Browsing by Subject "Yarrowia lipolytica"
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Item Engineering Yarrowia lipolytica for high lipid production(2014-12) Liu, Leqian; Hal, Alper S.; Appling, Dean; Contreras, Lydia; Iyer, Vishwanath; Whiteley, MarvinAmong potential value-added fuels and chemicals, fatty acid-based chemicals are important due to their wide use in industrial processes and in daily life. Fatty acids produced from microbial systems could provide a sustainable supply to replace the current costly and unsustainable process using plant oil or animal fat. The oleaginous yeast Yarrowia lipolytica naturally possesses moderate lipid production capacity and grows on different kinds of biomass and organic waste. However, fatty acid production from native, un-engineered strains is not economically viable. Therefore, this work develops strategies inspired from synthetic biology and metabolic engineering to expand the engineering potential of Y. lipolytica — helping to establish this organism as a premier platform for industrial-level, high lipid production as well as providing a platform for uncovering novel understanding of lipogenesis. To do so, first, novel synthetic promoters and high expression plasmid were necessary to achieve the ability to tune gene expression levels inside the cell. We developed a hybrid promoter engineering strategy to create a promoter library exhibiting a range of more than 400-fold in terms of mRNA levels as well as engineered plasmids with regulated centromeric function to achieve a 2.7 fold expression range. Next, a rational and evolutionary metabolic engineering approach was coupled with genomic and transcriptomic studies to both engineer and understand underlying lipogenesis in this organism. Through the engineering efforts, we successfully increased the lipid production titer to over 40 g/L in bioreactor as well as identified novel lipogenic enhancers and mechanisms. In addition, we identified and characterized a mutant mga2 protein with superior lipogenesis enhancing capacity, which can regulate fatty acid desaturation and carbon flux inside the cells. Collectively, these studies have facilitated the utilization of Y. lipolytica as an industrially relevant microbial lipid production platform and supplied novel understanding of its lipogenesis process. The methods and concepts developed here can also be adapted to other oleaginous microbes and serve as a template for enabling value-added chemical production in other nonconventional organism.Item Harnessing Yarrowia lipolytica’s potential as a lipid and alkane production platform(2013-08) Blazeck, John James; Alper, Hal S.; Contreras, Lydia; Ellington, Andrew; Georgiou, George; Maynard, JenniferEngineering cellular phenotype can enable the in vivo synthesis of renewable fuels, industrial precursors, and pharmaceuticals. Achieving economic viability requires the use of a cellular platform that generates high titers independent of fermentation condition, through either native or imported biosynthetic metabolism. While lacking fully developed genetic tools, the oleaginous yeast Yarrowia lipolytica has the native capacity to produce large titers of lipids and citric acid cycle intermediates. However, unlocking this biosynthetic capacity requires complete rewiring of native metabolism. To this end, this work focuses on the development and engineering of the yeast Y. lipolytica to rewire native metabolism and enable the production of lipids, alkanes, and itaconic acid. Precise control of gene expression is a requisite to enable metabolic and pathway engineering applications for any host organism. However, Y. lipolytica lacks promoter elements strong enough to manipulate intracellular metabolism. Thus, we utilized a hybrid promoter engineering approach to produce libraries of high-expressing, tunable promoters, seven-fold stronger than promoters previously characterized in Y. lipolytica 1,2. We successfully applied this approach to Saccharomyces cerevisiae, expanding transcriptional capacity of the strongest constitutive to highlight our hybrid approach as a generalizable method to increase expression capacity in eukaryotic organisms 3. We utilized our novel Y. lipolytica hybrid promoters to drive intracellular metabolism towards lipid production and to overexpress heterologous enzymes that enable alkane and itaconic acid production. Specifically, we implemented a global rewiring of Y. lipolytica’s native metabolism to increase lipogenesis more than sixty fold to 25.3g/L (the highest lipid production ever reported) and generated cells nearly 90% lipid content. We further expressed a lipoxygenase enzyme to catalyze the novel microbial production of the short-chain n-alkane, pentane. Finally, we exploited Y. lipolytica’s capacity to accumulate citric acid cycle intermediates by expressing a heterologous cis-aconitic acid decarboxylase enzyme to produce itaconic acid. Increasing substrate availability through media optimization and genomic engineering increased pentane and itaconic acid production threefold and eightfold, respectively 4. Collectively, these studies have facilitated the utilization of Y. lipolytica as an industrially relevant microbial platform, and represent a generic approach towards enabling biosynthetic control in microbial hosts will ill-defined gene expression technology.