The availability of the omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) is currently limited because they are produced mainly by marine fisheries that cannot keep pace with the demands of the growing market for these products. A sustainable non-animal source of EPA and DHA is needed. Metabolic engineering of the oleaginous yeast Yarrowia lipolytica resulted in a strain that produced EPA at 15% of dry cell weight. The engineered yeast lipid comprises EPA at 56.6% and saturated fatty acids at less than 5% by weight, which are the highest and the lowest percentages, respectively, among known EPA sources. Inactivation of the peroxisome biogenesis gene PEX10 was crucial in obtaining high EPA yields and may increase the yields of other commercially desirable lipid-related products. This technology platform enables the production of lipids with tailored fatty acid compositions and provides a sustainable source of EPA.

The yeast Saccharomyces cerevisiae senses glucose, its preferred carbon source, through multiple signal transduction pathways. In one pathway, glucose represses the expression of many genes through the Mig1 transcriptional repressor, which is regulated by the Snf1 protein kinase. In another pathway, glucose induces the expression of HXT genes encoding glucose transporters through two glucose sensors on the cell surface that generate an intracellular signal that affects function of the Rgt1 transcription factor. We profiled the yeast transcriptome to determine the range of genes targeted by this second pathway. Candidate target genes were verified by testing for Rgt1 binding to their promoters by chromatin immunoprecipitation and by measuring the regulation of the expression of promoter lacZ fusions. Relatively few genes could be validated as targets of this pathway, suggesting that this pathway is primarily dedicated to regulating the expression of HXT genes. Among the genes regulated by this glucose signaling pathway are several genes involved in the glucose induction and glucose repression pathways. The Snf3/Rgt2-Rgt1 glucose induction pathway contributes to glucose repression by inducing the transcription of MIG2, which encodes a repressor of glucose-repressed genes, and regulates itself by inducing the expression of STD1, which encodes a regulator of the Rgt1 transcription factor. The Snf1-Mig1 glucose repression pathway contributes to glucose induction by repressing the expression of SNF3 and MTH1, which encodes another regulator of Rgt1, and also regulates itself by repressing the transcription of MIG1. Thus, these two glucose signaling pathways are intertwined in a regulatory network that serves to integrate the different glucose signals operating in these two pathways.

The expression pattern of 1,529 yeast genes in response to sulfometuron methyl (SM) was analyzed by DNA microarray technology. SM, a potent herbicide, inhibits acetolactate synthase, a branched-chain amino acid biosynthetic enzyme. Exposure of yeast cells to 0.2 microg/ml SM resulted in 40% growth inhibition, a Gcn4p-mediated induction of genes involved in amino acid and cofactor biosynthesis, and starvation response. The accumulation of intermediates led to the induction of stress response genes and the repression of genes involved in carbohydrate metabolism, nucleotide biosynthesis, and sulfur assimilation. Extended exposure to SM led to a relaxation of the initial response and induction of sugar transporter and ergosterol biosynthetic genes, as well as repression of histone and lipid metabolic genes. Exposure to 5 microg/ml SM resulted in >98% growth inhibition and stimulated a similar initial expression change, but with no relaxation after extended exposure. Instead, more stress response and DNA damage repair genes become induced, suggesting a serious cellular consequence. Other salient features of metabolic regulation, such as the coordinated expression of cofactor biosynthetic genes with amino acid biosynthetic ones, were evident from our data. A potential link between SM sensitivity and ergosterol metabolism was uncovered by expression profiling and confirmed by genetic analysis.

Abstract. We previously identified a protein (p67) in the yeast, Saccharomyces cerevisiae, that specifically recognizes nuclear localization sequences. We report here the partial purification of p67, and the isolation, sequencing, and disruption of the gene (NSR1) encoding this protein, p67 was purified using an affinity column conjugated with a peptide containing the histone H2B nuclear localization sequence from yeast. Using antibodies against p67 we have cloned the gene for this protein. The protein encoded by the NSR1 gene recognizes the wild-type H2B nuclear localization sequence, but does not recognize a mutant H2B sequence that is incompetent for nuclear localization in vivo. Interestingly, the NSRI protein has two RNA recognition motifs, as well as an acidic NH2 terminus containing a series of serine clusters, and a basic COOH terminus containing arg-gly repeats. We have confirmed the nuclear localization of p67 by immunofluorescence and found that a restricted portion of the nucleus is highlighted. We have also shown that NSRI (p67) is required for normal cell growth.

