acuaculture industries. This pigments is mainly obtained from Phaffia rhodozyma and Haematococcus pluvialis and other organisms. The phycobilliproteins obtained from cyanobacteria and some group of algae, have recently been increased on the food industries. In the last years it has been used as fluorescent marker in biochemical assays. Our research group have carried out studies about the factors that improve the production of these pigments obtained from different microbial species as well as the methods for their extraction and application.