Browsing by Subject "carotenoids"
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Item Adaptive Evolution for the Study of Complex Phenotypes in Microbial Systems(2013-07-24) Reyes Barrios, Luis HumbertoMicrobial-based industrial production has experienced a revolutionary development in the last decades as chemical industry has shifted its focus towards more sustain- able production of fuels, building blocks for materials, polymers, chemicals, etc. The strain engineering and optimization programs for industrially relevant phenotypes tackle three challenges for increased production: optimization of titer, productivity, and yield. The yield of production is function of the robustness of the microbe, generally associated with complex phenotypes. The poor understanding of complex phenotypes associated with increased production poses a challenge for the rational design of strains of more robust microbial producers. Laboratory adaptive evolution is a strain engineering technique used to provide fundamental biological insight through observation of the evolutionary process, in order to uncover molecular determinants associated with the desired phenotype. In this dissertation, the development of different methodologies to study complex phenotypes in microbial systems using laboratory adaptive evolution is described. Several limitations imposed for the nature of the technique were discussed and tackled. Three different cases were studied. Initially, the n-butanol tolerance in Escherichia coli was studied in order to illustrate the effect of clonal interference in microbial systems propagated under selective pressure of an individual stressor. The methodology called Visualizing Evolution in Real Time (VERT) was developed, to aid in mapping out the adaptive landscape of n-butanol tolerance, allowing the uncovering of divergent mechanisms of tolerance. A second case involves the study of clonal interference of microbial systems propagated under several stressors. Using VERT, Saccharomyces cerevisiae was evolved in presence of hydrolysates of lignocellulosic biomass. Isolated mutants showed differential fitness advantage to individual inhibitors present in the hydrolysates; however, some mutants exhibited increased tolerance to hydrolysates, but not to individual stressors. Finally, dealing with the problem of using adaptive evolution to increase production of secondary metabolites, an evolutionary strategy was successfully designed and applied in S. cerevisiae, to increase the production of carotenoids in a short-term experiment. Molecular mechanisms for increased carotenoids production in isolates were identified.Item Environmental and genetic strategies to improve carotenoids and quality in watermelon(Texas A&M University, 2007-04-25) Bang, Hae JeenThe evaluation of environmental and genotypic effects on fruit physical and chemical characteristics enables assessment of the feasibility of selecting diploid and/or triploid cultivars for either specific or more diverse locations. Isolation and characterization of genes encoding enzymes in the carotenoid biosynthetic pathway provides fundamental genetic information which can facilitate breeding of watermelon cultivars having desirable flesh colors and enhanced beneficial carotenoids. For the environmental studies, the effects of deficit irrigation on lycopene content, total soluble solids, firmness, and yield of diploid and triploid watermelon were evaluated in different locations and growing seasons. Irrigation regimes were 1.0 evapotranspiration (ET), 0.75 ET, and 0.5 ET. To investigate if there is a consistent response in cultivars across diverse locations, studies were conducted in three distinct Texas regions. Deficit irrigation reduced total marketable yield, and increased the yield of small fruits. Location and irrigation regimes had major influences on yield. Soluble solids content increased with deficit irrigation at 0.5 ET in triploids, but not in diploids. Flesh firmness also increased in triploids compared to diploids. Lycopene content increased with maturity at all irrigation regimes and cultivars. This work confirms that deficit irrigation directly reduces yield, but does not reduce lycopene and fruit quality of the triploids used in this study. From the genetic studies, a total of eight genes encoding enzymes in the carotenoid biosynthetic pathway were isolated and characterized. Two members of the phytoene synthase (PSY) gene family were identified; PSY-A was expressed in all type of tissues, but PSY-B transcript was detected only in ovary, leaf, and root tissues. Gene expression of carotenoid isomerase (CRTISO) was not detected in salmon yellow. A color inheritance study of watermelon flesh indicated that a single gene might determine color difference between canary yellow and red without an inhibitory effect. A cleaved amplified polymorphic sequence (CAPS) marker developed from the SNP marker tagging two different lycopene ????-cyclase (LCYB) alleles cosegregated perfectly with color phenotypes. It was concluded that color determination may be due to a reduced activity of LCYB enzyme in red, whereby a phenylalanine is conserved among canary yellow and valine is conserved among red watermelon.Item Screening potato genotypes for antioxidant activity, identification of the responsible compounds, and differentiating Russet Norkotah strains using AFLP and microsatellite marker analysis(Texas A&M University, 2005-02-17) Hale, Anna LouiseTotal antioxidant activity and total carotenoid levels were evaluated for more than 100 common potato (Solanum tuberosum, L.) cultivars grown in the United States, advanced breeding lines from several Western U.S. breeding programs, and 47 related, tuber-bearing species. An initial assessment of variability for antioxidant activity provided baseline information to be used for potential potato promotion and for the development of new varieties with greater human health benefits. Wide variability in antioxidant levels provided evidence of genetic control of this trait, indicating that it could be possible to breed for enhanced levels of antioxidant compounds in potato. Accessions, varieties, and advanced breeding lines identified in the broad screen as having high antioxidant activity and high total carotenoid levels, were fine screened via HPLC to determine specific phenolic and carotenoid compounds present in potato. The objective of the study was to identify parents for use in the Texas breeding program to develop potato varieties containing increased levels antioxidant compounds. In the broad screen for total antioxidant activity, the 47 related, tuber-bearing species showed a wider range of variability than the cultivated varieties and breeding lines. Based on the DPPH assay, antioxidant activity ranged from 103-648 uM trolox equivalents in the cultivated varieties and advanced breeding lines, while that of the wild species was 42-892. HPLC analysis revealed that the phenolic content of the species, and their cultivated counterparts, was primarily composed of caffeic and chlorogenic acids. Other phenolics identified were p-coumaric acid, rutin hydrate, vanillic acid, epicatechin, t-cinnamic acid, gallic acid, and salicylic acid. The highest phenolic content discovered in the accessions was five-fold higher than the highest of the cultivated genotypes. Carotenoid analysis revealed lutein in the accessions, but the yellow-flesh breeding lines were much higher in carotenoids. In addition to the work conducted on antioxidants, an attempt was made to separate intraclonal variants of the potato cultivar Russet Norkotah. Eleven microsatellite primers and 112 AFLP primer combinations failed to produce any reproducible polymorphisms. The inability to detect differences between the clones could be due to the tetraploid nature of the clones or epigenetic differences not detected by the procedures utilized in this study.