No abstract

Oleaginous yeast Yarrowia lipolytica is an important host for the production of lipidderived compounds or heterologous proteins. Selection of strong promoters and effective expression systems is critical for heterologous protein secretion. To search for a strong promoter in Y. lipolytica, activities of FBA1, TDH1 and GPM1 promoters were compared to that of TEF1 promoter by constructing GUS reporter fusions. The FBA1 promoter activity was 2.2 and 5.5 times stronger than the TDH1 and GPM1 promoters, respectively. The FBA1 IN promoter (FBA1 sequence of À826 to +169) containing an intron (+64 to +165) showed five-fold higher expression than the FBA1 promoter (À831 to À1). The transcriptional enhancement by the 5′-region within the FBA1 gene was confirmed by GPM1::FBA1 chimeric promoter construction. Using the strong FBA1 IN promoter, four different S. cerevisiae SUC2 expression cassettes were tested for the SUC + phenotype in Y. lipolytica. Functional invertase secretion was facilitated by the Xpr2 prepro-region with an additional 13 amino acids of mature Xpr2, or by the native Suc2 signal sequence. However, these two secretory signals in tandem, or the mature Suc2 with no secretory signal, did not direct secretion of functional invertase. Unlike previously reported Y. lipolytica SUC + strains, our engineered stains secreted most of invertase into the medium. Copyright

Abstract. We have identified a gene (NPI46) encoding a new prolyl cis-trans isomerase within the nucleolus of the yeast Saccharomyces cerevisiae. The protein encoded by NPI46 was originally found by us in a search for proteins that recognize nuclear localization sequences (NLSs) in vitro. Thus, NPI46 binds to affinity columns that contain a wild-type histone H2B NLS but not a mutant H2B NLS that is incompetent for nuclear localization in vivo. NPI46 has two domains, a highly charged NH2 terminus similar to two other mammalian nucleolar proteins, nucleolin and Noppl40, and a COOH terminus with 45 % homology to a family of mammalian and yeast proline isomerases. NPI46 is capable of catalyzing the prolyl cistrans isomerization of two small synthetic peptides, succinyl-Ala-Leu-Pro-Phe-p-nitroanilide and succinylAla-Ala-Pro-Phe-p-nitroanilide, as measured by a chymotrypsin-coupled spectrophotometric assay. By indirect immunofluorescence we have shown that NPI46 is a nucleolar protein. NPI46 is not essential for cell viability.

ω-3 fatty acid desaturase is a key enzyme for the biosynthesis of ω-3 polyunsaturated fatty acids via the oxidative desaturase/elongase pathways. Here we report the identification of three ω-3 desaturases from oomycetes, Pythium aphanidermatum, Phytophthora sojae, and Phytophthora ramorum. These new ω-3 desaturases share 55 % identity at the amino acid level with the known Δ-17 desaturase of Saprolegnia diclina, and about 31 % identity with the bifunctional Δ-12/Δ-15 desaturase of Fusarium monoliforme. The three enzymes were expressed in either wild-type or codon optimized form in an engineered arachidonic acid producing strain of Yarrowia lipolytica to study their activity and substrate specificity. All three were able to convert the ω-6 arachidonic acid to the ω-3 eicosapentanoic acid, with a substrate conversion efficiency of 54–65 %. These enzymes have a broad ω-6 fatty acid substrate spectrum, including both C18 and C20 ω-6 fatty acids although they prefer the C20 substrates, and have strong Δ-17 desaturase activity but weaker Δ-15 desaturase activity. Thus, they belong to the Δ-17 desaturase class. Unlike the previously identified bifunctional Δ-12/Δ-15 desaturase from F. monoliforme, they lack Δ-12 desaturase activity. The newly identified Δ-17 desaturases could use fatty acids in both acyl-CoA and phospholipid fraction as substrates. The identification of these Δ-17 desaturases provides a set of powerful new tools for genetic engineering of microbes and plants to produce ω-3 fatty acids, such as eicosapentanoic acid and docosahexanoic acid, at high levels.