Project description:Total RNA versus genomic DNA hybridization on custom arrays designed for all Yarrowia lipolytica genes Overall design: Total RNA was collected in mid-log phase from Yarrowia lipolytica cells grown in rich medium (abbreviated CM, in house recipe). RNA was then converted to cDNA, Cy3-labeled and hybridized competitively against Cy5 labeled genomic DNA from Yarrowia lipolytica.
Project description:BACKGROUND:Yarrowia lipolytica is an ascomycete yeast used in biotechnological research for its abilities to secrete high concentrations of proteins and accumulate lipids. Genetic tools have been made in a variety of backgrounds with varying similarity to a comprehensively sequenced strain. RESULTS:We have developed a set of genetic and molecular tools in order to expand capabilities of Y. lipolytica for both biological research and industrial bioengineering applications. In this work, we generated a set of isogenic auxotrophic strains with decreased non-homologous end joining for targeted DNA incorporation. Genome sequencing, assembly, and annotation of this genetic background uncovers previously unidentified genes in Y. lipolytica. To complement these strains, we constructed plasmids with Y. lipolytica-optimized superfolder GFP for targeted overexpression and fluorescent tagging. We used these tools to build the "Yarrowia lipolytica Cell Atlas," a collection of strains with endogenous fluorescently tagged organelles in the same genetic background, in order to define organelle morphology in live cells. CONCLUSIONS:These molecular and isogenetic tools are useful for live assessment of organelle-specific protein expression, and for localization of lipid biosynthetic enzymes or other proteins in Y. lipolytica. This work provides the Yarrowia community with tools for cell biology and metabolism research in Y. lipolytica for further development of biofuels and natural products.
Project description:Total RNA versus genomic DNA hybridization on custom arrays designed for all Yarrowia lipolytica genes Total RNA was collected in mid-log phase from Yarrowia lipolytica cells grown in rich medium (abbreviated CM, in house recipe). RNA was then converted to cDNA, Cy3-labeled and hybridized competitively against Cy5 labeled genomic DNA from Yarrowia lipolytica.
Project description:Investigation of whole genome gene expression level changes in a Yarrowia lipolytica Y4184 snf1 mutant, compared to the Y4184U+. The Y4184 is an engineered strain to produce eicosapentaenoic acid (EPA) via expression of a Δ9 elongase/Δ8 desaturase pathway, and is derived from Yarrowia lipolytica ATCC#20362. A six chip study using total RNA recovered from three separate cultures of Yarrowia lipolytica Y4184Ura+ strains and three separate cultures of Y4184 snf1 mutant strains in which YlSNF1 gene is deleted entirely. Each chip measures the expression level of 6708 genes from Yarrowia lipolytica ATCC#20362 with fourteen 24-mer probe pairs (PM/MM) per gene, with three-fold technical redundancy.
Project description:Enzymes of the non-conventional yeast Yarrowia lipolytica seem to be tailor-made for the conversion of lipophilic substrates. Herein, we cloned and overexpressed the Zn-dependent alcohol dehydrogenase ADH2 from Yarrowia lipolytica in Escherichia coli. The purified enzyme was characterized in vitro. The substrate scope for YlADH2 mediated oxidation and reduction was investigated spectrophotometrically and the enzyme showed a broader substrate range than its homolog from Saccharomyces cerevisiae. A preference for secondary compared to primary alcohols in oxidation direction was observed for YlADH2. 2-Octanone was investigated in reduction mode in detail. Remarkably, YlADH2 displays perfect (S)-selectivity and together with a highly (R)-selective short chain dehydrogenase/ reductase from Yarrowia lipolytica it is possible to access both enantiomers of 2-octanol in >99% ee with Yarrowia lipolytica oxidoreductases.
Project description:To develop a direct microbial sugar conversion platform for the production of lipids, drop-in fuels and chemicals from cellulosic biomass substrate, we chose Yarrowia lipolytica as a viable demonstration strain. Y. lipolytica is known to accumulate lipids intracellularly and is capable of metabolizing sugars to produce lipids; however, it lacks the lignocellulose-degrading enzymes needed to break down biomass directly. While research is continuing on the development of a Y. lipolytica strain able to degrade cellulose, in this study, we present successful expression of several xylanases in Y. lipolytica. The XynII and XlnD expressing Yarrowia strains exhibited an ability to grow on xylan mineral plates. This was shown by Congo Red staining of halo zones on xylan mineral plates. Enzymatic activity tests further demonstrated active expression of XynII and XlnD in Y. lipolytica. Furthermore, synergistic action in converting xylan to xylose was observed when XlnD acted in concert with XynII. The successful expression of these xylanases in Yarrowia further advances us toward our goal to develop a direct microbial conversion process using this organism.
Project description:Background:Yarrowia lipolytica, a non-traditional oil yeast, has been widely used as a platform for lipid production. However, the production of other chemicals such as terpenoids in engineered Y. lipolytica is still low. ?-Farnesene, a sesquiterpene, can be used in medicine, bioenergy and other fields, and has very high economic value. Here, we used ?-farnesene as an example to explore the potential of Y. lipolytica for terpenoid production. Results:We constructed libraries of strains overexpressing mevalonate pathway and ?-farnesene synthase genes by non-homologous end-joining (NHEJ) mediated integration into the Y. lipolytica chromosome. First, a mevalonate overproduction strain was selected by overexpressing relevant genes and changing the cofactor specificity. Based on this strain, the downstream ?-farnesene synthesis pathway was overexpressed by iterative integration. Culture conditions were also optimized. A strain that produced 25.55 g/L ?-farnesene was obtained. This is the highest terpenoid titer reported in Y. lipolytica. Conclusions:Yarrowia lipolytica is a potentially valuable species for terpenoid production, and NHEJ-mediated modular integration is effective for expression library construction and screening of high-producer strains.
Project description:The yeast Yarrowia lipolytica Y-2 shown the capability to degrade OTA by the intracellular enzymes. However, the enzymes which risponsible for the degradation process was unkonown. Transcriptome change in response to mycotoxin OTA was analyzed. The molecular mechanism of Yarrowia lipolytica Y-2 withstand OTA was revealed. Overall design: Each two samples were analyzed , the yeast Yarrowia lipolytica Y-2 amending with ochratoxin A and the control which was not amending with OTA
Project description:Here, we report the genome sequence of the oleaginous yeast Yarrowia lipolytica H222. De novo genome assembly shows three main chromosomal rearrangements compared to that of strain E150/CLIB122. This genomic resource will help integrate intraspecies diversity into synthetic biology projects that utilize Yarrowia as a biotechnological chassis for value-added chemical productions.
Project description:Second part of the time-course transcriptomic profilling of the oleaginous yeast Yarrowia lipolytica, obtained during a controlled decellerostat (D-stat) setup. A nitrogen limitation was applied during the course of the D-stat to initiate and control de novo biolipid synthesis. Overall design: Yarrowia lipolytica Agilent microarray, one-color staining. Each time sample was spotted in triplicate (except for sample 26, for which a technical replicate of replicate r1 was performed).